CN113234089B

CN113234089B - Limonin compound, preparation method and application thereof as medicine for treating echinococcosis

Info

Publication number: CN113234089B
Application number: CN202110500091.5A
Authority: CN
Inventors: 王海; 李曹龙; 夏元铮
Original assignee: China Pharmaceutical University; Nanjing Medical University
Current assignee: China Pharmaceutical University; Nanjing Medical University
Priority date: 2021-05-08
Filing date: 2021-05-08
Publication date: 2022-07-15
Anticipated expiration: 2041-05-08
Also published as: WO2022237913A1; CN113234089A

Abstract

The limonin compounds shown in general formulas I, II and III can be efficiently prepared at normal temperature and normal pressure by adopting hydrazone forming reaction or alkali catalysis reaction, the preparation process is simple, the limonin compounds have excellent curative effect on echinococcosis treatment, the echinococcosis resistant inhibition rate reaches 95%, damaged liver cells can be repaired at the same time, the liver protection effect can be achieved, and the condition of liver and kidney injury caused by medicine application is avoided.

Description

Limonin compound, preparation method and application of limonin compound as medicine for treating echinococcus

Technical Field

The invention relates to the field of medicinal chemistry, in particular to a limonin compound, a preparation method and application thereof as a medicament for treating echinococcus.

Background

Echinococcosis, also known as echinococcosis, is a serious zoonosis caused by the parasitism of echinococcus larvae (echinococcosis) in human and animal bodies, is distributed worldwide, and is a high-incidence area in China. Echinococcus granulosus, Echinococcus multilocularis and Echinococcus shikoensis are prevalent in China, and among them, echinococcus granulosus larvae cause echinococcus cysticercosis. CE can cause death of human and livestock and cause direct economic loss of over 30 million yuan per year in animal husbandry in China. The CE epidemic areas in China are mainly distributed in vast pasturing areas and farming and pasturing areas in the north and the northwest, and in recent years, the disease has a tendency of spreading from pasturing areas to farming areas. The current treatment means comprises two aspects of surgical treatment and drug treatment, wherein the surgical treatment is often accompanied with the risk of relapse because of the existence of factors for insufficient removal of the small cysts, and the leakage of cyst fluid during puncture causes the sacs or the head nodes to pollute the abdominal cavity, or secondary hydatid cysts formed by rupture of the cysts in the surgical process. In the aspect of drug treatment, internationally adopted treatment drugs are praziquantel and benzimidazole compounds, albendazole is one of representative drugs, the drug has obvious advantages in the aspect of treating echinococcus granulosus and alveolar echinococcus, the survival time and the survival quality of patients are improved, but clinical studies show that the cure rate of the drugs is only 30%, the symptoms of the drugs are improved to a certain extent, the rate of the patients accounts for 30% -50%, and the problems of large individual difference, unstable curative effect and the like exist. Clinical studies show that the albendazole has serious side effects such as encephalitis syndrome, acute liver injury, hemolytic anemia, anaphylactic shock, renal failure and even death, and the like, and in addition, the albendazole mainly has a pest inhibiting effect without a pest killing effect in the process of treating echinococcus granulosus patients. Albendazole needs to be taken for a long time, which increases the economic burden on patients and even the whole society to some extent.

The limonin compounds have typical chemical structures, namely all compounds contain 4,4, 8-trimethyl-17-furan steroid skeletons or are derived from the steroid skeletons, are extracted and separated from natural plants, are plant secondary substances with special structures and functions, are four-carbon reduction products of triterpenoids, are mainly prepared in rutaceae plants and meliaceae plants, and all naturally-produced limonins are connected with a furan ring at a D ring C-17 position and are connected with oxygen-containing functional groups at C-3, C-4, C-7, C-16 and C-17 positions.

Disclosure of Invention

The invention aims to: aiming at the problems that the medicine for treating echinococcosis in the prior art has no insecticidal effect, needs to be taken for a long time and has great toxic and side effects, the invention provides a limonin compound, and also provides a preparation method of the limonin compound and application of the limonin compound as a medicine for treating echinococcosis.

The technical scheme is as follows: the structural general formula of the limonin compound is as follows:

wherein X represents: -O-, -CH₂-，Bond

R₁Represents: C1-C6 alkyl,

R₂Represents ortho-para-substituted alkyl of all C1-C4, -X, -CX₃、-OCH₃、-NH₂、-NO₂、

-OH。

R₃All alkyl groups representing C1-C2 ═ CH₂。

The preparation method of the limonin compound with the general formula I comprises the following steps:

wherein, when the X substituent in the limonin compound of the general formula I is-O-, Bond-CH respectively₂When the compound is IV, V and VI; in the process of preparing the compound V from the compound IV, the reaction reagent is hydroiodic acid; the reaction solvent is acetic acid. In the process of preparing the compound VI from the compound V, cuprous chloride is used as a catalyst, and diiodomethane and zinc powder are stirred in ether for 48 hours at the reaction temperature of 40 ℃.

The preparation method of the limonin compound with the general formula II comprises the following steps:

the limonin compound of the general formula II is prepared from the limonin compound of the general formula I, the reaction reagent is hydrazine corresponding to alkyl or acyl, the reaction solvent is corresponding methanol or ethanol, acetic acid is used as a catalyst, and the reaction is carried out for 48 hours at 70 ℃.

The preparation method of the limonin compound with the general formula III comprises the following steps:

the limonin compound of the general formula III is prepared from the limonin compound of the general formula I, reaction reagents are corresponding iodoalkane and pyrrolidine, solvents are ethanol and benzene, the reaction temperature is 20 ℃, and the reaction time is 3 hours.

The invention also discloses application of the limonin compound as a medicine for treating echinococcus.

Preferably, the pharmaceutical composition also comprises a pharmaceutically acceptable salt formed by the limonin compound and an acid and a pharmaceutically acceptable carrier, wherein the acid is one or more of hydrogen chloride, hydrogen bromide, sulfuric acid, carbonic acid, citric acid, succinic acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid or ferulic acid.

Has the advantages that: compared with the prior art, the invention has the following remarkable advantages:

the limonin compound prepared by the invention is used for treating echinococcosis, can efficiently resist echinococcosis, and has an inhibition rate of 95 percent, which is far higher than the inhibition rate of 77.4 percent of the existing commonly used albendazole; meanwhile, the liver protection effect is achieved, liver and kidney injury caused by medicines used in the prior art is avoided, damaged liver cells can be repaired, and the echinococcosis treatment effect is excellent.

Drawings

FIG. 1: eosin staining pattern of echinococcus granulosus metacercaria incubated with limonoid and albendazole, which are positive drugs, for 3 days in vitro;

FIG. 2 is a schematic diagram: eosin staining pattern of limonin compound and albendazole positive by incubating echinococcus granulosus metacercaria for 5 days in vitro;

FIG. 3: a histogram of in vitro inhibition results of the limonin compounds and albendazole on echinococcosis granulosa;

FIG. 4: the result of H.E staining of mouse liver tissue after the intervention of limonin and albendazole and the infection of echinococcus granulosus metacercaria;

FIG. 5: the limonin and albendazole intervene in the H.E staining result of the mouse kidney tissue after the echinococcus granulosus metacercaria is infected;

FIG. 6: the result of H.E staining of mouse cyst tissue after the intervention of limonin and albendazole and the infection of echinococcus granulosus metacercaria;

FIG. 7 is the results of limonin and albendazole intervention in cysts after echinococcus granulosus metacercaria infection;

FIG. 8 is a histogram of the wet weight of the bursa of each group after 3 weeks of drug treatment.

Detailed Description

The technical scheme of the invention is further explained by combining the attached drawings.

Example 1

Preparation of Compound II-1

Taking a compound limonin as a starting material, adding 0.4725g of limonin, 20ml of methanol, 0.200ml of glacial acetic acid and 0.2148g of thiophene formylhydrazine in a 50ml eggplant-shaped bottle in sequence, and stirring for 48h at 70 ℃. TLC detection till the reaction is complete, cooling and decompressing to evaporate the solvent. Crude product is extracted with dichloromethane: column chromatography on methanol (100:1) gave II-1 as a white solid, 0.2507g, 42% yield. The molecular formula is: c₃₁H₃₄N₂O₈S，HR-ESUMS m/z：595.2113[M+H]⁺，¹H NMR(300MHz,DMSO-d6)δ10.93(s,1H),7.93(s,1H),7.85(d,J＝5.0Hz,1H),7.75–7.73(m,1H),7.68(t,J＝1.7Hz,1H),7.20(dd,J＝5.0,3.7Hz,1H),6.51(dd,J＝2.0,0.8Hz,1H),5.47(s,1H),4.84(s,1H),4.42(d,J＝12.9Hz,1H),4.10(d,J＝3.6Hz,1H),4.02(s,1H),2.81(d,J＝16.4Hz,1H),2.62(dd,J＝16.4,3.9Hz,1H),2.42(d,J＝15.3Hz,1H),2.25–2.17(m,1H),1.84(d,J＝43.4Hz,4H),1.22(s,3H),1.18(s,3H),1.04(s,3H)。

Preparation of Compound II-2

Taking compound limonin as a starting material, sequentially adding 0.4041g of limonin, 10ml of methanol, 0.100ml of glacial acetic acid and p-methoxy methyl ether into a 50ml eggplant-shaped bottle0.2144g of benzoyl hydrazine is stirred for 48h at 70 ℃. TLC detection till the reaction is complete, cooling, and distilling off the solvent under reduced pressure. The crude product is extracted by dichloromethane: column chromatography on methanol (120:1) gave II-2 as a white solid, 0.3879g, 73% yield. The molecular formula is: c₃₄H₃₈N₂O₉，HR-ESUMS m/z：641.2482[M+Na]⁺，¹H NMR(300MHz,DMSO-d6)δ10.83(s,1H),7.77(d,J＝8.0Hz,2H),7.74(s,1H),7.68(t,J＝1.7Hz,1H),7.31(d,J＝7.9Hz,2H),6.51(s,1H),5.45(s,1H),4.84(d,J＝13.0Hz,1H),4.43(d,J＝12.8Hz,1H),4.09(s,2H),2.82(d,J＝16.5Hz,1H),2.62(dd,J＝16.4,4.0Hz,1H),2.38(s,3H),2.18(d,J＝14.2Hz,1H),1.77(s,4H),1.23(d,J＝3.6Hz,4H),1.17(s,3H),1.03(s,3H),0.99(s,3H)。

Preparation of Compound II-3

The compound limonin is taken as a starting material, 50ml of eggplant-shaped bottles are sequentially added with the limonin, 0.4730g, 20ml of methanol, 0.200ml of glacial acetic acid and 0.2300g of 3-hydroxybenzoyl hydrazine, and stirred for 48h at 70 ℃. TLC detection till the reaction is complete, cooling and decompressing to evaporate the solvent. Crude product is extracted with dichloromethane: column chromatography with methanol (80:1) gave II-3 as a white solid, 0.3042g, 50% yield. The molecular formula is: c₃₃H₃₆N₂O₉，HR-ESUMS m/z：627.2319[M+Na]⁺，¹H NMR(300MHz,DMSO-d6)δ10.81(s,1H),9.74(s,1H),7.74(d,J＝1.4Hz,1H),7.68(t,J＝1.7Hz,1H),7.29(d,J＝5.5Hz,2H),7.22(s,1H),6.97–6.92(m,1H),6.55–6.49(m,1H),5.45(s,1H),4.84(d,J＝13.0Hz,1H),4.43(d,J＝13.0Hz,1H),4.12–4.09(m,2H),3.18(s,1H),3.16(s,1H),2.82(d,J＝16.4Hz,1H),2.62(dd,J＝16.4,3.9Hz,1H),2.40(d,J＝14.6Hz,1H),2.18(d,J＝14.5Hz,1H),1.77(s,4H),1.20(d,J＝14.8Hz,6H),1.01(d,J＝13.7Hz,6H)。

Preparation of Compound II-4

The compound limonin is used as a starting material, 50ml of eggplant-shaped bottles are sequentially added with the limonin, 0.4729g, 20ml of methanol, 0.200ml of glacial acetic acid and 0.2304g of 4-hydroxybenzoyl hydrazine, and stirred for 48h at 70 ℃. TLC detection till the reaction is complete, cooling and decompressing to evaporate the solvent. Crude product is extracted with dichloromethane: column chromatography on methanol (100:1) gave II-4 as a white solid, 0.2193g, 36% yield. The molecular formula is:C₃₃H₃₆N₂O₉，HR-ESUMS m/z：627.2322[M+Na]⁺，¹H NMR(300MHz,DMSO-d6)δ10.65(s,1H),10.07(s,1H),7.76(s,1H),7.74(d,J＝1.6Hz,2H),7.68(t,J＝1.7Hz,1H),6.85–6.81(m,2H),6.51(dd,J＝2.0,0.8Hz,1H),5.45(s,1H),4.83(d,J＝13.0Hz,1H),4.42(d,J＝12.9Hz,1H),4.09(s,2H),2.82(d,J＝16.3Hz,1H),2.62(dd,J＝16.5,4.0Hz,1H),2.38(d,J＝14.7Hz,1H),2.22–2.14(m,1H),1.76(s,4H),1.23(d,J＝6.8Hz,5H),1.17(s,3H),1.03(s,3H),0.99(s,3H)。

preparation of Compound II-5

The compound limonin is used as a starting material, 50ml of eggplant-shaped bottles are sequentially added with the limonin, 0.4694g, 20ml of methanol, 0.200ml of glacial acetic acid and 0.2046g of benzoyl hydrazine, and the mixture is stirred for 48 hours at 70 ℃. TLC detection till the reaction is complete, cooling, and distilling off the solvent under reduced pressure. The crude product is extracted by dichloromethane: column chromatography on methanol (100:1) gave II-5 as a white solid, 0.2760g, 47% yield. The molecular formula is: c₃₃H₃₆N₂O₈，HR-ESUMS m/z：589.2541[M+H]⁺，¹H NMR(300MHz,DMSO-d6)δ10.92(s,1H),7.86(d,J＝7.4Hz,2H),7.74(s,1H),7.68(t,J＝1.7Hz,1H),7.61–7.55(m,1H),7.51(t,J＝7.3Hz,2H),6.52(s,1H),5.46(s,1H),4.85(d,J＝13.0Hz,1H),4.43(d,J＝13.0Hz,1H),4.10(d,J＝5.0Hz,2H),2.95(d,J＝14.2Hz,1H),2.82(d,J＝16.4Hz,1H),2.63(dd,J＝16.5,4.0Hz,1H),2.42(d,J＝14.8Hz,1H),2.19(d,J＝14.6Hz,1H),1.77(s,3H),1.25(d,J＝4.6Hz,3H),1.18(s,3H),1.04(s,3H),1.00(s,3H)。

Preparation of Compound II-6

The compound limonin is used as a starting material, 50ml of eggplant-shaped bottles are sequentially added with the limonin, 0.4707g, 20ml of methanol, 0.200ml of glacial acetic acid and 0.2258g of p-methyl benzoyl hydrazine, and the mixture is stirred for 48 hours at 70 ℃. TLC detection till the reaction is complete, cooling, and distilling off the solvent under reduced pressure. The crude product is extracted by dichloromethane: column chromatography with methanol (100:1) gave II-6 as a white solid, 0.3087g, 51% yield. The molecular formula is: c₃₄H₃₈N₂O₈，HR-ESUMS m/z：625.2526[M+Na]⁺，¹H NMR(300MHz,DMSO-d6)δ10.77(s,1H),7.85(d,J＝8.4Hz,2H),7.74(d,J＝1.5Hz,1H),7.68(t,J＝1.8Hz,1H),7.04(d,J＝8.7Hz,2H),6.51(d,J＝1.6Hz,1H),5.45(s,1H),4.84(d,J＝13.1Hz,1H),4.43(d,J＝13.0Hz,1H),4.09(s,2H),3.83(s,3H),2.62(dd,J＝16.6,4.0Hz,1H),2.40(d,J＝14.9Hz,1H),2.18(d,J＝14.6Hz,1H),1.76(s,4H),1.22(s,3H),1.17(s,3H),1.03(s,3H),0.99(s,3H)。

Preparation of Compound II-7

The compound limonin is used as a starting material, 50ml of eggplant-shaped bottles are sequentially added with the limonin, 0.4704g, 20ml of methanol, 0.200ml of glacial acetic acid and 0.2268g of 4-carbamyl hydrazine, and stirred for 48h at 70 ℃. TLC detection till the reaction is complete, cooling, and distilling off the solvent under reduced pressure. The crude product is extracted by dichloromethane: column chromatography with methanol (100:1) gave II-7 as a white solid, 0.3526g, 58% yield. The molecular formula is: c₃₃H₃₇N₃O₈，HR-ESUMS m/z：626.2480[M+Na]⁺，¹H NMR(300MHz,DMSO-d6)δ10.41(s,1H),7.75–7.72(m,1H),7.68(t,J＝1.7Hz,1H),7.61(d,J＝8.6Hz,2H),6.57(d,J＝8.6Hz,2H),6.51(dd,J＝2.0,0.8Hz,1H),5.73(s,2H),5.45(s,1H),4.82(d,J＝13.0Hz,1H),4.42(d,J＝12.9Hz,1H),4.13(s,1H),3.17(d,J＝5.2Hz,0H),2.91(d,J＝14.4Hz,1H),2.81(d,J＝16.4Hz,1H),2.62(dd,J＝16.5,4.0Hz,1H),2.36(d,J＝14.7Hz,1H),2.17(d,J＝14.5Hz,1H),1.91(s,1H),1.75(d,J＝9.0Hz,3H),1.22(s,3H),1.17(s,3H),1.03(s,3H),0.98(s,3H)。

Preparation of Compound II-8

The compound limonin is used as a starting material, 50ml of eggplant-shaped bottles are sequentially added with the limonin, 0.4032g, 20ml of methanol, 0.200ml of glacial acetic acid and 0.2204g of 4-chlorobenzoyl hydrazine, and the mixture is stirred for 48 hours at 70 ℃. TLC detection till the reaction is complete, cooling and decompressing to evaporate the solvent. The crude product is extracted by dichloromethane: column chromatography with methanol (100:1) gave II-8 as a white solid, 0.2721g, 51% yield. The molecular formula is: c₃₃H₃₅ClN₂O₈，HR-ESUMS m/z：645.1985[M+Na]⁺，¹H NMR(300MHz,DMSO-d6)δ10.99(s,1H),7.89(d,J＝8.2Hz,2H),7.73(s,1H),7.67(t,J＝1.7Hz,1H),7.58(d,J＝8.1Hz,2H),6.51(s,1H),5.45(s,1H),4.85(d,J＝13.0Hz,1H),4.42(d,J＝13.0Hz,1H),4.09(s,2H),2.87(d,J＝15.3Hz,0H),2.76(d,J＝16.2Hz,1H),2.63(dd,J＝16.5,4.0Hz,1H),2.41(d,J＝14.9Hz,1H),2.18(d,J＝14.2Hz,1H),1.77(s,4H),1.24(d,J＝3.6Hz,4H),1.17(s,3H),1.04(s,3H),0.99(s,3H)。

Preparation of Compound II-9

The compound limonin is taken as a starting material, 50ml of eggplant-shaped bottles are sequentially added with the limonin, 0.4011g, 20ml of methanol, 0.200ml of glacial acetic acid and 0.2147g of 3-chlorobenzoyl hydrazine, and stirred for 48h at 70 ℃. TLC detection till the reaction is complete, cooling and decompressing to evaporate the solvent. The crude product is extracted by dichloromethane: column chromatography with methanol (100:1) gave II-9 as a white solid, 0.2853g, 54% yield. The molecular formula is: c₃₃H₃₅ClN₂O₈，HR-ESUMS m/z：645.2021[M+Na]⁺，¹H NMR (300MHz, DMSO-d6) δ 11.03(s,1H),7.90(s,1H),7.83(d, J ═ 7.4Hz,1H),7.74(s,1H),7.68(t, J ═ 1.7Hz,1H),7.64(s,1H),7.55(t, J ═ 7.8Hz,1H),6.51(s,1H),5.45(s,1H),4.85(d, J ═ 12.9Hz,1H),4.43(d, J ═ 12.9Hz,1H),4.09(s,2H),2.87(d, J ═ 15.0Hz,0H),2.76(d, J ═ 16.7Hz,1H),2.63(dd 16.5, J ═ 0, 1H),2.42(d, J ═ 15.0Hz,0H),2.76(d, J ═ 16.7Hz,1H),2.63(d, J ═ 16.5, 1H), 14.42 (H), 14.14H, 3(s,1H), 3.3H), 3(s,1H), 3.3.7 (s,1H), 1H, 3.3.3 (d, 1H), 1H), 3.3.3.3 (d, 1H), 1H. The molecular formula is:

preparation of Compound II-10

The compound limonin is taken as a starting material, 50ml of eggplant-shaped bottles are sequentially added with the limonin, 0.4695g, 20ml of methanol, 0.200ml of glacial acetic acid and 0.2079g of 2-pyrazine formylhydrazine, and the mixture is stirred for 48h at 70 ℃. TLC detection till the reaction is complete, cooling and decompressing to evaporate the solvent. The crude product is extracted by dichloromethane: column chromatography with methanol (100:1) gave II-3 as a white solid, 0.1375g, 23% yield. The molecular formula is: c₃₁H₃₄N₄O₈，HR-ESUMS m/z：613.2276[M+Na]⁺，¹H NMR(300MHz,DMSO-d6)δ11.25(s,1H),9.20(d,J＝1.5Hz,1H),8.91(d,J＝2.5Hz,1H),8.79(dd,J＝2.5,1.5Hz,1H),7.75(s,1H),7.68(t,J＝1.8Hz,1H),6.52(d,J＝1.3Hz,1H),5.47(s,1H),4.84(d,J＝12.9Hz,1H),4.44(d,J＝13.4Hz,1H),4.11(d,J＝6.1Hz,2H),3.13(t,J＝15.1Hz,0H),2.91(d,J＝14.6Hz,1H),2.82(d,J＝16.3Hz,1H),2.65(d,J＝3.9Hz,1H),2.60(d,J＝3.1Hz,1H),2.45(s,0H),2.31(d,J＝3.1Hz,0H),2.26(d,J＝3.3Hz,0H),1.24(s,4H),1.18(d,J＝5.0Hz,4H),1.11(s,1H),1.05–0.99(m,7H)。

Preparation of Compound II-11

The compound limonin is used as a starting material, 50ml of eggplant-shaped bottles are sequentially added with the limonin, 0.4033g, 20ml of methanol, 0.200ml of glacial acetic acid and 0.1712g of isoniazide, and the mixture is stirred for 48 hours at 70 ℃. TLC detection till the reaction is complete, cooling and decompressing to evaporate the solvent. The crude product is extracted by dichloromethane: column chromatography on methanol (100:1) gave II-11 as a white solid, 0.1310g, 30% yield. The molecular formula is: c₃₂H₃₅N₃O₈，HR-ESUMS m/z：598.2541[M+H]⁺,1H NMR(400MHz,DMSO-d6)δ11.09(s,1H),9.01(s,1H),8.21(d,J＝8.0Hz,1H),7.74(s,1H),7.68(t,J＝1.8Hz,1H),7.56(d,J＝7.1Hz,1H),6.52(s,1H),5.46(s,1H),4.85(d,J＝13.0Hz,1H),4.43(d,J＝13.0Hz,1H),4.13–4.07(m,2H),2.99(d,J＝14.6Hz,1H),2.82(d,J＝16.4Hz,1H),2.63(dd,J＝16.4,4.0Hz,1H),2.43(d,J＝14.9Hz,1H),2.18(d,J＝14.1Hz,1H),1.77(s,3H),1.21(d,J＝21.3Hz,9H),1.05(s,3H),1.00(s,4H)。

Preparation of Compound II-12

The compound limonin is used as a starting material, 50ml of eggplant-shaped bottles are sequentially added with the limonin, 0.4061g, 20ml of methanol, 0.200ml of glacial acetic acid and 0.1702g of 3-pyridine formylhydrazine, and stirred for 48h at 70 ℃. TLC detection till the reaction is complete, cooling, and distilling off the solvent under reduced pressure. Crude product is extracted with dichloromethane: column chromatography on methanol (100:1) gave II-12 as a white solid, 0.1128g, 30% yield. The molecular formula is: c₃₂H₃₅N₃O₈，HR-ESUMS m/z：598.2541[M+H]⁺，¹H NMR(400MHz,DMSO-d6)δ11.08(s,1H),9.01(s,1H),8.74(d,J＝4.8Hz,1H),8.20(d,J＝7.9Hz,1H),7.73(s,1H),7.67(t,J＝1.8Hz,1H),7.55(d,J＝7.0Hz,1H),6.51(s,1H),5.45(s,1H),4.84(d,J＝13.1Hz,1H),4.42(d,J＝13.0Hz,1H),4.17–4.00(m,2H),2.96(s,0H),2.81(d,J＝16.4Hz,1H),2.62(dd,J＝16.4,4.0Hz,1H),2.50(p,J＝1.9Hz,9H),2.19(s,1H),1.76(s,3H),1.38–1.29(m,2H),1.24(d,J＝6.2Hz,3H),1.18(s,5H),1.04(s,3H),0.99(s,4H)。

Preparation of Compound II-13

The compound limonin is used as the starting material, and lemon is added into a 50ml eggplant-shaped bottle in sequenceLimonin, 0.4717g, methanol 20ml, glacial acetic acid 0.200ml, 4-trifluoromethyl benzoyl hydrazine 0.3062g, stirring 48h at 70 ℃. TLC detection till the reaction is complete, cooling, and distilling off the solvent under reduced pressure. The crude product is extracted by dichloromethane: column chromatography with methanol (100:1) gave II-13 as a white solid, 0.1908g, 30% yield. The molecular formula is: c₃₄H₃₅F₃N₂O₈，HR-ESUMS m/z：657.1222[M+H]⁺，¹H NMR(300MHz,DMSO-d₆)δ11.14(s,1H),8.06(d,J＝8.0Hz,2H),7.90(d,J＝8.1Hz,2H),7.74(s,1H),7.67(t,J＝1.7Hz,1H),6.52(s,1H),5.46(s,1H),4.86(d,J＝13.0Hz,1H),4.43(d,J＝13.0Hz,1H),4.10(s,2H),2.94(d,J＝14.4Hz,1H),2.82(d,J＝16.4Hz,1H),2.66(d,J＝4.0Hz,1H),2.60(d,J＝3.9Hz,0H),2.19(d,J＝14.6Hz,1H),1.78(s,4H),1.24(s,4H),1.18(s,3H),1.04(s,4H),1.02–0.99(m,3H)。

Example 2

Taking 0.1g of the compound prepared in the example 1, 0.5g of starch, 0.05g of talcum powder and a proper amount of CMC-Na as a wetting agent, granulating and tabletting.

Example 3

The application of the limonin compound as a medicine for treating echinococcus:

the experimental contents are as follows:

1.1 medicinal intervention for in vitro culturing Echinococcus granulosus metacercaria

Separating echinococcus granulosus metacercaria in vitro, carrying out aseptic culture in a 24-well plate, adding limonin and limonin derivatives with different concentrations, intervening by positive medicament albendazole, and dividing into a solvent group (0.1% DMSO control group), a limonin group (80 mu M), an obacunone group (80 mu M), a derivative group (80 mu M) and an albendazole group (40 mu M) for intervening for 0 day, 3 days and 5 days respectively, observing the drug action change of the echinococcus granulosus, and observing the metacercaria activity by using eosin. The results of the experiments are shown in FIG. 1 and FIG. 2, which are eosin staining graphs of echinococcus granulosus metacercaria incubated with the positive drug albendazole for 3 days and 5 days in vitro, respectively, wherein a: blank group, b: albendazole, c: limonin, d: compound II-5, e: compound II-10, f: the compound II-13 can see that the metacercaria treated by the limonin and the derivatives thereof has expanded volume, eversion of head nodes and abnormal overall shape, the in vitro inhibition rate after treatment is shown in figure 3 after eosin dyeing, the in vitro anti-echinococcosis activity of the limonin is superior to that of the albendazole treatment group, the synthesized part of the limonin derivatives presents a metering dependence relation on the anti-echinococcosis activity, and the experimental result shows that the limonin and the derivatives thereof have treatment effect on echinococcosis in vitro.

1.2 pharmacological intervention in vivo on Echinococcus granulosus infected mice

1.2.1 model inoculation

(1) Carefully treating hydatid cyst of a patient infected with echinococcosis with 75% ethanol, transferring into a biological safety cabinet, carefully absorbing cyst fluid into a sterile beaker by using a 50ml sterile injector, standing until protocephalic segments naturally settle, discarding supernatant, washing three times by using sterile PBS (phosphate buffer solution), adjusting the density of protocephalic segments to 10000 protocephalic segments/ml, performing intraperitoneal injection on 0.2ml protocephalic segment suspension in each KM mouse, infecting for 150 days, randomly taking the mouse, dissecting, and judging whether molding is successful or not.

(2) Pharmacodynamic tests are carried out on mice which are successfully modeled, the experiments are divided into a blank group, a positive drug group (albendazole group) and a treatment group (limonin group), and the three groups are total, and the administration dosage is 50 mg/kg/day and 15 days.

The specific implementation is as follows: the albendazole group and the limonin group were administered orally in a volume of 0.2ml, with the drug being 50 mg/kg/day per mouse daily intake, after grinding in a mortar with 0.4% CMC-Na. After continuously taking the medicine for 15 days, the mice are euthanized, cysts are collected from the abdominal cavity, the damp weight of the cysts is measured, the inhibition rate of the medicine to the cysts is calculated, the inhibition rate is (blank group cyst wet weight-treatment group cyst wet weight)/blank group cyst wet weight is 100%, the weight and the damp weight of the cysts of the mice are recorded, the liver, the kidney and the cysts are taken to be pathological sections, and the conditions of the change of the kidney and the liver, the thickness of the cyst wall, the thickness of the germinal layer and the like are observed. Finally, statistical analysis is carried out by using a sps 20.0, data are expressed by mean values +/-standard deviation, One-way ANOVA analysis is adopted among groups, and the p value is smaller than 0.05 and has statistical difference.

(3) The liver, kidney and cyst of each group of mice were fixed with 4% paraformaldehyde, embedded, sectioned, and observed for each experimental group, liver, kidney and cyst after H & E staining.

As shown in fig. 4, wherein a: a normal group; b: a model group; c: albendazole treatment group; d: limonin treatment group; the limonin group has normal liver group morphology, liver tissue cell parts of albendazole lose characteristic cell structures, and the limonin has certain liver protection effect and can repair damaged liver cells, and the albendazole has liver damage. As shown in fig. 5, wherein a: a normal group; b: a model group; c: albendazole treatment group; d: the cell parts of the kidney tissues of the limonin group and the albendazole lose characteristic cell structures and have certain damage.

As shown in fig. 6, the results of h.e staining of mouse cyst tissue after the intervention of limonin and albendazole with echinococcus granulosus metacercaria infection are shown, where a: model group, b: albendazole treatment group, c: in the limonin treatment group, the echinococcus granulosus tissues treated by the limonin are obviously changed, the horny layer is thinned or disappears, a large amount of large-area vacuoles appear in the whole outer membrane, the structure is seriously changed, and the germinal layer cells are thinned or disappear; therefore, the limonin compound can effectively treat echinococcosis and has excellent insecticidal effect

TABLE 1 cyst Wet weight and inhibition in groups of mice after 3 weeks of treatment

As shown in fig. 7, fig. 8 and table 1, the wet weight of the cysts of the mice in each group after 3 weeks of treatment was significantly reduced compared to the model group (4.1955 ± 2.7995g) in the albendazole group (0.9480 ± 0.5558g) and the limonin group (0.2037 ± 0.2259g), and the cysts of the mice in vivo almost disappeared (p ═ 0.017 and p ═ 0.03), and compared to the limonin group and the positive albendazole group, the therapeutic effect of the limonin was significantly higher than that of the albendazole group (p ═ 0.021), and the inhibition rate reached 95%.

The limonin and the derivatives thereof are novel natural compounds which can be used for developing anti-echinococcosis drugs, in-vivo pharmacodynamic data suggest that the limonin is a relatively efficient anti-echinococcosis drug molecule, and key members of the signal path can also be used as targets for screening novel echinococcosis drugs. The invention can provide a new way for treating echinococcosis.

Claims

1. The application of limonin compounds in preparing medicines for treating echinococcosis is disclosed, wherein the limonin compounds are represented by the general formula I or II:

wherein X is-O-, -CH₂One of the bonds;

R₁is C1-C6 alkyl,

One of (1);

R₂is ortho-, meta-or para-substituted C1-C4 alkyl, -OCH₃、-NH₂、-NO₂One of-Ph and-OH.

2. The use of claim 1, wherein the medicament further comprises a pharmaceutically acceptable carrier for the limonoid.