Praziquantel derivative PC-6 as well as preparation method and application thereof

Abstract

The invention discloses a praziquantel derivative PC-6 and a preparation method and application thereof.The praziquantel derivative PC-6 is obtained by introducing an active group coumarin into a praziquantel structure through a pharmacophore fusion strategy, and the praziquantel derivative PC-6 has the application advantages that the insect-resistant efficacy of praziquantel is improved, and parasite drug resistance is reduced. The 48-hour median inhibitory concentration (EC50) of the praziquantel derivative PC-6 to third-generation microforest worms is 1.94 mu M, and the pesticide effect of the praziquantel derivative PC-6 is improved by 3.99 times compared with that of praziquantel; the 96h median lethal concentration (LC50) of zebra fish is 70.49 mu M, and the toxicity is reduced by 3.20 times compared with praziquantel; the therapeutic index (TI) is 36.34, which is 12.78 times that of praziquantel. According to the invention, a pharmacophore fusion strategy is utilized to synthesize a high-efficiency and low-toxicity anti-homoparagonimus drug, the production cost is low, the use method is simple, and the drug has important development and application prospects.

Description

TECHNICAL FIELD

[0001] The present application relates to the fields of medicinal chemistry and aquaculture, and in particular to a praziquantel derivative PC-6, a preparation method and application thereof. BACKGROUND

[0002] Monogenean disease is a common parasitic disease in aquaculture, which can cause large-scale death of freshwater and marine fish and cause serious economic losses. In recent years, with the increase of market demand, the characteristic freshwater fish industry has developed rapidly. Compared with bulk freshwater fish, characteristic freshwater fish has a more scaled and intensive farming mode, and is a high economic benefit variety, but is also prone to suffer losses caused by disease outbreaks. According to statistics, monogenean infection can cause the annual mortality rate of Norwegian salmon fry to be as high as 86%, which seriously threatens the fry cultivation and fish farming of characteristic freshwater fish.

[0003] Praziquantel is a broad-spectrum antiparasitic drug, which has killing activity on a variety of trematodes, tapeworms and helminths. Compared with other drugs, praziquantel has lower toxicity, and has good killing effect on common fish parasites in aquaculture, and is one of the specific drugs for treating fish monogenean disease at present. However, due to the long-term and overdosage use of praziquantel, the drug resistance of parasites is generally enhanced; other drugs such as trichlorphon and benzimidazole have high toxicity to fish, and are easy to cause the death of host fish during the medication process. These factors increase the difficulty of preventing and treating monogenean disease. Therefore, it is of great significance to develop new type of high-efficiency and low-toxicity anti-monogenean drug.

[0004] Structural modification of existing drugs is an effective way for new drug research and development. Among them, multi-target drug design is one of the strategies for developing new drugs. Multi-target drugs usually have better therapeutic effect, lower toxicity and side effect, and less drug resistance. In recent years, important progress has been made in the synthesis of multi-target drugs based on natural product pharmacophore in the field of anti-parasite.

[0005] Coumarin is a kind of natural organic compound widely existing in nature, which has the characteristics of simple chemical structure and diverse biological activity, and has become a hot spot for drug research and development in various fields. Studies have shown that coumarin compounds have good anthelmintic activity on fish monogenean. Therefore, coumarin can be used as an ideal candidate molecule for new multi-target drug pharmacophore, and used for the research and development of drugs for treating fish monogenean disease.

[0006] At present, there is no report on the synthesis of anti-fish monogenean drugs by connecting coumarin molecules with praziquantel. SUMMARY

[0007] The present application aims to overcome the deficiencies of the prior art, and provides a praziquantel derivative PC-6, a preparation method and application thereof. The active group coumarin is introduced into the praziquantel structure through a pharmacophore fusion strategy to obtain the praziquantel derivative PC-6, which has the application advantages of improving the anti-parasite efficacy of praziquantel and reducing parasite drug resistance. The derivative PC-6 has significant anti-monogene activity and low fish toxicity, and provides a highly efficient and low-toxicity anti-monogene (especially trichodinid) treatment drug for the aquaculture industry, and has a wide application prospect.

[0008] To achieve the above-mentioned object, the technical scheme designed by the present application is as follows: The present application provides a praziquantel derivative PC-6, and the structure formula of the PC-6 is as follows: .

[0009] The present application also provides a preparation method of the above-mentioned praziquantel derivative PC-6, and the synthesis line is as follows: The specific steps are as follows: (1) preparing 10-amino praziquantel; (2) preparing 7-(6-bromohexyloxy) coumarin; (3) dissolving the above-mentioned 10-amino praziquantel in acetonitrile, adding anhydrous potassium carbonate, stirring at room temperature, then adding the 7-(6-bromohexyloxy) coumarin prepared in step (2), heating and refluxing, then filtering, extracting, drying, and column chromatography separation to obtain the praziquantel derivative PC-6 (yellow waxy compound).

[0010] Further, the 10-amino praziquantel is prepared by the following steps: a. dissolving praziquantel in concentrated sulfuric acid and stirring to dissolve, slowly adding concentrated nitric acid after ice bath, and obtaining a reaction solution; b. pouring the reaction solution into ice water, adding saturated sodium carbonate solution to adjust the pH to 7, extracting with dichloromethane, combining the organic layers and drying with anhydrous sodium sulfate overnight; distilling under reduced pressure, purifying (residue is separated by silica gel column chromatography), and obtaining 10-nitropraziquantel (which is a light yellow solid compound); c. dissolving 10-nitropraziquantel in methanol to obtain a reaction mixture, adding SnCl2•2H2O dissolved with hydrochloric acid dropwise to the reaction mixture, stirring and heating to reflux, removing methanol under reduced pressure, dissolving with water, adjusting the pH to 8 with saturated sodium carbonate solution, extracting, combining the organic layers and drying with anhydrous sodium sulfate, distilling under reduced pressure, and purifying (residue is separated by column chromatography), and obtaining 10-amino praziquantel (which is a white solid).

[0011] Further, the 7-(6-bromohexyloxy)coumarin is prepared by the following steps: Dissolve 7-hydroxycoumarin in acetone, add anhydrous potassium carbonate, stir at room temperature for 30 min, then slowly add 1,6-dibromohexane; heat to reflux at 70℃ for 24 h, remove acetone under reduced pressure, add deionized water, then extract with dichloromethane, combine the organic phases and dry overnight with anhydrous sodium sulfate, filter to remove the solids, and remove the liquid organic phase under reduced pressure; purify (residue is separated by silica gel column chromatography) to obtain white solid, which is 7-(6-bromohexyloxy)coumarin.

[0012] Further, in step (3), the heating reflux temperature is 85℃, and the time is 24 h.

[0013] The application also provides application of the above-mentioned praziquantel derivative PC-6 in anti-monogenea.

[0014] The application also provides application of the above-mentioned praziquantel derivative PC-6 in the medicine for preventing and treating monogenea.

[0015] The application also provides a medicine for preventing and treating monogenea, which contains the praziquantel derivative PC-6, wherein the content of the praziquantel derivative PC-6 is 1-8 μM.

[0016] Further, in the medicine, the content of the praziquantel derivative PC-6 is 4-8 μM.

[0017] Further, in the medicine, the content of the praziquantel derivative PC-6 is 8 μM.

[0018] The use method of the above-mentioned medicine is whole pond spraying or bait feeding.

[0019] The application has the following beneficial effects: (1) The 48 h half maximal inhibitory concentration (EC50) of the praziquantel derivative PC-6 of the application to clonorchis sinensis is 1.94 μM, the drug efficacy is 3.99 times higher than that of praziquantel; the 96 h half maximal lethal concentration (LC50) of the praziquantel derivative PC-6 of the application to zebra fish is 70.49 μM, the toxicity is 3.20 times lower than that of praziquantel; the treatment index (TI) is 36.34, which is 12.78 times of that of praziquantel; the drug efficacy is significantly improved by using the pharmacophore fusion strategy, the application advantage of reducing parasite drug resistance is achieved, the toxicity to the host is reduced, the safety factor is high, and the application value in actual production is high.

[0020] (2) The compound developed in the application has simple synthesis method, is suitable for industrialized production, and has good commercialization prospect. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Figure 3 is a graph showing survival of Trichinella spiralis after 48 hours of treatment with different concentrations of PC-6 in Example 3. DETAILED DESCRIPTION

[0022] The application will be further described in conjunction with specific examples so that those skilled in the art will understand.

[0023] Example 1 The chemical structure of the piperazine derivative PC-6 is as follows: .

[0024] The specific steps of the preparation method of the piperazine derivative PC-6 are as follows: (1) Synthesis of 10-amino piperazine: Step a (synthesis of 10-nitro piperazine): Take piperazine 3.12 g (10 mM) in a 100 mL round-bottom flask, add 20 mL of concentrated sulfuric acid, stir to dissolve, and cool to 0°C with an ice bath, then slowly add 1.8 mL (40 mM) of concentrated nitric acid solution, and keep the reaction at 0°C for 8 h. After the reaction is completed, pour the reaction liquid into 200 mL of ice water, add saturated sodium carbonate solution to adjust the pH to 7, then extract with dichloromethane (30 mL x 3), dry the combined organic layers with anhydrous sodium sulfate overnight. Distill under reduced pressure, and purify the residue by silica gel column chromatography (ethyl acetate: petroleum ether = 2:1) to obtain 1.6 g of light yellow solid, which is 10-nitro piperazine, with a yield of 45%.

[0025] Figure 2 shows the spectrum characterization 1 H NMR (600 MHz, Chloroform-d) δ 8.15 – 8.14 (m, 1H), 8.05 –8.03 (m, 1H), 7.33 (d, J = 8.4 Hz, 1H), 5.20 – 5.18 (m, 1H), 4.84 (d, J = 4.0Hz, 1H), 4.44 (s, 1H), 4.04 (d, J = 7.2 Hz, 1H), 3.01 (d, J = 5.2 Hz, 1H),2.88 (d, J = 5.1 Hz, 2H), 2.43 (tt, J = 11.7, 3.3 Hz, 2H), 1.98 (s, 2H), 1.77(d, J = 6.3 Hz, 2H), 1.50 (d, J = 9.5 Hz, 1H), 1.24 – 1.20 (m, 4H). 13C NMR (151 MHz, Chloroform-d) δ 174.88, 171.16, 164.42, 146.93, 142.45, 134.53, 130.46, 122.39, 120.91, 60.37, 54.69, 48.99, 44.69, 40.70, 38.53, 29.18, 25.62, 21.01, 14.16. ESI-MS m / z: 380.16 [M+Na] + ; the identification data show that the obtained compound is indeed the compound of interest.

[0026] Step b (synthesis of 10-amino-pyrantel): nitro-pyrantel (4.5 mM) was weighed into a 50 mL flask, dissolved in 15 mL of methanol, and SnCl2•2H2O 1.01 g (45 mM) dissolved in hydrochloric acid was added dropwise to the reaction solution. After stirring and heating to reflux for 6 h, the methanol was removed under reduced pressure, water was added for dissolution, the pH was adjusted to 8 with saturated sodium carbonate solution, and ethyl acetate (20 mL x 3) was used for extraction. The organic layers were combined and dried over anhydrous sodium sulfate. After distillation under reduced pressure, the residue was separated by column chromatography with an elution system of ethyl acetate: petroleum ether = 2:1, and 1.18 g of white solid was obtained, which was 10-amino-pyrantel, with a yield of 80%.

[0027] Figure characterization 1 H NMR (600 MHz, Chloroform-d) δ 7.02 – 6.93 (m, 1H), 6.61(s, 1H), 5.10 – 5.04 (m, 1H), 4.79 – 4.76 (m, 1H), 4.68 (dd, J = 10.7, 4.2Hz, 1H), 4.45 (d, J = 17.4 Hz, 1H), 4.09 (d, J = 10.0 Hz, 1H), 2.82 – 2.76(m, 2H), 2.67 – 2.63 (m, 1H), 2.45 (tt, J = 11.9, 3.3 Hz, 1H), 2.04 (s, 1H),1.79 – 1.72 (m, 3H), 1.56 – 1.49 (m, 2H), 1.38 – 1.28 (m, 2H), 1.28 – 1.20(m, 5H), 0.90 – 0.81 (m, 1H). 13C NMR (151 MHz, Chloroform-d) δ 174.93, 164.50, 145.01, 133.62, 130.24, 124.82, 115.08, 111.89, 60.53, 55.11, 49.14, 45.39, 40.91, 39.58, 29.37, 29.12, 27.98, 25.83, 14.32. ESI-MS m / z: 328.20[M+H] + ; the identification data indicate that the resulting 10-amino pamaquine is indeed the compound of interest.

[0028] (2) Synthesis of 7-(6-bromohexyloxy)coumarin: Step c: Compound 7-hydroxycoumarin (6.2 mmol, 1.00 g) was dissolved in 30 mL of acetone, anhydrous potassium carbonate (10 mmol, 1.38 g) was added, stirred at room temperature for 30 min, then 1,6-dibromohexane was slowly added. After heating at 70 °C for 24 h, the reaction solution was removed by evaporation under reduced pressure to remove acetone, 250 mL of deionized water was added to the round-bottom flask, the mixture was extracted with 150 mL of dichloromethane for 3 times, the organic phase was combined and dried with anhydrous sodium sulfate overnight. The solid was removed by filtration, the liquid was removed by evaporation under reduced pressure, and the residue was purified by silica gel column chromatography (elution system: petroleum ether: ethyl acetate = 1:5) to obtain white solid, which was 7-(6-bromohexyloxy)coumarin, with a yield of about 50%.

[0029] Figure characterization 1 H NMR (600 MHz, Chloroform-d) δ 7.62 (d, J = 9.4 Hz, 1H), 7.35 (d, J = 8.6 Hz, 1H), 6.82 (dd, J = 8.6, 2.4 Hz, 1H), 6.78 (d, J = 2.3 Hz, 1H), 6.23 (d, J = 9.4 Hz, 1H), 4.01 (t, J = 6.4 Hz, 2H), 3.42 (t, J = 6.8 Hz, 2H), 1.86 (dp, J = 40.9, 6.6 Hz, 4H), 1.51 (dq, J = 6.6, 3.3 Hz, 4H). 13CNMR (151 MHz, CDCl3) δ 162.42, 161.37, 156.02, 143.57, 128.85, 113.08,113.05, 112.55, 101.43, 68.48, 33.83, 32.70, 28.91, 27.95, 25.31. ESI-MS m / z:347.02[M+Na] + ; the identification data show that the obtained 7-(6-bromohexyloxy)coumarin is the desired compound.

[0030] 3) Synthesis of the praziquantel derivative PC-6: Dissolve 10-amino praziquantel (6 mmol) in 30 mL of acetonitrile, add 1.38 g of anhydrous potassium carbonate, stir at room temperature for 30 min, then add 7-(6-bromohexyloxy)coumarin (3 mmol), heat to reflux at 85°C for 24 h, filter the reaction liquid acetonitrile, add 100 mL of deionized water to a round-bottom flask, extract the mixture with 50 mL of dichloromethane 4 times, combine the organic phases, dry with anhydrous sodium sulfate overnight, filter to remove the solid, and remove the organic phase under reduced pressure. The residue is purified by silica gel column chromatography (elution system: petroleum ether: ethyl acetate = 1:4); a yellow waxy compound is obtained, which is the praziquantel derivative PC-6; the yield is about 45%.

[0031] Characterization of the spectrum: 1 H NMR (600 MHz, Chloroform-d) δ 7.62 (s, 1H), 7.35 (s, 1H),6.96 (d, J = 8.2 Hz, 1H), 6.81 (d, J = 2.4 Hz, 1H), 6.51 – 6.47 (m, 1H), 6.24(d, J = 7.0 Hz, 1H), 5.13 – 5.09 (m, 1H), 4.79 (d, J = 3.8 Hz, 1H), 4.44 (s,1H), 4.12 (q, J = 7.1 Hz, 1H), 4.01 (s, 1H), 3.10 (t, J = 7.1 Hz, 2H), 2.82(d, J = 11.2 Hz, 2H), 2.68 – 2.62 (m, 2H), 2.50 – 2.43 (m, 2H), 2.04 (s, 2H),1.82 (s, 2H), 1.64 – 1.63 (m, 4H), 1.41 (s, 1H). 13C NMR (151 MHz, CDCl3) δ 174.95, 164.46, 162.47, 161.40, 161.37, 156.04, 156.02, 143.58, 133.54, 130.12, 128.85, 113.07, 108.82, 101.46, 68.57, 60.50, 55.26, 53.55, 49.12, 45.47, 44.16, 40.89, 39.61, 29.80, 29.48, 29.37, 29.03, 27.92, 26.95, 25.93, 25.82, 14.54, 14.30. ESI-MS m / z: 572.31[M+H] + ; the identification data showed that the obtained praziquantel derivative PC-6 was indeed the target compound.

[0032] Example 2 Evaluation of in vivo insecticidal activity of praziquantel derivative PC-6 (1) Drug preparation: PC-6 was dissolved in DMSO to form a 20 μΜ stock solution. The stock solution was stored in a 4 °C refrigerator and used according to different dosages by diluting with DMSO before use.

[0033] (2) Healthy and strong-swimming goldfish were randomly selected from the fish tank infected with C. sinensis and anesthetized with 50 mg / L ethyl aminobenzoate mesylate. The number of C. sinensis on the tail fin was counted under a stereomicroscope, and the fish with an average of 40-60 C. sinensis were selected for in vivo efficacy evaluation test. The test was carried out in a fish tank with a volume of 2.0 L. 1.0 L of dechlorinated and well-aerated tap water and different volumes of PC-6 stock solution were added to the fish tank to make the drug concentration of the experimental group 0.5, 1, 2, 4 and 8 μΜ, respectively. After stirring evenly and standing for 10-15 min, 3 C. sinensis-infected goldfish were added to each fish tank, and the experiment was repeated 3 times. The blank control group was added with 0.2% DMSO to eliminate the influence of the solvent on the survival rate of C. sinensis. The water temperature was maintained at 21±0.5 °C during the test. The number of C. sinensis on the tail fin of each goldfish was counted before and 48 h after drug administration, and the insecticidal rate of PC-6 at different concentrations was calculated according to the above method.

[0034] Insecticidal rate (%) = (average number of C. sinensis before drug administration-average number of C. sinensis after drug administration) / average number of C. sinensis before drug administration x 100.

[0035] As Figure 1The results show that PC-6 can kill 100% of Trichinella spiralis at a concentration of 8 μM for 48 h, and the 48 h median effective concentration (EC50) is 1.94 μM. The drug efficacy is increased by 3.99 times compared with that of piperazine.

[0036] Example 3 Acute toxicity test of PC-6 on zebrafish (1) Drug preparation: the method is the same as that in Example 2; (2) The acute toxicity of piperazine derivatives on zebrafish was determined by 96 h static water test method. The test was carried out in several water tanks with a volume of 2.0 L. 1 L of aerated tap water was added to each water tank, and different volumes of mother liquor were added. A glass rod was used to stir uniformly, and 30 healthy zebrafish were placed in each tank. The test lasted for 96 h, and no feeding was carried out during the test. The poisoning or death of zebrafish was observed and recorded in time, and the dead zebrafish was fished out in time to avoid pollution of water quality.

[0037] The poisoning criteria are floating head, sinking bottom, lateral swimming or slow reaction when knocking, etc.; and the death criteria are gill cover stop moving or no stress response when the tail handle part of zebrafish is lightly tapped with a glass rod or other blunt objects.

[0038] The number of surviving zebrafish after 96 h of each treatment group was counted, and the mortality of goldfish was calculated by the following formula: Mortality (%) = (number of zebrafish released - number of surviving zebrafish) / number of zebrafish released x 100.

[0039] Table 1. 96 h acute toxicity of PC-6 on zebrafish As shown in Table 1, the 96 h median lethal concentration (LC 50 ) of PC-6 on zebrafish is 70.49 μM, which is 3.20 times lower than that of piperazine. The treatment index TI is 36.34, which is 12.78 times that of piperazine.

[0040] The other parts not specifically described are prior art. Although the above examples have made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained under the premise of no creativity according to the present embodiments, which all belong to the protection scope of the present application.

Claims

1. A praziquantel derivative PC-6, characterized in that: The PC-6 structure is as follows: 。 2. A method for preparing the praziquantel derivative PC-6 according to claim 1, characterized in that, The synthesis circuit is as follows: The specific steps are as follows: (1) Preparation of 10-aminopraziquantel; (2) Preparation of 7-(6-bromohexyloxy)coumarin; (3) Dissolve the above 10-aminopyridine in acetonitrile, add anhydrous potassium carbonate and stir at room temperature, then add 7-(6-bromohexyloxy)coumarin prepared in step (2), heat and reflux, then filter, extract, dry and separate by column chromatography to obtain pyridine derivative PC-6.

3. The preparation method according to claim 2, characterized in that: The 10-aminopraziquantel is prepared by the following steps: a. Dissolve praziquantel in concentrated sulfuric acid with stirring, then slowly add concentrated nitric acid after an ice bath to react and obtain a reaction solution; b. Pour the reaction solution into ice water, add saturated sodium carbonate solution to adjust the pH to 7, extract with dichloromethane, combine the organic layers and dry with anhydrous sodium sulfate overnight; distill under reduced pressure and purify to obtain 10-nitropraziquantel; c. Dissolve 10-nitropyraquinone in methanol to obtain a reaction mixture. Add SnCl2•2H2O dissolved in hydrochloric acid dropwise to the reaction mixture, stir and heat under reflux, remove methanol under reduced pressure, dissolve in water, adjust pH to 8 with saturated sodium carbonate solution, extract, combine organic layers and dry with anhydrous sodium sulfate, distill under reduced pressure and purify to obtain 10-aminopyraquinone.

4. The preparation method according to claim 2, characterized in that: The 7-(6-bromohexyloxy)coumarin was prepared by the following steps: 7-hydroxycoumarin was dissolved in acetone, anhydrous potassium carbonate was added, and the mixture was stirred at room temperature for 30 min. Then, 1,6-dibromohexane was slowly added dropwise. The mixture was heated under reflux at 70 °C for 24 h, and the acetone was removed by vacuum distillation. After adding deionized water, the mixture was extracted with dichloromethane. The organic phases were combined and dried overnight with anhydrous sodium sulfate. The solid was removed by filtration, and the organic phase of the liquid was removed by vacuum distillation. The purified liquid was 7-(6-bromohexoxy)coumarin.

5. The preparation method according to claim 2, characterized in that: In step (3), the heating reflux temperature is 85°C and the time is 24 hours.

6. The use of the praziquantel derivative PC-6 according to claim 1 in the treatment of monogenean trematodes.

7. The use of the praziquantel derivative PC-6 as described in claim 1 in a drug for the prevention and treatment of monogenean trematodes.

8. A drug for controlling monogenean trematodes, characterized in that: The drug contains a praziquantel derivative PC-6, wherein the content of the praziquantel derivative PC-6 is 1~8 μM.

9. The medicament according to claim 8, characterized in that: The drug contains 4-8 μM of the praziquantel derivative PC-6.

10. The medicament according to claim 8 or 9, characterized in that: The drug contains 8 μM of the praziquantel derivative PC-6.

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