Pyrazole derivative, preparation method therefor and use thereof

Abstract

The present invention belongs to the technical field of pharmaceuticals, and relates to a pyrazole derivative, a preparation method therefor and a use thereof. Specifically, disclosed are a pyrazole derivative represented by general formula (I), a pharmaceutical composition containing said derivative, and a use thereof in the preparation of an antiparasitic drug. The structural formula (I) of the pyrazole derivative is: [formula I]. The pyrazole derivative of the present invention has significant effects against parasites, agricultural and forestry pests or environmental pests, and has broad application prospects.

Description

A pyrazole derivative and its preparation method and application Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a pyrazole derivative and a preparation method and application thereof. Background Art

[0002] Cryptosporidium belongs to the genus Cryptosporidium, a group of parasitic protozoa in the order Coccidiales, encompassing multiple species that infect humans and animals. Cryptosporidium parvum is a major cause of diarrhea in humans and commercial animals. Cryptosporidium is a gastrointestinal parasite primarily transmitted through the fecal-oral route, with infection occurring after ingestion of contaminated food or water. While cryptosporidiosis is generally subtle in immunocompetent humans and animals, it can cause severe diarrhea and even death in immunocompromised hosts.

[0003] To date, there is no specific drug for Cryptosporidium. Nitazoxanide, a highly effective, broad-spectrum antiparasitic, bacterial, and fungal agent for both animal and human infections, is the first and only drug approved by the U.S. Food and Drug Administration (FDA) for the treatment of Cryptosporidium. However, it is only indicated for the treatment of immunocompetent children or adults and is ineffective in immunocompromised patients. Therefore, the development of new and effective anti-Cryptosporidium drugs is of great practical significance.

[0004] Pyrazole is a five-membered heterocyclic compound with two nitrogen atoms at positions 1 and 2. It exists in three forms and serves as the core structure in numerous compounds with important agrochemical and pharmaceutical properties. It exhibits low toxicity, high efficacy, and diverse structural transformations. Fusion of pyrazole with other cyclic groups can generate a large number of lead compounds. Pyrazole derivatives have been reported to exhibit antimicrobial, antitumor, antiviral, antituberculosis, antimalarial, antidiabetic, antidiuretic, anticonvulsant, antidepressant, antiobesity, and antipyretic activities. However, the therapeutic efficacy of pyrazole derivatives against Cryptosporidium is still unclear.

[0005] Therefore, it is necessary to explore and develop new pyrazole derivatives for use as drugs against Cryptosporidium.

[0006] Summary of the Invention

[0007] The present invention aims to solve the technical problems in the prior art, design and screen a series of novel pyrazole derivatives through high-throughput screening, and the derivatives have a wide range of anti-Cryptosporidium activity in the screening of new anti-Cryptosporidium drugs, laying a solid foundation for the development of new and highly effective anti-Cryptosporidium drugs.

[0008] In order to achieve the above purpose, the specific technical solutions of the present invention are as follows:

[0009] In the first aspect of the present invention, a pyrazole derivative represented by general formula (I) is provided, and its structure is as follows:

[0010] Wherein, X1 and X2 are each independently selected from nitrogen, oxygen, and carbon;

[0011] R1 is selected from F, Cl, OCH3, CH3, COOCH3, R5-substituted benzene ring, R5-substituted 5-7 membered aromatic heterocycle or condensed ring, R5-substituted C3-C6 aliphatic ring or aliphatic heterocycle;

[0012] R5 is selected from one or any combination of F, Cl, OCH3, NH2, COONH2, COOH, COOCH3, CN, CF3, C1-C5 alkane or cycloalkane;

[0013] R2 is selected from H, Cl, F, CH3 or OCH3;

[0014] R3 and R4 are each independently selected from H, CN, CF3;

[0015] W is selected from CH2, R6 substituted or unsubstituted benzene ring, pyridine, pyrimidine, pyrazole, imidazole or W is absent;

[0016] R6 is selected from H, Cl, F, CH3, NH2 or CN;

[0017] Z is selected from R7 substituted or unsubstituted benzene ring, 5-7 membered aromatic heterocycle;

[0018] R7 is selected from H, Cl, F, CH3, OCH3, NH2, COONH2, CN or CF3.

[0019] In certain preferred embodiments, R1 is selected from an R5-substituted benzene ring, pyridine, pyrazole, imidazole, indole, quinoline, benzocycloheptane, cyclopropane, cyclobutane, cyclopentane, or cyclohexane.

[0020] In certain preferred embodiments, Z is selected from an R7-substituted benzene ring, pyridine, indole, quinoline, benzocyclohexane, or benzocycloheptane.

[0021] Preferably, the pyrazole derivative includes but is not limited to one of structures 1-35:

[0022] The compounds of the general formula (I) described herein may also exist in the form of their salts, which are converted in vivo into compounds of the general formula I. For example, within the scope of the present invention, the compounds of the present invention may be converted into pharmaceutically acceptable salts according to processes known in the art and used in the form of salts.

[0023] All tautomeric forms of the compounds of formula (I) of the present invention are included within the scope of the present invention. The compounds of the present invention may exist in specific geometric or stereoisomer forms. Additional asymmetric carbon atoms may exist in substituents such as alkyl groups, and all such isomers and mixtures thereof are included within the scope of the present invention.

[0024] In some preferred embodiments, the pharmaceutically acceptable salts include but are not limited to salts formed by the compound of formula (I) with the following acids: hydrochloric acid, sulfuric acid, phosphoric acid, hydrobromic acid, acetic acid, trifluoroacetic acid, pyruvic acid, citric acid, tartaric acid, lactic acid, maleic acid, benzenesulfonic acid, succinic acid, methanesulfonic acid, p-toluenesulfonic acid, fumaric acid, salicylic acid or phenylacetic acid.

[0025] In a second aspect, the present invention provides a method for synthesizing a pyrazole derivative represented by general formula (I), comprising the following steps:

[0026] Solvent I, base, and starting material 2 were added to starting material 1 in sequence, the temperature was raised to reflux, the temperature was lowered to room temperature, and the filter cake was washed twice with water. The filter cake was dried and recrystallized with solvent II. After drying, an off-white solid was obtained, namely, a pyrazole derivative (compound 1-35).

[0027] The solvent I is one or more of acetonitrile, toluene, methanol, ethanol, ethyl acetate, N,N-dimethylformamide, acetone, and 1,4-dioxane.

[0028] The base is one or more of triethylamine, N,N-diisopropylethylamine, pyridine, potassium carbonate, cesium carbonate, sodium hydroxide, potassium hydroxide, and lithium hydroxide.

[0029] The solvent II is one or more of methanol, ethanol and ethyl acetate.

[0030] The compounds of the general formula (I) of the present invention can be obtained by the above or similar preparation methods, and the corresponding starting materials can be selected according to the different substituents and the different positions of the substituents. It should be appreciated by those skilled in the art that the above routes are helpful for understanding the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the variables are defined as mentioned in the general formula (I).

[0031] In a third aspect, the present invention provides a pharmaceutical composition comprising the pyrazole derivative represented by the general formula (I) or its enantiomers, diastereomers, racemates, pharmaceutically acceptable salts or mixtures thereof and one or more pharmaceutically acceptable carriers and / or excipients.

[0032] The pharmaceutical compositions of the present invention can be administered in various known ways, such as orally, parenterally, by spray, or via an implanted reservoir. The pharmaceutical compositions of the present invention can be administered alone or in combination with other drugs. Oral compositions can be in any orally acceptable dosage form, including but not limited to tablets, capsules, emulsions, suspensions, dispersions, and solutions. Commonly used pharmaceutically acceptable carriers or excipients include stabilizers, diluents, surfactants, lubricants, antioxidants, binders, colorants, fillers, emulsifiers, and the like.

[0033] Sterile injection compositions can be formulated using suitable dispersants or wetting agents and suspending agents according to techniques known in the art. Pharmaceutically acceptable carriers and solvents include water, mannitol, sodium chloride solution, and the like.

[0034] In a fourth aspect, the present invention provides the use of the pyrazole derivative represented by the general formula (I) or the pharmaceutical composition in the preparation of a drug for treating parasitic infections or preventing and controlling agricultural pests or pests in the living environment.

[0035] Preferably, the parasite is Cryptosporidium.

[0036] The agricultural pests are one or more of diamondback moth, armyworm, fall armyworm, cotton bollworm, corn borer, mosquito larvae, aphids, spider mites, whiteflies, psyllids, leafhoppers, scale insects and beetles.

[0037] The pests in the living environment are mosquitoes, flies and the like. Beneficial effects:

[0038] The present invention reports for the first time a pyrazole compound with antiparasitic activity. The novel pyrazole compound has significant antiprotozoal effects, especially against Cryptosporidium, laying a solid foundation for the development of new drugs. DETAILED DESCRIPTION

[0039] The following describes the preparation methods of the compounds of general formula (I) of the present invention in conjunction with specific examples, but these specific methods do not limit the present invention in any way. The compounds of the present invention can also be conveniently prepared by optionally combining various synthetic methods described in this specification or known in the art. Such combinations can be easily performed by those skilled in the art to which the present invention belongs.

[0040] The starting materials, reaction reagents, etc. used in the specific examples of the present invention are all commercially available. The present invention can be prepared into salt forms using salt-forming methods commonly used in the art.

[0041] The experimental methods in the examples of the present invention where specific conditions are not specified are generally based on conventional conditions or conditions recommended by the raw material or product manufacturers.

[0042] The structures of all compounds in the examples were determined by waters QDa mass spectrometry and H NMR spectra ( 1 H-NMR) characterization.

[0043] Example 1

[0044] Preparation of compound 1

[0045] 2.1 g of the starting reactant 1 was added to a three-necked flask, followed by 20 mL of acetonitrile, 3.8 g of N,N-diisopropylethylamine, and 1.9 g of the starting reactant 2. The reaction was refluxed at elevated temperature for 3 hours, cooled to room temperature, filtered, and the filter cake was washed twice with water. The filter cake was dried and recrystallized with methanol to obtain an off-white solid (i.e., compound 1). [M+H] + :381.2, 1 H-NMR(DMSO-d6,300MHz)δ:11.95(s,1H),7.92(s,1H),7.64-7.74(d,2H),7 .42-7.78(m,6H),7.38-7.40(m,2H),6.31(s,2H),2.12(s,3H),2.38(s,3H).

[0046] Example 2

[0047] Preparation of compound 2

[0048] 2.2 g of the starting reactant 1 was added to a three-necked flask, followed by 20 mL of acetonitrile, 3.3 g of N,N-diisopropylethylamine, and 1.9 g of the starting reactant 2. The reaction was refluxed at elevated temperature for 4 hours, cooled to room temperature, filtered, and the filter cake was washed twice with water. The filter cake was dried and recrystallized from methanol to obtain an off-white solid (compound 2). [M+H] + :411.1, 1 H-NMR (DMSO-d6, 300MHz) δ: 13.95 (s, 1H), 8.24 (s, 1H), 7.95 (s, 1H), 7.64-7.75 (m, 3H), 7.45-7.63 (m, 3H), 7.38-7.40 (m, 2H), 2.42 (s, 3H).

[0049] Example 3

[0050] Preparation of compound 3

[0051] 2.3 g of the starting reactant 1 was added to a three-necked flask, followed by 20 mL of acetonitrile, 3.8 g of N,N-diisopropylethylamine, and 2.2 g of the starting reactant 2. The reaction was heated and refluxed for 3.5 hours, cooled to room temperature, filtered, and the filter cake was washed twice with water. The filter cake was dried and recrystallized from ethanol to obtain an off-white solid (compound 3). [M+H] + :409.2, 1 H-NMR(DMSO-d6,300MHz)δ:12.67(s,1H),7.85(s,1H),7.65-7.79(m,4H),7.45-7.72(m,4H),7.15-7.21(d,1H),2.46(s,3H),2.39(s,3H).

[0052] Example 4

[0053] Preparation of compound 4

[0054] 2.4 g of the starting reactant 1 was added to a three-necked flask, followed by 20 mL of acetonitrile, 3.8 g of N,N-diisopropylethylamine, and 2.2 g of the starting reactant 2. The reaction was refluxed at elevated temperature for 3.5 hours, cooled to room temperature, filtered, and the filter cake was washed twice with water. The filter cake was dried and recrystallized from ethanol to obtain an off-white solid (Compound 4). [M+H] + :443.1, 1 H-NMR(DMSO-d6,300MHz)δ:12.65(s,1H),7.85(s,1H),7.65-7.79(m,2H),7 .45-7.62(m,4H),7.15-7.30(d,2H),7.11(s,1H),2.43(s,3H),2.47(s,3H).

[0055] Example 5

[0056] Preparation of compound 5

[0057] 2.5 g of the starting reactant 1 was added to a three-necked flask, followed by 20 mL of acetonitrile, 3.8 g of N,N-diisopropylethylamine, and 2.6 g of the starting reactant 2. The reaction was refluxed at elevated temperature for 3.5 hours, cooled to room temperature, filtered, and the filter cake was washed twice with water. The filter cake was dried and recrystallized from ethanol to obtain an off-white solid (Compound 5). [M+H] + :456.2, 1H-NMR(DMSO-d6,300MHz)δ:12.65(s,1H),7.87(s,1H),7.35-7.51(m,2H),7 .15-7.27(m,4H),6.91-7.05(m,3H),6.21(s,2H),3.82(s,3H),2.80(s,3H).

[0058] Example 6

[0059] Preparation of compound 6

[0060] 2.5 g of the starting reactant 1 was added to a three-necked flask, followed by 20 mL of acetonitrile, 3.8 g of N,N-diisopropylethylamine, and 2.5 g of the starting reactant 2. The reaction was heated and refluxed for 4 hours, cooled to room temperature, filtered, and the filter cake was washed twice with water. The filter cake was dried and recrystallized from ethanol to obtain an off-white solid (Compound 6). [M+H] + :442.2, 1 H-NMR(DMSO-d6,300MHz)δ:13.45(s,1H),8.23(s,1H),7.87-8.02(d,2H),7.30 -7.48(d,2H),7.25-3.30(d,2H),6.91-7.05(d,2H),6.27(s,2H),3.82(s,3H).

[0061] Example 7

[0062] Preparation of compound 7

[0063] 2.5 g of the starting reactant 1 was added to a three-necked flask, followed by 20 mL of acetonitrile, 3.8 g of N,N-diisopropylethylamine, and 3.6 g of the starting reactant 2. The reaction was heated and refluxed for 5 hours, cooled to room temperature, filtered, and the filter cake was washed twice with water. The filter cake was dried and recrystallized from ethanol:ethyl acetate (V:V=1:2) to obtain an off-white solid (Compound 7). [M+H] + :456.2, 1 H-NMR(DMSO-d6,300MHz)δ:12.55(s,1H),7.94(s,1H),7.48-7.60(m,3H),7.24-7. 32(d,2H),7.25(d,1H),6.89-7.05(d,2H),6.23(s,2H),3.86(s,3H),4.04(s,3H).

[0064] Example 8

[0065] Preparation of compound 8

[0066] 2.4 g of the starting reactant 1 was added to a three-necked flask, followed by 20 mL of acetonitrile, 3.8 g of N,N-diisopropylethylamine, and 3.4 g of the starting reactant 2. The reaction was heated and refluxed for 5 hours, cooled to room temperature, filtered, and the filter cake was washed twice with water. The filter cake was dried and recrystallized from ethanol:ethyl acetate (V:V=1:3) to obtain an off-white solid (Compound 8). [M+H] + :484.2, 1 H-NMR(DMSO-d6,300MHz)δ:12.57(s,1H),8.60(s,1H),8.02-8.13(d,2H),7.65(m, 1H),7.41-7.52(m,2H),7.11-7.20(m,2H),6.24(s,2H),3.86(s,3H),4.05(s,3H).

[0067] Example 9

[0068] Preparation of compound 9

[0069] 2.5 g of the starting reactant 1 was added to a three-necked flask, followed by 20 mL of acetonitrile, 3.8 g of N,N-diisopropylethylamine, and 2.7 g of the starting reactant 2. The reaction was refluxed at elevated temperature for 5 hours, cooled to room temperature, filtered, and the filter cake was washed twice with water. The filter cake was dried and recrystallized from ethanol to obtain an off-white solid (Compound 9), [M+H] + :463.2, 1 H-NMR(DMSO-d6,300MHz)δ:12.57(s,1H),8.15(s,1H),8.02-8.13(m,2H),7.85(s,1H),7 .72(m,1H),7.21-7.33(m,2H),6.85-6.97(m,2H),6.27(s,2H),3.87(s,3H),4.06(s,3H).

[0070] Example 10

[0071] Preparation of compound 10

[0072] 2.6 g of the starting reactant 1 was added to a three-necked flask, followed by 20 mL of acetonitrile, 3.8 g of N,N-diisopropylethylamine, and 2.7 g of the starting reactant 2. The reaction was heated and refluxed for 4 hours, cooled to room temperature, filtered, and the filter cake was washed twice with water. The filter cake was dried and recrystallized from ethanol to obtain an off-white solid (Compound 10). [M+H] + :510.2, 1H-NMR(DMSO-d6,300MHz)δ:12.57(s,1H),8.10(s,1H),7.94(s,1H),7.62-7.78(m, 2H),7.45-7.54(m,2H),7.40(m,1H),7.22-7.35(m,2H),6.27(s,2H),4.06(s,3H).

[0073] Example 11

[0074] Preparation of compound 11

[0075] 2.6 g of the starting reactant 1 was added to a three-necked flask, followed by 20 mL of acetonitrile, 3.8 g of N,N-diisopropylethylamine, and 2.4 g of the starting reactant 2. The reaction was refluxed at elevated temperature for 4 hours, cooled to room temperature, filtered, and the filter cake was washed twice with water. The filter cake was dried and recrystallized from ethanol to obtain an off-white solid (Compound 11). [M+H] + :469.2, 1 H-NMR(DMSO-d6,300MHz)δ:12.57(s,1H),12.20(s,1H),8.62(s,1H),8.12-8.25(m,2H),7.9 4(s,1H),7.71-7.80(m,1H),7.43-7.55(m,2H),7.22-7.35(m,2H),2.41(s,3H),2.45(s,3H).

[0076] Example 12

[0077] Preparation of compound 12

[0078] 2.6 g of the starting reactant 1 was added to a three-necked flask, followed by 20 mL of acetonitrile, 3.8 g of N,N-diisopropylethylamine, and 3.0 g of the starting reactant 2. The reaction was heated and refluxed for 5 hours, cooled to room temperature, filtered, and the filter cake was washed twice with water. The filter cake was dried and recrystallized from ethanol to obtain an off-white solid (Compound 12). [M+H] + :443.2, 1 H-NMR(DMSO-d6,300MHz)δ:12.57(s,1H),7.94(s,1H),7.42-7.60(m,3H),7.44-7.51(d,1H),7.02-7.22(m,4H),5.32(s,2H),2.45(s,3H).

[0079] Example 13

[0080] Preparation of compound 13

[0081] 3.0 g of the starting reactant 1 was added to a three-necked flask, followed by 20 mL of acetonitrile, 3.8 g of N,N-diisopropylethylamine, and 2.0 g of the starting reactant 2. The reaction was refluxed at elevated temperature for 5 hours, cooled to room temperature, filtered, and the filter cake was washed twice with water. The filter cake was dried and recrystallized from ethyl acetate to obtain an off-white solid (Compound 13). [M+H] + :450.2, 1 H-NMR(DMSO-d6,300MHz)δ:11.84(s,1H),7.47-7.56(m,1H),7.31-7.44(m,3H),7.28(s, 1H),6.41-6.53(m,1H),3.20(m,1H),2.47(s,3H),1.48-1.53(m,1H),1.13-1.25(m,1H).

[0082] Example 14

[0083] Preparation of compound 14

[0084] 2.7 g of the starting reactant 1 was added to a three-necked flask, followed by 20 mL of acetonitrile, 3.8 g of N,N-diisopropylethylamine, and 2.2 g of the starting reactant 2. The reaction was refluxed at elevated temperature for 5 hours, cooled to room temperature, filtered, and the filter cake was washed twice with water. The filter cake was dried and recrystallized from ethyl acetate to obtain an off-white solid (Compound 14). [M+H] + :439.2, 1 H-NMR(DMSO-d6,300MHz)δ:11.80(s,1H),7.94(s,1H),7.76(s,2H),7.31-7.48(m,2H),7.18-7.28(m,2H),3.75 -3.85(m,1H),3.11-3.23(m,1H),2.34-2.45(m,1H),3.20(m,1H),2.47(s,3H),2.13-2.21(m,1H),1.82(s,3H).

[0085] Example 15

[0086] Preparation of compound 15

[0087] 2.2 g of the starting reactant 1 was added to a three-necked flask, followed by 20 mL of acetonitrile, 3.8 g of N,N-diisopropylethylamine, and 2.5 g of the starting reactant 2. The reaction was refluxed at elevated temperature for 4 hours, cooled to room temperature, filtered, and the filter cake was washed twice with water. The filter cake was dried and recrystallized from ethyl acetate to obtain an off-white solid (Compound 15). [M+H] + :436.2, 1H-NMR(DMSO-d6,300MHz)δ:11.80(s,1H),7.94(s,1H),7.82-7.95(m,2H),7.43-7.58(m,2H) ,6.71(s,1H),3.42(m,1H),2.90(s,3H),2.71(m,1H),1.65-1.86(m,4H),1.50-1.70(m,4H).

[0088] Example 16

[0089] Preparation of compound 16

[0090] 1.3 g of the starting reactant 1 was added to a three-necked flask, followed by 20 mL of acetonitrile, 3.8 g of N,N-diisopropylethylamine, and 2.4 g of the starting reactant 2. The reaction was heated and refluxed for 6 hours, cooled to room temperature, filtered, and the filter cake was washed twice with water. The filter cake was dried and recrystallized from ethyl acetate to obtain an off-white solid (Compound 16). [M+H] + :352.2, 1 H-NMR(DMSO-d6,300MHz)δ:12.55(s,1H),7.94(s,1H),7.41-7.53(m,2H),7 .29(s,1H),7.03-7.29(m,4H),6.43-6.65(m,2H),5.28(s,2H),3.81(s,3H).

[0091] Example 17

[0092] Preparation of compound 17

[0093] 1.8 g of the starting reactant 1 was added to a three-necked flask, followed by 20 mL of acetonitrile, 3.8 g of N,N-diisopropylethylamine, and 2.4 g of the starting reactant 2. The reaction was refluxed at elevated temperature for 4 hours, cooled to room temperature, filtered, and the filter cake was washed twice with water. The filter cake was dried and recrystallized from ethanol to obtain an off-white solid (Compound 17). [M+H] + :366.2, 1 H-NMR(DMSO-d6,300MHz)δ:12.55(s,1H),8.79-8.84(d,1H),8.31-8.39(d,1H),7.94(s,1H),7.90 (s,1H),7.71-7.82(m,2H),7.45-7.50(m,1H),6.67(s,1H),5.80(s,2H),2.48(s,3H),2.43(s,3H).

[0094] Example 18

[0095] Preparation of compound 18

[0096] 1.7 g of the starting reactant 1 was added to a three-necked flask, followed by 20 mL of acetonitrile, 3.8 g of N,N-diisopropylethylamine, and 2.4 g of the starting reactant 2. The reaction was refluxed at elevated temperature for 5 hours, cooled to room temperature, filtered, and the filter cake was washed twice with water. The filter cake was dried and recrystallized from ethanol to obtain an off-white solid (Compound 18). [M+H] + :358.2, 1 H-NMR(DMSO-d6,300MHz)δ:12.55(s,1H),7.94(s,1H),7.42-7.61(m,3H),7 .12-7.22(m,2H),7.69(s,1H),6.48-6.55(d,1H),6.41(s,1H),2.48(s,3H).

[0097] Example 19

[0098] Preparation of compound 19

[0099] 2.8 g of the starting reactant 1 was added to a three-necked flask, followed by 20 mL of acetonitrile, 3.8 g of N,N-diisopropylethylamine, and 2.3 g of the starting reactant 2. The reaction was heated and refluxed for 4 hours, cooled to room temperature, filtered, and the filter cake was washed twice with water. The filter cake was dried and recrystallized from ethanol to obtain an off-white solid (Compound 19). [M+H] + :464.2, 1 H-NMR(DMSO-d6,300MHz)δ:11.85(s,1H),7.96(s,1H),7.94(s,1H),7.55-7.66(m,2H),7.25-7. 42(m,3H),6.87(s,1H),6.68-6.72(d,1H),6.25(s,2H),4.61(s,2H),2.48(s,3H),2.10(s,3H).

[0100] Example 20

[0101] Preparation of compound 20

[0102] 2.5 g of the starting reactant 1 was added to a three-necked flask, followed by 20 mL of acetonitrile, 3.8 g of N,N-diisopropylethylamine, and 2.2 g of the starting reactant 2. The reaction was refluxed at elevated temperature for 5 hours, cooled to room temperature, filtered, and the filter cake was washed twice with water. The filter cake was dried and recrystallized from ethanol to obtain an off-white solid (Compound 20). [M+H] + :439.2, 1H-NMR(DMSO-d6,300MHz)δ:11.85(s,1H),8.74(s,1H),8.12(s,1H),7.80(s,1H),7.69-7.74(m, 1H),7.30-7.50(m,2H),6.90-7.19(m,2H),6.90(s,1H),3.87(s,3H),2.45(s,3H),2.14(s,3H).

[0103] Example 21

[0104] Preparation of compound 21

[0105] 2.1 g of the starting reactant 1 was added to a three-necked flask, followed by 20 mL of acetonitrile, 3.8 g of N,N-diisopropylethylamine, and 3.2 g of the starting reactant 2. The reaction was refluxed at elevated temperature for 4.5 hours, cooled to room temperature, filtered, and the filter cake was washed twice with water. The filter cake was dried and recrystallized from ethanol to obtain an off-white solid (Compound 21). [M+H] + :450.3, 1 H-NMR(DMSO-d6,300MHz)δ:11.85(s,1H),7.94(s,1H),7.48-7.60(m,5H),6.80(s,1H),6.68(s,1H ),6.56(s,2H),6.27(s,2H),2.50-2.63(m,4H),2.50-2.63(m,4H),2.45(s,3H),1.62-1.75(m,4H).

[0106] Example 22

[0107] Preparation of compound 22

[0108] 2.6 g of the starting reactant 1 was added to a three-necked flask, followed by 20 mL of acetonitrile, 3.8 g of N,N-diisopropylethylamine, and 3.2 g of the starting reactant 2. The reaction was refluxed at elevated temperature for 5 hours, cooled to room temperature, filtered, and the filter cake was washed twice with water. The filter cake was dried and recrystallized from ethanol to obtain an off-white solid (Compound 22). [M+H] + :494.2, 1H-NMR(DMSO-d6,300MHz)δ:11.85(s,1H),8.12(s,1H),7.48-7.60(m,5H),7.45(s,2H),7.41(s,1H),6.31(s,1H ),6.27(s,2H),6.21(s,1H),2.93-3.04(m,2H),2.63-2.73(m,2H),2.15(s,3H),2.11(s,3H),1.82-1.95(m,2H).

[0109] Example 23

[0110] Preparation of compound 23

[0111] 2.1 g of the starting reactant 1 was added to a three-necked flask, followed by 20 mL of acetonitrile, 3.8 g of N,N-diisopropylethylamine, and 3.4 g of the starting reactant 2. The reaction was refluxed at elevated temperature for 5 hours, cooled to room temperature, filtered, and the filter cake was washed twice with water. The filter cake was dried and recrystallized from ethanol to obtain an off-white solid (Compound 23). [M+H] + :465.3, 1 H-NMR(DMSO-d6,300MHz)δ:11.85(s,1H),7.64(s,1H), 7.48-7.60(m,5H),7.45(s,2H),6.31(s,1H),6.27(s,2H),6.21(s,1H),5.04(s,1H) ,2.93-3.04(m,2H),2.63-2.73(m,2H),2.15(s,3H),2.11(s,3H),1.74-1.93(m,4H).

[0112] Example 24

[0113] Preparation of compound 24

[0114] 2.1 g of the starting reactant 1 was added to a three-necked flask, followed by 20 mL of acetonitrile, 3.8 g of N,N-diisopropylethylamine, and 2.5 g of the starting reactant 2. The reaction was refluxed at elevated temperature for 4 hours, cooled to room temperature, filtered, and the filter cake was washed twice with water. The filter cake was dried and recrystallized from ethanol to obtain an off-white solid (Compound 24). [M+H] + :434.2, 1H-NMR(DMSO-d6,300MHz)δ:11.85(s,1H),8.34-8.39(d,1H),7.74-7.84(d,1H),7.45-7.62(m,5H),7. 32(s,1H),6.92-7.03(d,1H),6.31-6.42(d,1H),6.25(s,2H),3.74(s,3H),2.47(s,3H),2.12(s,3H).

[0115] Example 25

[0116] Preparation of compound 25

[0117] 2.1 g of the starting reactant 1 was added to a three-necked flask, followed by 20 mL of acetonitrile, 3.8 g of N,N-diisopropylethylamine, and 1.9 g of the starting reactant 2. The reaction was refluxed at elevated temperature for 6 hours, cooled to room temperature, filtered, and the filter cake was washed twice with water. The filter cake was dried and recrystallized from ethanol to obtain an off-white solid (Compound 25). [M+H] + :323.2, 1 H-NMR (DMSO-d6, 300MHz) δ: 11.85 (s, 1H), 7.94 (s, 1H), 7.45-7.62 (m, 5H), 6.28 (s, 2H), 2.47 (s, 3H), 2.12 (s, 3H).

[0118] Example 26

[0119] Preparation of compound 26

[0120] 2.3 g of the starting reactant 1 was added to a three-necked flask, followed by 20 mL of acetonitrile, 3.8 g of N,N-diisopropylethylamine, and 2.2 g of the starting reactant 2. The reaction was refluxed at elevated temperature for 5 hours, cooled to room temperature, filtered, and the filter cake was washed twice with water. The filter cake was dried and recrystallized from ethanol to obtain an off-white solid (Compound 26). [M+H] + :361.1, 1 H-NMR(DMSO-d6,300MHz)δ:11.85(s,1H),9.29(s,1H),8.76(s,1H),7.94(s,1H),7.78-7.90(m ,2H),7.48-7.61(d,1H),7.23-7.35(m,1H),6.72-6.87(m,1H),6.42-6.54(d,1H),2.47(s,3H).

[0121] Example 27

[0122] Preparation of compound 27

[0123] 1.9 g of the starting reactant 1 was added to a three-necked flask, followed by 20 mL of acetonitrile, 3.8 g of N,N-diisopropylethylamine, and 2.3 g of the starting reactant 2. The reaction was refluxed at elevated temperature for 4.5 hours, cooled to room temperature, filtered, and the filter cake was washed twice with water. The filter cake was dried and recrystallized from ethanol to obtain an off-white solid (Compound 27). [M+H] + :314.1, 1 H-NMR(DMSO-d6,300MHz)δ:11.85(s,1H),7.94(s,1H),7.76-7.87(d,1H),7.55 -7.68(d,1H),7.01-7.27(m,2H),6.27-6.39(d,1H),4.06(s,3H),2.47(s,3H).

[0124] Example 28

[0125] Preparation of compound 28

[0126] 2.3 g of the starting reactant 1 was added to a three-necked flask, followed by 20 mL of acetonitrile, 3.8 g of N,N-diisopropylethylamine, and 1.6 g of the starting reactant 2. The reaction was refluxed at elevated temperature for 5 hours, cooled to room temperature, filtered, and the filter cake was washed twice with water. The filter cake was dried and recrystallized from ethanol to obtain an off-white solid (Compound 28). [M+H] + :339.1, 1 H-NMR(DMSO-d6,300MHz)δ:11.85(s,1H),7.94(s,1H),7.83(s,1H),7.16-7.34(m,3H),6.89(s,1H),2.44-2.51(m,6H),2.27(s,3H).

[0127] Example 29

[0128] Preparation of compound 29

[0129] 2.9 g of the starting reactant 1 was added to a three-necked flask, followed by 20 mL of acetonitrile, 3.8 g of N,N-diisopropylethylamine, and 1.9 g of the starting reactant 2. The reaction was refluxed at elevated temperature for 5 hours, cooled to room temperature, filtered, and the filter cake was washed twice with water. The filter cake was dried and recrystallized from ethanol to obtain an off-white solid (Compound 29). [M+H] + :442.1, 1H-NMR(DMSO-d6,300MHz)δ:11.85(s,1H),8.09(s,1H),7.94(s,1H),7.62-7.72(d ,1H),7.46-7.58(m,2H),7.35-7.41(t,1H),7.13-7.24(d,1H),3.77-3.82(m,6H).

[0130] Example 30

[0131] Preparation of compound 30

[0132] 2.2 g of the starting reactant 1 was added to a three-necked flask, followed by 20 mL of acetonitrile, 3.8 g of N,N-diisopropylethylamine, and 2.3 g of the starting reactant 2. The reaction was heated and refluxed for 5 hours, cooled to room temperature, filtered, and the filter cake was washed twice with water. The filter cake was dried and recrystallized from ethanol to obtain an off-white solid (Compound 30). [M+H] + :407.2, 1 H-NMR(DMSO-d6,300MHz)δ:11.85(s,1H),8.82(m,1H),8.21(s,1H),7.94(s,1H),7.63-7.79(m ,3H),7.46-7.58(m,3H),7.35-7.41(d,1H),7.13-7.24(m,2H),3.79(s,3H),2.43-2.52(m,6H).

[0133] Example 31

[0134] Preparation of compound 31

[0135] 2.7 g of the starting reactant 1 was added to a three-necked flask, followed by 20 mL of acetonitrile, 3.8 g of N,N-diisopropylethylamine, and 1.8 g of the starting reactant 2. The reaction was heated and refluxed for 6 hours, cooled to room temperature, filtered, and the filter cake was washed twice with water. The filter cake was dried and recrystallized from ethanol to obtain an off-white solid (Compound 31). [M+H] + :377.2, 1 H-NMR(DMSO-d6,300MHz)δ:11.85(s,1H),7.94(s,1H),6.73-6.91(m,3H),6.02 (s,2H),2.75(m,2H),2.70(m,2H),2.45(s,3H),2.10(s,3H),1.65-1.77(m,4H).

[0136] Example 32

[0137] Preparation of compound 32

[0138] 2.8 g of the starting reactant 1 was added to a three-necked flask, followed by 20 mL of acetonitrile, 3.8 g of N,N-diisopropylethylamine, and 2.1 g of the starting reactant 2. The reaction was refluxed at elevated temperature for 5 hours, cooled to room temperature, filtered, and the filter cake was washed twice with water. The filter cake was dried and recrystallized from ethanol to obtain an off-white solid (Compound 32). [M+H] + :390.2, 1 H-NMR(DMSO-d6,300MHz)δ:11.85(s,1H),7.94(s,1H),7.03-7.26(m,3H),2.73-2.86 (m,4H),2.75(s,3H),2.47(s,3H),2.12(s,3H),1.79-1.85(m,2H),1.63-1.76(m,4H).

[0139] Example 33

[0140] Preparation of compound 33

[0141] 2.3 g of the starting reactant 1 was added to a three-necked flask, followed by 20 mL of acetonitrile, 3.8 g of N,N-diisopropylethylamine, and 1.8 g of the starting reactant 2. The reaction was refluxed at elevated temperature for 5 hours, cooled to room temperature, filtered, and the filter cake was washed twice with water. The filter cake was dried and recrystallized from ethanol to obtain an off-white solid (Compound 33). [M+H] + :341.2, 1 H-NMR (DMSO-d6, 300MHz) δ: 11.85 (s, 1H), 7.94 (s, 1H), 8.38 (d, 2H), 6.88-6.99 (m, 2H), 2.75 (s, 3H), 2.47 (s, 3H), 2.12 (s, 3H).

[0142] Example 34

[0143] Preparation of compound 34

[0144] 2.5 g of the starting reactant 1 was added to a three-necked flask, followed by 20 mL of acetonitrile, 3.8 g of N,N-diisopropylethylamine, and 1.6 g of the starting reactant 2. The reaction was refluxed at elevated temperature for 5 hours, cooled to room temperature, filtered, and the filter cake was washed twice with water. The filter cake was dried and recrystallized from ethanol to obtain an off-white solid (Compound 34). [M+H] + :361.2, 1H-NMR(DMSO-d6,300MHz)δ:11.85(s,1H),10.84(s,1H),7.94(s,1H),7.85(d,1H),7 .64(s,1H),7.52(d,1H),6.84-7.03(m,2H),2.75(s,3H),2.47(s,3H),2.12(s,3H).

[0145] Example 35

[0146] Preparation of compound 35

[0147] 2.6 g of the starting reactant 1 was added to a three-necked flask, followed by 20 mL of acetonitrile, 3.8 g of N,N-diisopropylethylamine, and 1.8 g of the starting reactant 2. The reaction was heated and refluxed for 5 hours, cooled to room temperature, filtered, and the filter cake was washed twice with water. The filter cake was dried and recrystallized from ethanol to obtain an off-white solid (Compound 35). [M+H] + :373.2, 1 H-NMR(DMSO-d6,300MHz)δ:11.85(s,1H),8.91(d,1H),8.35(d,1H),7.94(s, 1H),7.85-7.97(m,3H),7.53(d,1H),2.75(s,3H),2.47(s,3H),2.12(s,3H).

[0148] Example 36 Human colon cancer (HCT-8) cells were tested for anti-Cryptosporidium in vitro activity

[0149] 1. Reagents: RPMI1640, trypsin, sodium deoxycholate, fetal bovine serum and other reagents were purchased from commercial sources.

[0150] 2. Experimental Cryptosporidium species: Bovine Cryptosporidium oocysts were collected from cow feces, sieved through 60 mesh, and stored in 2.5% potassium dichromate (K2Cr2O7) preservation solution at 4°C until use.

[0151] 3. Experimental Methods

[0152] 3.1 Oocyst purification

[0153] Pipette 10 mL of fecal suspension from 2.5% K2Cr2O7 preservative solution, wash twice with 3 volumes of purified water, and then resuspend the feces in 2 volumes of purified water to purify the oocysts. Add 30 mL of saturated sucrose solution to a 50 mL centrifuge tube, slowly add 10 mL of the fecal suspension dropwise, and centrifuge at 1500 g for 20 minutes at 4°C. Aspirate 5 mL of the surface liquid, wash twice with 3 volumes of purified water, and centrifuge at 1500 g for 20 minutes before use.

[0154] 3.2 Oocyst sterilization

[0155] 2×10 purified Cryptosporidium andersoni oocysts were drawn from 2.5% K2Cr2O7 storage solution. 5 Wash each oocyst twice with purified water to remove K2Cr2O7. Resuspend the washed oocysts in sterile purified water to 900 μL in a 2 mL EP tube. Cool on ice and then sterilize. For sterilization, add 100 μL of ice-cold 1.05% sodium hypochlorite solution to the oocyst suspension. Shake vigorously and place on ice for 5 minutes. Wash twice with sterile PBS buffer and resuspend to 500 μL.

[0156] 3.3 Excystation of Sterile Cryptosporidium Oocysts

[0157] To 500 μL of sterile oocyst suspension, add 12x the volume of oocyst excystment solution (50 mg of trypsin and 150 mg of sodium taurodeoxycholate dissolved in 10 mL of PBS, sterilized with a 0.22 μm filter) and excyst in a 37°C water bath for 40 minutes. After excystment, wash twice with sterile PBS buffer and resuspend to 1 mL in RPMI-1640 basal medium containing 10% FCS for later use.

[0158] 3.4 Cell culture

[0159] HCT-8 cells were cultured in RPMI-1640 medium containing 5% fetal calf serum (FCS) in a 37°C 5% CO2 incubator. After the cells had grown to the bottom of the culture dish, they were digested with cell digestion solution and transferred to new culture dishes.

[0160] 3.5 HCT-8 cell infection

[0161] HCT-8 cells were cultured in 6-well plates using RPMI-1640 medium containing 5% fetal bovine serum and placed in a 37°C 5% CO2 cell culture incubator for 48 hours. 3 The excysted oocysts were added in the appropriate number and cultured for 8 hours, followed by incubation with complete medium for 48 hours.

[0162] 3.6 Anti-Cryptosporidium Activity of Test Compounds against HCT-8 Cells Infected

[0163] Nitazoxanide was used as the control drug. Positive control group and blank control group were set up. The test compound was administered according to 6 concentration gradients (200 μg / mL, 40 μg / mL, 8 μg / mL, 1.6 μg / mL, 0.32 μg / mL, 0.064 μg / mL) and added to infected HCT-8 cells. The cells were incubated for 48 hours to evaluate the pharmacodynamic activity.

[0164] Table 1 Results of in vitro anti-Cryptosporidium activity test on HTC-8 cells

[0165] Experimental conclusion: The in vitro anti-Cryptosporidium activity of the compounds in the examples of the present invention is better than or equivalent to that of nitazoxanide, and has development value.

[0166] Example 37 In vivo anti-Cryptosporidium activity test in animals

[0167] 1. Experimental animals: 3-week-old ICR mice were purchased from Shanghai JST Laboratory Animal Co., Ltd.

[0168] 2. Test method:

[0169] 2.1 Mouse Grouping and Inoculation: The experimental mice were divided into groups, and nitazoxanide was used as a control drug. A positive control group and a blank control group were set up and inoculated with the sterilized Cryptosporidium oocysts prepared in Example 36.

[0170] 2.2 Dosing regimen for mice: Mice were gavaged 4 days after inoculation, with each mouse receiving 200 μL of nitazoxanide at a dose of 100 mg / kg and the test compound at doses of 100 mg / kg and 200 mg / kg for 15 consecutive days. The blank control group received no treatment.

[0171] 2.3 Collection of mouse fecal samples:

[0172] One day after inoculation, mouse feces were collected and collected continuously for 20 days. Two portions of feces were collected from each group every day, each weighing 1 g. The feces were transferred to 5 mL centrifuge tubes and a small amount of water was added. One portion was stored at 4°C for subsequent oocyst counting, and the other portion was stored at -20°C for future use.

[0173] 2.4 Cryptosporidium oocyst count:

[0174] Dissolve the mouse feces collected daily from each group in 40 mL of water. Stir thoroughly with a tongue depressor and filter through four layers of gauze. Take 5 mL of the filtrate and centrifuge at 3000 rpm for 10 minutes. Discard the supernatant. Add 10 mL of saturated sugar solution to the precipitate, mix thoroughly, and centrifuge at 1500 rpm for 10 minutes. Add a drop of the top layer of the solution to a glass slide, cover with a slide, and examine under a 400x microscope. Record the number of oocysts with a counter.

[0175] Calculate the oocyst reduction rate of each experimental group as follows: oocyst reduction rate (%) = (total number of oocysts expelled in the infection group - total number of oocysts expelled in the experimental group) ÷ total number of oocysts expelled in the infection group × 100%.

[0176] Table 2 Oocyst reduction rate in mice

[0177] Experimental conclusion: The anti-Cryptosporidium activity of the compounds in the examples of the present invention in vivo is better than or equivalent to that of nitazoxanide.

[0178] Example 38 In vitro toxicity test on diamondback moth

[0179] Diamondback moth larvae were randomly divided into blank control, flurella control, and experimental groups. The test drug concentration was set at 100 μg / mL. Thirty larvae were placed on 12-day-old radish plants in each experimental group and the drug was sprayed onto each radish plant. After 48 hours, plant feeding damage was assessed based on leaf feeding, and diamondback moth mortality was counted. The diamondback moth toxicity results are shown in Table 3.

[0180] Table 3 Detection rate of drug toxicity to diamondback moth

[0181] The results show that the compounds of the present invention have activity in killing Plutella xylostella, and the activity of the compounds in the examples is better than or equivalent to that of flurella.

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