A quinoline chalcone derivative, a synthesis method and its application

Abstract

The invention discloses a quinoline chalcone derivative, a synthesis method and an application thereof. The method has a short route and a high yield. The quinoline chalcone derivative provided by the invention is combined with fluconazole to produce a synergistic anti-drug resistant Candida albicans effect, which is expected to further solve the drug resistance problem of deep fungal infection.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical chemistry, and specifically relates to a quinoline chalcone derivative, a synthesis method and application thereof. Background Art

[0002] In recent years, with the development of broad-spectrum antibiotics, immunosuppressants, anti-tumor drugs, immunodeficiency diseases, and the popularization of transplantation surgery, the incidence of deep fungal infections (IFIs) has increased year by year, seriously threatening human health and life. More than 1.5 million people die from IFIs each year. Candida albicans ) is the main pathogen causing IFIs. Candida albicans is a commensal bacterium that exists in the human mucosa and is usually harmless to health. When the human immune system is weakened, Candida albicans can cause superficial infections and even IFIs. It is the most common nosocomial fungal infection and has become the fourth largest pathogen in the world.

[0003] Azoles represented by fluconazole have strong antifungal inhibitory effects and low toxicity and are widely used in clinical practice. However, azoles are only antibacterial agents without direct killing effects, which provides a strong condition for the surviving strains to develop drug resistance. In the long-term treatment process, repeated administration will aggravate the development of fungal fluconazole resistance. Faced with the emergence of a large number of drug-resistant strains, the current stage of antifungal drugs for the treatment of fungal infectious diseases has reached a bottleneck.

[0004] The quinoline chalcone derivative provided by the invention has a good effect on resisting drug-resistant Candida albicans. Summary of the invention

[0005] The purpose of the present invention is to provide a quinoline chalcone derivative, a synthesis method and an application thereof.

[0006] The structural formula is as follows: , where R=H, , , , , , , , , , , , , , , , , , , .

[0007] The synthetic route is as follows:

[0008] .

[0009] The specific steps are:

[0010] (1) Preparation of compound 1: Weigh 2-chloroquinoline-3-carboxaldehyde (3.84 g, 20 mmol) and potassium carbonate (5.52 g, 40 mmol) in a 100 mL round-bottom flask, add 50 mL of dried acetonitrile to dissolve; then add N-methylpiperazine (4.0 g, 40 mmol), and heat under reflux to react overnight; after the raw material disappears as detected by thin layer chromatography, cool to room temperature and pour into 200 mL of cold water; extract with 120 mL of dichloromethane three times, dry the dichloromethane layer over anhydrous sodium sulfate and filter; concentrate in vacuo, and the residue is compound 1;

[0011] (2) Preparation of compound 2: 2.762 g 4-fluoroacetophenone (20 mmol) and 3.438 g anhydrous piperazine (40 mmol) were weighed in a 100 mL round-bottom flask, 60 mL N,N-dimethylformamide dried over potassium hydroxide was added, and finally 5.522 g potassium carbonate (40 mmol) was added. The mixture was placed in an oil bath at 115°C for one day. After the reaction, the mixture was poured into 200 mL ice water and stirred to precipitate a light yellow solid. The solid was filtered and washed with water (50 mL×3), and vacuum dried to obtain a light-colored solid compound 2.

[0012] (3) Synthesis of key intermediate PK-1: Weigh 2.55 g of prepared compound 1 (10 mmol) and 2.04 g of compound 2 (10 mmol) in a 100 mL round-bottom flask, add 40 mL of anhydrous methanol to dissolve; then add 4 g of sodium hydroxide in batches under ice-water bath cooling; after the solid is completely dissolved, remove the ice-water bath and continue to react at room temperature for 24 hours; after thin-layer chromatography detection shows that the reaction raw materials are basically gone, pour into 200 mL of cold water and extract with dichloromethane (30 mL×3); after the organic phases are combined, dry with anhydrous sodium sulfate, filter, and the filtrate is concentrated in vacuo; the residue is separated by column chromatography (using methanol / dichloromethane = 3:97 as the eluent) to obtain a yellow-brown solid key intermediate PK-1;

[0013] (4) Preparation of derivatives PK-2~PK20: Weigh 88 mg of the key intermediate PK-1 (0.2 mmol) into a 10 mL dry round-bottom flask, then add 69 mg of potassium carbonate (0.6 mmol) and 5 mL of dry dichloromethane in sequence to dissolve; finally, add 0.3 mmol of acyl chloride, acid anhydride, sulfonyl chloride or halogenated hydrocarbon dropwise, and react at room temperature for 5-12 hours; after the reaction is completed by thin layer chromatography, add 5 mL of water and stir for 30 minutes; the dichloromethane layer is directly chromatographed using methanol / dichloromethane = 2:98 as the eluent to separate the quinoline chalcone derivative as claimed in claim 1.

[0014] The acid chloride, acid anhydride, sulfonyl chloride or halogenated hydrocarbon described in step (4) are: acetic anhydride, , , , , , , , , , , , , , , , , , .

[0015] Application of quinoline chalcone derivatives in combination with antifungal drugs against drug-resistant fungi.

[0016] The resistant fungus is resistant Candida albicans.

[0017] The resistant fungus was resistant Candida albicans (CA23).

[0018] The antifungal drug is fluconazole.

[0019] Advantages of the present invention:

[0020] The invention discloses a novel quinoline chalcone derivative.

[0021] The invention also discloses a synthesis method of the derivative, which has a short route and high yield.

[0022] The quinoline chalcone derivative provided by the present invention is combined with fluconazole to produce a synergistic anti-drug resistant Candida albicans effect, which is expected to further solve the drug resistance problem of deep fungal infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the structural formula of the quinoline chalcone derivatives PK-1 to PK-8 of the present invention;

[0024] Figure 2 is the structural formula of the quinolinechalcone derivatives PK-9 to PK-16 of the present invention;

[0025] Figure 3 is the structural formula of the quinolinechalcone derivatives PK-17 to PK-20 of the present invention;

[0026] Figure 4 It is the synthetic route of quinoline chalcone derivatives PK-1~PK-20;

[0027] Figure 5 It is a time-killing curve of the quinoline chalcone derivative PK-10 of the present invention and fluconazole used alone or in combination against Candida albicans;

[0028] Figure 6 The growth of Candida albicans hyphae in SD+10% FBS medium was affected by quinoline chalcone derivative PK-10 and fluconazole alone or in combination (×20);

[0029] Figure 7 The growth of Candida albicans hyphae in Spider medium was affected by quinoline chalcone derivative PK-10 and fluconazole alone or in combination (×20);

[0030] Figure 8 To investigate the effects of quinolinechalcone derivative PK-10 and fluconazole alone or in combination on the efflux pump of Candida albicans;

[0031] Fig. 9 The effects of quinolinechalcone derivative PK-10 and fluconazole alone or in combination on the mitochondrial membrane potential in Candida albicans cells;

[0032] Fig.10 To investigate the effects of quinolinechalcone derivative PK-10 and fluconazole alone or in combination on reactive oxygen species in Candida albicans cells;

[0033] Fig.11 To investigate the effects of quinolinechalcone derivative PK-10 and fluconazole alone or in combination on intracellular ATP in Candida albicans.

[0034] Fig.12 The effects of quinoline chalcone derivative PK-10 and FLC alone or in combination on the expression of genes related to the RAS1-cAMP-EFG1 signaling pathway;

[0035] Fig.13 The effect of exogenous cAMP on hyphae formation of Candida albicans in SD+10% FBS medium;

[0036] Fig.14The effect of exogenous cAMP on hyphae formation of Candida albicans in Spider medium;

[0037] Fig.15 To investigate the effects of quinolinechalcone derivative PK-10 and fluconazole alone or in combination on the survival rate of mice infected with systemic Candida albicans.

[0038] Fig.16 To investigate the effects of quinoline chalcone derivative PK-10 and fluconazole alone or in combination on the changes in body weight of mice before and after administration during an experiment on mice infected with systemic Candida albicans.

[0039] Fig.17 To investigate the effects of quinolinechalcone derivative PK-10 and fluconazole alone or in combination on the colony counts in the kidney, lung and liver of mice infected with systemic Candida albicans.

[0040] Fig.18 To investigate the effects of quinoline chalcone derivative PK-10 and fluconazole alone or in combination on spleen and thymus indexes in mice infected with systemic Candida albicans. DETAILED DESCRIPTION

[0041] The present invention is further described below, but the present invention is not limited in any way. Any changes made based on the present invention belong to the protection scope of the present invention.

[0042] The structural formula of quinoline chalcone involved in the present invention is as follows Figure 1 , Figure 2 and Figure 3 shown.

[0043] The synthetic route of quinoline chalcone involved in the present invention is as follows Figure 4 shown.

[0044] The preparation steps of quinoline chalcone involved in the present invention are:

[0045] (1) Preparation of compound 1: Weigh 2-chloroquinoline-3-carboxaldehyde (3.84 g, 20 mmol) and potassium carbonate (5.52 g, 40 mmol) in a 100 mL round-bottom flask, add 50 mL of dried acetonitrile to dissolve; then add N-methylpiperazine (4.0 g, 40 mmol), and heat under reflux to react overnight; after the raw material disappears as detected by thin layer chromatography, cool to room temperature and pour into 200 mL of cold water; extract with 120 mL of dichloromethane three times, dry the dichloromethane layer over anhydrous sodium sulfate and filter; concentrate in vacuo, and the residue is compound 1;

[0046] (2) Preparation of compound 2: 2.762 g 4-fluoroacetophenone (20 mmol) and 3.438 g anhydrous piperazine (40 mmol) were weighed in a 100 mL round-bottom flask, 60 mL N,N-dimethylformamide dried over potassium hydroxide was added, and finally 5.522 g potassium carbonate (40 mmol) was added. The mixture was placed in an oil bath at 115°C for one day. After the reaction, the mixture was poured into 200 mL ice water and stirred to precipitate a light yellow solid. The solid was filtered and washed with water (50 mL×3), and vacuum dried to obtain a light-colored solid compound 2.

[0047] (3) Synthesis of key intermediate PK-1: Weigh 2.55 g of prepared compound 1 (10 mmol) and 2.04 g of compound 2 (10 mmol) in a 100 mL round-bottom flask, add 40 mL of anhydrous methanol to dissolve; then add 4 g of sodium hydroxide in batches under ice-water bath cooling; after the solid is completely dissolved, remove the ice-water bath and continue to react at room temperature for 24 hours; after thin-layer chromatography detection shows that the reaction raw materials are basically gone, pour into 200 mL of cold water and extract with dichloromethane (30 mL×3); after the organic phases are combined, dry with anhydrous sodium sulfate, filter, and the filtrate is concentrated in vacuo; the residue is separated by column chromatography (using methanol / dichloromethane = 3:97 as the eluent) to obtain a yellow-brown solid key intermediate PK-1;

[0048] (4) Preparation of derivatives PK-2~PK20: Weigh 88 mg of the key intermediate PK-1 (0.2 mmol) into a 10 mL dry round-bottom flask, then add 69 mg potassium carbonate (0.6 mmol) and 5 mL dry dichloromethane in sequence to dissolve; finally, add 0.3 mmol of acyl chloride, acid anhydride, sulfonyl chloride or halogenated hydrocarbon dropwise, and react at room temperature for 5-12 h; after the reaction is completed by thin layer chromatography, add 5 mL of water and stir for 30 min; the dichloromethane layer is directly chromatographed using methanol / dichloromethane = 2:98 as the eluent to separate the derivatives PK-2~PK20.

[0049] The acid chloride, acid anhydride, sulfonyl chloride or halogenated hydrocarbon described in step (4) are: acetic anhydride, , , , , , , , , , , , , , , , , , .

[0050] Application of quinoline chalcone derivatives in combination with antifungal drugs against drug-resistant fungi.

[0051] The resistant fungus is resistant Candida albicans.

[0052] The resistant fungus was resistant Candida albicans (CA23).

[0053] The antifungal drug is fluconazole.

[0054] Embodiment 1:

[0055] Synthesis of key intermediate PK-1

[0056] Preparation of compound 1: Weigh 2-chloroquinoline-3-carboxaldehyde (3.84 g, 20 mmol) and potassium carbonate (5.52 g, 40 mmol) in a 100 mL round-bottom flask, add 50 mL of dried acetonitrile to dissolve. Then add N-methylpiperazine (4.0 g, 40 mmol) and heat to reflux overnight. After the raw material disappears by thin layer chromatography, cool to room temperature and pour into 200 mL of cold water. Extract with 120 mL of dichloromethane three times, and filter the dichloromethane layer after drying over anhydrous sodium sulfate. Concentrate in vacuo, and the residue does not need to be purified and can be used for the next step.

[0057] Preparation of compound 2: 2.762 g 4-fluoroacetophenone (20 mmol) and 3.438 g anhydrous piperazine (40 mmol) were weighed in a 100 mL round-bottom flask, 60 mL N,N-dimethylformamide dried over potassium hydroxide was added, and finally 5.522 g potassium carbonate (40 mmol) was added, and the mixture was placed in an oil bath at 115°C for one day. After the reaction, the mixture was poured into 200 mL of ice water and stirred to precipitate a light yellow solid. The solid was filtered and washed with water (50 mL×3), and vacuum dried to obtain a light-colored solid. 1 H NMR (400MHz, CDCl 3 ) δ: 7.19(d, J =8.7Hz, 2H), 6.95(d, J =8.9Hz, 2H), 3.40(t, J =4.7Hz, 4H), 3.11 (t, J =4.8Hz, 4H), 2.07(s, 1H), 2.02(s, 3H); 13 C NMR (100 MHz, CDCl 3) δ: 197.21, 146.26, 134.22, 122.31, 118.48, 46.70, 44.34,27.76.

[0058] Synthesis of key intermediate PK-1: Weigh 2.55g of prepared compound 1 (10mmol) and 2.04g of compound 2 (10mmol) in a 100 mL round-bottom flask, add 40 mL of anhydrous methanol to dissolve. Then add 4g of sodium hydroxide in batches under ice-water bath cooling. After the solid is completely dissolved, remove the ice-water bath and continue to react at room temperature for 24 hours. After the reaction raw materials are basically disappeared by thin layer chromatography, pour into 200 mL of cold water and extract with dichloromethane (30 mL×3). After the organic phases are combined, dry with anhydrous sodium sulfate, filter, and the filtrate is concentrated in vacuo. The residue is separated by column chromatography (using methanol / dichloromethane = 3:97 as eluent) to obtain 3.74g of yellow-brown solid with a yield of 85%.

[0059] Embodiment 2:

[0060] Preparation of PK-2~PK20: Weigh 88 mg of intermediate 3 (0.2 mmol) into a 10 mL dry round-bottom flask, then add 69 mg potassium carbonate (0.6 mmol) and 5 mL dry dichloromethane in order to dissolve. Finally, add 0.3 mmol of acyl chloride, anhydride, sulfonyl chloride or halogenated hydrocarbon dropwise, and react at room temperature for 5-12 hours. After the reaction is completed by thin layer chromatography, add 5 mL of water and stir for 30 minutes. The dichloromethane layer is directly chromatographed with methanol / dichloromethane = 2:98 as the eluent to separate the corresponding target product.

[0061] Compound PK-9: 1 H NMR (400MHz, CDCl 3 ) δ :8.22(s, 1H), 8.02(d, J =8.1Hz, 2H),7.95(d, J =8.6Hz, 2H), 7.75(d, J =8.4Hz, 2H), 7.62-7.66(m, 6H), 7.50(d, J =7.9Hz, 2H), 7.44(t, J =7.8Hz, 1H), 6.85(d, J =8.6Hz, 2H), 5.51(s, 2H), 3.99(s,4H), 3.89(d, J =14.7Hz, 2H), 3.69(d, J<h2 style=";text-align:left;direction:ltr">=10.9Hz, 2H), 3.61(s, 2H), 3.51(s, 3H),3.37(s, 4H), 2.57(s, 4H);<h2 style=";text-align:left;direction:ltr"> 13 <h2 style=";text-align:left;direction:ltr"> CNMR(101MHz,CDCl<h2 style=";text-align:left;direction:ltr"> 3 <h2 style=";text-align:left;direction:ltr"> )<h2 style=";text-align:left;direction:ltr"> δ <h2 style=";text-align:left;direction:ltr"> :186.9, 157.4, 154.3, 146.8, 143.8, 138.1, 137.2, 134.6, 132.7, 132.2, 131.9, 130.9, 130.8, 129.5, 127.9, 127.7, 127.2, 125.7, 125.5, 124.4, 122.7, 118.9, 117.8, 114.5, 113.4, 111.0, 62.3, 58.9, 53.5, 52.7, 47.1, 44.2.<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0062] <h2 style=";text-align:left;direction:ltr"> Download the latest version of PK-10:<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> H NMR (400MHz, CDCl<h2 style=";text-align:left;direction:ltr"> 3 <h2 style=";text-align:left;direction:ltr"> )<h2 style=";text-align:left;direction:ltr"> δ <h2 style=";text-align:left;direction:ltr"> :8.14(s, 1H)), 7.92(d,<h2 style=";text-align:left;direction:ltr"> J <h2 style=";text-align:left;direction:ltr"> =8.1Hz,2H), 7.90(d,<h2 style=";text-align:left;direction:ltr"> J <h2 style=";text-align:left;direction:ltr"> =9.0Hz, 2H), 7.72(d,<h2 style=";text-align:left;direction:ltr"> J <h2 style=";text-align:left;direction:ltr"> =16.0Hz, 1H), 7.70(d,<h2 style=";text-align:left;direction:ltr"> J <h2 style=";text-align:left;direction:ltr"> =7.7Hz, 2H), 7.56-7.58(m, 6H), 7.47(d,<h2 style=";text-align:left;direction:ltr"> J <h2 style=";text-align:left;direction:ltr"> =8.0Hz, 2H), 7.39(t,<h2 style=";text-align:left;direction:ltr"> J <h2 style=";text-align:left;direction:ltr"> =7.2Hz, 1H), 6.78(d,<h2 style=";text-align:left;direction:ltr"> J <h2 style=";text-align:left;direction:ltr"> =9.0Hz,2H), 5.46(s,2H), 4.01(d,<h2 style=";text-align:left;direction:ltr"> J <h2 style=";text-align:left;direction:ltr"> =9.6Hz, 2H), 3.94(d,<h2 style=";text-align:left;direction:ltr"> J <h2 style=";text-align:left;direction:ltr"> =12.4Hz, 2H), 3.86(d,<h2 style=";text-align:left;direction:ltr"> J <h2 style=";text-align:left;direction:ltr"> =14.8Hz, 2H), 3.66(d,<h2 style=";text-align:left;direction:ltr"> J <h2 style=";text-align:left;direction:ltr"> =11.0Hz, 2H), 3.57(s, 2H), 3.48(s, 3H), 3.32(t,<h2 style=";text-align:left;direction:ltr"> J <h2 style=";text-align:left;direction:ltr"> =4.4Hz, 4H), 2.55(t,<h2 style=";text-align:left;direction:ltr"> J <h2 style=";text-align:left;direction:ltr"> =4.6Hz, 4H);<h2 style=";text-align:left;direction:ltr"> 13 <h2 style=";text-align:left;direction:ltr"> CNMR(101MHz,CDCl<h2 style=";text-align:left;direction:ltr"> 3 <h2 style=";text-align:left;direction:ltr"> )<h2 style=";text-align:left;direction:ltr"> δ4, 58.9, 52.7, 47.1,46.2, 44.3.

[0063] Compound PK-13: 1 H NMR (400MHz, CDCl 3 ) δ :8.23(s, 1H), 8.06(d, J =9.0Hz, 2H),8.01(d, J =15.9Hz, 1H), 7.93(d, J =8.2Hz, 2H), 7.85(d, J =8.6Hz, 1H), 7.74(s,1H), 7.72(d, J =7.3Hz, 1H), 7.63(t, J =7.0Hz, 1H), 7.38(t, J =5.9Hz, 1H), 7.28(d, J =7.2Hz, 2H), 6.96(d, J =9.1Hz, 2H), 3.87(s, 2H), 3.50(t, J =5.0Hz, 8H),2.79(t, J =5.0Hz, 4H), 2.68(s, 4H), 2.42(s, 3H), 2.39(s, 3H); 13 CNMR (101MHz, CDCl 3 ) δ :195.8, 187.7, 159.8, 154.3, 147.7, 144.5, 140.7, 136.9, 133.6, 130.9,130.4, 129.5, 128.4, 128.2, 127.8, 127.7, 125.0, 124.5, 123.4, 122.9, 113.7,64.3, 55.2, 53.3, 50.4, 47.3, 46.3, 21.8.

[0064] Compound PK-14:1 H NMR (400 MHz, CDCl 3 ) δ : 8.22 (s, 1H), 8.14 (d, J J = 8.6 Hz, 2H), 8.06 (d, J J = 9.0 Hz, 2H), 8.00 (d, J J = 15.7 Hz, 1H), 7.85 (d, J J = 8.4 Hz, 1H), 7.79 (d, J J = 8.6 Hz, 2H), 7.73 (s, 1H), 7.71 (d, J J = 8.6 Hz, 1H), 7.59 - 7.63 (m, 1H), 7.33 - 7.37 (m, 1H), 6.95 (d, J J = 9.0 Hz, 2H), 3.87 (s, 2H), 3.47 (t, J J = 4.8 Hz, 8H), 2.78 (t, J J = 5.0 Hz, 4H), 2.69 (s, 4H), 2.37 (s, 3H); 13 C NMR (101 MHz, CDCl 3 ) δ : 195.2, 187.7, 159.8, 154.2, 147.7, 140.8, 138.9, 136.9, 132.6, 131.0, 130.9, 130.4, 128.9, 128.4, 127.8, 127.7, 125.0, 124.5, 123.4, 122.8, 117.9, 116.8, 113.8, 111.0, 64.8, 55.2, 53.2, 50.4, 47.4, 46.3, 40.2。

[0065] Compound PK-17: 1 H NMR (400 MHz, CDCl 3 ) δ : 8.22 (s, 1H), 8.07 (d, J J = 9.0 Hz, 2H), 8.01 (d, J J = 15.7 Hz, 1H), 7.85 (d, J J = 8.5 Hz, 1H), 7.68 - 7.73 (m, 2H), 7.63 (t, J J = 8.4 Hz, 1H), 7.38 (t, J<h2 style=";text-align:left;direction:ltr">=1.1Hz, 1H), 6.96(d,<h2 style=";text-align:left;direction:ltr"> J <h2 style=";text-align:left;direction:ltr"> =9.0Hz, 2H), 3.45-3.50(m, 8H),3.37-3.39(m, 4H), 2.83(s, 3H), 2.66(s, 4H), 2.38(s, 3H);<h2 style=";text-align:left;direction:ltr"> 13 <h2 style=";text-align:left;direction:ltr"> CNMR(101MHz,CDCl<h2 style=";text-align:left;direction:ltr"> 3 <h2 style=";text-align:left;direction:ltr"> )<h2 style=";text-align:left;direction:ltr"> δ <h2 style=";text-align:left;direction:ltr"> :187.8, 159.8, 153.7, 147.8, 141.1, 137.0, 130.8, 130.5, 129.3, 127.8, 127.7, 125.0, 124.6, 123.2, 122.6, 114.6, 55.2, 50.4, 47.7, 46.2, 45.5, 34.8.<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0066] <h2 style=";text-align:left;direction:ltr"> Description: PK-18:<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> H NMR (400MHz, CDCl<h2 style=";text-align:left;direction:ltr"> 3 <h2 style=";text-align:left;direction:ltr"> )<h2 style=";text-align:left;direction:ltr"> δ <h2 style=";text-align:left;direction:ltr"> : 8.20(s, 1H), 8.03(d,<h2 style=";text-align:left;direction:ltr"> J <h2 style=";text-align:left;direction:ltr"> =9.0Hz,2H), 7.99(d,<h2 style=";text-align:left;direction:ltr"> J <h2 style=";text-align:left;direction:ltr"> =15.6Hz, 1H), 7.84(d,<h2 style=";text-align:left;direction:ltr"> J <h2 style=";text-align:left;direction:ltr"> =8.5Hz, 1H), 7.70(d,<h2 style=";text-align:left;direction:ltr"> J <h2 style=";text-align:left;direction:ltr"> =10.6Hz, 1H), 7.67(d,<h2 style=";text-align:left;direction:ltr"> J <h2 style=";text-align:left;direction:ltr"> =8.2Hz, 2H), 7.62(t, 1H), 7.32-7.34(m, 3H), 6.87(d,<h2 style=";text-align:left;direction:ltr"> J <h2 style=";text-align:left;direction:ltr"> =9.0Hz, 2H), 3.43(s, 8H), 3.15(d,<h2 style=";text-align:left;direction:ltr"> J <h2 style=";text-align:left;direction:ltr"> =4.7Hz, 4H), 2.64(s, 4H), 2.42(s, 3H), 2.36(s, 3H);<h2 style=";text-align:left;direction:ltr"> 13 <h2 style=";text-align:left;direction:ltr"> CNMR(101MHz,CDCl<h2 style=";text-align:left;direction:ltr"> 3 <h2 style=";text-align:left;direction:ltr"> )<h2 style=";text-align:left;direction:ltr"> δ <h2 style=";text-align:left;direction:ltr"> :187.6, 159.7, 153.5, 147.7, 144.1, 140.9, 136.9, 132.3,130.7, 130.3, 129.9, 128.9, 127.9, 127.8, 127.6, 124.9, 124.5, 123.1, 122.5,114.3, 55.1, 50.3, 47.2, 46.2, 45.7.<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0067] Compound PK-19: 1 H NMR (400MHz, CDCl 3 ) δ :8.21(s, 1H), 8.04(d, J =9.0Hz,2H), 8.00(d, J =15.8Hz, 1H), 7.80-7.84(m, 3H), 7.70(d, J =15.6Hz, 1H), 7.63(t, J =7.0Hz, 1H), 7.37(t, J =8.0Hz, 1H), 7.27(d, J =4.6Hz, 1H), 7.23(d, J =8.6Hz,1H), 6.90(d, J =9.0Hz, 2H), 3.47(t, J =5.1Hz, 8H), 3.19(t, J =5.1Hz, 4H), 2.64(s, 4H), 2.37(s, 3H); 13 CNMR (101MHz, CDCl 3 ) δ 54.2, 114.5, 123.2, 122.5, 116.8, 116.5, 114.5, 55.2, 50.4, 47.3, 46.3, 45.7.

[0068] Compound PK-20: 1 HNMR (400MHz, CDCl 3 ) δ :8.24(s, 1H), 8.02(d, 3H), 7.90-7.93(m, 3H), 7.83(d, J =8.3Hz, 3H), 7.75(d, J =7.7Hz, 1H), 7.67(d, J =15.6Hz, 1H),7.63(d, J =8.1Hz, 2H), 7.37-7.41(m, 1H), 6.89(d,J =9.0Hz, 2H), 3.60(s, 4H),3.47(t, J =4.6Hz, 4H), 3.21(t, J =5.0Hz, 4H), 3.03(s, 4H), 2.66(s, 3H); 13 CNMR (101MHz, CDCl 3 ) δ :187.5, 158.7, 153.5, 147.4, 140.0, 137.1, 130.8, 130.6,129.0, 128.3, 127.8, 127.7, 126.6, 126.5, 126.4, 125.5, 125.4, 125.1, 125.0,123.1, 122.9, 114.5, 54.4, 48.9, 47.2, 46.1, 45.7, 45.2.

[0069] Embodiment 3:

[0070] Antifungal activity of quinolinechalcone derivatives PK-1~PK20 against ATCC14053

[0071] I. Materials and Methods

[0072] 1. Drugs and Culture Media

[0073] Quinoline chalcone derivatives PK-1 to PK20 were prepared by Example 1 and Example 2.

[0074] Prepare samples PK-1~PK20 into 50mg / ml stock solution with DMSO and store in a refrigerator at 4℃ for later use. Grind fluconazole dispersible tablets, fully dissolve with DMSO, sonicate for 30min, centrifuge at 7000rpm for 5min, take the supernatant, prepare 50mg / mL stock solution and store in a refrigerator at 4℃ for later use.

[0075] Sabouraud liquid medium (SDB): weigh 50.1g of this product, add 1L of distilled water or deionized water, stir and heat to boil until completely dissolved, divide into test tubes or Erlenmeyer flasks, autoclave at 115℃ for 15min. Store at 4℃ for future use.

[0076] Sabouraud solid culture medium (SDA): weigh 65.1g of this product, add 1L of distilled water or deionized water, stir and heat to boil until completely dissolved, divide into test tubes or Erlenmeyer flasks, autoclave at 115℃ for 15min, when the temperature of the autoclave drops to 60℃, take out, mix well, divide into sterile culture dishes, store at 4℃ for later use.

[0077] SD culture medium: Take 255, 15, and 30 mL of pure water into three reagent bottles respectively, sterilize them at 115℃ for 15 min, cool the pure water to 60℃, pour 2.01g 1×YNB into the reagent bottle with 10 mL of pure water, and pour 4g glucose into the reagent bottle with 20 mL of pure water. Mix them separately, then mix the three different solutions together, shake them well, cool them to room temperature, and store them in a 4℃ refrigerator for later use.

[0078] Spider medium: accurately weigh 2g of ready-to-use nutrient broth, add 200mL of deionized water and mix thoroughly until completely dissolved, sterilize at 115℃ for 30min, cool to about 60℃, add 2g of mannose and 0.4g of potassium dihydrogen phosphate, cool to room temperature, and store at 4℃ for later use.

[0079] PBS phosphate buffer: Add appropriate amount of deionized distilled water to PBS buffer powder to fully dissolve it, make up to 2L, sterilize it by high pressure at 121℃ for 30min, cool it to room temperature, and store it in a 4℃ refrigerator for later use.

[0080] 2. Experimental Methods

[0081] Take the sensitive strains of Candida albicans SC5314 and CA23 resistant strains frozen at -80℃ and inoculate them on Sabouraud agar medium. Activate them twice continuously. Pick 1-3 colonies and incubate them in Sabouraud liquid medium at 30℃ and 150 rpm for 16-17 h to activate them in the logarithmic growth phase. Wash them three times with PBS phosphate buffer and adjust the concentration of the bacteria to 2×10 5CFU / mL. Prepare the working solutions of PK-1~PK-24, fluconazole (FLC) single drugs with a final concentration of 400 µg / mL and PK-1~PK-24 combined with fluconazole with a final concentration of 400 µg / mL+400 µg / mL. Take a sterile 96-well flat-bottom culture plate, add 125 µL of the above-mentioned single drug and combined drug solutions in the first row, and set up 3 replicates for each drug and each concentration. From the second row, 100 µL of Sabouraud liquid medium was added to wells 1-12, and 200 µL of Sabouraud liquid medium was added to the last row as a blank control; the seventh row was the growth control well (Control, 100µL Sabouraud liquid medium + 100µL bacterial solution). Use a pipette to transfer 25 µL from the first row to the wells in the next row of the corresponding column. After mixing, transfer 25 µL from each well in the second row to the wells in the corresponding column of the third row and mix well. Repeat this process, and discard 25 µL after 5-fold dilution to the sixth row. The drug concentrations of PK-1~PK-24 and fluconazole in the first to sixth rows are 200, 40, 8, 1.6, 0.32, and 0.064 µg / mL, respectively. The drug concentrations of PK-1~PK-20 combined with fluconazole in the wells are 200+200, 40+40, 8+8, 1.6+1.6, 0.32+0.32, and 0.064+0.064µg / mL, respectively, which is a 5-fold decrease. The DMSO content in each well is less than 1%. Add 100 µl of the prepared 2×10 5 CFU / mL of Candida albicans liquid was added to make the final total volume of each well 200 µL. The prepared culture plate was placed in a constant temperature incubator at 30 ℃ and cultured for 24 h. The OD value at 630 nm was measured by an ELISA reader. The above experiment was repeated 3 times.

[0082] 3. Calculation formula

[0083] Minimum inhibitory concentration (MIC 80 ) End point determination: Compared with the growth control group, the lowest drug concentration corresponding to ≥80% growth inhibition is its MIC 80 . Inhibition rate and MIC 80 Calculation: The absorbance value was used to calculate the inhibition rate of each drug-treated well against Candida albicans according to the following calculation method: Inhibition rate = [100 - (OD 样品组吸光度 -OD 空白对照组吸光度 )] / (OD 对照组吸光度 -OD 空白对照组吸光度 )× 100%, MIC 80The values ​​were calculated by GraPhpad Prism 8.0 software. Evaluation of the interaction between the two drugs when used in combination: The Fractional Inhibitory Concentration Index (FICI) was used for determination.

[0084] FICI=FICIA+FICIB=MICcomb A / MICAlone A+ MICcomb B / MICAlone B, where MICAloneA and MICAlone B are the MIC values ​​of drug A and drug B when used alone; MICcombA and MICcombB are the MIC values ​​of drug A and drug B when used simultaneously. When the MIC value is higher than the upper limit of detection, the FICI is calculated by taking twice the upper limit concentration. The judgment criteria are: when FICI<0.5, the two drugs are synergistic; when 0.5≤FICI<4, the two drugs are unrelated; when FICI≥4, the two drugs are antagonistic.

[0085] 4. Test results

[0086] Table 1 Inhibitory activity of PK-1~PK-20 and fluconazole alone and in combination against Candida albicans SC5314 and CA23

[0087]

[0088]

[0089]

[0090] As can be seen from Table 1, PK-10 alone has an inhibitory effect on both the sensitive strain of Candida albicans SC5314 and the resistant strain of CA23, and the MIC 80 PK-1, PK-4, PK-7, PK-8, PK-9, and PK-10 all had inhibitory effects on the sensitive strain of Candida albicans SC5314 when used alone, and the MIC 80The results were 172.03, 155.70, 155.78, 106.53, 182.11, and 27.77 µg / mL, respectively. The samples that had synergistic inhibitory effects on both SC5314-sensitive and CA23-resistant strains of Candida albicans when used in combination with fluconazole were PK-1, PK-5, PK-6, PK-10, and PK-21. The sample that had synergistic inhibitory effects on only SC5314-sensitive strains of Candida albicans when used in combination with fluconazole was PK-12, and the samples that had synergistic inhibitory effects on only CA23-resistant strains of Candida albicans when used in combination with fluconazole were PK-4, PK-7, PK-8, PK-9, PK-13, PK-14, PK-15, PK-16, PK-17, PK-18, PK-19, PK-20, PK-22, and PK-23. From the above results, it can be seen that PK-6, PK-10 combined with fluconazole have the strongest synergistic antibacterial effect on SC5314 and CA23.

[0091] Take Candida albicans CA187, CA808, CA381, CA602, CA550, CA800, CA799, CA3816, CA4508, CA3511, CA4574, ATCC10231 frozen at -80℃ and inoculate them on Sabouraud agar medium. Activate them twice continuously. Pick 1-3 colonies and place them in Sabouraud liquid medium. Activate them at 30℃ and 150 rpm for 16-17 h to make them in logarithmic growth phase. Wash them three times with PBS phosphate buffer and adjust the concentration of bacteria to 2×10 5CFU / mL. PK-6 and PK-10 were prepared to a final concentration of 80 µg / mL, fluconazole was prepared to a final concentration of 80 µg / mL, and PK-6, PK-10 combined with fluconazole were prepared to a final concentration of 80+80 µg / mL. Take a sterile 96-well flat-bottom culture plate, add 125 µL of the above-mentioned single drug and combined drug solutions in the first row, and set up 3 replicates for each drug and each concentration. From the second row, 25 µL of Sabouraud liquid medium was added to wells 1-12, and 200 µL of Sabouraud liquid medium was added to the last row as a blank control; the seventh row was the growth control well (Control, 100 µL Sabouraud liquid medium + 100 µL bacterial solution). Use a pipette to transfer 25 µL from the first row to the wells in the next row of the corresponding column. After mixing, transfer 25 µL from each well in the second row to the wells in the corresponding column of the third row and mix well. Repeat this process, perform 5-fold dilution to the sixth row and discard 25 µL. The drug concentrations of PK-6, PK-10 and fluconazole in the first to sixth rows are 40, 8, 1.6, 0.32, 0.064, 0.0128 µg / mL, respectively. The drug concentrations of PK-6, PK-10 combined with fluconazole in the wells are 40+40, 8+8, 1.6+1.6, 0.32+0.32, 0.064+0.064, 0.0128+0.0128 µg / mL, respectively, which is a 5-fold decrease. The DMSO content in each well is less than 1%. 100 µL of the prepared solution with a concentration of 2×10 5 CFU / mL of Candida albicans liquid was added to make the final total volume of each well 200 µL. The prepared culture plate was placed in a constant temperature incubator at 30 ℃ and cultured for 24 h. The OD value at 630 nm was measured by an ELISA reader. The above experiment was repeated 3 times.

[0092] Table 2 Inhibitory activity of PK-10 and fluconazole alone and in combination against different clinical Candida albicans

[0093]

[0094] As can be seen from Table 2, PK-6 and PK-10 combined with fluconazole have inhibitory effects on different strains of Candida albicans, and the mode of action is synergistic. The synergistic antibacterial effect of PK-10 combined with fluconazole is better than that of PK-6 combined with fluconazole. Therefore, PK-10 will be used as the research object for mechanism exploration in the future.

[0095] Time-killing curves of quinoline chalcone derivative PK-10 and fluconazole alone or in combination against Candida albicans ATCC14053

[0096] MIC 80The cells were divided into 6 groups, namely, growth control group (Control), PK-10 (final concentration of 1µg / mL), fluconazole (final concentration of FLC, 1µg / mL), PK-10+fluconazole low-dose group (PK-10+FLC (L), final concentration of 0.5µg / mL+1µg / mL), PK-10+fluconazole medium-dose group (PK-10+FLC (M), final concentration of 1µg / mL+1µg / mL), PK-10+fluconazole high-dose group (PK-10+FLC (H), final concentration of 2µg / mL+1µg / mL), the final volume of each group was 10mL, and Candida albicans culture in the logarithmic growth phase was added to make the final concentration of each group of culture liquid 1×10 5 CFU / mL. After mixing, place in a 30℃ 150rpm constant temperature shaker for culture, take out at 0, 4, 8, 12, 24, 36, and 48h, mix, take 200µL of each group in a 96-well culture plate, three replicates, and measure the OD value at 630nm with an enzyme marker. 630 The time-killing curve was plotted with the value as the ordinate. The experiment was repeated three times.

[0097] from Figure 5 It can be seen that compared with the control group, the PK-10 single-use group had no inhibitory effect on Candida albicans ATCC14053. The antibacterial activity of the drug combination group was better than that of the fluconazole group starting from 8 hours, and it showed a certain dose dependence. It could still inhibit the growth of Candida albicans ATCC14053 at 48 hours, and the difference was statistically significant ( **** P <0.0001).

[0098] Effects of quinolinechalcone derivative PK-10 and fluconazole alone or in combination on morphological transformation of Candida albicans ATCC14053

[0099] Collect Candida albicans in the logarithmic growth phase and adjust the bacterial concentration to 1×10 5 CFU / mL. 1 mL of Candida albicans solution containing PK-10 (final concentration of 1µg / mL), fluconazole (final concentration of 1µg / mL), PK-10+fluconazole (final concentration of 1µg / mL+1µg / mL) and drug-free solution (final concentration of 1×10 5 CFU / mL) were added to a 24-well culture plate and placed in a 30°C constant temperature incubator for 2, 4, and 8 hours, respectively. The culture plate was then taken out and the hyphae formation of Candida albicans was observed and photographed under an inverted microscope. The experiment was repeated three times.

[0100] Candida albicans hyphae induced by SD+10% FBS and Spider medium Figure 6 As shown in Figure 7, the results showed that compared with the control, after 2 hours of culture, the PK-10 group, the fluconazole group alone and the PK-10 combined with fluconazole group all had smaller germ tubes with no significant difference, and all had no inhibitory effect. However, after 4 hours of culture, the control and PK-10 groups formed slender and loose hyphae, the fluconazole group alone and the PK-10 combined with fluconazole group formed long hyphae, which were shorter than those of the control group and the PK-10 group, and the amount of hyphae in the field of view of the PK-10 combined with fluconazole group was less than that of the fluconazole group. After 8 hours of culture, the control and PK-10 groups formed very long and interlaced hyphae, and many yeast phase and pseudohyphal phase Candida albicans were attached to the hyphae. The fluconazole group alone and the PK-10 combined with fluconazole group did not form long hyphae, and there were still many pseudohyphal cells, and the pseudohyphal clusters of the PK-10 combined with fluconazole group were less than those of the fluconazole group.

[0101] Effects of quinolinechalcone derivative PK-10 and fluconazole alone or in combination on the efflux pump function of Candida albicans

[0102] The effect of each group of drugs on the efflux pump function of Candida albicans was determined using rhodamine 6G (R6G), a fluorescent pigment substrate of the efflux pump. Candida albicans in the logarithmic growth phase were collected and the bacterial concentration was adjusted to 1×10 7 CFU / mL. Set up Candida albicans growth group (Control); fluconazole group (FLC, 1µg / mL); PK-10 group (PK-10, 1µg / mL); PK-10 combined with fluconazole group (PK-10+FLC, (1µg / mL+1µg / mL). Each group had a volume of 10mL and was placed in a 30℃ constant temperature shaker for 4h. Wash three times with PBS phosphate buffer and resuspend with sterile PBS phosphate buffer to adjust the bacterial concentration of each group to 1×10 7CFU / mL. Continue to incubate in a constant temperature shaker for 1 hour to fully deplete the glucose in Candida albicans, add Rh6G under light-proof conditions, so that the final concentration of each group reaches 10uM, and continue to incubate in a constant temperature shaker in the dark for 2 hours to allow the Candida albicans cells to fully contact and fuse with the Rh 6G fluorescent dye. After the incubation is completed, each group is placed in an ice water bath and incubated in the dark for 10 minutes to stop the Candida albicans cells from absorbing Rh 6G. After the ice water bath, the bacterial cells of each group were collected by centrifugation, and the bacterial suspension was washed 3 times with pre-cooled PBS to remove the exogenous Rh6G in the Candida albicans cells. The cells were collected by centrifugation and resuspended with PBS phosphate buffer containing 5% glucose. After mixing, place in a constant temperature shaker at 30℃ and 150rpm for incubation. Take out at 0, 30, 60, 90, and 120min, mix, take 1mL of each group into an EP tube, centrifuge at 4℃ and 12000g for 2min, take 100uL of the supernatant into a 96-well culture plate black board, set 3 duplicate wells for each group, and use a multifunctional microplate reader to measure its fluorescence value (excitation wavelength 488nm, emission wavelength 525nm). Pay attention to light protection during operation. Repeat the experiment three times.

[0103] from Figure 8 It can be seen that the MFI values ​​of each group showed an upward trend, indicating that the drug efflux activity of each group in Candida albicans cells increased over time. Compared with the control group, the PK-10 group, the fluconazole group or the PK-10 combined with fluconazole group could significantly inhibit this increase after 30 minutes, and this inhibitory effect was still significant during the 60-120 minute period ( **** P <0.0001). This indicates that PK-10 alone or in combination with fluconazole reduced the efflux capacity of the strain.

[0104] Effects of quinoline chalcone derivative PK-10 and fluconazole alone or in combination on mitochondrial membrane potential in Candida albicans cells

[0105] The mitochondrial membrane potential was measured using the fluorescent probe JC-1. The Candida albicans suspension in the logarithmic growth phase was treated with fluconazole (FLC, 1µg / mL); PK-10 (PK-10, 1µg / mL); PK-10 combined with fluconazole (PK-10+FLC, (1µg / mL+1µg / mL)). According to the instructions provided by the manufacturer, JC-1 fluorescent probe was added to each group of bacterial suspension to make the final concentration of 10 µg / mL under light-proof conditions, and the mixture was fully mixed and incubated in a constant temperature shaker at 30°C, 150rpm for 20 min. After incubation, the bacterial suspensions in each group were washed twice with PBS and centrifuged (12000g, 4°C, 2min). 100µL of the supernatant was placed in a 96-well plate with three replicates in each group. The red (excitation wavelength 525nm, emission wavelength 590nm) and green fluorescence values ​​(excitation wavelength 490nm, emission wavelength 530nm) were measured using a full-wavelength multifunctional microplate reader. The membrane potential was determined by the ratio of red and green fluorescence.

[0106] from Fig. 9 It can be seen that compared with the control group, PK-10 did not reduce the expression of JC-1 MFI in the cells, while the fluconazole group and the PK-10 combined with fluconazole group significantly reduced the expression of JC-1 MFI in the Candida albicans cells, indicating that the mitochondrial membrane potential in the Candida albicans cells was unstable and significantly downregulated, and the difference was statistically significant ( **** P <0.0001).

[0107] Effects of quinoline chalcone derivative PK-10 and fluconazole alone or in combination on mitochondrial membrane potential in Candida albicans cells

[0108] DCFH-DA was used as a fluorescent probe to detect the level of reactive oxygen in Candida albicans cells. The suspension of Candida albicans in the logarithmic growth phase was treated with fluconazole (FLC, 1µg / mL); PK-10 (PK-10, 1µg / mL); PK-10 combined with fluconazole (PK-10+FLC, (1µg / mL+1µg / mL)). According to the instructions provided by the manufacturer, DCFH-DA was added and incubated at 30°C and 150rpm for 30min to allow the probe to fully fuse with Candida albicans. The cells were washed twice with PBS and resuspended. The cells were centrifuged (12000g, 4°C, 2min) and 100 µL of the supernatant was placed in a 96-well plate with three replicate wells in each group. The fluorescence value was determined using a multifunctional microplate reader (excitation wavelength 488nm, emission wavelength 525nm). The experiment was repeated three times.

[0109] from Fig.10 It can be seen that compared with the control group, the intracellular ROS accumulation of Candida albicans in the fluconazole group and the PK-10 combined with fluconazole group was significantly higher than that in the control group (**** P <0.0001), while the accumulation of ROS in the PK-10 group did not change significantly. The results suggest that the good antibacterial effect of PK-10 combined with fluconazole is related to the induction of ROS accumulation in Candida albicans cells, while the lack of antibacterial effect of PK-10 group is related to its inability to induce ROS accumulation.

[0110] Effects of quinolinechalcone derivative PK-10 and fluconazole alone or in combination on intracellular ATP in Candida albicans

[0111] The enhanced ATP detection kit was used to detect the ATP level in the cells. The final concentration of the Candida albicans in the logarithmic growth phase was adjusted to 1×10 5 CFU / mL, treated with different drugs, the drug groups in each group are as follows: Candida albicans group (Control); Fluconazole treatment group (FLC, 1µg / mL);

[0112] PK-10 treatment group (PK-10, 1µg / mL): PK-10 combined with fluconazole treatment group (PK-10+FLC, (1µg / mL+1µg / mL). Incubate in a 30°C constant temperature shaker for 16 h, wash three times with PBS phosphate buffer, and adjust the bacterial concentration of each group to 1×10 7 CFU / mL, cells were collected by centrifugation and the manufacturer's instructions were followed.

[0113] ATP detection lysis solution was added to each group, and the lysis solution was vortexed vigorously to fully contact and lyse the cells. After lysis, centrifuge at 4ºC12000g for 5 minutes, and the supernatant was taken for subsequent determination. According to the requirement of each sample / standard, 100µL detection working solution was added to the 96-well blackboard detection well, placed at room temperature for 5 minutes, and then 20µL sample / standard was added to the detection well, mixed quickly, and 3 replicate wells were set for each group. The chemiluminescence was detected by a multifunctional microplate reader Luminometer. The experiment was repeated three times. In order to eliminate the error caused by the difference in protein amount during sample preparation, the protein concentration in the sample was determined using the BCA protein concentration determination kit.

[0114] from Fig.11 It can be seen that compared with the Control, PK-10 alone had no effect on the intracellular ATP content of Candida albicans, but the intracellular ATP content was significantly reduced after combined use with fluconazole, and the difference was statistically significant ( **** P <0.0001).

[0115] Effects of quinoline chalcone derivative PK-10 and fluconazole alone or in combination on genes related to the RAS1-cAMP-EFG1 signaling pathway

[0116] To evaluate the molecular mechanism of PK-10, real-time quantitative PCR (qRT-PCR) was used to measure the expression of RAS1

[0117] -The expression of cAMP-EFG1 signaling pathway related genes RAS1, CYR1, EFG1, ECE1, ALS3, and HWP1. Candida albicans suspension 1×10 5 CFU / mL was incubated with FLC (final concentration 1µg / mL) or PK-10 (final concentration 1µg / mL) or FLC+PK-10 (final concentration 1+1µg / mL) and shaken at 30℃ for 16h. A drug-free growth control was set up. Centrifuge at 3500rpm for 5 minutes and wash three times with PBS. The fungal cells were collected and ground with liquid nitrogen, and total RNA was extracted using Trizol reagent. The concentration and purity of RNA were determined using a NanoDroplite spectrophotometer, and the integrity of RNA was determined by electrophoresis. RNA (1µg) was reverse transcribed into cDNA using the GoScript reverse transcription kit (Promega). The expression of related genes was detected by qPCR (Thermo). 2 -(∆∆Ct) Methods: Relative gene expression was calculated. Primers were designed by NCBI and synthesized by Qingke Biotechnology Co., Ltd. The primer sequences are shown in Table 3.

[0118]

[0119] from Fig.12 It can be seen that compared with the growth control group (Control), the PK-10+FLC group can downregulate RAS1、CYR1、 EFG1, ECE1, ALS3, HWP1 The expression levels of protein genes were statistically different ( **** P <0.0001).

[0120] Rescue Trial

[0121] To verify the effect of cAMP on the cAMP-EFG1 pathway after PK-10 treatment, the Candida albicans cell culture medium (1×105 CFU / mL) was prepared with SD+10% FBS and Spider liquid medium, respectively, and co-cultured with PK-10 (final concentration of 1μg / mL) or FLC (final concentration of 1μg / mL) or PK-10+FLC (final concentration of 1+1μg / mL) solution in 24-well plates and incubated at 30℃ for 4h and 8h with or without dibutyl-cAMP (db-cAMP). The group without db-cAMP served as the control group. Images were acquired using an inverted microscope.

[0122] like Fig.13 , 14As shown in the figure, after the addition of exogenous cAMP, the formation of Candida albicans hyphae became more dense, and the formation of hyphae in the drug combination group in SD+10%FBS and Spider liquid culture medium was restored to a certain extent compared with the case without adding exogenous cAMP, indicating that the addition of exogenous cAMP can partially rescue the inhibitory effect of PK-10 combined with fluconazole on Candida albicans.

[0123] Evaluation of the in vivo antifungal activity of quinoline chalcone derivatives PK-10 and FLC in a mouse systemic Candida albicans infection model

[0124] BALB / C male mice (6-8 weeks; weight 18-22g) were used to establish a mouse systemic Candida albicans infection model. First, cyclophosphamide (100mg / Kg, 0.1mL / 10g, once a day) was intraperitoneally injected for 3 consecutive days to cause systemic immunodeficiency in mice. Subsequently, the mice were randomly assigned to the following experimental groups:

[0125]

[0126] Then, on the fourth day, mice were inoculated via the caudal vein with 5×10 5 CFU / mouse Candida albicans suspension. 2h after infection, each treatment group was gavaged with 0.2 mL / 10g, once a day. The blank group, control group, and model group were gavaged with 0.2 mL / 10g of water, once a day. The administration was continued for 6 consecutive days. During the 6-day treatment, the general condition of the mice (activity, hair condition, body weight, and survival) was observed and recorded. After anesthesia, the mice were killed by cervical dislocation, and appropriate amounts of kidneys, lungs, and livers were weighed, added with 0.9% saline, and ground with grinding fluid to make a homogenate. 10μL was spread on a Sabouraud solid plate containing 1% penicillin and streptomycin, placed in a 30℃ constant temperature incubator for 48h, and the number of colonies was counted.

[0127] from Fig.15 It can be seen that there was no death in the Blank group and the Control group, and the survival rate was 100%; the survival rate of the Model group was 20%, the survival rate of the FLC group and the PK-10 group was 30%, and the survival rate of the PK-10+FLC combination low and medium dose groups was 90%. Both combination groups significantly improved the survival rate of mice infected with systemic Candida albicans, and the effect was better than that of the FLC and PK-10 groups alone.

[0128] from Fig.16 It can be seen that there was no significant difference in the body weight of mice in the PK-10 and fluconazole combination group compared with before administration, indicating that the combined drug treatment can restore the survival status of infected mice.

[0129] from Fig.17It can be seen that compared with the Model group or FLC group, PK-10 alone or in combination with fluconazole can significantly reduce the number of bacterial colonies in the kidneys of mice (compared with the Model group, **** P <0.0001; compared with FLC group, #### P <0.0001). However, there was no significant change in the colony counts in the lungs and liver.

[0130] from Fig.18 It can be seen that compared with the Model group or FLC group, the combination of PK-10 and fluconazole at low and medium doses can significantly increase the spleen coefficient of mice (compared with the Model group, **** P <0.0001; compared with FLC group, #### P <0.0001). Compared with the Model group, the combination of PK-10 and fluconazole at low and medium doses can significantly increase the thymus index of mice (compared with the Model group, **** P <0.0001); compared with the FLC group, the combination of PK-10 and fluconazole at a medium dose can significantly increase the thymus index of mice (compared with the FLC group, #### P <0.0001).

Claims

1. A quinolinechalcone derivative, characterized in that: The structural formula is as follows: , where R=H, , , , , , , , , , , , , , , , , , , .

2. A method for synthesizing a quinolinechalcone derivative as claimed in claim 1, characterized in that The synthetic route is as follows: ， The specific steps are: (1) Preparation of compound 1: 3.84 g, 20 mmol, of 2-chloroquinoline-3-carboxaldehyde and 5.52 g, 40 mmol, of potassium carbonate were weighed into a 100 mL round-bottom flask, and 50 mL of dried acetonitrile was added to dissolve; then 4.0 g, 40 mmol, of N-methylpiperazine was added, and the mixture was heated under reflux to react overnight; after the disappearance of the raw material by thin layer chromatography, the mixture was cooled to room temperature and poured into 200 mL of cold water; the mixture was extracted with 120 mL of dichloromethane three times, and the dichloromethane layer was dried over anhydrous sodium sulfate and filtered; the mixture was concentrated in vacuo, and the residue was compound 1; (2) Preparation of Compound 2: 2.762 g, 20 mmol, of 4-fluoroacetophenone and 3.438 g, 40 mmol, of anhydrous piperazine were weighed in a 100 mL round-bottom flask, 60 mL of N,N-dimethylformamide dried over potassium hydroxide was added, and finally 5.522 g, 40 mmol, of potassium carbonate was added. The mixture was placed in an oil bath at 115°C for one day. After the reaction, the mixture was poured into 200 mL of ice water and stirred to precipitate a light yellow solid. The solid was filtered and washed three times with 50 mL of water and dried in vacuo to obtain a light-colored solid Compound 2. (3) Synthesis of key intermediate PK-1: Weigh 2.55 g, 10 mmol of the prepared compound 1 and 2.04 g, 10 mmol of the compound 2 in a 100 mL round-bottom flask, add 40 mL of anhydrous methanol to dissolve; then add 4 g of sodium hydroxide in batches under ice-water bath cooling; after the solid is completely dissolved, remove the ice-water bath and continue to react at room temperature for 24 hours; after thin-layer chromatography detection shows that the reaction raw materials are basically gone, pour into 200 mL of cold water and extract with 30 mL of dichloromethane three times; after the organic phases are combined, dry with anhydrous sodium sulfate, filter, and the filtrate is concentrated in vacuo; the residue is separated by column chromatography using methanol / dichloromethane = 3:97 as the eluent to obtain a yellow-brown solid key intermediate PK-1; (4) Preparation of derivatives PK-2~PK20: Weigh 88 mg and 0.2 mmol of the key intermediate PK-1 into a 10 mL dry round-bottom flask, then add 69 mg and 0.6 mmol of potassium carbonate and 5 mL of dry dichloromethane in sequence to dissolve; finally, add 0.3 mmol of acyl chloride, acid anhydride, sulfonyl chloride or halogenated hydrocarbon dropwise, and react at room temperature for 5~12 h; after the reaction is completed by thin layer chromatography, add 5 mL of water and stir for 30 min; the dichloromethane layer is directly chromatographed using methanol / dichloromethane = 2:98 as the eluent to separate the derivatives PK-2~PK20.

3. A method for synthesizing a quinolinechalcone derivative as claimed in claim 2, characterized in that: The acid chloride, acid anhydride, sulfonyl chloride or halogenated hydrocarbon described in step (4) are: acetic anhydride, , , , , , , , , , , , , , , , , , .

4. Use of the quinoline chalcone derivative as claimed in claim 1 in combination with fluconazole in the preparation of an anti-drug resistant Candida albicans drug.

5. The use according to claim 4, characterized in that: The drug-resistant Candida albicans is a Candida albicans CA23 resistant strain.

Images