Use of a 2,6-dibenzylidenecyclohexanone analogue in the preparation of an anti-schistosome drug

Abstract

The application belongs to the technical field of medicine, and particularly relates to application of 2,6-benzhydryl cyclohexanone analogues in preparation of anti-schistosome drugs. The application provides application of 2,6-benzhydryl cyclohexanone analogues in preparation of anti-schistosome drugs, wherein the 2,6-benzhydryl cyclohexanone analogue has a structure shown in formula 1. The 2,6-benzhydryl cyclohexanone analogue is synthesized by introducing a cyclohexanone structure (substituting a methylene structure), thereby improving stability of the compound, and improving lipophilicity (ClogP is increased from 2.64 to 3.70) through the cyclohexanone structure, which is helpful to make the compound be absorbed by a schistosome body membrane more quickly, further improve anti-schistosome activity of the compound, especially in vivo anti-worm activity, and then play a killing effect earlier.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of a 2,6-diphenylmethylenecyclohexanone analog in the preparation of antischistosomiasis drugs. Background Technology

[0002] Schistosomiasis is an infectious disease that seriously endangers human health.

[0003] Currently, chemical drugs remain the primary method for treating schistosomiasis. Since Kikuth et al. began systematic research on drugs for treating schistosomiasis in the mid-1920s, dozens of chemotherapeutic drugs with insecticidal activity have emerged. However, only a few, such as praziquantel and oxaniquine, have been commercially produced. Praziquantel is currently the only effective drug widely used to prevent and control infections of various adult schistosomiasis worms in humans and animals. However, due to its extensive and long-term use, drug resistance has developed. According to the World Health Organization, 200 million people worldwide are infected with schistosomiasis, approximately 600 million are at risk of infection, 20 million have a high incidence rate, and at least 280,000 people die from schistosomiasis each year. In my country, counties where schistosomiasis transmission has not yet been controlled are concentrated in marshy and mountainous areas with numerous animal hosts, widespread distribution of Oncomelania snails, and significant environmental influences, making prevention and control particularly difficult, and reinfection remains a serious problem.

[0004] Given the large number of people infected with schistosomiasis, relying solely on praziquantel as a chemical drug is disproportionate. Therefore, finding other or alternative anti-schistosomiasis drugs has become a public health priority. Summary of the Invention

[0005] The purpose of this invention is to provide an application of a 2,6-diphenylmethylenecyclohexanone analog in the preparation of anti-schistosomiasis drugs. The 2,6-diphenylmethylenecyclohexanone analog with the structure shown in Formula 1 provided by this invention has strong anti-schistosomiasis activity and low cytotoxicity. It can repair liver fibrosis in mammals (mice) suffering from schistosomiasis and protect the host liver.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides the application of a 2,6-diphenylmethylenecyclohexanone analog in the preparation of an anti-schistosomiasis drug, wherein the 2,6-diphenylmethylenecyclohexanone analog has the structure shown in Formula 1:

[0008]

[0009] In Formula 1: R is one or more of hydroxyl, alkoxy, halogen, haloalkyl and cyano groups; n is an integer from 0 to 5.

[0010] Preferably, R is one or more of hydroxyl, methoxy, halogen, halomethyl and cyano.

[0011] Preferably, R is one or more of hydroxyl, methoxy, fluorine, bromine, chlorine, CF3 and cyano.

[0012] Preferably, the 2,6-diphenylmethylenecyclohexanone analogue has any one of the following structures:

[0013]

[0014] Preferably, the method for preparing the 2,6-diphenylmethylenecyclohexanone analogue includes the following steps:

[0015] The compound with the structure shown in Formula 2, cyclohexanone, organic solvent and inorganic strong acid were mixed and subjected to aldol condensation reaction to obtain the 2,6-diphenylmethylenecyclohexanone analog with the structure shown in Formula 1.

[0016] In Formula 2: R is one or more of hydroxyl, alkoxy, halogen, haloalkyl and cyano groups; n is an integer from 0 to 5.

[0017] Preferably, the molar ratio of the compound with the structure shown in Formula 2 to cyclohexanone is 2:1.

[0018] Preferably, the inorganic strong acid is hydrochloric acid, and the mass content of HCl in the hydrochloric acid is 36-38%.

[0019] Preferably, after the aldol condensation reaction is completed, the aldol condensation reaction solution is obtained, and the process further includes: adjusting the pH of the aldol condensation reaction solution to neutral, extracting the obtained neutral reaction solution with dichloromethane, combining the organic phases, and then sequentially drying, concentrating, and purifying by column chromatography to obtain a pure product of the 2,6-diphenylmethylenecyclohexanone analogue with the structure shown in Formula 1. The eluent used in the column chromatography purification is petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is (1-20):1.

[0020] This invention provides an anti-schistosomiasis drug comprising a 2,6-diphenylmethylenecyclohexanone analog of Formula 1 and a pharmaceutically acceptable carrier;

[0021]

[0022] In Formula 1: R is one or more of hydroxyl, alkoxy, halogen, haloalkyl and cyano groups; n is an integer from 0 to 5.

[0023] Preferably, the dosage form of the antischistosomiasis drug includes tablets, capsules, granules, oral liquids, or injections.

[0024] This invention provides the application of a 2,6-diphenylmethylenecyclohexanone analog in the preparation of antischistosome drugs, wherein the 2,6-diphenylmethylenecyclohexanone analog has the structure shown in Formula 1. This invention synthesizes a 2,6-diphenylmethylenecyclohexanone analog by introducing a cyclohexanone structure (substituting the methylene structure), which improves the stability of the compound and enhances its lipophilicity (ClogP increases from 2.64 to 3.70) through the cyclohexanone structure. This facilitates faster absorption by the schistosome membrane, improves the compound's antischistosome activity, especially its in vivo antiparasitic activity, and thus achieves its insecticidal effect earlier. The results of the examples show that the 2,6-diphenylmethylenecyclohexanone analog provided by this invention improves the compound's in vitro antischistosome activity through the cyclohexanone structure. Within the suitable lipid solubility range of the compounds (ClogP 3-4), the introduction of methoxy groups and strong electron-withdrawing compounds effectively improved their anti-insect activity. The position of the substituents also modulated the activity, with dimethoxy (compound 3, compound 18) and 4-cyano (compound 20) phenyl showing the best activity. Cytotoxicity experiments evaluated compounds 3 and 20 as having low cytotoxicity. Compound 20, in particular, exhibited outstanding in vivo insecticidal effects; continuous administration at 500 mg / kg for 5 days resulted in an 86.7% reduction rate of parasites, a 90.1% reduction rate of liver eggs, and a 95.3% reduction rate of small intestinal eggs. Furthermore, two mice (40%) were completely treated. Simultaneously, compound 20 significantly protected the liver of mice with schistosomiasis (the host) and reversed liver fibrosis, repairing the liver, far superior to praziquantel, which did not show significant anti-liver fibrosis activity. Attached Figure Description

[0025] Figure 1 The flowchart for the preparation of 2,6-diphenylmethylenecyclohexanone analogs provided in the examples;

[0026] Figure 2 The diagram shows the antischistosomiasis activity results of compounds 1, 2, 3 and 4 prepared for the examples;

[0027] Figure 3 The results of the antischistosomiasis activity of compounds 5, 6, 7 and 8 prepared for the examples are shown in the figure.

[0028] Figure 4 The results of the antischistosomiasis activity of compounds 9, 10, 11 and 12 prepared for the examples are shown in the figure.

[0029] Figure 5 The results of the antischistosomiasis activity of compounds 13, 14, 15 and 16 prepared for the examples are shown in the figure.

[0030] Figure 6The results of the antischistosomiasis activity of compounds 17, 18, 19 and 20 prepared for the examples are shown in the figure.

[0031] Figure 7 The effects of cisplatin, compound 3, compound 18 and compound 20 on the survival and growth of L929 cells;

[0032] Figure 8 The changes in liver and spleen indices after gavage administration of compounds 3 and 20 in in vivo experiments;

[0033] Figure 9 The images show histopathological sections of mouse liver tissue (×100) after gavage administration of compound 20 in in vivo experiments; the blank group ( Figure 9 A), negative control group ( Figure 9 B), administered via gavage at a dose of 500 mg / kg praziquantel ( Figure 9 C) and compound 20 ( Figure 9 D);

[0034] Figure 10 The changes in hydroxyproline in the liver of mice after treatment with compound 20 and praziquantel. Detailed Implementation

[0035] This invention provides the application of a 2,6-diphenylmethylenecyclohexanone analog in the preparation of an anti-schistosomiasis drug, wherein the 2,6-diphenylmethylenecyclohexanone analog has the structure shown in Formula 1:

[0036]

[0037] In Formula 1: R is one or more of hydroxyl, alkoxy, halogen, haloalkyl and cyano groups; n is an integer from 0 to 5.

[0038] In this invention, unless otherwise specified, all raw materials / components used in the preparation are commercially available products well known to those skilled in the art.

[0039] In this invention, R in Formula 1 is preferably one or more of hydroxyl, methoxy, halogen, halomethyl and cyano, more preferably one or more of hydroxyl, methoxy, fluorine, bromine, chlorine, CF3 and cyano, and even more preferably one or more of methoxy, hydroxyl and cyano.

[0040] In this invention, n in Formula 1 is preferably an integer from 0 to 3, and more preferably an integer from 1 to 3.

[0041] In this invention, the 2,6-diphenylmethylenecyclohexanone analogue preferably has any one of the following structures:

[0042]

[0043] In this invention, the 2,6-diphenylmethylenecyclohexanone analogue is more preferably compound 3, compound 18 or compound 20, further preferably compound 3 or 20, and most preferably compound 20.

[0044] In this invention, the method for preparing the 2,6-diphenylmethylenecyclohexanone analog preferably includes the following steps:

[0045] The compound with the structure shown in Formula 2, cyclohexanone, organic solvent and inorganic strong acid were mixed and subjected to aldol condensation reaction to obtain the 2,6-diphenylmethylenecyclohexanone analog with the structure shown in Formula 1.

[0046] In Formula 2: R is one or more of hydroxyl, alkoxy, halogen, haloalkyl and cyano groups; n is an integer from 0 to 5.

[0047] In this invention, the organic solvent is preferably ethanol. The inorganic strong acid is preferably hydrochloric acid, and the mass content of HCl in the hydrochloric acid is preferably 36-38%. The molar ratio of the compound with the structure shown in Formula 2 to cyclohexanone is preferably 2:1. The mass ratio of cyclohexanone to the volume ratio of the inorganic strong acid is preferably 0.29 g: 5 mL. This invention does not have special requirements on the amount of the organic solvent used, as long as the aldol condensation reaction proceeds smoothly. The mixing preferably includes: dissolving the compound with the structure shown in Formula 2 and cyclohexanone in the organic solvent to obtain a mixed solution; mixing the mixed solution with the inorganic strong acid. The temperature of the aldol condensation reaction is preferably room temperature, the time is preferably 6 hours, and the aldol condensation reaction is carried out under stirring. In this invention, after the aldol condensation reaction is completed, an aldol condensation reaction solution is obtained. Preferably, this invention further includes: adjusting the pH of the aldol condensation reaction solution to neutral, extracting the obtained neutral reaction solution with dichloromethane, combining the organic phases, and then sequentially drying, concentrating, and purifying by column chromatography to obtain a pure product of the 2,6-diphenylmethylenecyclohexanone analogue with the structure shown in Formula 1. The eluent used in the column chromatography purification is petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is preferably (1-20):1, specifically preferably 20:1, 3:1, 10:1, 2:1, 5:1, 1:1, or 15:1.

[0048] This invention provides an anti-schistosomiasis drug comprising a 2,6-diphenylmethylenecyclohexanone analog of Formula 1 and a pharmaceutically acceptable carrier;

[0049]

[0050] In Formula 1: R is one or more of hydroxyl, alkoxy, halogen, haloalkyl and cyano groups; n is an integer from 0 to 5.

[0051] In this invention, the dosage form of the antischistosomiasis drug includes tablets, capsules, granules, oral liquids, or injections.

[0052] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0053] The following embodiments are all in accordance with Figure 1 The preparation was carried out according to the process shown. Some of the chemical reagents used in the following examples—2,4,6-trimethoxybenzaldehyde, 4-bromobenzaldehyde, 2-fluorobenzaldehyde, 3-2-fluorobenzaldehyde, 3,4-difluorobenzaldehyde, 2-methylbenzaldehyde, and 2-trifluoromethylbenzaldehyde—were purchased from Aladdin Reagent Co., Ltd. Ethanol, cyclohexanone, and concentrated hydrochloric acid were purchased from Xilong Scientific Co., Ltd. All the above reagents were of analytical grade; other reagents were chemically pure.

[0054] Example 1

[0055] 1.06 g (10 mmol) of benzaldehyde and 0.49 g (5 mmol) of cyclohexanone were dissolved in 25 mL of ethanol. After the reactants were completely dissolved, 5 mL of concentrated hydrochloric acid was added, and the mixture was stirred at room temperature for 6 h. After the reaction was completed, the pH was adjusted to neutral with dilute NaOH solution (10 wt%), and the mixture was extracted with dichloromethane. The organic phases were combined, dried over anhydrous Na₂SO₄, filtered, and concentrated. The crude product was purified by column chromatography (V petroleum ether:V ethyl acetate = 20:1) to give a yellow solid 1. Melting point 117.0-117.9 °C, yield 54.7%.

[0056] 1 H NMR (400MHz, Chloroform-d) δ7.80(s,2H),7.48(d,J=7.5Hz,4H,4H),7.43(t,J=7.5Hz,4H),7.36(dd,J=8.3,6.1Hz,2H),2.96(m,4H),1.83(m,2H). 13 C NMR (101MHz, Chloroform-d) δ190.31,136.87,136.16,135.94,130.31,128.53,128.33,28.41,22.98.

[0057] Example 2

[0058] 1.36 g (10 mmol) of anisaldehyde and 0.49 g (5 mmol) of cyclohexanone were dissolved in 25 mL of ethanol. After the reactants were completely dissolved, 5 mL of concentrated hydrochloric acid was added, and the mixture was stirred at room temperature for 6 h. After the reaction was completed, the pH was adjusted to neutral with dilute NaOH solution (10 wt%), and the mixture was extracted with dichloromethane. The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography (V petroleum ether:V ethyl acetate = 10:1) to give a yellow solid 2 with a melting point of 166.3-167.7 °C and a yield of 70.0%.

[0059] 1 H NMR (400MHz, Chloroform-d) δ7.79 (s, 2H), 7.48 (d, J = 8.8Hz, 4H), 6.96 (d, J = 8.8Hz, 4H), 3.86 (s, 6H), 2.95 (t, J = 6.1Hz, 4H), 1.85 (p, J = 6.5Hz, 2H). 13 C NMR (101MHz, Chloroform-d) δ190.11,159.86,136.44,134.28,132.17,128.69,113.84,55.26,28.47,22.99.

[0060] Example 3

[0061] 1.66 g (10 mmol) of 3,4-dimethoxybenzaldehyde and 0.49 g (5 mmol) of cyclohexanone were dissolved in 25 mL of ethanol. After the reactants were completely dissolved, 5 mL of concentrated hydrochloric acid was added, and the mixture was stirred at room temperature for 6 h. After the reaction was completed, the pH was adjusted to neutral with dilute NaOH solution (10 wt%), and the mixture was extracted with dichloromethane. The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography (V petroleum ether:V ethyl acetate = 5:1) to give a yellow solid 3 with a melting point of 149.1-149.8 °C and a yield of 68.0%.

[0062] 1 H NMR (400MHz, Chloroform-d) δ7.74 (s, 2H), 7.11 (dd, J = 8.4, 1.8Hz, 2H), 7.01 (d, J = 1. 8Hz,2H),6.90(d,J=8.4Hz,2H),3.91(s,6H),3.90(s,6H),2.95(t,4H),1.84(m,2H). 13C NMR (101MHz, Chloroform-d) δ190.13,149.71,148.76,136.89,134.61,129.08,124.02,113.83,111.00,56.03,28.62,23.14.

[0063] Example 4

[0064] 1.96 g (10 mmol) of 3,4,5-trimethoxybenzaldehyde and 0.49 g (5 mmol) of cyclohexanone were dissolved in 25 mL of ethanol. After the reactants were completely dissolved, 5 mL of concentrated hydrochloric acid was added, and the mixture was stirred at room temperature for 6 h. After the reaction was completed, the pH was adjusted to neutral with dilute NaOH solution (10 wt%), and the mixture was extracted with dichloromethane. The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography (petroleum ether: ethyl acetate = 5:1) to give a yellow solid 4 with a melting point of 207.2-208.0 °C and a yield of 61.6%.

[0065] 1 H NMR (400MHz, Chloroform-d) δ7.72 (s, 2H), 6.71 (s, 4H), 3.89 (s, 6H), 3.89 (s, 12H), 2.97 (m, 4H), 1.86 (p, J = 6.3Hz, 2H). 13 C NMR (101MHz, Chloroform-d) δ189.99,153.09,138.92,137.17,135.50,131.52,108.02,61.04,56.31,28.54,23.10.

[0066] Example 5

[0067] 1.74 g (10 mmol) of 4-trifluoromethylbenzaldehyde and 0.49 g (5 mmol) of cyclohexanone were dissolved in 25 mL of ethanol. After the reactants were completely dissolved, 5 mL of concentrated hydrochloric acid was added, and the mixture was stirred at room temperature for 6 h. After the reaction was completed, the pH was adjusted to neutral with dilute NaOH solution (10 wt%), and the mixture was extracted with dichloromethane. The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography (V petroleum ether:V ethyl acetate = 20:1) to give a yellow solid 5 with a melting point of 153.2-153.6 °C and a yield of 55.2%.

[0068] 1H NMR (400MHz, Chloroform-d) δ7.79 (s, 2H), 7.67 (d, J = 8.3Hz, 4H), 7.56 (d, J = 8.2Hz, 4H), 2.94 (m, 4H), 1.85 (p, J = 6.3Hz, 2H). 13 C NMR (101MHz, Chloroform-d) δ189.70,139.29,137.72,135.54,130.46,130.31,130.14,125.38,125.34,125.30,125.26,122.58,28.31,22.75.

[0069] Example 6

[0070] 1.24 g (10 mmol) of 4-fluorobenzaldehyde and 0.49 g (5 mmol) of cyclohexanone were dissolved in 25 mL of ethanol. After the reactants were completely dissolved, 5 mL of concentrated hydrochloric acid was added, and the mixture was stirred at room temperature for 6 h. After the reaction was completed, the pH was adjusted to neutral with dilute NaOH solution (10 wt%), and the mixture was extracted with dichloromethane. The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography (V petroleum ether:V ethyl acetate = 20:1) to give a yellow solid 6 with a melting point of 155.0-155.9 °C and a yield of 67.8%.

[0071] 1 H NMR (400MHz, Chloroform-d) δ7.75 (s, 2H), 7.47 (dd, J = 8.3, 5.2, 2.4Hz, 4H), 7.12 (m, 4H), 2.91 (m, 4H), 1.83 (p, J = 6.3Hz, 2H). 13 C NMR (101MHz, Chloroform-d) δ189.95,163.87,161.38,135.82,135.70,135.68,132.26,132.17,132.03,131.99,115.59,115.38,28.31,22.87.

[0072] Example 7

[0073] 1.40 g (10 mmol) of 4-chlorobenzaldehyde and 0.49 g (5 mmol) of cyclohexanone were dissolved in 25 mL of ethanol. After the reactants were completely dissolved, 5 mL of concentrated hydrochloric acid was added, and the mixture was stirred at room temperature for 6 h. After the reaction was completed, the pH was adjusted to neutral with dilute NaOH solution (10 wt%), and the mixture was extracted with dichloromethane. The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography (V petroleum ether:V ethyl acetate = 20:1) to give a yellow solid 7 with a melting point of 141.6-142.1 °C and a yield of 58.2%.

[0074] 1 H NMR (400MHz, Chloroform-d) δ7.72(s,2H),7.38(s,8H),2.90(t,J=6.1Hz,4H),1.83(p,J=6.5Hz,2H). 13 C NMR (101MHz, Chloroform-d) δ189.81,136.39,135.75,134.59,134.28,131.55,128.66,28.35,22.79.

[0075] Example 8

[0076] 1.84 g (10 mmol) of 4-bromobenzaldehyde and 0.49 g (5 mmol) of cyclohexanone were dissolved in 25 mL of ethanol. After the reactants were completely dissolved, 5 mL of concentrated hydrochloric acid was added, and the mixture was stirred at room temperature for 6 h. After the reaction was completed, the pH was adjusted to neutral with dilute NaOH solution (10 wt%), and the mixture was extracted with dichloromethane. The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography (V petroleum ether:V ethyl acetate = 20:1) to give a yellow solid 8 with a melting point of 166.4-166.8 °C and a yield of 37.0%.

[0077] 1 H NMR (400MHz, Chloroform-d) δ7.70 (s, 2H), 7.55 (d, J = 8.5Hz, 4H), 7.33 (d, J = 8.5Hz, 4H), 2.90 (m, 4H), 1.83 (p, J = 6.3Hz, 2H). 13 C NMR (101MHz, Chloroform-d) δ189.77,136.48,135.78,134.69,131.76,131.61,122.91,28.34,22.76.

[0078] Example 9

[0079] 1.20 g (10 mmol) of 4-methylbenzaldehyde and 0.49 g (5 mmol) of cyclohexanone were dissolved in 25 mL of ethanol. After the reactants were completely dissolved, 5 mL of concentrated hydrochloric acid was added, and the mixture was stirred at room temperature for 6 h. After the reaction was completed, the pH was adjusted to neutral with dilute NaOH solution (10 wt%), and the mixture was extracted with dichloromethane. The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography (V petroleum ether:V ethyl acetate = 20:1) to give a yellow solid 9 with a melting point of 168.8-169.5 °C and a yield of 75.0%.

[0080] 1 H NMR (400MHz, Chloroform-d) δ7.79 (s, 2H), 7.39 (d, J = 8.1Hz, 4H), 7.23 (d, J = 8.0Hz, 4H), 2.94 (m, 4H), 2.39 (s, 6H), 1.82 (p, J = 6.3Hz, 2H). 13 CNMR(101MHz,Chloroform-d)δ190.33,138.75,136.81,135.46,133.18,130.43,129.09,28.49,22.99,21.37.

[0081] Example 10

[0082] 1.24 g (10 mmol) of 2-fluorobenzaldehyde and 0.49 g (5 mmol) of cyclohexanone were dissolved in 25 mL of ethanol. After the reactants were completely dissolved, 5 mL of concentrated hydrochloric acid was added, and the mixture was stirred at room temperature for 6 h. After the reaction was completed, the pH was adjusted to neutral with dilute NaOH solution (10 wt%), and the mixture was extracted with dichloromethane. The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography (V petroleum ether:V ethyl acetate = 20:1) to give a yellow solid 10, mp 101.4-102.4℃, yield: 35.9%.

[0083] 1 H NMR (400MHz, Chloroform-d) δ7.83 (s, 2H), 7.4 (m, 4H), 7.18 (m, 4H), 2.83 (t, J = 5.6Hz, 4H), 1.81 (p, J = 6.3Hz, 2H). 13C NMR(101MHz,Chloroform-d)δ189.50,162.07,159.58,138.21,130.70,130.67,130.36, 130.28,129.73,129.70,123.94,123.80,123.71,123.67,115.83,115.61,28.54,22.96.

[0084] Example 11

[0085] 1.40 g (10 mmol) of 2-chlorobenzaldehyde and 0.49 g (5 mmol) of cyclohexanone were dissolved in 25 mL of ethanol. After the reactants were completely dissolved, 5 mL of concentrated hydrochloric acid was added, and the mixture was stirred at room temperature for 6 h. After the reaction was completed, the pH was adjusted to neutral with dilute NaOH solution (10 wt%), and the mixture was extracted with dichloromethane. The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography (petroleum ether: ethyl acetate 20:1) to give a yellow solid 11, mp 111.6-111.9℃, yield: 35.0%.

[0086] 1 H NMR (400MHz, Chloroform-d) δ7.91 (s, 2H), 7.46-7.42 (m, 2H), 7.36-7.32 (m, 2H), 7.30 (dd, J = 5.9, 3.5Hz, 4H), 2.79 (m, 4H), 1.80 (p, J = 6.2Hz, 2H). 13 C NMR (101MHz, Chloroform-d) δ189.67,137.74,134.98,134.40,134.06,130.47,129.72,129.54,126.22,28.36,23.15.

[0087] Example 12

[0088] 1.84 g (10 mmol) of 2-bromobenzaldehyde and 0.49 g (5 mmol) of cyclohexanone were dissolved in 25 mL of ethanol. After the reactants were completely dissolved, 5 mL of concentrated hydrochloric acid was added, and the mixture was stirred at room temperature for 6 h. After the reaction was completed, the pH was adjusted to neutral with dilute NaOH solution (10 wt%), and the mixture was extracted with dichloromethane. The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography (V petroleum ether:V ethyl acetate = 20:1) to give a yellow solid 12, mp 131.2-131.6℃, yield: 28.0%.

[0089] 13C NMR (101MHz, Chloroform-d) δ189.67,137.74,134.98,134.40,134.06,130.47,129.72,129.54,126.22,28.36,23.15. 13 C NMR (101MHz, Chloroform-d) δ189.67,137.74,134.98,134.40,134.06,130.47,129.72,129.54,126.22,28.36,23.15.

[0090] Example 13

[0091] 1.36 g (10 mmol) of 2-methoxybenzaldehyde and 0.49 g (5 mmol) of cyclohexanone were dissolved in 25 mL of ethanol. After the reactants were completely dissolved, 5 mL of concentrated hydrochloric acid was added, and the mixture was stirred at room temperature for 6 h. After the reaction was completed, the pH was adjusted to neutral with dilute NaOH solution (10 wt%), and the mixture was extracted with dichloromethane. The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography (petroleum ether: ethyl acetate 10:1) to give a yellow solid 13, mp 101.9-102.9℃, yield: 22.8%.

[0092] 1 HNMR(400MHz,Chloroform-d)δ7.97(s,2H),7.34-7.29(m,4H),6.96(t,J=7.3Hz,2 H),6.93-6.89(m,2H),3.86(s,6H),2.84(td,J=6.5,1.9Hz,4H),1.79-1.71(m,2H). 13 C NMR (101MHz, Chloroform-d) δ190.50,158.30,136.51,132.41,130.27,129.90,125.12,119.84,110.55,55.42,28.70,23.49.

[0093] Example 14

[0094] 1.20 g (10 mmol) of 2-methylbenzaldehyde and 0.49 g (5 mmol) of cyclohexanone were dissolved in 25 mL of ethanol. After the reactants were completely dissolved, 5 mL of concentrated hydrochloric acid was added, and the mixture was stirred at room temperature for 6 h. After the reaction was completed, the pH was adjusted to neutral with dilute NaOH solution (10 wt%), and the mixture was extracted with dichloromethane. The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography (V petroleum ether:V ethyl acetate = 15:1) to give a yellow solid 14, mp 101.9-102.9℃, yield: 59.5%.

[0095] 1 H NMR (400MHz, Chloroform-d) δ7.90 (s, 2H), 7.26-7.18 (m, 8H), 2.78 (td, J = 6.5, 1.9Hz, 4H), 2.35 (s, 6H), 1.74 (p, J = 6.2Hz, 2H). 13 C NMR (101MHz, Chloroform-d) δ190.32,137.88,136.72,135.98,135.10,130.14,128.95,128.36,125.33,28.39,23.52,20.02.

[0096] Example 15

[0097] 1.52 g (10 mmol) of 4-hydroxy-3-methoxybenzaldehyde and 0.49 g (5 mmol) of cyclohexanone were dissolved in 25 mL of ethanol. After the reactants were completely dissolved, 5 mL of concentrated hydrochloric acid was added, and the mixture was stirred at room temperature for 6 h. After the reaction was completed, the pH was adjusted to neutral with dilute NaOH solution (10 wt%), and the mixture was extracted with dichloromethane. The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography (V petroleum ether:V ethyl acetate = 3:1) to give a yellow solid 15. Melting point: 178.0–178.6 °C, yield: 65.5%.

[0098] 1 H NMR (400MHz, DMSO-d6) δ9.52(s,2H),7.56(s,2H),7.12(d,J=1.7Hz,2H),7.04(dd,J=8.3,1 .7Hz,2H),6.86(d,J=8.2Hz,2H),3.81(s,6H),2.91(t,J=5.3Hz,4H),1.76(p,J=6.4Hz,2H). 13C NMR (101MHz, DMSO-d6) δ188.95,148.29,147.91,136.62,133.98,127.41,124.69,116.02,115.28,56.13,28.44,23.06.

[0099] Example 16

[0100] 1.22 g (10 mmol) of 4-hydroxybenzaldehyde and 0.49 g (5 mmol) of cyclohexanone were dissolved in 25 mL of ethanol. After the reactants were completely dissolved, 5 mL of concentrated hydrochloric acid was added, and the mixture was stirred at room temperature for 6 h. After the reaction was completed, the pH was adjusted to neutral with dilute NaOH solution (10 wt%), and the mixture was extracted with dichloromethane. The organic phases were combined, dried over anhydrous Na₂SO₄, filtered, and concentrated. The crude product was purified by column chromatography (V petroleum ether:V ethyl acetate = 1:1) to give a yellow solid 16. Melting point >300℃, yield 91.4%.

[0101] 1 H NMR (400MHz, DMSO-d6) δ9.91 (s, 2H), 7.54 (s, 2H), 7.42 (d, J = 8.7Hz, 4H), 6.85 (d, J = 8.6Hz, 4H), 2.87 (t, J = 5.3Hz, 4H), 1.74 (p, J = 6.2Hz, 2H). 13 C NMR (101MHz, DMSO-d6) δ188.98,158.79,136.26,133.79,132.92,126.94,116.01,28.46,23.02.

[0102] Example 17

[0103] 1.42 g (10 mmol) of 3,4-difluorobenzaldehyde and 0.49 g (5 mmol) of cyclohexanone were dissolved in 25 mL of ethanol. After the reactants were completely dissolved, 5 mL of concentrated hydrochloric acid was added, and the mixture was stirred at room temperature for 6 h. After the reaction was completed, the pH was adjusted to neutral with dilute NaOH solution (10 wt%), and the mixture was extracted with dichloromethane. The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography (V petroleum ether:V ethyl acetate = 20:1) to give a yellow solid 17. mp 111.6-112.5℃, yield: 66.4%.

[0104] 1 H NMR (400MHz, Chloroform-d) δ7.67 (s, 2H), 7.31-7.23 (m, 2H), 7.21-7.18 (m, 4H), 2.91 (m, 4H), 1.86 (p, J = 6.3Hz, 2H).13 C NMR(101MHz,Chloroform-d)δ189.46,151.41(dd),148.92(dd),136.47(d),134.89,132.80(dd),126.98(dd),118.78(d),117.36(d),28.17,22.60.

[0105] Example 18

[0106] 1.82 g (10 mmol) of 4-hydroxy-3,5-dimethoxybenzaldehyde and 0.49 g (5 mmol) of cyclohexanone were dissolved in 25 mL of ethanol. After the reactants were completely dissolved, 5 mL of concentrated hydrochloric acid was added, and the mixture was stirred at room temperature for 6 h. After the reaction was completed, the pH was adjusted to neutral with dilute NaOH solution (10 wt%), and the mixture was extracted with dichloromethane. The organic phases were combined, dried over anhydrous Na₂SO₄, filtered, and concentrated. The crude product was purified by column chromatography (V petroleum ether:V ethyl acetate = 1:1) to give a yellow solid 18. Melting point: 176.6–177.0 °C, yield: 19.0%.

[0107] 1 H NMR (400MHz, DMSO-d6) δ8.91(s,2H),7.57(s,2H),6.85(s,4H),3.81(s,12H),2.95(t,J=5.3Hz,4H),1.78(p,J=6.5Hz,2H). 13 C NMR (101MHz, DMSO-d6) δ188.95,148.22,137.59,136.95,134.24,126.22,109.09,56.57,28.35,23.08.

[0108] Example 19

[0109] 1.66 g (10 mmol) of 3,5-dimethoxybenzaldehyde and 0.49 g (5 mmol) of cyclohexanone were dissolved in 25 mL of ethanol. After the reactants were completely dissolved, 5 mL of concentrated hydrochloric acid was added, and the mixture was stirred at room temperature for 6 h. After the reaction was completed, the pH was adjusted to neutral with dilute NaOH solution (10 wt%), and the mixture was extracted with dichloromethane. The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography (V petroleum ether:V ethyl acetate = 5:1) to give a yellow solid 19 with a melting point of 141.1-141.3 °C and a yield of 45.6%.

[0110] 1H NMR (400MHz, Chloroform-d) δ7.71 (s, 2H), 6.60 (d, J = 2.2Hz, 4H), 6.47 (t, J = 2.2Hz, 2H), 3.82 (s, 12H), 2.94 (m, 4H), 1.81 (p, J = 6.3Hz, 2H). 13 CNMR(101MHz,Chloroform-d)δ190.17,160.54,137.70,136.89,136.56,108.28,100.76,55.36,28.46,22.86.

[0111] Example 20

[0112] 1.31 g (10 mmol) of 4-cyanobenzaldehyde and 0.49 g (5 mmol) of cyclohexanone were dissolved in 25 mL of ethanol. After the reactants were completely dissolved, 5 mL of concentrated hydrochloric acid was added, and the mixture was stirred at room temperature for 6 h. After the reaction was completed, the pH was adjusted to neutral with dilute NaOH solution (10 wt%), and the mixture was extracted with dichloromethane. The organic phases were combined, dried over anhydrous Na₂SO₄, filtered, and concentrated. The crude product was purified by column chromatography (V petroleum ether:V ethyl acetate = 3:1) to give a yellow solid 20. Melting point: 228.8–229.3 °C, yield: 12.3%.

[0113] 1 H NMR (400MHz, Chloroform-d) δ7.75 (s, 2H), 7.70 (d, J = 8.4Hz, 4H), 7.53 (d, J = 8.2Hz, 4H), 2.91 (td, J = 6.5, 2.1Hz, 4H), 1.83 (p, J = 6.3Hz, 2H). 13 C NMR (101MHz, Chloroform-d) δ189.26,140.20,138.24,135.18,132.12,130.56,118.49,112.00,28.30,22.56.

[0114] Comparative Example 1

[0115] 1.06 g (10 mmol) of benzaldehyde and 0.29 g (5 mmol) of acetone were dissolved in 25 mL of ethanol. After the reactants were completely dissolved, 5 mL of concentrated hydrochloric acid was added, and the mixture was stirred at room temperature for 6 h. After the reaction was completed, the pH was adjusted to neutral with dilute NaOH solution (10 wt%), and the mixture was extracted with dichloromethane. The organic phases were combined, dried over anhydrous Na₂SO₄, filtered, and concentrated. The crude product was purified by column chromatography (V petroleum ether:V ethyl acetate = 10:1) to give a pale yellow solid B1. Melting point: 117.0–117.9 °C, yield: 54.7%.

[0116] 1 H NMR (400MHz, Chloroform-d) δ7.77(d,J=16.0Hz,2H),7.64-7.61(m,4H),7.44(qd,J=3.6,1.6Hz,6H),7.11(d,J=16.0Hz,2H). 13 C NMR (101MHz, Chloroform-d) δ189.02,143.43,134.93,130.61,129.09,128.51,125.56.

[0117]

[0118] Comparative Example 2

[0119] 1.52 g (10 mmol) of 4-hydroxy-3-methoxybenzaldehyde and 0.29 g (5 mmol) of acetone were dissolved in 25 mL of ethanol. After the reactants were completely dissolved, 5 mL of concentrated hydrochloric acid was added, and the mixture was stirred at room temperature for 6 h. After the reaction was completed, the pH was adjusted to neutral with dilute NaOH solution (10 wt%), and the mixture was extracted with dichloromethane. The organic phases were combined, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography (V petroleum ether:V ethyl acetate = 2:1) to give a yellow solid B2. Melting point: 73.3–77.7 °C, yield: 30.1%.

[0120] 1 H NMR (400MHz, DMSO-d6) δ9.66(s,2H),7.67(d,J=16.0Hz,2H),7.37(d,J=2.0Hz,2H),7. 21(dd,J=8.0,1.8Hz,2H),7.13(d,J=16.0Hz,2H),6.84(d,J=8.0Hz,2H),3.85(s,6H). 13 C NMR (101MHz, DMSO-d6) δ188.01,149.41,147.97,142.75,126.35,123.34,123.02,115.67,111.42,79.17,55.73.

[0121]

[0122] Test Example 1: Compound Purity Analysis

[0123] Furthermore, the purity of the 2,6-diphenylmethylenecyclohexanone analogues was determined by HPLC and RP-HPLC, respectively. As shown in Table 1, the purities of compounds 1-9, 15, 16, 18, 19, and 20 are all greater than 92%, which meets the requirements for bioactivity testing.

[0124]

[0125] Table 1 Purity analysis of the compounds prepared in the examples

[0126]

[0127]

[0128] a: Normal-phase high-performance liquid chromatography. Detector: SPD-16; Column: SunFire. @ Silice Prep 4.6 × 250 nm, 10 μm, eluent: n-hexane-isopropanol, flow rate: 1 mL / min. Wavelength range: 200-800 nm. b: Reversed-phase high-performance liquid chromatography. Detector: Alliance E2695, column: Compound 18, 4.6 × 250 nm, 5 μm. Eluent: methanol-water, flow rate: 1 mL / min. Wavelength range: 200-800 nm.

[0129] Test Example 2: Antischistosomiasis Activity of 2,6-Diphenylmethylenecyclohexanone Analog

[0130] Following the in vitro schistosomiasis-killing experimental procedures of the compounds prepared in Examples 1-20, the anti-schistosomiasis activity of 2,6-diphenylmethylenecyclohexanone analogues was evaluated sequentially. RPMI 1640 medium was used as the negative control group, RPMI 1640 medium containing 1% DMSO was used as the solvent control group, and praziquantel was used as the positive control.

[0131] Preparation of culture medium: Prepare the culture medium by mixing RPMI 1640 medium (88%), penicillin-streptomycin (2%) and newborn calf serum (10%) in the appropriate proportions and store at 4°C for later use.

[0132] 2,6-Diphenylmethylenecyclohexanone analogues (compounds 1-20) stock solution: Weigh 0.04 mmol of 2,6-diphenylmethylenecyclohexanone and dissolve it in 1 mL of DMSO to prepare a 0.04 μmol / L stock solution. For small amounts of compounds that cannot be completely dissolved, vortexing or sonication can be used to promote complete dissolution. Store at 4°C. Mix well before use and prepare the required concentration according to the appropriate ratio.

[0133] In vitro anti-Schistosoma japonicum experiment

[0134] 1980 μL of culture medium containing 10% newborn calf serum was added to each 24-well plate and incubated at 37°C with 5% CO2 for 30 min. Five pairs of active adult worms with intact body membranes were picked into each well, and then different concentrations of compound solutions (20 μL) were added. Changes in the worms were observed at regular intervals, such as whether the body membrane swelled, whether the worms were curved, and the degree of decrease in activity frequency. The experiment was repeated twice.

[0135] In vitro anti-schistosomiasis activity experiment grouping:

[0136] Control group: RPMI 1640 medium containing only 10% newborn calf serum

[0137] Negative control group: Culture medium containing 2% DMSO RPMI 1640. The concentration of DMSO was the highest among all experimental groups.

[0138] Positive control group: Praziquantel, used at a concentration of 10 μM during the experiment.

[0139] Experimental group: Compound concentration used: 2.5-40 μM.

[0140] 2,6-Diphenylmethylenecyclohexanone analogues were used in culture media at concentrations ranging from 1 to 40 μM. Each experiment was repeated twice, and the results are shown below. Figures 2-6 Meanwhile, the results showed that no schistosomiasis deaths occurred in the blank control group and the negative control group, meaning the mortality rate was 0%.

[0141] Figure 2 2,6-Diphenylmethylenecyclohexanone compound 1 ( Figure 2 Compound A in the group, 2,6-diphenylmethylenecyclohexanone, is 2 ( Figure 2 Compound B), 2,6-diphenylmethylenecyclohexanone, 3 ( Figure 2 C) and 2,6-diphenylmethylenecyclohexanone compounds 4 ( Figure 2 The diagram shows the anti-schistosomiasis activity of D); by Figure 2 It is known that a 40 μM compound can cause 100% mortality of schistosomes after 148 hours, a 20 μM compound can cause 50% mortality after 72 hours, and a 10 μM compound can cause 45% mortality after 48 hours. Figure 2 Compound A). 240 μM for 72 hours caused 55% mortality of schistosomes. Figure 2 Compound B). 340 μM of the compound caused 75% mortality of schistosomes after 24 hours, and 20 μM after 72 hours caused 90% mortality. Figure 2 (C). Compound 440 μM 36h caused 70% mortality of schistosomes, and 20 μM 72h caused 85% mortality of schistosomes. Figure 2 (D).

[0142] Figure 3 5,6-Diphenylmethylenecyclohexanone compounds Figure 3 Compound A in the group, 2,6-diphenylmethylenecyclohexanone, 6 ( Figure 3 Compound B), 2,6-diphenylmethylenecyclohexanone, 7 ( Figure 3 C) and 2,6-diphenylmethylenecyclohexanone compounds 8 ( Figure 3 The diagram shows the anti-schistosomiasis activity of D); by Figure 3 It is known that a 40 μM compound can cause 100% mortality of schistosomes after 572 hours, a 20 μM compound can cause 90% mortality after 72 hours, and a 10 μM compound can cause 35% mortality after 72 hours. Figure 3 Compound A). 640 μM of compound induced the death of 95% of schistosomes after 72 hours. Figure 3 Compound B). 740 μM of the compound caused 100% mortality of schistosomes after 72 hours, while 20 μM after 72 hours caused 55% mortality. Figure 3 (C). Compound 840 μM 36h caused 80% mortality of schistosomes, and 20 μM 72h caused 60% mortality of schistosomes. Figure 3 (D).

[0143] Figure 4 2,6-Diphenylmethylenecyclohexanone compounds 9 ( Figure 4 Compound A), 2,6-diphenylmethylenecyclohexanone, 10 ( Figure 4 Compound B), 2,6-diphenylmethylenecyclohexanone, compound 11 ( Figure 4 C) and 2,6-diphenylmethylenecyclohexanone compounds 12 ( Figure 4 The diagram shows the anti-schistosomiasis activity of D); by Figure 4 It is known that a 40 μM compound can cause 40% mortality of schistosomes after 972 hours, and a 20 μM compound can cause 20% mortality after 72 hours. Figure 4 Compound A). 1040 μM of the compound caused 100% mortality of schistosomes after 72 hours. Figure 4 Compound B). At 1140 μM for 72 hours, compound B caused the death of 90% of schistosomes, and at 20 μM for 72 hours, it caused the death of 65% of schistosomes. Figure 4 (C). Compound 1240 μM 36h caused 50% mortality of schistosomes, and 20 μM 72h caused 55% mortality of schistosomes. Figure 4 (D).

[0144] Figure 5 It is a 2,6-diphenylmethylenecyclohexanone compound 13 ( Figure 5Compound A), 2,6-diphenylmethylenecyclohexanone, 14 ( Figure 5 Compound B), 2,6-diphenylmethylenecyclohexanone, 15 ( Figure 5 C) and 2,6-diphenylmethylenecyclohexanone compounds 17 ( Figure 5 The diagram shows the anti-schistosomiasis activity of D); by Figure 5 It is known that a 40 μM compound can cause 90% mortality of schistosomes after 1372 hours, and a 20 μM compound can cause 30% mortality after 72 hours. Figure 5 Compound A). Compound 1420 μM for 72 hours caused 35% mortality of schistosomes. Figure 5 Compound B). 1540 μM of the compound caused 100% mortality of schistosomes after 72 hours, and 20 μM of the compound also caused 100% mortality of schistosomes after 72 hours. Figure 5 (C). Compound 1640 μM 36h caused 100% mortality of schistosomes, and 20 μM 72h caused 45% mortality of schistosomes. Figure 5 (D).

[0145] Figure 6 2,6-Diphenylmethylenecyclohexanone compound 17 ( Figure 6 Compound A), 2,6-diphenylmethylenecyclohexanone, 18 ( Figure 6 Compound B), 2,6-diphenylmethylenecyclohexanone, 19 ( Figure 6 C) and 2,6-diphenylmethylenecyclohexanone compounds 20 ( Figure 6 The diagram shows the anti-schistosomiasis activity of D); by Figure 6 It is known that a 40 μM compound can cause 100% mortality of schistosomes after 1772 hours, a 20 μM compound can cause 90% mortality after 72 hours, and a 10 μM compound can cause 70% mortality after 72 hours. Figure 6 Compound A). 1840 μM of this compound caused 100% mortality of schistosomes after 72 hours. Figure 6 Compound B). At 1940 μM for 72 hours, compound B caused 100% mortality of schistosomes, and at 20 μM for 72 hours, it caused 90% mortality of schistosomes. Figure 6 (C). Compound 2040 μM 36h caused 95% mortality of schistosomes, and 20 μM 72h caused 100% mortality of schistosomes. Figure 6 (D).

[0146] The polar anti-schistosomiasis activity of compounds 1-20 prepared in Examples 1-20 was determined in this invention, and the adult mortality rate test results are shown in Table 2.

[0147] Table 2. Polar antischistosomiasis activity of compounds 1–20

[0148]

[0149]

[0150]

[0151]

[0152] Test Example 3: Toxicity Evaluation of 2,6-Diphenylmethylenecyclohexanone Analog

[0153] In vitro anti-schistosomiasis experiments using 2,6-diphenylmethylenecyclohexanone analogs showed that compounds 3, 18, and 20 exhibited the best anti-schistosomiasis activity. Therefore, compounds 3, 18, and 20 were initially selected for this invention to evaluate cytotoxic activity and assess preliminary safety.

[0154] (1) Cytotoxicity of 2,6-diphenylmethylenecyclohexanone analogues

[0155] By evaluating the effects of compounds on the survival and growth of normal cells (L929), compounds that have no effect on normal cells were screened.

[0156] Using cisplatin as a positive control, compounds 3, 18, and 20 were co-incubated with normal L929 (mouse epithelial fibroblasts) cells for 48 hours at concentrations ranging from 5 to 40 μM. Cell viability was then observed. After 48 hours of co-incubation with the compounds, at concentrations below 40 μM, the cell viability of compounds 3 and 20 was higher than 80%. At concentrations above 10 μM, the cell viability of compound 18 was lower than 80%. Figure 7 Therefore, within the concentration range of 5-40 μM, compounds 3 and 20 exhibit better cell biocompatibility and lower toxicity. Therefore, compounds 3 and 20 were selected for in vivo experimental studies in this invention. Figure 7 The effects of cisplatin, compound 3, compound 18 and compound 20 on the survival and growth of L929 cells.

[0157] Test Example 4: In vivo anti-schistosomiasis effects of 2,6-diphenylmethylenecyclohexanone analogs compound 3 and compound 20

[0158] A detailed evaluation of the in vivo control efficacy against adult Schistosoma worms (28 days) was conducted using compounds 3 and 20, which are low in toxicity and exhibit excellent in vitro performance, with praziquantel as a control.

[0159] Mice were randomly divided into a blank control group (uninfected mice), a negative control group (infected but untreated mice), a positive control group (praziquantel), and a treatment group (compound 3 and compound 20), with five mice in each group. After 28 days of cercarial infection (adult stage), mice were administered compounds at a dose of 500 mg / kg via gavage for five consecutive days, followed by 14 days of routine feeding (a total of 47 days of feeding). The mice were then dissected. The number of adult worms parasitizing the portal vein and mesentery was counted, along with the total number of worms, female worms, and male worms. Simultaneously, worm eggs on the liver and small intestine were counted. Due to space limitations, the experiment was conducted in two batches: negative control group 1 served as a control for praziquantel and compound 20, and negative control group 2 served as a control for compound 3. The results are shown in Table 3.

[0160] Table 3. In vivo killing effects of compounds 3 and 20 on 28-day-old adults.

[0161]

[0162] a Control 1: Infected mice (untreated), blank control for praziquantel and compound 20; b Control 2: Infected mice (untreated), 6 as a control; c The group in which no adult worms were found in the portal vein, mesentery and other parts of the mouse were classified as the complete treatment group. d Mice died abnormally after being administered compound 3 by gavage for 6 days; data not included. e The calculation uses the corresponding average value.

[0163] The control group mice showed strong activity and normal weight gain during the in vivo experiment, without exhibiting piloerection or restlessness. The negative control group mice (1 and 2) showed poor activity, continuous weight loss, restlessness, and piloerection during the experiment, but no mice died. Mice treated with 500 mg / kg praziquantel by gavage for 5 days showed good activity during the experiment, without weight loss or piloerection, and no mice died. In the compound 3 treatment group, mice were lethargic, significantly less active, and exhibited weight loss, restlessness, and piloerection during the observation period. In the compound 20 treatment group, mice were active, without restlessness or piloerection, and their weight continued to increase.

[0164] After 47 days of rearing, mice were dissected. In the negative control groups 1 and 2, a large number of adult worms were found, mainly concentrated in the portal vein and mesentery, with the livers riddled with eggs. In the praziquantel group, no adult worms were found in the portal vein and mesentery, and there were virtually no eggs. This is mainly because praziquantel effectively kills adult worms, preventing egg production. In the compound 3 treatment group, a large number of adult worms were found in the portal vein and mesentery, with a reduction rate of 60.4%. The livers were riddled with eggs, and the spleens contained a small number of eggs. The egg reduction rates in the liver and small intestine were 28.8% and 58.5%, respectively. In the compound 20 treatment group, a small number of adult worms were found in the portal vein and mesentery, with a reduction rate of 86.7%. The egg reduction rates in the liver and small intestine were 90.1% and 95.3%, respectively (Table 3).

[0165] Therefore, compounds 3 and 20 exhibit varying degrees of schistosomiasis control, reducing worm load and egg production. Compound 20 shows better control, consistent with its in vitro anti-worm activity. Compound 20 demonstrates insecticidal efficacy close to that of praziquantel, with near-moderate egg reduction efficiency.

[0166] Furthermore, the present invention calculated the liver index and spleen index of mice in each treatment group. Figure 8 The liver index after gavage administration of compounds 3 and 20 in in vivo experiments ( Figure 8 Changes in A and spleen index (in the text) Figure 8 (B) Compared to the negative control, the liver and spleen indices of mice treated with compound 20 and praziquantel were significantly reduced, approaching those of the normal control group. The liver and spleen indices of compound 3 were comparable to those of the negative control, indicating no protective effect on liver and spleen tissues. Therefore, compound 20 has a better protective effect on the liver and spleen. Figure 8 ).

[0167] same, Figure 9 The images show histopathological sections of mouse liver tissue (×100) after gavage administration of compound 20 in in vivo experiments; the blank group ( Figure 9 A), negative control group ( Figure 9 B), administered via gavage at a dose of 500 mg / kg praziquantel ( Figure 9 C) and compound 20 ( Figure 9 (D). H&E staining of liver sections from diseased mice showed that after treatment with compound 20, there were no obvious granulomas, no egg deposition, no inflammatory response, and the liver parenchyma was normal. Figure 9 It showed good ability to protect and repair the liver, significantly better than praziquantel, and effectively reduced liver fibrosis.

[0168] After treatment with compound 20, the level of hydroxyproline (an important indicator of liver fibrosis) in mice was significantly reduced compared to the negative control, almost approaching the normal blank value. This indicates that compound 20 can delay liver fibrosis in mice and has a good anti-liver fibrosis effect. Figure 10 The hydroxyproline levels in mice treated with praziquantel were comparable to those in the control group, showing no improvement in liver fibrosis. This indicates that compound 20 has a significant protective effect on the liver of mice infected with Schistosoma, maintaining normal liver tissue morphology and resisting liver fibrosis, thus preventing liver lesions and effectively avoiding the drawback of praziquantel's lack of anti-liver fibrosis effect during the 28-day worm stage in vivo.

[0169] This invention synthesizes several 2,6-diphenylmethylenecyclohexanone analogs, compounds 1 to 20, by designing cyclohexanone to replace the methylene group, thereby improving the in vitro anti-schistosomiasis activity of the compounds. Within the suitable lipid solubility range (ClogP 3-4), the introduction of methoxy groups and strong electron-withdrawing groups effectively enhances the antiparasitic activity. The position of the substituents also modulates the activity, with dimethoxy (compound 3, compound 18) and 4-cyano (compound 20) phenyl showing the best activity. Toxicological evaluation showed that compounds 3 and 20 exhibited low cytotoxicity. In particular, compound 20 demonstrated outstanding in vivo insecticidal effects; continuous administration at 500 mg / kg for 5 days achieved an 86.7% reduction rate of parasites, a 90.1% reduction rate of liver eggs, and a 95.3% reduction rate of small intestinal eggs, with two mice (40%) achieving complete treatment. Simultaneously, compound 20 significantly protected the mouse (host) liver and reversed liver fibrosis, far superior to praziquantel, which did not show significant anti-fibrotic activity.

[0170] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The use of a 2,6-diphenylmethylenecyclohexanone analog in the preparation of antischistosomiasis drugs, characterized in that, The 2,6-diphenylmethylenecyclohexanone analogue has the structure shown in Formula 1: Formula 1; In Formula 1: R is selected from one or more of hydroxyl, methoxy, fluorine, bromine, chlorine, CF3 and cyano; n is an integer from 0 to 3.

2. The application according to claim 1, characterized in that, The 2,6-diphenylmethylenecyclohexanone analogue has any one of the following structures: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。 3. The application according to claim 1 or 2, characterized in that, The method for preparing the 2,6-diphenylmethylenecyclohexanone analogue includes the following steps: The compound with the structure shown in Formula 2, cyclohexanone, organic solvent and inorganic strong acid were mixed and subjected to aldol condensation reaction to obtain the 2,6-diphenylmethylenecyclohexanone analog with the structure shown in Formula 1. Formula 2; In Formula 2: R is one or more of hydroxyl, alkoxy, halogen, haloalkyl and cyano groups; n is an integer from 0 to 3.

4. The application according to claim 3, characterized in that, The molar ratio of the compound with the structure shown in Formula 2 to cyclohexanone is 2:

1.

5. The application according to claim 3, characterized in that, The inorganic strong acid is hydrochloric acid, and the mass content of HCl in the hydrochloric acid is 36-38%.

6. The application according to claim 3, characterized in that, After the aldol condensation reaction is completed, an aldol condensation reaction solution is obtained. The process further includes: adjusting the pH of the aldol condensation reaction solution to neutral; extracting the obtained neutral reaction solution with dichloromethane; combining the organic phases and then sequentially drying, concentrating, and purifying by column chromatography to obtain a pure product of the 2,6-diphenylmethylenecyclohexanone analogue with the structure shown in Formula 1. The eluent used in the column chromatography purification is petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is (1~20):

1.

7. The application according to claim 1, characterized in that, The dosage forms of the antischistosomiasis drugs include tablets, capsules, granules, oral liquids, or injections.

Images