Application of combination of small organic molecule compound and bivalent copper ions in preparation of medicine for treating schistosomiasis japonicas

Abstract

The invention belongs to the technical field of medicine treatment, and particularly relates to application of combination of an organic small molecule compound and bivalent copper ions in preparation of a medicine for treating schistosomiasis japonicas. The invention provides an application of combination of an organic small molecule compound and bivalent copper ions in preparation of a medicine for treating schistosomiasis japonicas. The organic small molecule compound is selected from one or more of a compound with a structure as shown in a formula 1-1, a compound with a structure as shown in a formula 2-2 and a compound with a structure as shown in a formula 3. According to the invention, development of schistosoma japonicum at multiple juvenile stages in vivo can be blocked in a targeted manner through combination, the schistosoma load and the spawning rate are reduced, compared with praziquantel, the drug has better schistosoma killing activity, and preparation and application of the drug for treating schistosoma japonicum with efficient insect killing can be realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical treatment, and particularly relates to the application of the combination of an organic small molecule compound and divalent copper ions in the preparation of a drug for treating Schistosomiasis japonica. Background Art

[0002] Schistosomiasis japonica is a zoonotic parasitic disease that seriously endangers public health. This disease can cause lesions such as hepatosplenomegaly in infected individuals, thereby reducing labor capacity.

[0003] Praziquantel (PZQ) is currently the only drug for the prevention and control of schistosomiasis globally, and its clinical application still faces multiple limitations. In particular, it is almost ineffective against schistosomula (immature worms) and eggs, which leads to multiple problems. (1) Unable to block the transmission chain: Eggs are the core link in the transmission of schistosomiasis. After treatment with PZQ, viable eggs may still be continuously excreted in the feces of patients, contaminating water sources and maintaining the infection cycle of the intermediate host (snails). (2) Risk of repeated infection: In endemic areas, patients may be reinfected with cercariae after contact with contaminated water after being cured, that is, PZQ cannot prevent repeated infection, and repeated administration (usually 1 - 2 times a year) is required, increasing the treatment cost and burden. (3) Limited effect on patients with acute infection: After using PZQ, patients with acute infection (caused by the migration of a large number of schistosomula) may still experience symptoms such as fever and hepatosplenomegaly due to the failure to clear schistosomula. Based on the limitations of current anti-schistosomal drugs, there is an urgent need to develop new anti-schistosomal drugs targeting the schistosomula stage. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of the combination of an organic small molecule compound and divalent copper ions in the preparation of a drug for treating Schistosomiasis japonica. The combination of the present invention can effectively reduce the worm burden and egg-laying rate during the schistosomula stage compared with PZQ, has better schistosomicidal activity, and can realize the preparation and application of a drug for treating Schistosomiasis japonica with high-efficiency insecticidal effect.

[0005] In order to achieve the above purpose, the present invention provides the following technical solutions:

[0006] The present invention provides the application of the combination of an organic small molecule compound and divalent copper ions in the preparation of a drug for treating Schistosomiasis japonica; the organic small molecule compound is selected from one or more of the compounds having the structures shown in Formula 1 - 1, the compounds having the structures shown in Formula 2 - 2, and the compounds having the structures shown in Formula 3:

[0007]

[0008] Preferably, the molar ratio of the organic small molecule compound to divalent copper ions is 2:(1 - 4).

[0009] Preferably, when applied, the divalent copper ions are used in the form of water-soluble divalent copper salts.

[0010] Preferably, the water-soluble divalent copper salts include one or more of copper sulfate, copper chloride, and copper acetate.

[0011] The present invention provides a pharmaceutical composition comprising an organic small molecule compound and a water-soluble divalent copper salt; the molar ratio of the organic small molecule compound to the divalent copper ions in the water-soluble divalent copper salt is ≤2.

[0012] The organic small molecule compound is selected from one or more of the compounds having the structure shown in Formula 1-1, the compounds having the structure shown in Formula 2-2, and the compounds having the structure shown in Formula 3:

[0013]

[0014] Preferably, the water-soluble divalent copper salts include one or more of copper sulfate, copper chloride, and copper acetate.

[0015] Preferably, the molar ratio of the organic small molecule compound to the divalent copper ions in the water-soluble divalent copper salt is 2:(1-4).

[0016] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable solvent.

[0017] Preferably, in the pharmaceutical composition: the molar concentration of the organic small molecule compound is 20 μmol / L; the molar concentration of the water-soluble divalent copper salt is 10-40 μmol / L.

[0018] The present invention provides the use of the pharmaceutical composition according to the above technical solution in the preparation of a drug for treating schistosomiasis japonica.

[0019] The present invention provides the use of the combination of an organic small molecule compound and divalent copper ions in the preparation of a drug for treating schistosomiasis japonica, and the organic small molecule compound is selected from one or more of the compounds having the structure shown in Formula 1-1, the compounds having the structure shown in Formula 2-2, and the compounds having the structure shown in Formula 3. The present invention can target and block the development of schistosomes at multiple juvenile stages in vivo by combination, reduce the worm burden and egg-laying rate, has better schistosomicidal activity, and can realize the preparation and application of a drug for treating schistosomiasis japonica with high-efficiency insecticidal effect.

[0020] The combination of divalent copper ions and organic small molecule compounds provided by the present invention can provide basic data for breaking through the problem of insufficient efficacy of the existing drug PZQ against juvenile schistosomiasis japonica based on the metal-organic small molecule synergistic strategy. Description of the Drawings

[0021] Figure 1The reduction rates of viability and mortality of adult worms in RPMI 1640 medium, RPMI 1640 medium containing 1% DMSO, PZQ (10 μM), and CuSO4 (40 μM) respectively;

[0022] Figure 2 The survival rates of paired adult worms, female worms, and male worms after co-incubation of the compound of the structure shown in Formula 1-1 or the compound of the structure shown in Formula 1-2 with CuSO4; Figure 2 A, B, and C in [description] are the survival rates of Schistosoma japonicum after co-incubation of the compound of the structure shown in Formula 1-1 or with CuSO4; Figure 2 D, E, and F in [description] are the survival rates of Schistosoma japonicum after co-incubation of the compound of the structure shown in Formula 1-2 with CuSO4;

[0023] Figure 3 The survival rates of paired adult worms, female worms, and male worms after co-incubation of the compound of the structure shown in Formula 2-2, the compound of the structure shown in Formula 2-1, and the compound of the structure shown in Formula 2-3 with CuSO4;

[0024] Figure 4 The result characterization diagram of the survival rates of paired adult worms, female worms, and male worms after co-incubation of the compound of the structure shown in Formula 3 with CuSO4;

[0025] Figure 5 The synthetic route diagram of the compound of the structure shown in Formula 3 in Example 3;

[0026] Figure 6 The worm loads in the negative control group and each dosing group of mice in the in vivo experiment of the compound of the structure shown in Formula 1-1;

[0027] Figure 7 The worm loads in the negative control group and each dosing group of mice in the in vivo experiment of the compound of the structure shown in Formula 2-2;

[0028] Figure 8 The worm loads in the negative control group and each dosing group of mice in the in vivo experiment of the compound of the structure shown in Formula 3. Detailed implementation methods

[0029] The present invention provides the application of the combination of an organic small molecule compound and divalent copper ions in the preparation of a drug for treating schistosomiasis japonica; the organic small molecule compound is selected from one or more of the compounds of the structure shown in Formula 1-1, the compounds of the structure shown in Formula 2-2, and the compounds of the structure shown in Formula 3:

[0030]

[0031] The present invention has no special requirements for the sources of the compounds with the structures shown in Formula 1-1, the compounds with the structures shown in Formula 2-2, and the compounds with the structures shown in Formula 3. They can be prepared by the preparation methods well-known to those skilled in the art or directly purchased.

[0032] In the present invention, when used, the divalent copper ions are preferably used in the form of water-soluble divalent copper salts. The water-soluble divalent copper salts preferably include one or more of copper sulfate, copper chloride, and copper acetate, and can be copper sulfate in the examples. The molar ratio of the organic small molecule compound to the divalent copper ions is preferably 2:(1-4), and can be 2:1, 1:1, or 1:2 in the examples.

[0033] The present invention provides a pharmaceutical composition, comprising an organic small molecule compound and a water-soluble divalent copper salt; the molar ratio of the organic small molecule compound to the divalent copper ions in the water-soluble divalent copper salt ≤ 2;

[0034] The organic small molecule compound is selected from one or more of the compounds with the structures shown in Formula 1-1, the compounds with the structures shown in Formula 2-2, and the compounds with the structures shown in Formula 3:

[0035]

[0036] In the present invention, the water-soluble divalent copper salts preferably include one or more of copper sulfate, copper chloride, and copper acetate, and can be copper sulfate in the examples. The molar ratio of the organic small molecule compound to the divalent copper ions in the water-soluble divalent copper salt is preferably 2:(1-4), and can be 2:1, 1:1, or 1:2 in the examples.

[0037] In the present invention, the pharmaceutical composition preferably further comprises a pharmaceutically acceptable solvent, and can be physiological saline in the examples.

[0038] In the present invention, in the pharmaceutical composition: the molar concentration of the organic small molecule compound is preferably 20 μmol / L. The molar concentration of the water-soluble divalent copper salt is preferably 10-40 μmol / L, and can be 10 μmol / L, 20 μmol / L, or 40 μmol / L in the examples.

[0039] The present invention provides the use of the pharmaceutical composition according to the above technical solution in the preparation of a drug for treating schistosomiasis japonica.

[0040] In order to further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below in conjunction with examples, but they should not be construed as limiting the protection scope of the present invention.

[0041] The main chemical reagents used in the following examples and comparative examples: substituted benzaldehyde was purchased from Shanghai Aladdin Reagent Technology Co., Ltd.; acetophenone was purchased from Shanghai Adamas Reagent Co., Ltd.; CuSO4 and CuCl2 were purchased from Shanghai Sigma-Aldrich Trading Co., Ltd., all of which were chemically pure. C11-BODIPY and CS1 were purchased from Shanghai Haoyuan Biomedical Technology Co., Ltd. Neutral gum was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. Bathoxaline disulfonic acid disodium salt was purchased from Tanmo Quality Inspection Technology Co., Ltd. Other reagents were analytically pure and used directly.

[0042] 3,4,5-Trimethoxycinnamic acid was purchased from Shanghai Haohong Biopharmaceutical Technology Co., Ltd., and oxalyl chloride, tetrahydrofuran and lithium diisopropylamide (LDA) were purchased from Shanghai Adamas Reagent Co., Ltd.

[0043] Embodiment 1:

[0044] Synthesis of the compound of the structure shown in Formula 1-1: Dissolve 0.6g NaOH in 10mL methanol, add 0.68g (5mmol) 2-hydroxyacetophenone, stir for 15min, add 0.53g (5mmol) benzaldehyde, and detect the reaction by TLC. After the reaction is complete, adjust the pH to neutral, extract with dichloromethane, dry with anhydrous sodium sulfate, and separate and purify by column chromatography to obtain the target compound. Yield: 43.8%, melting point: 87.5-88.3℃. 1 HNMR(400MHz,Chloroform-d)δ12.83(s,1H),7.95-7.87(m,2H),7.71-7.61(m,3H),7.50(dd d,J=8.6,7.1,1.6Hz,1H),7.47-7.41(m,3H),7.04(dd,J=8.4,1.2Hz,1H),6.99-6.92(m,1H). 13 C NMR (101MHz, Chloroform-d) δ193.85,163.73,145.58,136.52,134.73,131.04,129.78,129.16,128.78,120.25,120.15,118.97,118.76. HPLC-MS (ESI - ):Calcd for C 15 H 12 O2H - [MH] - 223.07645, found 223.07638.

[0045] In Example 1, the purity of the compound was determined using a Waters Alliance E2695 reversed-phase high-performance liquid chromatograph, with a diode array detector, a wavelength range of 200 - 800 nm, a C18 chromatographic column (4.6×250 nm, 5 μm), a column temperature of 25 °C, an eluent of chromatographic-grade methanol and water, and a flow rate of 1 mL / min. After purity analysis, the purity of the compound with the structure shown in Formula 1-1 was greater than 95%.

[0046]

[0047] Comparative Example 1:

[0048] Synthesis of the compound with the structure shown in Formula 1-2: Dissolve 2,4-dihydroxyacetophenone (1.52 g, 10 mmol) and 4-hydroxybenzaldehyde (1.71 g, 14 mmol) in diethylene glycol (5 mL). Then add a 40 wt% KOH solution (10 mL), stir and react under nitrogen protection at 60 °C for 2 - 6 h, and monitor the reaction process by thin-layer chromatography. After the reaction is completed, cool to room temperature, add 1:1 (v / v) hydrochloric acid, acidify to pH = 3, extract with ethyl acetate, dry over anhydrous sodium sulfate, filter by suction, concentrate the liquid, and purify by column chromatography (V petroleum ether:V ethyl acetate = 3:1). After recrystallization from ethyl acetate - petroleum ether, a pale yellow powdery solid was obtained

[83] . Yield: 40.1%, melting point: 194.9 - 198.9 °C. 1 H NMR (400 MHz, DMSO-d6) δ 13.60 (s, 1H), 10.36 (s, 2H), 8.16 (d, J = 8.9 Hz, 1H), 7.80 - 7.71 (m, 4H), 6.88 - 6.81 (m, 2H), 6.41 (dd, J = 8.8, 2.4 Hz, 1H), 6.29 (d, J = 2.4 Hz, 1H). 13 C NMR (101 MHz, DMSO-d6) δ 191.58, 165.86, 165.00, 160.32, 144.31, 132.83, 131.22, 125.82, 117.47, 115.93, 113.09, 108.18, 102.69. HPLC-MS (ESI + ): Calcd for C 15 H 12 O2H + [M + H] + 255.06628, found 255.06665.

[0049] The purity of the compound in Comparative Example 1 was determined using a Waters Alliance E2695 reversed-phase high-performance liquid chromatograph, with a diode array detector, a wavelength range of 200 - 800 nm, a C18 chromatographic column (4.6×250 nm, 5 μm), a column temperature of 25 °C, an eluent of chromatographic-grade methanol and water, and a flow rate of 1 mL / min. After purity analysis, the purity of the compound with the structure shown in Formula 1-2 was greater than 95%.

[0050]

[0051] Example 2:

[0052] The compound with the structure shown in Formula 2-2 (3-hydroxyflavone) was purchased from Shanghai Aoboxing Biopharmaceutical Co., Ltd., with a purity greater than 98%.

[0053]

[0054] Comparative Example 2:

[0055] The compound with the structure shown in Formula 2-1 (biochanin A) was purchased from Shanghai Aoboxing Biopharmaceutical Co., Ltd., with a purity greater than 98%.

[0056]

[0057] Comparative Example 3:

[0058] The compound with the structure shown in Formula 2-3 (5-hydroxyflavone) was purchased from Ambeed China, with a purity of 98.0%.

[0059]

[0060] Example 3:

[0061] Synthesis of the compound with the structure shown in Formula 3: According to the preparation process shown in Figure 5 , 3,4,5-trimethoxycinnamic acid (0.95 g, 4 mmol) was dissolved in 10 mL of dichloromethane, 2 mL of oxalyl chloride was added dropwise. After the addition was complete, the mixture was stirred and refluxed for 4 h, then vacuum-dried to obtain cinnamoyl chloride. 2,3-Dihydro-cyclohexanone (0.39 g, 4 mmol) was dissolved in 10 mL of anhydrous tetrahydrofuran, cooled to -78 °C, stirred for 15 min, 2.6 mL of LDA (2 mol / L) was added dropwise, and then stirred for 1 h. Then cinnamoyl chloride was dissolved in 10 mL of anhydrous tetrahydrofuran solution and added dropwise to the reaction flask, and stirred at -78 °C for another 45 min, then transferred to room temperature and stirred overnight. 10 mL of saturated ammonium chloride solution was added to quench the reaction, and the mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate, concentrated, column-purified (Rf), and recrystallized from chloroform and n-hexane to obtain a light yellow powdery solid. Yield: 39.5%, melting point: 132.5 - 137.7 °C.1 1H NMR (400 MHz, Chloroform-d) δ 15.76 (d, J = 1.3 Hz, 1H), 7.54 (dd, J = 15.5, 3.0 Hz, 1H), 6.90 - 6.68 (m, 5H), 6.14 (dd, J = 10.1, 2.2 Hz, 1H), 3.88 (dd, J = 8.6, 2.0 Hz, 12H), 2.75 (t, J = 7.2 Hz, 2H), 2.40 (dt, J = 4.4, 2.4 Hz, 2H). 13 13C NMR (101 MHz, Chloroform-d) δ 190.22, 170.05, 153.58, 147.03, 139.86, 139.21, 131.39, 129.98, 117.86, 105.92, 105.21, 61.09, 56.33, 24.79, 22.13. HPLC-MS (ESI + ): Calcd for C 18 H 20 O5H + [M + H] + 317.13835, found 317.13990.

[0062] The purity of the compound in Example 3 was determined by a Waters Alliance E2695 reversed-phase high-performance liquid chromatograph, using a diode array detector, with a wavelength range of 200 - 800 nm, a C18 chromatographic column (4.6 × 250 nm, 5 μm), a column temperature of 25°C, and the eluent being chromatographic-grade methanol and water, with a flow rate of 1 mL / min. After purity analysis, the purity of the compound with the structure shown in Formula 3 was greater than 95%.

[0063]

[0064] Test Example 1: Insecticidal Activity in Vitro

[0065] The in vitro schistosomicidal adult activity of the compound with the structure shown in Formula 1-1 and CuSO4, and the in vitro schistosomicidal adult activity of the compound with the structure shown in Formula 1-2 and CuSO4:

[0066] (1) Solution Preparation

[0067] Preparation of the culture medium: The culture medium was prepared according to the following volume ratio; RPMI 1640 medium (88%), double-antibiotic (penicillin-streptomycin, 2%), and newborn bovine serum (10%). After mixing, a complete culture medium was obtained and stored at 4°C for later use.

[0068] Preparation of the compound with the structure shown in Formula 1-1, the compound with the structure shown in Formula 1-2, and the copper stock solution: Weigh the compound with the structure shown in Formula 1-1 or the compound with the structure shown in Formula 1-2 and the metal compound (0.004 mmol), and dissolve them in 1 mL of DMSO or physiological saline respectively to prepare a stock solution with a concentration of 0.004 mol / L. The prepared stock solution should be stored in a refrigerator at 4°C. Before use, it needs to be fully mixed and diluted to the corresponding working concentration according to the experimental requirements.

[0069] (2) In vitro anti-schistosomal activity experiment

[0070] Collection of adult Schistosoma japonicum: 35 days after infecting the mice, collect the adult worms from the portal-mesenteric vein, wash them three times with physiological saline, and set aside for use.

[0071] Pre-distribute the RPMI 1640 medium containing 10% fetal bovine serum (FBS) (1980 mL / well, and 1960 mL / well when adding two compounds) into 24-well culture plates, and preheat them in an incubator at 37°C and 5% CO2 for 30 min. Randomly select 5 pairs of adult worms with good vitality and intact worm membranes and transfer them to the well plates, and then add the test compound solutions with different concentrations respectively. Use a pipette to gently and evenly mix the liquid in the wells to ensure uniform distribution of the drug. During the experiment, regularly observe and record indicators such as changes in worm morphology (such as curling, swelling) and the degree of decrease in movement frequency. Set up parallel controls and conduct two independent repeated experiments. The experimental groups are as follows:

[0072] Blank control group: RPMI 1640 medium containing 10% newborn bovine serum (FBS).

[0073] Negative control group: RPMI 1640 medium containing 1% DMSO and 10% newborn bovine serum (FBS), and the concentration of DMSO is the highest concentration used to dilute the compound in the experimental group of this study.

[0074] Positive control group: PZQ with a final experimental concentration of 10 μM.

[0075] Experimental group: CuSO4 with a final experimental concentration of 40 μM; chalcone analog with a final experimental concentration of 20 μM; complexes of chalcone analog and CuSO4 with final concentrations of 20 μM:10 μM, 20 μM:20 μM, and 20 μM:40 μM (compound with the structure shown in Formula 1-1 or compound with the structure shown in Formula 1-2:CuSO4) Average worm viability = (1 - average viability of the experimental group / average viability of the control group) × 100% Reference standard for worm viability scoring:

[0076] Table 1 Evaluation table for the viability of adult Schistosoma japonicum

[0077]

[0078] Specific experimental procedure: After the mice infected with cercariae were conventionally raised for 35 days, the mice were dissected to obtain adult Schistosoma japonicum (hereinafter simply referred to as adults). Five pairs of intact adults were selected and placed into a 24-well plate, and different concentrations of the compound and CuSO4 were added (the compound and CuSO4 were respectively: 20 μM + 10 μM, 20 μM + 20 μM, 20 μM + 40 μM). At regular intervals (6 h, 12 h, 24 h, 36 h, 48 h, 72 h), the viability status and morphological changes of the adult schistosomes were observed, and their death situations were counted. The effects of the compounds with the structures shown in Formula 1-1, the compounds with the structures shown in Formula 1-2 and CuSO4 used in combination on the survival of adult Schistosoma japonicum were evaluated.

[0079] First, the effects of different concentrations of CuCl2 and CuSO4 on the survival status of adults were evaluated respectively. At 40 μM, CuCl2 could cause the death of schistosomes, while CuSO4 had no effect on the activity of adults at this time. Therefore, in the subsequent tests of the present invention, CuSO4 was used in combination with the compound with the structure shown in Formula 1-1 or the compound with the structure shown in Formula 1-2. In RPMI 1640 medium (negative control) and RPMI 1640 medium containing 1% DMSO (solvent control), the viability status of the adults did not decrease significantly and no death occurred. It shows that RPMI 1640 medium and RPMI 1640 medium containing 1% DMSO (solvent control) have no effect on the adults and can meet the nutritional requirements for in vitro experiments of schistosomes at the same time. When CuSO4 was at 10 - 40 μM, the effect on the viability of adults was similar to that of the solvent control group, indicating that CuSO4 would not affect the viability of schistosomes at this time. After the positive control (PZQ = 10 μM) was co-incubated with the adults for 6 h, the adults showed swelling and bending phenomena, the viability was completely lost, and all died. Figure 1 In which A is the in vitro viability reduction rate of adults in RPMI 1640 medium, RPMI 1640 medium containing 1% DMSO, PZQ (10 μM) and CuSO4 (40 μM) respectively, Figure 1 In which B is the in vitro mortality rate of adults in RPMI 1640 medium, RPMI 1640 medium containing 1% DMSO, PZQ (10 μM) and CuSO4 (40 μM) respectively. The results of the control groups in other in vitro test examples of the present invention are the same. To avoid repetition, they will not be elaborated.

[0080] When incubating adult worms with the compound of the structure shown in Formula 1-1 alone at 20 μM, adult worms start to die at 36 h, and the mortality rate of adult worms is only 35% after 72 h of incubation. Fix the compound of the structure shown in Formula 1-1 at 20 μM, and co-incubate with adult worms at molar ratios of compound of the structure shown in Formula 1-1:CuSO4 = 2:1, 1:1, and 1:2, respectively. It is found that adult worms start to die after co-incubation with the compound of the structure shown in Formula 1-1:CuSO4 at 20 μM:10 μM for 24 h. However, when the incubation time is extended to 72 h, the mortality rate of adult worms is only 40%, showing no significant difference from the anti-schistosome activity of using the compound of the structure shown in Formula 1-1 alone (20 μM). When the compound of the structure shown in Formula 1-1:CuSO4 is 20 μM:20 μM, the mortality rates of adult worms at 36 h, 48 h, and 72 h are 20%, 50%, and 60%, respectively. Compared with using 20 μM of the compound of the structure shown in Formula 1-1 alone, the insecticidal effect in the early stage (6 - 24 h) of combining the compound of the structure shown in Formula 1-1 with CuSO4 is not improved much, but the insecticidal effect is significantly improved at 36 h, 48 h, and 72 h, with increases of 300%, 400%, and 71.4%, respectively. When the compound of the structure shown in Formula 1-1:CuSO4 is 20 μM:40 μM, the insecticidal activity from 36 h to 72 h is further improved, and the mortality rate at 72 h reaches 70%, which is the best insecticidal effect among all ratios, as shown in Figure 2 shown in A in Figure 2 Survival rates of paired adult worms, female worms, and male worms after co-incubating the compound of the structure shown in Formula 1-1 or the compound of the structure shown in Formula 1-2 with CuSO4. Figure 2 A, B, and C in Figure 2 are the survival rates of schistosomes after co-incubating the compound of the structure shown in Formula 1-1 with CuSO4,

[0081] Furthermore, compare the death situations of male and female worms after administration. As shown in B in Figure 2 and C in Figure 2 shown, at the same time and dose, male worms start to die first and are more sensitive to the drug. For example, when the compound of the structure shown in Formula 1-1:CuSO4 is 20 μM:40 μM, the mortality rate of male worms reaches 100% at 48 h, while the mortality rate of female worms is only 40% at 72 h. It can be seen that after combining the compound of the structure shown in Formula 1-1 with CuSO4, the insecticidal activity is significantly improved, showing a concentration and time-dependent relationship, and the killing effect on male worms is better.

[0082] As shown in D in Figure 2 and E in Figure 2 and F in Figure 2The F in [Figure] shows that for the compound with the structure shown in Formula 1-2 with two more hydroxyl groups introduced, its in vitro insecticidal activity is significantly lower than that of the compound with the structure shown in Formula 1-1. And after co-incubation with CuSO4, its in vitro anti-insect activity has not been significantly improved. The adult mortality rate at 72 h is about 40%, and it has basically no activity against female insects. It shows that increasing the number of phenolic hydroxyl groups on the benzene ring is not conducive to enhancing the insecticidal activity.

[0083] Test Example 2: In vitro insecticidal activity

[0084] Referring to the experimental procedure of Test Example 1, the in vitro anti-Schistosoma japonicum activities of the compounds with the structures shown in Formula 2-2, Formula 2-1, and Formula 2-3 in combination with CuSO4 were evaluated respectively. Adult worms were isolated from the portal vein system of mice infected with Schistosoma japonicum, and the adult worms were randomly divided into the following experimental groups (10 worms in each group): negative control group (containing only RPMI 1640 medium), solvent control group (RPMI 1640 medium containing 1% DMSO), positive control group (PZQ = 10 μM), flavonoid analog (20 μM) single treatment group, CuSO4 (40 μM) single treatment group, flavonoid analog and CuSO4 combination group (flavonoid analog concentration is 20 μM, CuSO4 concentrations are 10 μM, 20 μM, and 40 μM). The phenomena of Schistosoma in the control group are the same as those in Test Example 1 and will not be repeated.

[0085] Figure 3 Survival rates of paired adult worms, female worms, and male worms after co-incubation of the compounds with the structures shown in Formula 2-2, Formula 2-1, and Formula 2-3 with CuSO4. Figure 3 A, B, and C in [Figure] are the survival rates of Schistosoma japonicum after co-incubation of the compound with the structure shown in Formula 2-1 with CuSO4. Figure 3 D, E, and F in [Figure] are the survival rates of Schistosoma japonicum after co-incubation of the compound with the structure shown in Formula 2-2 with CuSO4. Figure 3 G, H, and I in [Figure] are the survival rates of Schistosoma japonicum after co-incubation of the compound with the structure shown in Formula 2-3 with CuSO4.

[0086] After 72 h of incubation, the survival rate of adult worms treated with the compound with the structure shown in Formula 2-1 (20 μM) is 60% ( Figure 3 A in [Figure]). When 40 μM CuSO4 is further added, the survival rate of adult worms shows a certain degree of decrease and finally is only 40% ( Figure 3 A in [Figure]). In all experimental groups, the survival rate of female worms at 72 h can reach 80% or even higher ( Figure 3In B). For male worms, after adding CuSO4, their death time was earlier than when using the compound alone. However, as the incubation time was extended to 72 h, the difference in the survival rate of male worms in each group gradually decreased ( Figure 3 in C).

[0087] Similarly, as the concentration of CuSO4 increased, the insecticidal activity of the compound shown in Formula 2-2 against Schistosoma japonicum was significantly enhanced. The insecticidal effect of the compound shown in Formula 2-3 at 72 h was weak, and the mortality rate was only 20%. After adding 10 μM and 20 μM of CuSO4, the adult worm mortality rate increased by 2-3 times; when 40 μM of CuSO4 was added, the adult worm mortality rate was as high as 90%. It was shown that after the compound shown in Formula 2-3 was combined with CuSO4, its in vitro activity against adult Schistosoma japonicum was greatly improved. Further comparison of the survival status of female and male worms found that female worms had relatively higher drug tolerance, and only in the highest concentration group at 72 h [compound shown in Formula 2-3 + CuSO4 = (20 + 40) μM], the survival rate of female worms showed an obvious decrease. While male worms all died at 24 h in the highest concentration group; in the group with compound shown in Formula 2-3 + CuSO4 being (20 + 20) μM, male worms also all died at 48 h ( Figure 3 in D, E and F).

[0088] Different from the significant improvement in anti-schistosome activity when the compound shown in Formula 2-2 was combined with CuSO4, as the concentration of CuSO4 added to the compound shown in Formula 2-3 increased, the survival rate of adult worms at 72 h was close to that of the group with only the compound shown in Formula 2-3, and the adult worm mortality rate was less than 50% after adding 40 μM of CuSO4. This indicated that after the compound shown in Formula 2-3 was combined with CuSO4, the improvement in its activity against adult Schistosoma japonicum was limited. Comparative analysis of female and male worms showed that female worms were hardly affected by the drug, and the survival rate was close to 100%, and only in the experimental group with compound shown in Formula 2-3 + CuSO4 concentration of (20 + 10) μM, the mortality rate of female worms was 10%. While male worms were sensitive to the drug and the survival rate was significantly reduced ( Figure 3 in G, H and I).

[0089] From the above experimental results of in vitro anti-schistosome activity, it could be seen that only when 2-2 was used in combination with CuSO4, its in vitro activity against adult Schistosoma japonicum was greatly improved. Therefore, 2-2 was used to carry out in vivo anti-schistosome activity experiments subsequently.

[0090] Test Example 3: In vitro insecticidal activity

[0091] In vitro anti-adult Schistosoma japonicum activity of the compound shown in Formula 3 combined with CuSO4:

[0092] To evaluate the in vitro killing effect of the compound with the structure shown in formula 3 in combination with CuSO4 on adult Schistosoma japonicum, an in vitro activity evaluation was carried out with reference to the experimental procedure in Test Example 1. Figure 4 Survival rate results characterization diagrams of paired adult worms ( Figure 4 A in Figure 4 ), female worms ( Figure 4 B in

[0093] ) and male worms ( Figure 4 C in

[0094] ) after co-incubation of the compound with the structure shown in formula 3 and CuSO4.

[0095] Establishment of positive mice for in vivo anti-Schistosoma japonicum activity experiment: Select 15 - 30 laboratory-confirmed positive Oncomelania hupensis, place them in a 100 mL conical flask with light-shielding treatment (wrapped with tin foil), inject natural water to 1 cm from the bottle mouth, and irradiate with tungsten filament lamp for about 1 h to release cercariae. Fix the mice on a special operation board and infect the abdominal hair-removal area by the patch method. Each mouse is infected with 50 ± 5 cercariae and kept in contact for 15 minutes. After the infection is completed, randomly divide the mice into groups (n = 5 mice / cage) and raise them conventionally.

[0096] An ICR mouse was used to establish a schistosome skin schistosomulum infection model (judged as the skin schistosomulum stage 24 h after infection). One day after infection (skin schistosomulum stage), intraperitoneal injection was used. PZQ positive control group: 10 mg / kg (solvent: 10% DMSO + 10% Tween 80 / saline); CuSO4 single drug group: 5 mg / kg (solvent: saline); the compound with the structure shown in Formula 1-1 single drug group: 10 mg / kg (solvent: the same as the PZQ group); combined administration group: the compound with the structure shown in Formula 1-1 + CuSO4 (10 + 5) mg / kg (solvent: the same as the PZQ group). Administration was carried out on the 1st - 2nd day after infection, and then the mice were conventionally raised for 40 days (total 45 days of raising). After the experiment, the mice were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.1 mL / 10 g body weight) solution, and the schistosomes in the hepatic portal vein and mesenteric vein were collected. The number of worms was recorded and the worm reduction rate was calculated. At the same time, the heart, liver, spleen, lung, and kidney were collected and the wet weights of the heart, liver, spleen, lung, and kidney were accurately weighed, and the organ index was calculated.

[0097] Liver schistosomulum stage: On the 14th day after infection (in the liver schistosomulum stage), drug treatment was started, and the remaining experimental steps were the same as those in the 1-day in vivo anti-schistosome activity experiment.

[0098] Adult stage: On the 28th day after infection (adult stage), drug treatment was started, and the remaining experimental steps were the same as those in the 1-day in vivo anti-schistosome activity experiment.

[0099] Liver index = liver weight / mouse body weight × 100; heart, spleen, lung, and kidney index = spleen weight / mouse body weight × 1000.

[0100] Counting the number of eggs in the liver and small intestine of mice: After dissecting the mice, accurately weigh the liver (about 1 g) and small intestine and place them in a conical flask. Then add 40 mL of 2 wt% KOH solution, and place the conical flask in a shaker at 37 °C and 180 rpm for 12 h to digest the tissue. After shaking, transfer the digestion solution to a 50 mL centrifuge tube, centrifuge at 3000 rpm for 30 minutes, and discard the supernatant. Then add distilled water to make the volume of the solution up to 30 mL. Use a McMaster counting chamber to count, and repeat three times.

[0101] The calculation formula for the number of eggs per gram of liver (EPG) is: EPG (OPG) = A × (60 ÷ 0.15) ÷ 2 = A × 200

[0102] (1) The effect of the combination of the compound with the structure shown in Formula 1-1 and CuSO4 on worm reduction and egg reduction in the liver stage of 14-day infection in vivo

[0103] After 14 days (hepatic schistosomula stage) of cercaria infection, mice were administered a single dose continuously for 5 days by intraperitoneal injection. The mice were divided into a blank group (non-infected with Schistosoma group), a negative control group (infected with Schistosoma but untreated group), a positive control group (i.e., PZQ group, 10 mg / kg), a CuSO4 group (5 mg / kg), a compound group with the structure shown in Formula 1-1 (10 mg / kg), and a treatment group (i.e., compound with the structure shown in Formula 1-1 + CuSO4 group, (10 + 5) mg / kg), with 5 mice in each group. Each mouse was infected with (50 ± 5) cercariae. After 1 day (cutaneous schistosomula stage) of infection, the drugs were administered by intraperitoneal injection respectively for 5 consecutive days, and then the mice were conventionally raised for 40 days (total raising for 45 days). On the 46th day, the mice were sacrificed, and the number of adult worms parasitized in the hepatic portal vein and mesentery of the mice was counted respectively, and the total number of worms, the total number of female worms, and the total number of male worms were counted.

[0104] During the 45 days of the in vivo experiment, all the mice in the experimental groups survived. The effect of reducing worms in the PZQ administration group was the worst, with the total worm reduction rate only being 6.5%, and the total female worm reduction rate was only 6.7%, that is, PZQ was ineffective against the schistosomula in the 14-day hepatic stage. Compared with the PZQ group, the effect of reducing worms in the CuSO4 group was doubled, but the total worm reduction rate was 16.7% and the total female worm reduction rate was 14.4%, which was still not good. When the compound with the structure shown in Formula 1-1 was administered alone at a dose of 10 mg / kg, it had basically no effect on the schistosomula in the 14-day hepatic stage, with a total worm reduction rate of 7.8% and a total female worm reduction rate of 8.7%. However, when the compound with the structure shown in Formula 1-1 was combined with CuSO4, the effect of reducing worms was greatly improved, with a total worm reduction rate of 53.4%, reaching a medium worm reduction effect, which was 3.1 times that of the CuSO4 group and 6.8 times that of the compound group with the structure shown in Formula 1-1. Its total female worm reduction rate was also the highest, reaching 57.9%, which was 4 times that of the CuSO4 group and 6.6 times that of the compound group with the structure shown in Formula 1-1 (Table 2). At the same time, through statistical analysis, it was found that there was no statistical difference in the total worm burden among the three groups of the PZQ group, the CuSO4 group, and the compound group with the structure shown in Formula 1-1 compared with the non-administered infected group. However, when the compound with the structure shown in Formula 1-1 was combined with CuSO4, the total worm burden decreased significantly, and there were statistical differences compared with both the non-administered infected group and the 1-1 group ( Figure 6 ). It shows that the combination of the compound with the structure shown in Formula 1-1 and CuSO4 has certain in vivo anti-schistosomal activity against the schistosomula in the 14-day hepatic stage.

[0105] Table 2 In vivo anti-worm activities of the negative control group and each administration group against the schistosomula in the 14-day hepatic stage

[0106]

[0107] In Table 2: a: Infected mice (non-administered); b: Calculation using the corresponding average value

[0108] Meanwhile, by counting the number of eggs in the livers and small intestines of mice in each infected group (Table 3), it was found that the egg reduction rate in the PZQ group was only 9.5%, and the egg reduction rate in the small intestine was 13.9%, indicating a poor egg reduction effect; the liver egg reduction rate in the CuSO4 group was only 7.6%, and the small intestine egg reduction rate was 32.5%; the liver and small intestine egg reduction rates in the compound group with the structure shown in Formula 1-1 were 11.7% and 29.8% respectively, and the egg reduction effect was also not ideal; when the compound with the structure shown in Formula 1-1 was combined with CuSO4, it had the highest liver egg reduction rate (41.2%) and small intestine egg reduction rate (68.0%). This shows that the egg reduction effect was improved after the two were combined.

[0109] Table 3 Egg reduction effects of each administration group on 14-day liver-stage schistosomula

[0110]

[0111] (2) Effects of the compound with the structure shown in Formula 2-2 combined with CuSO4 on worm reduction and egg reduction in the body of mice infected with 14-day liver-stage schistosomula

[0112] Referring to the in-vivo experimental protocol of the compound with the structure shown in Formula 1-1 and CuSO4, after 14 days of mouse infection (liver-stage schistosomula stage), intraperitoneal injection was used for administration for 5 consecutive days. The mice were dissected after 45 days of feeding.

[0113] After statistical analysis of the schistosomes in each infected group, it was found that the worm reduction rates of the PZQ group, CuSO4 group, and the compound group with the structure shown in Formula 2-2 alone were 8.3%, 1.1%, and 8.7% respectively compared with the untreated infected group, and the worm reduction effects were not significant. However, the worm reduction effect of the compound with the structure shown in Formula 2-2 combined with CuSO4 was more prominent, and its worm reduction rate (total worm reduction rate and total female worm reduction rate) was nearly 4 times that of the single administration group (Table 4).

[0114] At the same time, from Figure 7 it can be seen that when the compound with the structure shown in Formula 2-2 was used in combination with CuSO4, its total worm burden showed a significant decrease compared with the infected non-administered group, CuSO4 group, and the compound group with the structure shown in Formula 2-2. It was preliminarily judged that the combined application of the compound with the structure shown in Formula 2-2 and CuSO4 had a certain in-vivo worm reduction effect on 14-day liver-stage schistosomula, and was significantly better than PZQ.

[0115] Table 4 In-vivo anti-schistosome activities of the negative control group and each administration group against 14-day liver-stage schistosomula

[0116]

[0117]

[0118] 1 in Table 4a : Infected mice (untreated with drugs); b: Calculated using the corresponding average value

[0119] Table 5 The effect of each drug - administered group on reducing the number of eggs of schistosomula in the liver stage at 14 days

[0120]

[0121] By comparing the effect of each treatment group on reducing the number of eggs, it was found that, similar to the effect on reducing worms, the group of mice treated with the compound of the structure shown in Formula 2 - 2 + CuSO₄ had a significantly better effect on reducing the number of eggs than the PZQ group, the CuSO₄ group, and the single 2 - 2 group. The egg - reducing rate in the liver was 38.9%, and the egg - reducing rate in the small intestine was 51.2% (Table 5).

[0122] (3) The in - vivo effect of the compound of the structure shown in Formula 3 combined with CuSO₄ on schistosomula in the 1 - day skin stage

[0123] This invention studied the in - vivo anti - schistosomal activity of the compound of the structure shown in Formula 3 combined with CuSO₄ on schistosomula in the 1 - day skin stage. Mice were divided into 5 groups, with 5 mice in each group, namely the blank group (not infected with schistosomes), the negative control group (infected with schistosomes without treatment), the positive control group (PZQ), the drug control group (CuSO₄, the compound of the structure shown in Formula 3), and the treatment group (the compound of the structure shown in Formula 3+CuSO₄). One day after the mice were infected, they were intraperitoneally injected with drugs in the PZQ (10 mg / kg) group, CuSO₄ (5 mg / kg) group, the compound of the structure shown in Formula 3 (10 mg / kg) group, and the compound of the structure shown in Formula 3 + CuSO₄ [(10 + 5) mg / kg] group for 5 consecutive days. After a total of 45 days of feeding, the mice were dissected.

[0124] The group treated with the compound of the structure shown in Formula 3 combined with CuSO₄ showed significant advantages in in - vivo anti - schistosomal activity. As Figure 8 shown, the worm count in this combined - treatment group was significantly lower than that of the blank control group. By calculating the worm - reducing rate, it was found that the combined - drug regimen showed a synergistic effect, with a total worm - reducing rate of 42.8% and a female - worm clearance rate of 53.1% (Table 6). It is worth noting that the curative effect of the combined - drug group was 2 - 3 times higher than that of the single - drug group, indicating that the drug combination had an obvious synergistic effect.

[0125] Table 6 The in - vivo anti - worm activity of the negative control group and each drug - administered group on schistosomula in the 1 - day skin stage

[0126]

[0127] Control 1 in Table 6 a: Infected mice (without drug administration); b: One mouse died on the 43rd day; c: One mouse died on the 5th day; d: One mouse died on the 27th and 42nd days respectively; e: One mouse died on the 43rd day; f: The corresponding average values were calculated.

[0128] Statistical analysis of the number of eggs in the livers and small intestines of mice found that the compound with the structure shown in Formula 3 in combination with CuSO4 had the most significant inhibitory effect on the egg load of mice, and was significantly better than the positive control group PZQ. As shown in Table 7, this combination treatment achieved a 31.0% and 54.3% decrease in egg load in the liver and small intestine tissues respectively, and the anti-egg deposition effect in the small intestine was particularly prominent. In contrast, although the single compound with the structure shown in Formula 3 administration group showed a 29.5% egg reduction rate in the liver, its egg reduction rate in the small intestine was only 5.7%. The anti-egg effects of the PZQ and CuSO4 single-drug groups were weaker than those of the combination drug group.

[0129] Table 7 Egg reduction effects of each administration group on 1-day schistosomula in the skin stage

[0130]

[0131]

[0132] From the above examples, it can be seen that the compound with the structure shown in Formula 1-1 provided by the present invention can effectively complex with CuSO4 in a molar ratio of 2:1. Among them, the combination of the compound with the structure shown in Formula 1-1 and CuSO4 has better in vitro schistosomicidal activity. The compound with the structure shown in Formula 1-1 and CuSO4 are of low toxicity to cells and mice at a dose of (10 + 5) mg / kg, and continuous intraperitoneal injection was carried out for 5 days at this dose, which has different in vivo killing abilities for schistosomes at different development stages. Among them, the in vivo worm reduction rate and egg reduction rate for 14-day schistosomula in the liver stage were increased by 8.2 times and 4.9 times respectively compared with the positive control PZQ (in vivo experimental results), and were better than the single component of the compound with the structure shown in Formula 1-1 or CuSO4, showing the best insecticidal effect.

[0133] The above experiments showed that the compound with the structure shown in Formula 2-2 in combination with CuSO4 at (20 + 40) μM for 72 h had an adult worm mortality rate of 90%, which was significantly better than the single compound with the structure shown in Formula 2-2 (20%) and CuSO4 (inactive), and male worms were more sensitive. In vivo experiments showed that the compound with the structure shown in Formula 2-2 and CuSO4 were of low toxicity at (10 + 5) mg / kg, and single-dose continuous intraperitoneal injection was carried out for 5 days at this dose, and the curative effects on 1-day schistosomula in the skin stage and 14-day schistosomula in the liver stage were significant (the total worm reduction rates were 39.6% and 39.4% respectively), both better than the PZQ group (11.8% and 8.3%).

[0134] The inventors of the present invention have found that the compound with the structure shown in Formula 3 in combination with CuSO4 can synergistically enhance the in vitro anti-schistosome activity. When the compound with the structure shown in Formula 3 and CuSO4 are used in combination at (10 + 5) mg / kg, the total worm reduction rate and female worm reduction rate of the in vivo anti-worm activity against 1-day-old skin-stage schistosomula are 42.8% and 53.1% respectively, which are significantly higher than those of the compound with the structure shown in Formula 3 in the single-dose group (11.1%, 28.6%), CuSO4 (16.7%, 26.0%) and the positive control PZQ group (0%, 20.9%). At the same time, it has a certain ability to reduce egg production.

[0135] In summary, the compound with the structure shown in Formula 1-1, the compound with the structure shown in Formula 2-2 or the compound with the structure shown in Formula 3 provided by the present invention in combination with CuSO4 can target and block the development of schistosomes at multiple schistosomula stages in vivo to varying degrees, reducing the worm burden and egg-laying rate. The present invention provides basic data for breaking through the problem of insufficient efficacy of the existing drug PZQ against schistosomula based on the metal-organic small molecule synergistic strategy, and also provides new ideas for the research and development of anti-schistosome drugs through the metal ion-mediated pathway.

[0136] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all embodiments. Other embodiments can be obtained based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of an organic small molecule compound in combination with divalent copper ions in the preparation of a medicament for treating Schistosomiasis japonica; the organic small molecule compound is selected from one or more of the compounds having the structures shown in Formula 1-1, the compounds having the structures shown in Formula 2-2, and the compounds having the structures shown in Formula 3:

2. The application according to claim 1, characterized in that, The molar ratio of the organic small molecule compound to divalent copper ions is 2:(1-4).

3. The application according to claim 1 or 2, characterized in that, When used, the divalent copper ions are used in the form of a water-soluble divalent copper salt.

4. The application according to claim 3, wherein The water-soluble divalent copper salt includes one or more of copper sulfate, copper chloride, and copper acetate.

5. A pharmaceutical composition, characterized in that, It includes an organic small molecule compound and a water-soluble divalent copper salt; the molar ratio of the organic small molecule compound to divalent copper ions in the water-soluble divalent copper salt is ≤2; The organic small molecule compound is selected from one or more of the compounds having the structures shown in Formula 1-1, the compounds having the structures shown in Formula 2-2, and the compounds having the structures shown in Formula 3:

6. The pharmaceutical composition according to claim 5, characterized in that, The water-soluble divalent copper salt includes one or more of copper sulfate, copper chloride, and copper acetate.

7. The pharmaceutical composition according to claim 5 or 6, characterized in that, The molar ratio of the organic small molecule compound to divalent copper ions in the water-soluble divalent copper salt is 2:(1-4).

8. The pharmaceutical composition according to claim 5, wherein The pharmaceutical composition further includes a pharmaceutically acceptable solvent.

9. The pharmaceutical composition according to claim 8, wherein In the pharmaceutical composition: the molar concentration of the organic small molecule compound is 20 μmol / L; the molar concentration of the water-soluble divalent copper salt is 10-40 μmol / L.

10. Use of the pharmaceutical composition according to any one of claims 5-9 in the preparation of a medicament for treating Schistosomiasis japonica.

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