Substituted benzoyl piperazine compounds and uses thereof

Abstract

The application discloses a substituted benzoyl piperazine compound or a pharmaceutical salt thereof, and has the following general structure: wherein the definitions of the substituents are described in the description. The application further discloses an application of the substituted benzoyl piperazine compound or the pharmaceutical salt thereof in preparation of an antifungal drug or an application of the substituted benzoyl piperazine compound or the pharmaceutical salt thereof in preparation of a synergistic fluconazole antifungal drug.

Description

[0001] This application is a divisional application of the following original divisional application: filed on November 24, 2020, application number 202210949547.0, entitled "A Class of Substituted Benzoylpiperazine Compounds and Their Applications"; that original divisional application is a divisional application of the following parent application: filed on November 24, 2020, application number 202011329548.2, entitled "A Class of Substituted Benzoylpiperazine Compounds and Their Applications". This application is a further divisional application filed by the applicant in accordance with the examination opinion on unity of invention in the first examination opinion of the original divisional application. Technical Field

[0002] This invention belongs to the field of pharmaceutical technology, specifically, it relates to a class of substituted benzoylpiperazine compounds and their applications. Background Technology

[0003] The antifungal effects of natural products have attracted widespread attention. Natural products, with their novel structures, strong biological activity, and low toxicity, have become a research hotspot in recent years for the search of new antifungal drugs and their lead compounds. Gallic acid, also known as 3,4,5-trihydroxybenzoic acid, is an organic acid found in plants such as gallnut, lacquer tree, and tea. Literature reports its antibacterial, antiviral, and antitumor effects. Based on this, a series of substituted benzoylpiperazine compounds were designed and synthesized, and their individual and synergistic antifungal effects were evaluated, providing a foundation for further research and development of antifungal drugs. Summary of the Invention

[0004] The first objective of this invention is to provide a class of substituted benzoylpiperazine compounds.

[0005] A second objective of this invention is to provide the use of the substituted benzoylpiperazine compound in the preparation of antifungal drugs.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] The first aspect of the present invention provides a class of substituted benzoylpiperazine compounds or pharmaceutical salts thereof, having the following general structural formula:

[0008]

[0009] R1, R2, R3, R4, and R5 are each independently selected from hydrogen, hydroxyl, C1-C10 alkyl, C1-C10 alkoxy, and halogen;

[0010] X is selected from C or N;

[0011] Y is selected from one of the following groups:

[0012]

[0013]

[0014] R6, R7, R8, R9, R 10 R 11 R 12 R 13 R 14 R 15 Each is independently selected from hydrogen, hydroxyl, halogen (fluorine, chlorine, bromine, iodine), C1-C10 alkyl, and C1-C10 alkoxy.

[0015] Preferably, the substituted benzoylpiperazine compound has one of the following structures:

[0016]

[0017] R1, R2, R3, R4, and R5 are each independently selected from hydrogen, hydroxyl, and C1-C10 alkoxy groups;

[0018] R6, R7, R8, R9, R 10 R 11 R 12 R 13 R 14 R 15 Each is independently selected from hydrogen, hydroxyl, halogen (fluorine, chlorine, bromine, iodine), C1-C10 alkyl, and C1-C10 alkoxy.

[0019] Preferably, in the substituted benzoylpiperazine compound:

[0020] R1, R2, and R3 are all hydroxyl groups, and R4 and R5 are all hydrogens;

[0021] Alternatively, R1 and R2 may both be hydroxyl groups, and R3, R4, and R5 may all be hydrogen.

[0022] Alternatively, R2 and R3 may both be hydroxyl groups, and R1, R4, and R5 may all be hydrogen.

[0023] Alternatively, R2, R3, and R4 may all be hydroxyl groups, and R1 and R5 may all be hydrogen groups.

[0024] Alternatively, R2 is a methoxy group, and R1, R4, R5, and R3 are all hydrogen atoms.

[0025] More preferably, in the substituted benzoylpiperazine compound:

[0026] R6, R7, R8, R9, R 10 R 11 R 12 R13 R 14 R 15 Each is independently selected from hydrogen, fluorine, and chlorine.

[0027] Most preferably, the substituted benzoylpiperazine compound has one of the following structures:

[0028]

[0029]

[0030]

[0031] A third aspect of the invention provides the use of the substituted benzoylpiperazine compound or a pharmaceutical salt thereof in the preparation of an antifungal drug.

[0032] A fourth aspect of the present invention provides the use of the substituted benzoylpiperazine compound or a pharmaceutically acceptable salt thereof in the preparation of a synergistic antifungal drug with fluconazole.

[0033] The fungus in question is Candida albicans.

[0034] The substituted benzoylpiperazine compounds of the present invention can be prepared as pharmaceutical salts using conventional methods.

[0035] The pharmaceutical salts of substituted benzoylpiperazine compounds described in this invention are salts formed from pharmaceutically acceptable inorganic and organic acids, wherein preferred inorganic acids include: hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, and sulfuric acid; preferred organic acids include: formic acid, acetic acid, propionic acid, succinic acid, naphthalene disulfonic acid (1,5), linalool, glycyrrhizic acid, glycyrrhetinic acid, oleanolic acid, hawthorn acid, ursolic acid, corosolic acid, betulinic acid, boswellic acid, oxalic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, valeric acid, diethylacetic acid, malonic acid, succinic acid, fumaric acid, pimelic acid, adipic acid, maleic acid, malic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, nicotinic acid, isonicotinic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, citric acid, and amino acids.

[0036] By adopting the above technical solution, the present invention has the following advantages and beneficial effects:

[0037] The substituted benzoylpiperazine compounds of this invention exhibit good antifungal activity, especially against drug-resistant Candida albicans, showing significant effects when used alone. Furthermore, when used in combination with fluconazole, they can re-sensitize fungi that were previously resistant to azoles, reducing the dosage of azoles and enhancing their efficacy, thus achieving a synergistic effect. Detailed Implementation

[0038] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0039] The materials used in this invention embodiment are as follows: Bruker Spectmspin AC-P300 (Bruker, Switzerland); AC-P600 nuclear magnetic resonance spectrometer (Bruker, Switzerland); Agilent 6120 LC-MS mass spectrometer (Agilent Technologies, USA); GF254 silica gel plates (Yantai Huanghai Chemical, China); darkroom UV analyzer (ZF-20D model); rotary evaporator (Buchi Rotavapor R-3 model); SHB-III circulating multi-purpose vacuum pump; vacuum drying oven (DZF-6021 model); ultrasonic cleaner (KQ-500E model); reagents (analytical grade) and raw materials used in the experiment were purchased from Anaiji Reagent Co., Ltd., Bailingwei Technology Co., Ltd., Bide Pharmaceutical Technology Co., Ltd., and Exploration Platform, etc.

[0040] Example 1

[0041] Preparation of compound 1B-1:

[0042]

[0043] 3,4,5-Trimethoxybenzoic acid (compound 1) (100.0 mg, 0.5 mmol) was placed in a 100 mL round-bottom flask and dissolved in DCM (10.0 mL, dichloromethane). Then, 1-(2-chlorophenyl)piperazine (120.0 mg, 0.6 mmol), DCC (124.0 mg, 0.6 mmol, N,N'-dicyclohexylcarboimide), and DMAP (244.0 mg, 2.0 mmol) were added sequentially. The mixture was stirred at room temperature for 6 h, and the solution changed from clear to turbid. TLC was used to detect the change (DCM:MeOH = 10:1). After the reaction was complete, the mixture was filtered, and the filtrate was evaporated to dryness under reduced pressure. Recrystallization with 95% EtOH yielded compound 1A-1 (111.0 mg, yield: 56%).

[0044] Compound 1A-1 (100.0 mg, 0.3 mmol) was placed in a 100 mL round-bottom three-necked flask. Anhydrous DCM (10.0 mL) was added under anhydrous and oxygen-free argon atmosphere. A DCM solution of BBr3 (6.0 mL, 3.0 mmol) was added dropwise at a constant rate of two drops per second at -30 °C. After the addition was complete, the reaction apparatus was moved to room temperature and stirred overnight. TLC was used for monitoring (DCM:MeOH = 10:1). After the reaction was complete, 100.0 mL of water was added to quench the reaction. The mixture was stirred at room temperature for 1 h. The presence of solid precipitate was then observed. If solid precipitate was present, the mixture was filtered, and the filter cake was dried in an oven. If no solid precipitate was present, the mixture was extracted three times with EA. The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness. The organic phase was then recrystallized with DCM or EtOH to obtain compound 1B-1.

[0045] Example 2

[0046] Preparation of compound 1B-2:

[0047]

[0048] The 1-(2-chlorophenyl)piperazine used in Example 1 was replaced with 1-(3-chlorophenyl)piperazine (120.0 mg, 0.6 mmol), and all other procedures were the same as in Example 1, to obtain compound 1A-2 (123.0 mg, yield: 61%). The preparation of compound 1B-2 was performed in accordance with the preparation of compound 1B-1 in Example 1.

[0049] Example 3

[0050]

[0051] The 1-(2-chlorophenyl)piperazine used in Example 1 was replaced with 1-(2,3-dichlorophenyl)piperazine (140.0 mg, 0.6 mmol), and all other procedures were the same as in Example 1, to obtain compound 1A-3 (107.0 mg, yield: 58%). The preparation of compound 1B-3 was performed in accordance with the preparation of compound 1B-1 in Example 1.

[0052] Example 4

[0053]

[0054] 3,4,5-Trimethoxybenzoic acid (compound 1) (100.0 mg, 0.5 mmol) was placed in a 100 mL round-bottom flask, and DMF (10.0 mL) was added to dissolve it. Then, 4,4'-difluorodiphenylpiperazine (173.0 mg, 0.6 mmol), PyBOP (312.2 mg, 0.6 mmol), and DIEA (1.0 mL, 2.0 mmol) were added sequentially. After the addition was complete, the mixture was stirred at room temperature for 4 h, and the reaction was monitored by TLC (DCM:MeOH = 10:1). After the reaction was complete, 100.0 mL of water was added to quench the reaction, and the mixture was stirred at room temperature for 1 h. If solid precipitation occurred, the mixture was filtered, and the filter cake was dried in an oven. After recrystallization with 95% EtOH, compound 1A-4 (118.0 mg, yield: 60%) was obtained.

[0055] The preparation of compound 1B-4 is based on the preparation of compound 1B-1 in Example 1.

[0056] Example 5

[0057]

[0058] 3,4,5-Trimethoxybenzoic acid (compound 1) (100.0 mg, 0.5 mmol), N-Boc-piperazine (150.0 mg, 0.6 mmol), PyBOP (312.2 mg, 0.6 mmol), and DIEA (1.0 ml, 2.0 mmol) were added. After the addition was complete, the mixture was stirred at room temperature for 4 h, and the reaction was monitored by TLC (DCM:MeOH = 10:1). After the reaction was complete, 100.0 ml of water was added to quench the reaction, and the mixture was stirred at room temperature for 1 h. The mixture was filtered, and the filter cake was recrystallized from 95% EtOH to obtain intermediate a. The product was weighed and dissolved in hydrochloric acid-ethyl acetate solution at a 1:3 equivalent ratio. The mixture was stirred at room temperature, and the reaction was monitored by TLC (DCM:MeOH = 10:1). After the reaction was complete, the mixture was filtered, and the product was recrystallized from 95% EtOH to obtain intermediate b.

[0059] Intermediate b (140.0 mg, 0.5 mmol), 2,3-dichlorobenzoic acid (115.0 mg, 0.6 mmol), PyBOP (312.2 mg, 0.6 mmol) and DIEA (1.0 ml, 2.0 mmol) were administered in the same manner as compound 1A-4 to give compound 1A-5 121.0 mg (yield: 59%).

[0060] The preparation of compound 1B-5 is the same as that of compound 1B-1 in Example 1.

[0061] Example 6

[0062]

[0063] The 2,3-dichlorobenzoic acid used in Example 5 was replaced with 3,4-dichlorobenzoic acid (115.0 mg, 0.6 mmol), and all other procedures were the same as in Example 5, to obtain compound 1A-6 (115.0 mg, yield: 58%). The preparation of compound 1B-6 was performed in accordance with the preparation of compound 1B-1 in Example 1.

[0064] Example 7

[0065]

[0066] The 2,3-dichlorobenzoic acid used in Example 5 was replaced with 3,4,5-trifluorobenzoic acid (110.0 mg, 0.6 mmol), and all other procedures were the same as in Example 5, to obtain compound 1A-7 (120.0 mg, yield: 65%). The preparation of compound 1B-7 was performed in accordance with the preparation of compound 1B-1 in Example 1.

[0067] Example 8

[0068]

[0069] By replacing 2,3-dichlorobenzoic acid used in Example 5 with trans-cinnamic acid (100.0 mg, 0.6 mmol), and otherwise following the procedure of Example 5, compound 1A-8 (116.0 mg, yield: 59%) was obtained. The preparation of compound 1B-8 was performed in accordance with the preparation of compound 1B-1 in Example 1.

[0070] Example 9

[0071]

[0072] By replacing 2,3-dichlorobenzoic acid used in Example 5 with phenoxyacetic acid (100.0 mg, 0.6 mmol), and otherwise following the procedure of Example 5, compound 1A-9 (130.0 mg, yield: 75%) was obtained. The preparation of compound 1B-9 was performed in accordance with the preparation of compound 1B-1 in Example 1.

[0073] Example 10

[0074]

[0075] The 2,3-dichlorobenzoic acid used in Example 5 was replaced with 2,4-dichlorophenoxyacetic acid (135.0 mg, 0.6 mmol), and all other procedures were the same as in Example 5, to obtain compound 1A-10 (109.0 mg, yield: 58%). The preparation of compound 1B-10 was performed in accordance with the preparation of compound 1B-1 in Example 1.

[0076] Example 11

[0077]

[0078] Compound c (163.0 mg, 0.6 mmol) was placed in a 100 mL round-bottom flask and dissolved in DMF (10.0 mL). Then, 3,4,5-trimethoxybenzoic acid (compound 1) (100.0 mg, 0.5 mmol), PyBOP (312.2 mg, 0.6 mmol), and DIEA (1.0 mL, 2.0 mmol) were added sequentially. After the addition was complete, the mixture was stirred at room temperature for 2 h and monitored by TLC (DCM:MeOH = 10:1). Once the reaction was complete, 100.0 mL of water was added to quench the reaction, and the mixture was stirred at room temperature for 1 h. If solid precipitation occurred, the mixture was filtered, and the filter cake was dried in an oven. After recrystallization with 95% EtOH, 1A-11 (138.0 mg, yield: 78%) was obtained.

[0079] The preparation of compound 1B-11 is based on the preparation of compound 1B-1 in Example 1.

[0080] Example 12

[0081]

[0082] 2,3,4-Trimethoxybenzoic acid (compound 4) (100.0 mg, 0.5 mmol), 1-(2-chlorophenyl)piperazine (120.0 mg, 0.6 mmol), DCC (124.0 mg, 0.6 mmol), DMAP (244.0 mg, 2.0 mmol), were administered in the same manner as 1A-1 in Example 1 to obtain compound 1C-1 (102.0 mg, yield: 48%).

[0083] Compound 1C-1 (100.0 mg, 0.3 mmol), BBr3 in DCM solution (6.0 ml, 3.0 mmol), were prepared using the same method as compound 1B-1 in Example 1 to obtain compound 1D-1.

[0084] Example 13

[0085]

[0086] By replacing the 1-(2-chlorophenyl)piperazine used in Example 12 with 1-(3-chlorophenyl)piperazine (120.0 mg, 0.6 mmol), and otherwise referring to Example 12, compound 1C-2 (110.0 mg, yield: 54%) was obtained.

[0087] The preparation of compound 1D-2 is based on the preparation of compound 1D-1 in Example 12.

[0088] Example 14

[0089]

[0090] The compound 1C-3 (109.0 mg, yield: 50%) was obtained by replacing 1-(2-chlorophenyl)piperazine (140.0 mg, 0.6 mmol) in Example 12 with 1-(2-chlorophenyl)piperazine (140.0 mg, 0.6 mmol) in the same manner as in Example 12.

[0091] The preparation of compound 1D-3 is based on the preparation of compound 1D-1 in Example 12.

[0092] Example 15

[0093]

[0094] 2,3,4-Trimethoxybenzoic acid (compound 4) (100.0 mg, 0.5 mmol), 4,4'-difluorodiphenylpiperazine (173.0 mg, 0.6 mmol), PyBOP (312.2 mg, 0.6 mmol), DIEA (1.0 ml, 2.0 mmol), were prepared by the same method as 1A-4 in Example 4 to obtain compound 1C-4 (117.0 mg, yield: 58%).

[0095] The preparation of compound 1D-4 is based on the preparation of compound 1D-1 in Example 12.

[0096] Example 16

[0097]

[0098] Take 2,3,4-trimethoxybenzoic acid (compound 4) (100.0 mg, 0.5 mmol), N-Boc-piperazine (150.0 mg, 0.6 mmol), PyBOP (312.2 mg, 0.6 mmol), and DIEA (1.0 ml, 2.0 mmol) and proceed with the same method as intermediate b in Example 5 to obtain intermediate e.

[0099] Weigh intermediate e (140.0 mg, 0.5 mmol), 2,3-dichlorobenzoic acid (115.0 mg, 0.6 mmol), PyBOP (312.2 mg, 0.6 mmol), and DIEA (1.0 ml, 2.0 mmol), and proceed as in Example 4, 1A-4, to obtain 1C-5 (135.0 mg, yield: 79%).

[0100] The preparation of compound 1D-5 is based on the preparation of compound 1D-1 in Example 12.

[0101] Example 17

[0102]

[0103] By replacing 2,3-dichlorobenzoic acid used in Example 16 with 3,4-dichlorobenzoic acid (115.0 mg, 0.6 mmol), and otherwise referring to Example 16, compound 1C-6 (124.0 mg, yield: 68%) was obtained.

[0104] The preparation of compound 1D-6 is based on the preparation of compound 1D-1 in Example 12.

[0105] Example 18

[0106]

[0107] By replacing 2,3-dichlorobenzoic acid used in Example 16 with 3,4,5-trifluorobenzoic acid (110.0 mg, 0.6 mmol), and otherwise referring to Example 16, compound 1C-7 (132.0 mg, yield: 70%) was obtained.

[0108] The preparation of compound 1D-7 is based on the preparation of compound 1D-1 in Example 12.

[0109] Example 19

[0110]

[0111] By replacing 2,3-dichlorobenzoic acid used in Example 16 with trans-cinnamic acid (100.0 mg, 0.6 mmol), and otherwise referring to Example 16, compound 1C-8 (130.0 mg, yield: 78%) was obtained.

[0112] The preparation of compound 1D-8 is based on the preparation of compound 1D-1 in Example 12.

[0113] Example 20

[0114]

[0115] By replacing 2,3-dichlorobenzoic acid used in Example 16 with phenoxyacetic acid (100.0 mg, 0.6 mmol), and otherwise referring to Example 16, compound 1C-9 (139.0 mg, yield: 75%) was obtained.

[0116] The preparation of compound 1D-9 is based on the preparation of compound 1D-1 in Example 12.

[0117] Example 21

[0118]

[0119] By replacing 2,3-dichlorobenzoic acid used in Example 16 with 2,4-dichlorophenoxyacetic acid (135.0 mg, 0.6 mmol), and otherwise referring to Example 16, compound 1C-10 (125.0 mg, yield: 70%) was obtained.

[0120] The preparation of compound 1D-10 is based on the preparation of compound 1D-1 in Example 12.

[0121] Example 22

[0122]

[0123] Weigh intermediate c (163.0 mg, 0.6 mmol), 2,3,4-trimethoxybenzoic acid (compound 4) (100.0 mg, 0.5 mmol), PyBOP (312.2 mg, 0.6 mmol), and DIEA (1.0 ml, 2.0 mmol), and proceed as with compound 1A-4 to obtain 1C-11 (134.0 mg, yield: 70%).

[0124] The preparation of compound 1D-11 is based on the preparation of compound 1D-1 in Example 12.

[0125] Example 23

[0126]

[0127] 3,4-Dimethoxybenzoic acid (compound 5) (100.0 mg, 0.5 mmol), 1-(2-chlorophenyl)piperazine (120.0 mg, 0.6 mmol), PyBOP (312.2 mg, 0.6 mmol), DIEA (1.0 ml, 2.0 mmol), were administered according to the same method as compound 1A-4 to give compound 2A-1 (126.0 mg, yield: 71%).

[0128] Compound 2A-1 (100.0 mg, 0.3 mmol) and a DCM solution of BBr3 (6.0 ml, 3.0 mmol) were prepared using the same method as compound 1B-1 in Example 1 to obtain 2B-1.

[0129] Example 24

[0130]

[0131] By replacing the 1-(2-chlorophenyl)piperazine used in Example 23 with 1-(3-chlorophenyl)piperazine (120.0 mg, 0.6 mmol), and otherwise referring to Example 23, compound 2A-2 (121.0 mg, yield: 60%) was obtained.

[0132] The preparation of compound 2B-2 is based on the preparation of compound 2B-1 in Example 23.

[0133] Example 25

[0134]

[0135] By substituting 1-(2-chlorophenyl)piperazine (140.0 mg, 0.6 mmol) for 1-(2-chlorophenyl)piperazine used in Example 23, and otherwise referring to Example 23, compound 2A-3 (135.0 mg, yield: 72%) was obtained.

[0136] The preparation of compound 2B-3 is based on the preparation of compound 2B-1 in Example 23.

[0137] Example 26

[0138]

[0139] By replacing 1-(2-chlorophenyl)piperazine used in Example 23 with 4,4'-difluorobenzylpiperazine (173.0 mg, 0.6 mmol), and otherwise referring to Example 23, compound 2A-4 (125.0 mg, yield: 50%) was obtained.

[0140] The preparation of compound 2B-4 is based on the preparation of compound 2B-1 in Example 23.

[0141] Example 27

[0142]

[0143] 3,4-Dimethoxybenzoic acid (compound 5) (100.0 mg, 0.5 mmol), N-Boc-piperazine (150.0 mg, 0.6 mmol), PyBOP (312.2 mg, 0.6 mmol), DIEA (1.0 ml, 2.0 mmol), were prepared by recrystallization from 95% EtOH, as in compounds 1A-4, to give intermediate g.

[0144] Intermediate g (125.0 mg, 0.5 mmol), 2,3-dichlorobenzoic acid (115.0 mg, 0.6 mmol), PyBOP (312.2 mg, 0.6 mmol), DIEA (1.0 ml, 2.0 mmol), were prepared according to the same method as compound 1A-4 to give 2A-5 (128.0 mg, yield: 65%).

[0145] The preparation of compound 2B-5 is based on the preparation of compound 2B-1 in Example 23.

[0146] Example 28

[0147]

[0148] By replacing the 2,3-dichlorobenzoic acid used in Example 27 with 3,4-dichlorobenzoic acid (115.0 mg, 0.6 mmol), and otherwise referring to Example 27, compound 2A-6 (112 mg, yield: 70%) was obtained.

[0149] The preparation of compound 2B-6 is based on the preparation of compound 2B-1 in Example 23.

[0150] Example 29

[0151]

[0152] By replacing 2,3-dichlorobenzoic acid used in Example 27 with 3,4,5-trifluorobenzoic acid (110.0 mg, 0.6 mmol), and otherwise referring to Example 27, compound 2A-7 (126.0 mg, yield: 66%) was obtained.

[0153] The preparation of compound 2B-7 is based on the preparation of compound 2B-1 in Example 23.

[0154] Example 30

[0155]

[0156] By replacing 2,3-dichlorobenzoic acid used in Example 27 with trans-cinnamic acid (100.0 mg, 0.6 mmol), and otherwise referring to Example 27, compound 2A-8 (104.0 mg, yield: 50%) was obtained.

[0157] The preparation of compound 2B-8 is based on the preparation of compound 2B-1 in Example 23.

[0158] Example 31

[0159]

[0160] By replacing 2,3-dichlorobenzoic acid used in Example 27 with phenoxyacetic acid (100.0 mg, 0.6 mmol), and otherwise referring to Example 27, compound 2A-9 (130.0 mg, yield: 66%) was obtained.

[0161] The preparation of compound 2B-9 is based on the preparation of compound 2B-1 in Example 23.

[0162] Example 32

[0163]

[0164] By replacing 2,3-dichlorobenzoic acid used in Example 27 with 2,4-dichlorophenoxyacetic acid (135.0 mg, 0.6 mmol), and otherwise referring to Example 27, compound 2A-10 (116.0 mg, yield: 60%) was obtained.

[0165] The preparation of compound 2B-10 is based on the preparation of compound 2B-1 in Example 23.

[0166] Example 33

[0167]

[0168] 2,3-Dimethoxybenzoic acid (compound 6) (100.0 mg, 0.5 mmol), 1-(2-chlorophenyl)piperazine (120.0 mg, 0.6 mmol), PyBOP (312.2 mg, 0.6 mmol), DIEA (1.0 ml, 2.0 mmol), were prepared by the same method as compound 1A-4 in Example 4 to give 2C-1 (116.0 mg, yield: 65%).

[0169] Weigh 100.0 mg (0.3 mmol) of 2C-1 and a DCM solution of BBr3 (6.0 ml, 3.0 mmol), and obtain 2D-1 by the same method as compound 1B-1 in Example 1.

[0170] Example 34

[0171]

[0172] The compound 2C-2 (113.0 mg, yield: 60%) was obtained by replacing 1-(2-chlorophenyl)piperazine (120.0 mg, 0.6 mmol) in Example 33 with 1-(3-chlorophenyl)piperazine (120.0 mg, 0.6 mmol) in other ways as in Example 33.

[0173] The preparation of compound 2D-2 is the same as that of compound 2D-1 in Example 33.

[0174] Example 35

[0175]

[0176] The compound 2C-3 (136.0 mg, yield: 77%) was obtained by replacing 1-(2-chlorophenyl)piperazine (140.0 mg, 0.6 mmol) in Example 33 with 1-(2,3-dichlorophenyl)piperazine (140.0 mg, 0.6 mmol) in other ways as in Example 33.

[0177] The preparation of compound 2D-3 is based on the preparation of compound 2D-1 in Example 33.

[0178] Example 36

[0179]

[0180] The compound 2C-4 (126.0 mg, yield: 50%) was obtained by replacing 1-(2-chlorophenyl)piperazine used in Example 33 with 4,4'-difluorodiphenylpiperazine (173.0 mg, 0.6 mmol), otherwise following the same procedure as in Example 33.

[0181] The preparation of compound 2D-4 is based on the preparation of compound 2D-1 in Example 33.

[0182] Example 37

[0183]

[0184] 2,3-Dimethoxybenzoic acid (compound 6) (100.0 mg, 0.5 mmol), N-Boc-piperazine (150.0 mg, 0.6 mmol), PyBOP (312.2 mg, 0.6 mmol), DIEA (1.0 ml, 2.0 mmol) were administered in the same manner as compounds 1A-4 in Example 4 to obtain intermediate i.

[0185] Weigh intermediate i (150.0 mg, 0.5 mmol), 2,3-dichlorobenzoic acid (compound 5) (115.0 mg, 0.6 mmol), PyBOP (312.2 mg, 0.6 mmol), and DIEA (1.0 ml, 2.0 mmol), and proceed as in compound 1A-4 in Example 4 to obtain 2C-5 (129.0 mg, yield: 70%).

[0186] The preparation of compound 2D-5 is based on the preparation of compound 2D-1 in Example 33.

[0187] Example 38

[0188]

[0189] By replacing 2,3-dichlorobenzoic acid used in Example 35 with 3,4,5-trifluorobenzoic acid (110.0 mg, 0.6 mmol), and otherwise referring to Example 35, compound 2C-7 (114.0 mg, yield: 72%) was obtained.

[0190] The preparation of compound 2D-7 is based on the preparation of compound 2D-1 in Example 33.

[0191] Example 39

[0192]

[0193] By replacing 2,3-dichlorobenzoic acid used in Example 35 with trans-cinnamic acid (100.0 mg, 0.6 mmol), and otherwise following the same procedure as in Example 35, compound 2C-8 (126.0 mg, yield: 66%) was obtained.

[0194] The preparation of compound 2D-8 is based on the preparation of compound 2D-1 in Example 33.

[0195] Example 40

[0196]

[0197] By replacing 2,3-dichlorobenzoic acid used in Example 35 with 2,4-dichlorophenoxyacetic acid (135.0 mg, 0.6 mmol), and otherwise referring to Example 35, compound 2C-10 (123.0 mg, yield: 60%) was obtained.

[0198] The preparation of compound 2D-10 is based on the preparation of compound 2D-1 in Example 33.

[0199] Example 41

[0200] Synthetic method of compound (4-(2,3-dichlorophenyl)piperazin-1-yl)(3-methoxyphenyl)methanone (3B-3)

[0201]

[0202] 3-Methoxybenzoic acid (compound 9) (100.0 mg, 0.65 mmol), 1-(2,3-dichlorophenyl)piperazine (139.0 mg, 0.8 mmol), PyBOP (416.3 mg, 0.8 mmol), DIEA (1.0 ml, 2.0 mmol), were administered in the same manner as compound 1A-4 in Example 4 to give 3B-3 (126 mg, yield: 79%).

[0203] The structures, NMR, and MS data of the compounds prepared in Examples 1–41 are shown in Table 1:

[0204] Table 1

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216] Example 42

[0217] Pharmacological Experimental Materials and Methods

[0218] Instruments and reagents: Multiskan MK3 ELISA reader (Labsystems, Finland); SW-CT-IF ultra-clean workbench (Suzhou Antai Air Technology Co., Ltd.); inverted microscope (Amersham Pharmacia); micropipette (Finnpette, Finland); 96-well cell culture plate (Nunclon, Denmark); MJX intelligent mold incubator (Ningbo Jiangnan Instrument Factory); water-jacketed electric thermostatic incubator (Shanghai Yuejin Medical Instrument Factory); THZ-82A benchtop thermostatic shaker (Shanghai Yuejin Medical Instrument Factory).

[0219] Fluconazole (FLC) injection (specification: 5ml: 0.2g; Pfizer, Inc., USA); dimethyl sulfoxide (DMSO, China National Pharmaceutical Group Chemical Reagent Co., Ltd.); baicalein (BE, Anaiji Reagent Co., Ltd.); gallic acid (GA, Adamas Pharmaceuticals).

[0220] Test strains: Drug-resistant Candida albicans (C. albicans 901, C. albicans 904) were donated to the Microbial Preservation Center of Changhai Hospital and underwent biological and morphological identification. All test strains were activated by streak plating on Sabouraud dextrose agar (SDA), and after incubation at 30℃ for 2 weeks, they were reactivated by single-clone streak plating. The single-clone Sabouraud dextrose agar slants obtained after the second activation were selected, cultured using the above method, and then stored at 4℃ for later use.

[0221] Culture medium

[0222] 1. Sabouraud dextrose agar solid medium (SDA)

[0223] Sabouraud dextrose agar (SDA) composition (g / L): glucose (40.0g), peptone (10.0g), agar (15.0g). Adjust the pH of the medium to 7.0±0.1, dissolve in 900.0ml of sterile triple-distilled water by heating and stirring, bring the volume to 1.0L, autoclave at 121℃ for 20min, cool to about 30℃, transfer to sterile petri dishes, and store at 4℃ for later use.

[0224] 2. RPMI 1640 liquid culture medium

[0225] RPMI-1640 liquid culture medium (g / L): Morpholine propanesulfonic acid (17.25g), RPMI 1640 (5.0g), and NaHCO3 (1.0g) were dissolved in 450.0ml of sterile triple-distilled water by heating and stirring, and the volume was adjusted to 500.0ml. The solution was then microfiltered through a 0.22μm microporous membrane and dispensed into 250.0ml Erlenmeyer flasks. The solution was stored at 4℃ for later use.

[0226] 3. YEPD culture medium

[0227] YEPD culture medium (g / L): Peptone (20.0g), glucose (20.0g), and yeast extract (10.0g) are heated and stirred and dissolved in 900.0ml of sterile triple-distilled water. The volume is adjusted to 1.0L, autoclaved at 121℃ for 20min, cooled to about 30℃, transferred to sterile culture dishes, and stored in a refrigerator at 4℃ for later use.

[0228] Experimental methods

[0229] Strain activation: Use an inoculation loop to take a small amount of the -80℃ frozen bacterial solution and inoculate it onto sterile SDA medium stored at 4℃. Incubate at 30℃ for 48 hours, then return to the 4℃ refrigerator for storage.

[0230] Preparation of fungal suspension: Before the experiment, activated Candida albicans was picked from sterile SDA medium stored at 4°C using an inoculation loop and inoculated into 1.0 ml of sterile YEPD liquid culture medium. The suspension was then incubated at 30°C with shaking at 200 rpm for 16 hours to activate the fungus into the late exponential growth phase. The suspension was then pipetted into 1.0 ml of sterile YEPD medium, and the above method was repeated for another 16 hours of activation and incubation. The number of fungi was counted using a hemocytometer. The suspension concentration could be adjusted to 1 × 10⁻⁶ using sterile RPMI-1640 liquid culture medium. 3 -5×10 3 It should be within the range of CFU / ml.

[0231] Preparation of the drug susceptibility test plate: In each row of a sterile 96-well plate, add 100.0 μl of sterile RPMI 1640 liquid culture medium as a blank control to well 1; add 40.0 μl of the test compound solution and 160.0 μl of bacterial suspension to well 2; add 100.0 μl of bacterial suspension to wells 3-12, except for well 12 which only contains 100.0 μl of bacterial suspension as a positive control. Dilute the drug sequentially in wells 2-11 to achieve final drug concentrations of 64.0, 32.0, 16.0, 8.0, 4.0, 2.0, 1.0, 0.5, 0.25, and 0.125 μg / ml, respectively, with DMSO content below 1.0% in all wells. The final concentration of fluconazole solution was 8.0 μg / ml. The MIC values ​​for the test compound combined with fluconazole (8.0 μg / ml) were obtained. Under the same conditions, a batch of drug sensitivity plates for quality control bacteria needs to be prepared. All drug sensitivity plates are incubated in a constant temperature incubator at 30℃ for 24 hours.

[0232] Determination of the minimum inhibitory concentration (MIC): The OD value of each well of the antimicrobial susceptibility plate cultured under isothermal conditions was measured using an enzyme-linked immunosorbent assay (ELISA) analyzer at a wavelength of 620 nm. The OD value of the positive control well 12 was approximately 0.2. Compared to this, the lowest concentration at which the OD values ​​of all other wells decreased by more than 80% was the MIC value (the minimum drug concentration at which 80% fungal growth is inhibited). If the MIC value of the drug was outside the test range, it was counted according to the following rules: when the MIC value was lower than the minimum concentration of 0.125 μg / ml, it was counted as "≤0.125 μg / ml"; when the MIC value exceeded the maximum concentration of 64.0 μg / ml, it was counted as ">64.0 μg / ml". The above parallel operation was repeated three times. If the adopted MIC value could be accurately reproduced or differed by one concentration unit, the relatively higher concentration was used as the MIC value. If the MIC values ​​differed by more than two concentration units, the above operation needed to be repeated until the requirements were met.

[0233] Quality control strain: *Candida glabrata* ATCC18062 was used as the quality control strain in this experiment. Its reference MIC values ​​are: FLC: 0.25-1.0 μg / ml; AmB: 0.5-2.0 μg / ml. This strain was used as a reference in each experiment. If the actual measured MIC values ​​remained within the above range, the experimental procedure was confirmed to be accurate and the results reliable. Therefore, the experiment was considered successful and the results reliable only when both the experimental and control strains grew well under identical conditions.

[0234] Efficacy evaluation of combined medication: In this experiment, the fractional inhibitory concentration index (FICI) specified in the CLSI M27-A3 and M38-A2 standards was referenced and adopted to evaluate the combined effect of the two drugs. The fractional inhibitory concentration index (FICI), as a main parameter for the interaction mode of two drugs in combined medication, can be used to interpret the interaction mode of antifungal drugs. The calculation formula of the FICI index: FICI index = MIC (group A combined use) / MIC (group A single use) + MIC (group B combined use) / MIC (group B single use) (when MIC 80 value is higher than the maximum detection limit, twice the maximum limit concentration is used to calculate FICI). The latest standard adopted by foreign journals at present: when FICI ≤ 0.5, the effect of the two drugs is synergistic; when 0.5 < FICI ≤ 1, the effect of the two drugs is additive; when 1 < FICI ≤ 4, the effect of the two drugs is indifferent; when FICI > 4, the effect of the two drugs is antagonistic.

[0235] Antifungal activity and structure-activity relationship of compounds: The in vitro activity of the target compounds prepared in Examples 1 to 41 when used alone and in synergy with fluconazole against drug-resistant Candida albicans was tested, and the results are shown in Table 2.

[0236] Table 2. Activity test results of the target compounds combined with fluconazole against drug-resistant Candida albicans

[0237]

[0238]

[0239] a Combined with fluconazole (FLC, 8.0 μg / ml) to act on drug-resistant Candida albicans 901; b Combined with fluconazole (FLC, 8.0 μg / ml) to act on drug-resistant Candida albicans 904.

[0240] A total of 25 target compounds (1B-1, 1D-1, 2D-1, 1B-2, 1D-2, 2D-2, 1B-3, 1D-3, 2D-3, 1B-4, 1D-4, 2B-4, 1B-5, 1D-5, 1B-6, 1D-6, 1D-7, 1B-8, 1D-8, 2D-8, 1B-9, 1D-9, 1B-10, 1D-10, 2D-10) showed individual antifungal activity (MIC < 128.0 μg / ml), among which compounds (2D-2, 2D-10) showed the strongest single-use antifungal activity, MIC = 2.0 μg / ml. There are a total of 37 target compounds (1B-1, 1D-1, 2D-1, 1B-2, 1D-2, 2D-2, 1B-3, 1D-3, 2D-3, 1B-4, 1D-4, 2B-4, 1B-5, 1D-5, 1B-6, 1D-6, 1D-7, 1B-8, 1D-8, 2D-8, 1B-9, 1D-9, 1B-10, 1D-10, 2D-10, 2 Compounds B-1, 2B-2, 2B-3, 2B-5, 2D-5, 2B-6, 1B-7, 2B-7, 2D-7, 2B-8, 1B-11, and 1D-11 exhibited synergistic activity with fluconazole against drug-resistant fungi, with compounds (2D-5, 2B-5, and 1B-10) showing the strongest synergistic activity against fluconazole resistance, MIC = 0.5 μg / ml and FICI = 0.066. In addition, two other target compounds (1B-10 and 1D-10) showed addition activity.

[0241] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A class of substituted benzoylpiperazine compounds or their pharmaceutical salts, characterized in that, The substituted benzoylpiperazine compound has one of the following structures: ； 。 2. The use of a substituted benzoylpiperazine compound of claim 1 or a pharmaceutical salt thereof in the preparation of an antifungal drug.

3. The use of a substituted benzoylpiperazine compound as described in claim 1 or a pharmaceutical salt thereof in the preparation of a synergistic antifungal drug with fluconazole.

4. The application according to claim 2 or 3, characterized in that, The fungus in question is Candida albicans.

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