Anti-hydatid disease drug, preparation method and medical use thereof

Abstract

The present invention relates to the field of pharmaceutical chemistry, and relates to an anti-echinococcosis drug, a preparation method thereof, and medical uses thereof, and particularly relates to a β-carboline anti-echinococcosis compound, a preparation method thereof, the compound, and the medical use of a pharmaceutical composition containing the compound in preparing a drug for preventing or treating echinococcosis.

Description

Technical Field

[0001] The present invention relates to the field of medicinal chemistry, and specifically to a series of β-carboline derivatives, their preparation methods, and medical uses. The present invention modifies β-carboline at different sites to design and synthesize a series of β-carboline analogs, as well as the medical uses of these compounds, particularly their use in preparing drugs for preventing or treating echinococcosis. Background Art

[0002] Hydatid disease, also known as echinococcosis, is caused by infection with adult or juvenile worms of the tapeworm Echinococcus and is a zoonosis affecting numerous hosts (Lancet, 2003, 362:1295-304). In my country, there are two main types: cystic echinococcosis (CE), caused by the tapeworm Echinococcus granulosus (Eg), and alveolar echinococcosis (AE), caused by Echinococcus multilocularis. The disease is predominant in areas with developed livestock farming and is a global public health concern (Int. J. Infec. Des. 2019, 79, 89). In my country, cystic echinococcosis is mainly distributed in the northwest and southwest regions, with the Qinghai-Tibet Plateau being the most prevalent area of ​​the disease (Adv. Parasitol. 2017, 95, 315-493). To date, surgical treatment is the preferred treatment for echinococcosis, but this method has certain risks and requires high medical conditions and doctor skills (Acta Trop. 2020, 203: 105-283). Therefore, drug therapy as an auxiliary treatment method has attracted more and more attention.

[0003] Peganum harmala L. is a perennial herbaceous plant in the family Tribulus, genus Peganum, primarily distributed in North China and Northwest my country (Food CH&Em. Toxicol. 2010, 48(3), 839-845; Food CH&Em. Toxicol. 2017, 103, 261-269; Toxins (Basel). 2015, 7(11), 4507-4518). β-Carboline is a three-membered ring alkaloid found in the seeds of Peganum harmala L., a member of the family Harmaceae. It is a benzindole-like alkaloid that inhibits topoisomerase, cyclin-dependent kinase, and DNA synthesis, and can intercalate into DNA, thus exhibiting a wide range of biological activities. Studies have shown that the β-carboline derivative harmine (HM) has multiple pharmacological effects, including antiparasitic (TetraH&Edron Lett. 2010, 51(4), 583-585; J. Drug Target. 2004, 12(3), 165-175), bactericidal (Fitoterapia. 2010, 81(7), 779-782), antitumor (Oncol. Rep. 2017, 38(5), 2927-2934; Phytomedicine. 2017, 28, 10-18; Altern. Med. 2017, 1-7), and antidepressant (Prog. Neuropsychopharmacol. Biol. Psychiatry. 2017, 79(Pt B), 258-267; Brain Res. Bull. 2018, 137, 294-300). The research team found that HM has good anti-echinococcosis activity and that its combination with dehydrogenated harmaline and albendazole (ABZ), a commonly used anti-echinococcosis drug, can exert a synergistic effect. However, HM has certain neurotoxicity, causing vomiting, tremors, hallucinations, and even death in patients, thus limiting its clinical application and development.

[0004]

[0005] Based on this, the research team modified the structure of five structural sites, including β-carboline ring 1, 2, 3, 7, and 9, around the HM mother core, aiming to improve the activity of treating cystic echinococcosis while reducing its neurotoxicity (patent number: CN105998014B). The team's previous research found that HM derivatives DH-330 and DH-004 have good efficacy against CE, and their LC 50 The values ​​were 41.55±9.48μM and 47.77±18.99μM, respectively, which were better than HM(LC 50=250.39±92.11μM, Patent No.: CN105998014A). However, at a low dose of 25mg / kg, DH-330 only inhibited the growth of Echinococcus granulosus in mice by 32.91%. As we all know, the treatment of echinococcosis is a long-term medication process. Therefore, further research is needed to discover more effective derivatives to further reduce the dosage or shorten the medication course, improve patient compliance, and reduce the occurrence of adverse reactions. Summary of the Invention

[0006] Based on the above background, the present invention modifies different sites of β-carboline to design and synthesize a class of anti-echinococcosis compounds that have a killing effect on Echinococcus granulosus. The present invention also provides a preparation method of the above-mentioned compounds, as well as pharmaceutical compositions containing these compounds, pharmaceutically acceptable salts thereof, and medical uses thereof.

[0007] Technical solution:

[0008] In a first aspect, the present invention discloses a compound represented by Formula I or II, or a pharmaceutically acceptable salt thereof:

[0009]

[0010] Among them, R 1 Selected from or -CH3;

[0011] R 2 Selected from

[0012] R 3 Selected from

[0013] R 4 Selected from

[0014] In some embodiments, the compound represented by Formula I is selected from: 1a: 4-((4-hydroxypiperidin-1-yl)methyl)-N-(1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl)benzamide

[0015]

[0016] 1b: N-(1-(3-methoxyphenyl)-9H-pyridin[3,4-b]indol-3-yl)-4-((4-methyl-1,4-diazepin-1-yl)methyl)benzamide

[0017]

[0018] 1c: 4-([1,4'-bipiperidinyl]-1'-ylmethyl)-N-(1-(3,4-dimethoxyphenyl)-9H-pyridinyl[3,4-b]indol-3-yl)benzamide

[0019]

[0020] 1d: N-(1-(3,4-dimethoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl)-4-((4-methyl-1,4-diazepin-1-yl)methyl)benzamide

[0021]

[0022] 1e: 4-([1,4'-bipiperidinyl]-1'-ylmethyl)-N-(1-methyl-9H-pyridinyl[3,4-b]indol-3-yl)benzamide

[0023]

[0024] 1f: 4-([1,4'-bipiperidinyl]-1'-ylmethyl)-N-(1-(p-tolyl)-9H-pyridinyl[3,4-b]indol-3-yl)benzamide

[0025]

[0026] 1g: 4-((4-methyl-1,4-diazepin-1-yl)methyl)-N-(1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl)benzamide

[0027]

[0028] In some embodiments, the compound represented by Formula II is selected from: 2a: N-(4-([1,4'-bipiperidinyl]-1'-ylmethyl)phenyl)-1-(4-methoxyphenyl)-9H-pyridinyl[3,4-b]indole-3-carboxamide

[0029]

[0030] 2b: N-(4-([1,4'-bipiperidinyl]-1'-ylmethyl)phenyl)-1-(3-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxamide

[0031]

[0032] 2c: N-(4-([1,4'-bipiperidinyl]-1'-ylmethyl)phenyl)-1-(3,4-dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxamide

[0033]

[0034] 2d: N-(4-([1,4'-bipiperidinyl]-1'-ylmethyl)phenyl)-1-(2-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxamide

[0035]

[0036] 2e: N-(4-([1,4'-bipiperidinyl]-1'-ylmethyl)phenyl)-1-(2,3-dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxamide

[0037]

[0038] 2f: N-(4-([1,4'-bipiperidinyl]-1'-ylmethyl)phenyl)-1-(2,4-dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxamide

[0039]

[0040] 2g: N-(4-([1,4'-bipiperidinyl]-1'-ylmethyl)phenyl)-1-(3,5-dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxamide

[0041]

[0042] In a second aspect, the present invention also provides a method for preparing the compound.

[0043] The preparation method of compound I comprises:

[0044] Compound 1-1R 1 CHO and L-tryptophan undergo a Pictet-Spengler reaction under acidic conditions to obtain intermediate 1-2;

[0045] The carboxyl group of intermediate 1-2 is converted into methyl ester to give intermediate 1-3;

[0046] Intermediate 1-3 is oxidized to obtain intermediate 1-4; intermediate 1-4 is converted to intermediate 1-5 after treatment with hydrazine hydrate; the hydrazine group of intermediate 1-5 is converted to an azide group in the presence of NaNO2 to generate intermediate 1-6; intermediate 1-6 is converted to an amino group by Curtis rearrangement to generate intermediate 1-7; intermediate 1-7 reacts with p-chloromethylbenzoyl chloride to generate intermediate 1-8; intermediate 1-8 reacts with compound 1-9R 2 H undergoes nucleophilic substitution reaction to obtain target compound Ⅰ;

[0047]

[0048] In some embodiments, the compound of formula I can be prepared by the following method: L-tryptophan is used as a raw material and compound 1-1 (corresponding aldehyde R 1 CHO) undergoes a Pictet-Spengler reaction to produce intermediate 1-2, which is then converted to the methyl ester intermediate 1-3. Intermediate 1-3 is oxidized by KMnO4 in DMF to produce intermediate 1-4, which is converted to intermediate 1-5 after treatment with hydrazine hydrate. The hydrazine group of 1-5 is converted to an azide group in the presence of NaNO2 to produce 1-6, which is converted to an amino group in the presence of HAc in H2O via Curtis rearrangement to produce intermediate 1-7. 1-7 reacts with p-chloromethylbenzoyl chloride in DCM in the presence of TEA to produce intermediate 1-8. In the presence of K2CO3 and KI, intermediate 1-8 reacts with compound 1-9 (different secondary amines R 2 H) Nucleophilic substitution reaction occurs in acetonitrile to form the target compound I.

[0049] In some embodiments, the synthetic route of Compound I includes:

[0050]

[0051] The preparation method of compound II comprises:

[0052] Compound 2-1R 3 CHO and L-tryptophan undergo Pictet-Spengler reaction under acidic conditions to obtain intermediate 2-2.

[0053] Intermediate 2-2 is oxidized to give intermediate 2-3;

[0054] 4-Nitrobenzyl bromide and compound 2-4R 4 H undergoes a nucleophilic substitution reaction to give intermediate 2-5, and the nitro group of intermediate 2-5 is reduced to an amino group to give intermediate 2-6;

[0055] Intermediate 2-3 and intermediate 2-6 undergo amide condensation reaction to generate target compound II;

[0056]

[0057] In some embodiments, the compound represented by formula II can be prepared by the following method: L-tryptophan is used as a raw material and compound 2-1 (corresponding aldehyde R 3 CHO) was subjected to Pictet-Spengler reaction to generate 2-2. Intermediate 2-2 was oxidized by KMnO4 in DMF to obtain intermediate 2-3. In the presence of K2CO3 and KI, 4-nitrobenzyl bromide was reacted with compound 2-4R in acetonitrile. 4The H secondary amine undergoes nucleophilic substitution to give intermediate 2-5, which is then reduced with Pd / C and H2 in CH3OH to give intermediate 2-6. In the presence of EDCI and DMAP, intermediate 2-3 reacts with intermediate 2-6 in DCM via amide condensation to produce the target compound II.

[0058] In some embodiments, the synthetic route of Compound II includes:

[0059]

[0060] In a third aspect, the present invention further provides a pharmaceutical composition comprising a therapeutically effective amount of the compound or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

[0061] The dosage forms of the pharmaceutical compositions of the present invention can be prepared by those skilled in the art according to conventional methods in the pharmaceutical field. For example, the active ingredient can be mixed with one or more carriers (also known as excipients) and then formulated into the desired dosage form, including tablets, capsules, granules, and aerosols. Intravenous injection or intravenous lyophilized formulations can also be prepared according to conventional injection production methods.

[0062] In a fourth aspect, the present invention also provides the use of the compound and pharmaceutical composition in the preparation of a drug for preventing or treating echinococcosis.

[0063] Beneficial effects: The compounds of the present invention have the following excellent properties: (1) The compounds are highly stable. (2) The compounds are readily available for synthesis. (3) Compounds 1a, 1c, and 1e can effectively inhibit the growth of Echinococcus granulosus. (4) Compound 1a inhibits the growth of Echinococcus granulosus in a concentration-dependent manner and can completely inhibit the growth of Echinococcus granulosus even at low concentrations. (5) Compound 1a can inhibit the growth of Echinococcus granulosus and is superior to the positive drug albendazole (ABZ) and compound DH-003. (6) Compared with dehydrogenated echinococcus, compound 1a has a good safety profile.

[0064] Experimental validation revealed that the compounds of this invention exhibited the following excellent properties: 1a was shown to be the optimal compound. At an initial concentration of 1 μM, derivative 1a was found to cause over 50% death of Echinococcus granulosus prosegments (PSCs) after two days of treatment, in a dose-dependent manner. Furthermore, 1a exhibited significant morphological changes compared to the ABZ and HM control groups. Finally, in experiments involving derivatives treating Echinococcus granulosus prosegments, 1a was found to cause prosegmental damage and loss of viability. In this study, the in vitro activity of β-carboline derivative 1a was superior to that of the active agent albendazole (ABZ) and the lead compound dehydrogenated harmine (HM). Subsequently, 1a was subjected to in vivo pharmacodynamic studies. Results demonstrated that mice treated with 1a showed a statistically significant reduction in cyst weight compared to the model control group, and after 28 days of treatment, this reduction was also statistically significant compared to the ABZ and HM treatment groups. After 14 days of treatment, the wet weight reduction rate of cysts in the low-dose 1a group (12.5 mg / kg) was comparable to that in the high-dose ABZ and HM groups (50 mg / kg). After 28 days of treatment, the wet weight reduction rate of cysts in the 1a group reached a maximum of 76.87%, which was 1.39 times and 1.35 times that of the ABZ and HM groups at the same dose, respectively. Therefore, 1a has a very significant anti-hydatid effect. Overall, our study shows that 1a is a very promising candidate for anti-hydatid drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 Schematic diagram of the results of a study on the lethality of compounds 1a-1g and 2a-2g against Echinococcus granulosus PSCs in vitro.

[0066] Figure 2 This is a schematic diagram of the in vitro study results on the anti-Echinococcus granulosus PSCs of compounds 1a-1g and 2a-2g. It was found that compounds 1a, 1c, and 1e could significantly inhibit PSCs and change their morphology in a dose-dependent manner.

[0067] Figure 3 This is a schematic diagram of the results of pharmacokinetic studies on compounds 1a, 1c, and 1e.

[0068] Figure 4 This is a schematic diagram of the in vivo therapeutic results of compound 1a on mice infected with Echinococcus granulosus after 14 days and 30 days of intervention.

[0069] Figure 5 This is a schematic diagram of the results of observing the effect of compound 1a on the hydatid vesicle structure in mice at the ultramicroscopic level using transmission electron microscopy.

[0070] Figure 6 This is a schematic diagram of the research results on the toxicity of compound 1a on the brain tissue and hippocampal neuronal cell structure of mice infected with Echinococcus granulosus. DETAILED DESCRIPTION

[0071] The following are specific implementation methods shown in the form of examples to further illustrate the content of the present invention in detail.

[0072] 1. Synthesis of intermediates

[0073] Example 1: The synthesis steps of intermediate 1-2 are as follows: the raw material L-tryptophan (10.20 g, 50 mmol, 1.0 eq) is dissolved in 80 ml of CH3COOH solution, and compound 1-1R is added 1 CHO (55 mmol, 1.1 eq) was added and stirred at 90°C for 3 hours. After the reaction was completed, the reaction solution was adjusted to pH 5-6 with 2M NaOH solution to precipitate a large amount of yellow solid, which was filtered and dried in vacuo to obtain a yellow solid, namely intermediate 1-2.

[0074] Example 2: The synthesis steps of intermediate 1-3 are as follows: intermediate 1-2 (50 mmol, 1.0 eq) is dissolved in 140 ml of methanol, and SOCl2 (17.85 g, 150 mmol, 3.0 eq) is slowly added dropwise under vigorous stirring at -5°C. After 0.5 hour, the reaction solution is heated to reflux for 4 hours. After the reaction is completed, the solvent is removed by concentration under reduced pressure. An appropriate amount of water is added to the concentrate to dissolve it, and the pH is adjusted to 8 with 1 M NaOH solution to precipitate a yellow solid. The filter cake is dried under reduced pressure to obtain a yellow solid, namely intermediate 1-3.

[0075] Example 3: The synthesis steps of intermediate 1-4 are as follows: intermediate 1-3 (50 mmol, 1.0 eq) is dissolved in 150 ml of DMF solvent, and KMnO4 (11.05 g, 70 mmol, 1.4 eq) is added in batches under mechanical stirring at -5°C. Stirring is continued for 1 hour. After the reaction is completed, the reaction solution is filtered, and the filtrate is added to 200 ml of cold water to precipitate. A light brown solid, namely intermediate 1-4, is obtained by filtration.

[0076] Example 4: Synthesis of Intermediate 1-5 Intermediate 1-4 (50 mmol, 1.0 eq) was dissolved in 130 ml of methanol, and 88.20 ml of 85% hydrazine hydrate (75.00 g, 1500 mmol, 30.0 eq) was added thereto. The mixture was heated under reflux for 4 hours. After the reaction was completed, the mixture was cooled to 0° C. and filtered to obtain a light brown solid. A large amount of cold water was then added to the filtrate to continue to precipitate solids. The solids were filtered and the filter cake was dried under reduced pressure to obtain the product, Intermediate 1-5.

[0077] Example 5: The synthesis steps of intermediate 1-6 are as follows: intermediate 1-5 (50 mmol, 1.0 eq) is dissolved in 100 ml of 2M HCl solution, and then NaNO2 (10.35 g, 150 mmol, 3.0 eq) is dissolved in 80 ml of H2O. Under mechanical stirring at -5°C, the NaNO2 solution is slowly dripped into the 1-5 solution. After continuing stirring for 1 hour, the pH of the reaction solution is adjusted to 8 with 1M NaOH solution, the precipitated solid is filtered, and dried to obtain a light yellow solid, namely intermediate 1-6.

[0078] Example 6: The synthesis steps of intermediate 1-7 are as follows: intermediate 1-6 (50 mmol, 1.0 eq) is dissolved in a mixture of 150 ml of H2O and glacial acetic acid (1:1), heated under reflux at 90°C for 5 hours, and after completion of the reaction, the solvent is removed by concentration under reduced pressure. The crude product is purified by column chromatography to obtain a light yellow solid, namely intermediate 1-7.

[0079] Example 7: The synthesis of intermediate 1-8 is as follows: intermediate 1-7 (2 mmol, 1.0 eq) was dissolved in 5 ml of anhydrous dichloromethane, triethylamine (0.81 g, 8 mmol, 4.0 eq) was added, and p-chloromethylbenzoyl chloride (0.38 g, 2 mmol, 1.0 eq) was dissolved in 3 ml of anhydrous dichloromethane. The mixture was slowly added dropwise to the 1-7 solution under a -5°C ice bath. After 0.5 hours, the reaction was completed. The dichloromethane solvent in the reaction solution was removed by concentration under reduced pressure, 100 mL of water was added, and the mixture was extracted with ethyl acetate (3×20 mL). The organic phases were combined, washed with clean water (30 mL×2), washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a light yellow solid, namely intermediate 1-8.

[0080] Example 8: The synthesis steps of intermediate 2-2 were as described in Example 1, using raw materials L-tryptophan and compound 2-1R 3 CHO.

[0081] Example 9: The synthesis steps of intermediate 2-3 refer to Example 3.

[0082] Example 10: The synthesis steps of intermediate 2-5 are as follows: 4-nitrobenzyl bromide (1.0 eq) is dissolved in anhydrous acetonitrile, and compound 2-4R is added. 4 The reaction mixture was stirred at room temperature for 5 h. After completion of the reaction, the solvent was removed by concentration under reduced pressure. Water and ethyl acetate were added to the residue for extraction. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a yellow solid, Intermediate 2-5.

[0083] Example 11: Intermediate 2-6 was synthesized by dissolving Intermediate 2-5 (1.0 eq) in a mixture of ethanol and water (approximately 1.7:1), adding iron powder (5.0 eq) and ammonium chloride (10.0 eq), and reacting at 85°C for 2 hours. After completion of the reaction, the ethanol was removed by concentration under reduced pressure. The aqueous phase was extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography to obtain Intermediate 2-6.

[0084] 2. Synthesis of target compounds

[0085] Example 12: The synthesis of target compound 1a was carried out by dissolving 4-(chloromethyl)-N-(1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl)benzamide (1 mmol, 0.44 g) in acetonitrile (8 ml), followed by the addition of 4-hydroxypiperidine (1.5 mmol, 0.15 g), potassium carbonate (2.0 mmol, 0.28 g), and potassium iodide (0.1 mmol, 0.02 g). The mixture was allowed to react overnight at room temperature. After completion of the reaction, the acetonitrile was removed by concentration under reduced pressure, and the crude product was purified by column chromatography (dichloromethane:methanol = 100:1) to obtain 0.48 g of a light yellow solid in a yield of 94%. 1 H NMR (DMSO-d6, 300 MHz): 1 H NMR (400MHz, DMSO) δ11.4(s,1H,NH),10.5(s,1H,NH),8.7(s,1H,Ar-H),8.2(d,J=7.9Hz,1H,Ar-H ),8.0(d,J=8.0Hz,4H,Ar-H),7.6(d,J=8.3Hz,1H,Ar-H),7.5(m,1H,Ar-H),7.4(d,J=7.8Hz,2H,A r-H),7.2(m,1H,Ar-H),7.1(d,J=8.4Hz,2H,Ar-H),4.6(s,1H,OH),3.8(s,3H,OCH3),3.5(s,2H,C H2),2.9(m,2H,CH2),2.4(s,1H,CH),2.0(s,2H,CH2),1.7(s,2H,CH2),1.4(d,J=11.0Hz,2H,CH2). 13 C NMR (101MHz, DMSO) δ165.7,160.2,143.3,142.6,140.1,131.7,130.9,130.6,130.3,129.2,1 28.7,128.3,122.0,121.5,119.8,114.5,112.9,105.0,55.8,55.4,51.2,45.9.HRMS(ESI)m / z calcd for C31 H 31 N4O3,507.2396; found,507.2239[M+H] + .

[0086] Example 13: The synthesis of target compound 1b was performed by dissolving 4-(chloromethyl)-N-(1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl)benzamide (1 mmol, 0.44 g) in acetonitrile (8 ml). N-methylhomopiperazine (1.5 mmol, 0.17 g), potassium carbonate (2.0 mmol, 0.28 g), and potassium iodide (0.1 mmol, 0.02 g) were then added sequentially. The mixture was allowed to react overnight at room temperature. After completion of the reaction, the acetonitrile was removed by concentration under reduced pressure, and the crude product was purified by column chromatography (dichloromethane:methanol = 100:1) to obtain 0.49 g of a light yellow solid in a yield of 94%. 1 H NMR(400MHz,DMSO-d6)δ11.42(s,1H,NH),10.55(s,1H,NH),8.68(s,1H,Ar-H),8.17( d,J=7.9Hz,1H,Ar-H),8.01(m,4H,Ar-H),7.59(d,J=8.2Hz,1H,Ar-H),7.51–7.43(m,3 H,Ar-H),7.18(m,1H,Ar-H),7.11(d,J=8.3Hz,2H,Ar-H),3.81(s,3H,OCH3),3.69(s,2 H,CH2),3.24–3.12(m,6H,CH2),2.97(m,2H,CH2),2.79(m,3H,CH3),1.93(m,2H,CH2). 13 C NMR (101MHz, DMSO) δ165.6,160.2,143.3,142.6,140.1,133.9,131.7,130.9,130.3,128.9,128.4,122.0,12 1.5,119.8,114.5,113.0,105.0,61.4,56.4,55.8,54.7,53.8,49.5,45.7,44.0,23.9.MS(ESI)m / z:574[M+H] + .HRMS(ESI)m / z calcd forC 32 H 34 N5O2,520.2712; found,520.2705[M+H] + .

[0087] Example 14: The synthesis of target compound 1c was performed by dissolving 4-(chloromethyl)-N-(1-(3,4-dimethoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl)benzamide (1 mmol, 0.47 g) in acetonitrile (8 ml). 4-piperidinylpiperidine (1.5 mmol, 0.25 g), potassium carbonate (2.0 mmol, 0.28 g), and potassium iodide (0.1 mmol, 0.02 g) were then added sequentially. The mixture was allowed to react overnight at room temperature. After completion of the reaction, the acetonitrile was removed by concentration under reduced pressure, and the crude product was purified by column chromatography (dichloromethane:methanol = 100:1) to obtain 0.56 g of a light yellow solid in a 93% yield. 1 H NMR (400MHz, DMSO-d6) δ11.46(s,1H,NH),10.60(s,1H,NH),8.75(s,1H,NH),8.24(d,J=7.9Hz,1H,Ar-H),8.08(d, J=8.0Hz,2H,Ar-H),7.65(d,J=8.4Hz,3H,Ar-H),7.56(t,J=7.6Hz,1H,Ar-H),7.46(d,J=8.0Hz,2H,Ar-H),7.35(s, 1H, Ar-H), 7.25 (t, J = 7.4Hz, 1H, Ar-H), 7.20 (d, J = 8.1Hz, 1H, Ar-H), 3.90 (d, J = 13.7Hz, 6H, OCH3), 3.58 (s, 2H), 2. 98–2.90(m,4H,CH2),2.00(d,J=11.8Hz,4H,CH2),1.78–1.63(m,7H,CH2),1.52(s,1H,CH),1.26–1.16(m,3H,CH2). 13 C NMR(101MHz,DMSO-d6)δ170.4,154.6,154.0,148.0,147.3,145.1,138.6,136.4,135.7,135.5,133.9,133.5,133.1,132.1,126 .3,126.2,124.6,117.7,117.3,116.9,67.7,66.2,60.9,60.7,56.8,54.2,50.7,34.2,31.0,28.2,27.1.MS(ESI)m / z:604[M+H] + .HRMS(ESI)m / z calcd for C 37 H 42 N5O3,604.3287; found,604.3279[M+H] + .

[0088] Example 15: The synthesis of target compound 1d was as follows: 4-(chloromethyl)-N-(1-(3,4-dimethoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl)benzamide (1 mmol, 0.47 g) was dissolved in acetonitrile (8 ml). N-methylhomopiperazine (1.5 mmol, 0.17 g), potassium carbonate (2.0 mmol, 0.28 g), and potassium iodide (0.1 mmol, 0.02 g) were added sequentially. The mixture was allowed to react overnight at room temperature. After completion of the reaction, the acetonitrile was removed by concentration under reduced pressure, and the crude product was purified by column chromatography (dichloromethane:methanol = 100:1) to obtain 0.52 g of a light yellow solid in a 95% yield. 1 H NMR(400MHz,DMSO-d6)δ11.46(s,1H,NH),10.60(s,1H,NH),8.75(s,1H,NH),8.24(d,J=7.9Hz,1H,Ar-H),8.0 7(d,J=8.0Hz,2H,Ar-H),7.68–7.61(m,3H,Ar-H),7.55(t,J=7.6Hz,1H,Ar-H),7.50(d,J=7.9Hz,2H,Ar-H),7. 39–7.34(m,1H,Ar-H),7.25(t,J=7.5Hz,1H,Ar-H),7.20(d,J=8.1Hz,1H,Ar-H),3.90(d,J=13.8Hz,6H,OCH3) ,3.73(s,2H),2.98–2.88(m,6H,CH2),2.77–2.72(m,2H,CH2),2.70–2.62(m,3H,CH3),1.88–1.83(m,2H,CH2). 13 C NMR(101MHz,DMSO-d6)δ170.4,154.6,154.0,148.2,148.0,147.3,145.1,140.3,138.5,136.4,135.7,135.5,133.6,133.5, 133.1,132.2,124.6,117.7,117.3,116.9,66.5,62.0,61.9,60.9,60.7,60.5,58.8,58.7,50.3,30.5.MS(ESI)m / z:550[M+H] + .HRMS(ESI)m / z calcd for C 33 H 36 N5O3,550.2818; found,550.2811[M+H] + .

[0089] Example 16: The synthesis of target compound 1e was as follows: 4-chloromethyl-N-(1-methyl-9H-pyrido[3,4-b]indol-3-yl)benzamide (1 mmol, 0.35 g) was dissolved in acetonitrile (8 ml), followed by the addition of 4-piperidinylpiperidine (1.5 mmol, 0.25 g), potassium carbonate (2.0 mmol, 0.28 g), and potassium iodide (0.1 mmol, 0.02 g). The mixture was allowed to react overnight at room temperature. After completion of the reaction, the acetonitrile was removed by concentration under reduced pressure, and the crude product was purified by column chromatography (dichloromethane:methanol = 100:1) to afford 0.45 g of a pale yellow solid in a 94% yield. 1 H NMR (400MHz, DMSO-d6) δ11.54(s,1H,NH),10.52(s,1H,NH),8.67(s,1H,Ar-H),8.19(d,J=7.9H z,1H,Ar-H),8.06(d,J=8.0Hz,2H,Ar-H),7.62–7.49(m,2H,Ar-H),7.42(d,J=8.0Hz,2H,Ar-H) ,7.27–7.18(m,1H,Ar-H),3.52(s,2H,CH2),2.87(d,J=11.1Hz,2H,CH2),2.77(s,3H,CH3),2.6 1(s,3H,CH,CH2),1.98–1.91(m,2H,CH2),1.75(d,J=11.6Hz,2H,CH2),1.61–1.19(m,10H,CH2). 13 C NMR (101MHz, DMSO-d6) δ165.4,142.9,142.8,141.8,140.4,133.7,132.7,129.5,129.0,128.5,128.2,122. 2,121.7,119.6,112.4,103.9,63.5,62.6,62.0,53.0,50.0,31.4,27.5,25.6,20.4.MS(ESI)m / z:482[M+H] + .HRMS(ESI)m / z calcd for C 30 H 36 N5O,482.2920; found,482.2912[M+H] + .

[0090] Example 17: The synthesis of target compound 1f was performed by dissolving 4-chloromethyl-N-(1-(p-tolyl)-9H-pyrido[3,4-b]indol-3-yl)benzamide (1 mmol, 0.43 g) in acetonitrile (8 ml), followed by the addition of 4-piperidinylpiperidine (1.5 mmol, 0.25 g), potassium carbonate (2.0 mmol, 0.28 g), and potassium iodide (0.1 mmol, 0.02 g). The mixture was allowed to react overnight at room temperature. After completion of the reaction, the acetonitrile was removed by concentration under reduced pressure, and the crude product was purified by column chromatography (dichloromethane:methanol = 100:1) to obtain 0.54 g of a light yellow solid in a 97% yield. 1 H NMR (400MHz, DMSO-d6) δ11.41(s,1H,NH),10.57(s,1H,NH),8.76(s,1H,Ar-H),8.24(d,J=7.9Hz,1H,Ar-H),8.06( d,J=8.2Hz,2H,Ar-H),8.02–7.98(m,2H,Ar-H),7.63(d,J=8.2Hz,1H,Ar-H),7.57–7.52(m,1H,Ar-H),7.44(d,J=7 .8Hz,4H,Ar-H),7.25(t,J=7.5Hz,1H,Ar-H),3.53(s,2H,CH2),2.95(s,1H,CH),2.87(d,J=11.0Hz,2H,CH2),2.45 (s,3H,CH3),2.00–1.90(m,2H,CH2),1.71(d,J=11.4Hz,2H,CH2),1.54–1.36(m,8H,CH2),1.32–1.18(m,4H,CH2). 13 C NMR(101MHz,DMSO-d6)δ165.7,143.3,142.9,142.6,140.2,138.6,135.4,133.7,131.8,131.1,129.7,129.0,12 8.9,128.3,122.0,121.5,119.9,112.9,105.4,62.6,62.1,53.1,50.0,27.7,26.0,21.5.MS(ESI)m / z:558[M+H] + .HRMS(ESI)m / z calcd for C 36 H 40 N5O,558.3233; found,558.3223[M+H] + .

[0091] Example 18: The synthesis of the target compound 1g was performed by dissolving 4-chloromethyl-N-(1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl)benzamide (1 mmol, 0.50 g) in acetonitrile (8 ml). N-methylhomopiperazine (1.5 mmol, 0.17 g), potassium carbonate (2.0 mmol, 0.28 g), and potassium iodide (0.1 mmol, 0.02 g) were then added sequentially. The mixture was allowed to react overnight at room temperature. After completion of the reaction, the acetonitrile was removed by concentration under reduced pressure, and the crude product was purified by column chromatography (dichloromethane:methanol = 100:1) to obtain 0.54 g of a light yellow solid in a yield of 94%. 1 H NMR(400MHz,DMSO-d6)δ11.48(s,1H,NH),10.61(s,1H,NH),8.77(s,1H,Ar-H),8.25( d,J=7.9Hz,1H,Ar-H),8.09(d,J=7.8Hz,2H,Ar-H),7.68–7.45(m,4H,Ar-H),7.27(d, J=10.5Hz,3H,Ar-H),3.94(s,6H,OCH3),3.78(s,3H,OCH3),3.21(d,J=28.8Hz,4H,CH 2),2.84(s,3H,CH3),2.72(d,J=13.6Hz,4H,CH2),1.96(s,2H,CH2),1.23(s,2H,CH2). 13 C NMR (101MHz, DMSO-d6) δ165.7,153.5,143.5,143.3,142.6,140.4,138.4,133.7,133.6,131.7,131.1,128.8,128. 3,122.1,121.5,119.9,112.9,106.4,105.5,61.7,60.6,57.3,56.4,55.8,54.1,45.6,25.9.MS(ESI)m / z:580[M+H] + .HRMS(ESI)m / z calcd for C 34 H 38 N5O4,580.2924; found,580.2916[M+H] + .

[0092] Example 19: The synthesis of target compound 2a was performed by dissolving 1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxylic acid (1 mmol, 0.32 g) in anhydrous dichloromethane (10 ml), adding EDCI (2 mmol, 0.38 g) and DMAP (0.5 mmol, 0.06 g), and then adding 4-(1,4'-bipiperidinyl]-1'-ylmethyl)aniline (1 mmol, 0.27 g). The mixture was allowed to react overnight at room temperature. After completion of the reaction, the dichloromethane was removed by concentration under reduced pressure, and the crude product was purified by column chromatography to yield 0.45 g (92%) of a light yellow solid. 1 H NMR (400MHz, DMSO-d6) δ11.94(s,1H,NH),10.46(s,1H,NH),8.92(s,1H,Ar-H),8.45(d,J=8.1Hz,1H,Ar -H),8.21(d,J=8.3Hz,2H,Ar-H),7.87(d,J=8.1Hz,2H,Ar-H),7.73(d,J=8.3Hz,1H,Ar-H),7.62(t,J=7. 8Hz,1H,Ar-H),7.28(dd,J=38.3,8.3Hz,5H,Ar-H),3.91(s,3H,OCH3),2.99(t,J=15.2Hz,6H,CH2),1.9 9(t,J=12.4Hz,4H,CH2),1.71(d,J=21.9Hz,7H,CH2,CH),1.51(s,2H,CH2),1.21(d,J=18.6Hz,2H,CH2). 13 C NMR(101MHz,DMSO-d6)δ165.7,160.2,143.3,142.6,140.1,133.8,131.7,130.9,130.6,130.3,129.1,128.7,128.3,122 .0,121.5,119.8,114.5,112.9,105.0,73.0,63.6,62.0,55.8,55.0,52.8,45.9,30.9,29.5,22.6.MS(ESI)m / z:574[M+H] + .HRMS(ESI)m / z calcd forC 36 H 40 N5O2,574.3182; found,574.3171[M+H] + .

[0093] Example 20: The synthesis of target compound 2b was performed by dissolving 1-(3-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxylic acid (1 mmol, 0.32 g) in anhydrous dichloromethane (10 ml), adding EDCI (2 mmol, 0.38 g) and DMAP (0.5 mmol, 0.06 g), and then adding 4-(1,4'-bipiperidinyl]-1'-ylmethyl)aniline (1 mmol, 0.27 g). The mixture was allowed to react overnight at room temperature. After completion of the reaction, the dichloromethane was removed by concentration under reduced pressure, and the crude product was purified by column chromatography to yield 0.54 g (94%) of a light yellow solid. 1 H NMR (400MHz, DMSO-d6) δ11.96(s,1H,NH),10.45(s,1H,NH),8.98(s,1H,Ar-H),8.47(d,J=7.9Hz,1H,Ar-H),7.85(d,J=8.1H z,2H,Ar-H),7.79–7.71(m,3H,Ar-H),7.65–7.58(m,2H,Ar-H),7.34(t,J=7.5Hz,1H,Ar-H),7.29(d,J=8.1Hz,2H,Ar-H),7. 17(dd,J=8.2,2.6Hz,1H,Ar-H),3.95(s,3H,OCH3),3.41(s,2H,CH2),2.95(s,1H,CH),2.86(d,J=11.1Hz,2H,CH2),1.89(t, J=11.5Hz,3H,CH2),1.70(d,J=12.0Hz,2H,CH2),1.50(d,J=8.7Hz,7H,CH2),1.41–1.35(m,2H,CH2),1.27–1.19(m,2H,CH2). 13 C NMR(101MHz,DMSO-d6)δ163.7,160.1,142.2,141.2,139.8,139.2,137.8,135.0,134.1,130.6,130.5,129.7,129.2,122.6,121.6, 121.5,120.8,120.4,115.4,114.6,114.1,113.3,62.6,62.1,55.7,53.0,50.0,31.4,27.6,25.9,24.5,22.5.MS(ESI)m / z:574[M+H] + .HRMS(ESI)m / z calcdfor C 36 H 40 N5O2,574.3182; found,574.3172[M+H] + .

[0094] Example 21: The synthesis of target compound 2c was performed by dissolving 1-(3,4-dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxylic acid (1 mmol, 0.35 g) in anhydrous dichloromethane (10 ml), adding EDCI (2 mmol, 0.38 g) and DMAP (0.5 mmol, 0.06 g), and then adding 4-(1,4'-bipiperidinyl]-1'-ylmethyl)aniline (1 mmol, 0.27 g). The mixture was allowed to react overnight at room temperature. After completion of the reaction, the dichloromethane was removed by concentration under reduced pressure, and the crude product was purified by column chromatography to yield 0.55 g (91%) of a light yellow solid. 1 H NMR (400MHz, DMSO-d6) δ11.93(s,1H,NH),10.48(s,1H,NH),8.92(s,1H,Ar-H),8.45(d,J=7.9Hz,1H,Ar-H),7.88–7.82(m,2H, Ar-H),7.79(d,J=2.1Hz,1H,Ar-H),7.77–7.70(m,2H,Ar-H),7.64–7.58(m,1H,Ar-H),7.37–7.27(m,3H,Ar-H),7.24(d,J=8.4 Hz,1H,Ar-H),3.97(s,3H,OCH3),3.91(s,3H,OCH3),3.44(s,2H,CH2),2.88(d,J=11.0Hz,2H,CH2),2.61(s,2H,CH2),1.92(m, 2H,CH2),1.75(d,J=12.1Hz,2H,CH2),1.58–1.47(m,6H,CH2),1.43–1.37(m,2H,CH2),1.24(m,1H,CH),0.91–0.77(m,2H,CH2). 13 C NMR(101MHz,DMSO-d6)δ163.7,150.2,149.5,142.1,141.4,139.7,137.9,134.8,130.4,130.3,129.8,12 1.8,121.7,120.3,113.5,112.8,112.3,62.7,62.0,56.2,56.1,52.8,49.9,27.4.MS(ESI)m / z:604[M+H] + .HRMS(ESI)m / z calcd for C 37 H 42 N5O3,604.3287; found,604.3276[M+H] + .

[0095] Example 22: The synthesis of target compound 2d was as follows: 1-(2-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxylic acid (1 mmol, 0.32 g) was dissolved in anhydrous dichloromethane (10 ml), EDCI (2 mmol, 0.38 g) and DMAP (0.5 mmol, 0.06 g) were added, and then 4-(1,4'-bipiperidinyl]-1'-ylmethyl)aniline (1 mmol, 0.27 g) was added. The mixture was allowed to react overnight at room temperature. After completion of the reaction, the dichloromethane was removed by concentration under reduced pressure, and the crude product was purified by column chromatography to yield 0.45 g (92%) of a light yellow solid. 1 H NMR (400MHz, DMSO-d6) δ11.51(s,1H,NH),10.38(s,1H,NH),8.97(s,1H,Ar-H),8.44(d,J=8.0H z,1H,Ar-H),7.85(d,J=8.2Hz,2H,Ar-H),7.72(d,J=7.4,1.8Hz,1H,Ar-H),7.66(m,3H,Ar-H),7 .32(m,J=8.0Hz,4H,Ar-H),7.24(m,1H,Ar-H),3.82(s,3H,OCH3),3.55(s,2H,CH2),2.68(m,2H, CH2),2.33(m,2H,CH2),1.98(m,2H,CH2),1.86(m,6H,CH2),1.35(m,4H,CH2),0.90(m,2H,CH2). 13 C NMR (101MHz, DMSO-d6) δ163.7,157.6,149.7,141.7,140.4,139.4,137.7,136.2,134.2,132.3,131.0,129.6,122. 6,121.4,120.4,120.2,114.0,112.9,112.0,62.6,62.0,55.6,53.2,50.1,27.9,26.4,24.9.MS(ESI)m / z:574[M+H] + .HRMS(ESI)m / z calcd for C 36 H 40 N5O2,574.3182; found,574.3173[M+H] + .

[0096] Example 23: The synthesis of target compound 2e was performed by dissolving 1-(2,3-dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxylic acid (1 mmol, 0.35 g) in anhydrous dichloromethane (10 ml), adding EDCI (2 mmol, 0.38 g) and DMAP (0.5 mmol, 0.06 g), and then adding 4-(1,4'-bipiperidinyl]-1'-ylmethyl)aniline (1 mmol, 0.27 g). The mixture was allowed to react overnight at room temperature. After completion of the reaction, the dichloromethane was removed by concentration under reduced pressure, and the crude product was purified by column chromatography to yield 0.53 g (90%) of a light yellow solid. 1 H NMR(400MHz,DMSO-d6)δ11.49(s,1H,NH),10.34(s,1H,NH),9.00(s,1H,Ar-H),8.45(d,J=7.8Hz, 1H,Ar-H),7.80(d,J=8.5Hz,2H,Ar-H),7.65(d,J=8.2Hz,1H,Ar-H),7.59(m,1H,Ar-H),7.35(m,6 H,Ar-H),3.95(s,3H,OCH3),3.62(s,3H,OCH3),3.40(s,2H,CH2),2.84(d,J=11.1Hz,2H,CH2),2. 41(m,4H,CH2),1.88(m,2H,CH2),1.65(d,J=12.1Hz,2H,CH2),1.46(m,6H,CH2),1.37(m,2H,CH2). 13 C NMR(101MHz,DMSO-d6)δ165.3,146.2,144.4,138.6,134.2,133.9,131.5,129.9,129.5 ,129.4,126.4,120.5,115.9,73.0,63.5,62.6,53.3,28.0,26.6.MS(ESI)m / z:604[M+H] + .HRMS(ESI)m / z calcd for C 37 H 42 N5O3,604.3287; found,604.3280[M+H] + .

[0097] Example 24: The synthesis of target compound 2f was performed by dissolving 1-(2,4-dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxylic acid (1 mmol, 0.35 g) in anhydrous dichloromethane (10 ml), adding EDCI (2 mmol, 0.38 g) and DMAP (0.5 mmol, 0.06 g), and then adding 4-(1,4'-bipiperidinyl]-1'-ylmethyl)aniline (1 mmol, 0.27 g). The mixture was allowed to react overnight at room temperature. After completion of the reaction, the dichloromethane was removed by concentration under reduced pressure, and the crude product was purified by column chromatography to yield 0.57 g (93%) of a light yellow solid. 1 H NMR(400MHz,DMSO-d6)δ11.45(s,1H,NH),10.37(s,1H,NH),8.93(s,1H,Ar-H),8.42(d,J=7.9Hz,1H, Ar-H),7.86(m,2H,Ar-H),7.65(m,2H,Ar-H),7.58(m,1H,Ar-H),7.35(m,3H,Ar-H),6.84(d,J=2.3Hz ,1H,Ar-H),6.79(d,J=8.4,2.4Hz,1H,Ar-H),3.92(s,3H,OCH3),3.81(s,3H,OCH3),3.48(s,2H,CH2) ,2.93(d,J=11.1Hz,4H,CH2),2.01(m,4H,CH2),1.72(m,6H,CH2),1.25(m,2H,CH2),0.96(m,2H,CH2). 13 C NMR(101MHz,DMSO-d6)δ168.4,163.8,162.1,158.7,141.6,140.5,139.3,138.0,136.3,133.0,129.9,128.9,128.8,122.5,121.5,120.4, 120.2,119.7,113.6,112.9,106.1,98.9,62.9,61.5,56.1,56.0,55.9,55.9,52.0,49.6,26.41,23.86,22.8,21.7.MS(ESI)m / z:604[M+H] + .HRMS(ESI)m / zcalcd for C 37 H 42 N5O3,604.3287; found,604.3276[M+H] + .

[0098] Example 25: The target compound 2g was synthesized by dissolving 1-(3,5-dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxylic acid (1 mmol, 0.35 g) in anhydrous dichloromethane (10 ml), adding EDCI (2 mmol, 0.38 g) and DMAP (0.5 mmol, 0.06 g), and then adding 4-(1,4'-bipiperidinyl]-1'-ylmethyl)aniline (1 mmol, 0.27 g). The mixture was allowed to react overnight at room temperature. After completion of the reaction, the dichloromethane was removed by concentration under reduced pressure, and the crude product was purified by column chromatography to yield 0.55 g (91%) of a light yellow solid. 1 H NMR(400MHz,DMSO-d6)δ11.94(s,1H,NH),10.46(s,1H,NH),8.97(s,1H,Ar-H),8.46(d, J=7.9Hz,1H,Ar-H),7.86(m,2H,Ar-H),7.72(d,J=8.2Hz,1H,Ar-H),7.62(m,1H,Ar-H), 7.34(m,5H,Ar-H),6.73(m,1H,Ar-H),3.92(s,6H,OCH3),3.51(s,2H,CH2),2.95(d,J=1 1.5Hz,4H,CH2),2.09(m,4H,CH2),1.71(m,6H,CH2),1.25(m,2H,CH2),0.96(m,2H,CH2). 13 C NMR(101MHz,DMSO-d6)δ163.6,161.2,149.0,142.1,141.2,139.7,135.0,133.9,130.5,129.7,129.2,122.6,121.6,1 20.8,120.4,114.2,113.3,107.3,107.2,101.6,62.7,62.0,55.9,52.7,49.0,27.3,25.4,24.1.MS(ESI)m / z:604[M+H] + .HRMS(ESI)m / z calcdfor C 37 H 42 N5O3,604.3287; found,604.3277[M+H] + .

[0099] 3. Effect Verification

[0100] Example 26: In vitro anti-Echinococcus granulosus prosegmental heads (PSCs) activity study

[0101] 1. Experimental Methods

[0102] Approximately 250 PSCs were seeded in each well of a 96-well plate at 37°C and 5% CO₂. The derivatives were dissolved in DMSO and added to each well (containing 200 μL of culture medium) at a final concentration of 1 μM. The cells were incubated for 2 days for preliminary screening. Derivatives with promising therapeutic effects were further investigated. The dose-dependence of the derivatives was tested at final concentrations of 0.5, 1, and 5 μM. PSCs cultured in medium containing 1% DMSO served as a control. For 4 consecutive days, the effects of the derivatives on PSC morphology and structural integrity were observed microscopically, and their viability was assessed using an eosin exclusion assay. A total of 100 μL of 1% eosin was added to each well. After 2 minutes, PSCs were observed under an inverted microscope (dead PSCs stained red, live PSCs were colorless). The experiment was repeated three times, and the average value was calculated. After drug treatment, treated PSCs were harvested and observed using a scanning electron microscope. At the same time, PSCs were collected into 1.5 mL EP tubes, fixed with 4% paraformaldehyde solution, dehydrated, paraffin-embedded, and sliced. After staining, histopathological changes were observed under an inverted microscope.

[0103] 2. Experimental Results

[0104] First, the effects of target compounds 1a-1g and 2a-2g on Echinococcus granulosus prosegments in vitro were tested. ABZ and HM were used as positive controls. Echinococcus granulosus prosegments were first administered at an initial concentration of 1 μM, and the prosegments were collected 2 days after intervention. The survival rates of the 1% DMSO, ABZ, and HM groups were higher, while the activity of the prosegments of Echinococcus granulosus in the derivative groups showed a downward trend. Among them, the most significant in vitro effects were compounds 1a, 1c, and 1e ( Figure 1 ). In addition, we tested the LC of all target compounds against Echinococcus granulosus prosegments. 50 The results are shown in Table 1. After introducing different substituents at positions 1 and 3 of the β-carboline ring, the LC 50 The concentrations ranged from 1.61 to 18.13 μM, outperforming the positive agents ABZ and HM. Among them, compounds 1a, 1c, and 1e showed the most significant anti-Echinococcus granulosus activity, significantly outperforming the HM derivative DH-330. Based on this, compounds 1a, 1c, and 1e were selected as preferred compounds for further study.

[0105] Table 1 In vitro anti-Echinococcus granulosus activity studies of HM and target compounds

[0106]

[0107] Based on the above in vitro evaluation, the SAR of target compounds 1a-1g and 2a-2g was preliminarily analyzed. The in vitro anti-Echinococcus granulosus activity of the compounds was studied, and the structure-activity relationship of the target compounds was preliminarily obtained. It is encouraging that after introducing different substituents at the 1- and 3-positions of the β-carboline ring, the LC 50 The target compounds have an anti-PSCs ability ranging from 1.61 to 18.13 μM, which is superior to the positive drugs ABZ, HM, DH-330 and DH-004. This shows that compared with the prior art, the compounds of the present application can significantly improve the growth inhibition rate of Echinococcus granulosus in mice at low doses, thereby further reducing the dose or shortening the course of treatment.

[0108] On this basis, compounds 1a, 1c, and 1e were selected as preferred compounds for further study.

[0109] Based on the above results, at three doses of high (5μM), medium (1μM), and low (0.5μM), the protosegments of Echinococcus granulosus were collected on the 1st, 2nd, 3rd, and 4th day after drug treatment to study the dose- and time-dependence of the target compound's killing effect on the protosegments of Echinococcus granulosus. Compounds 1a, 1c, and 1e all showed dose- and time-dependence. At a concentration of 5μM, the three compounds were able to kill most of the protosegments of Echinococcus granulosus after four days of intervention ( Figure 2 A).

[0110] The morphological observation results under light microscope were consistent with the results of vitality determination. Figure 2 Figure B shows the morphological changes of Echinococcus granulosus prosegments after 4 days of intervention with compounds 1a, 1c, and 1e at a dose of 5 μM. The darker the staining, the more worms died. The prosegments of Echinococcus granulosus cultured for 4 days after drug intervention were further collected for HE staining to observe the pathological changes of Echinococcus granulosus. Figure 2 Figure C shows that the calcium granules in the 1% DMSO group were clear, the overall structure was intact, and the boundaries were clear. The worms in the HM and ABZ groups were shrunken, the edges were irregular, and some of them fell off. The worms in the compound 1a group were completely vacuolated, the matrix dissolved and disappeared, and the edge structure collapsed. In contrast, the worms in groups 1c and 1e were shrunken, the small hooks on the top process partially fell off, the edge structure was blurred, and some vacuoles were formed. Scanning electron microscopy results Figure 2 Figure D shows that the protoscols of Echinococcus granulosus in the blank control and 1% DMSO groups were morphologically intact, with intact apical processes and clearly visible microvilli. After two days of treatment with HM and ABZ (5 μM), the protoscol suckers of Echinococcus granulosus were relatively intact, with only the outer skin and hooks detached. Figures 1a, 1c, and 1e, treated with the same dose and duration, revealed significant ultrastructural changes in PSCs, including degeneration of the protoscol cuticle and collapse or loss of hooks and suckers. These three derivatives demonstrate significant in vitro anti-Echinococcus granulosus activity.

[0111] Based on the effectiveness of derivatives 1a, 1c, and 1e on PSCs, derivatives 1a, 1c, and 1e were further used at a concentration of 100 μM to intervene in hydatid cysts for 4 h. Figure 2 Figure E shows that the 1% DMSO-treated vesicles maintained a normal shape, a smooth surface, and intact germinal and stratum corneum layers. HM and ABZ groups showed less pronounced damage to hydatid cysts, demonstrating only a slight decrease in vesicle swelling. In contrast, vesicles treated with compounds 1a, 1c, and 1e exhibited complete destruction, with the germinal stratum collapsing significantly and separating from the stratum corneum, forming dense aggregates within the vesicles. In summary, modification of positions 1 and 3 of the β-carboline ring is beneficial for enhancing the anti-hydatid activity of compounds, with compounds 1a, 1c, and 1e worthy of further investigation.

[0112] Example 27: Pharmacokinetic Study of 1a, 1c, and 1e

[0113] 1. Experimental Methods

[0114] The pharmacokinetic properties of the selected derivatives were studied in Wistar rats. Derivatives 1a, 1c, and 1e were administered intravenously and orally to rats (230-250 g, three animals per time point). Rats were dosed with 5 mg / kg and 50 mg / kg intravenously and orally, respectively. The derivatives were dissolved in 5% DMSO and 95% saline for intravenous and intraperitoneal administrations. Blood samples (1.0 mL) were collected at 0.033, 0.083, 0.17, 0.33, 0.5, 1, 2, 4, 6, and 12 hours after intravenous administration and at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours after intraperitoneal administration. The samples were placed in centrifuge tubes containing sodium heparin and centrifuged at 4°C to separate plasma. Plasma samples were extracted with acetonitrile containing a universal internal standard (carbamazepine). The samples were centrifuged at 12,000 rpm for 20 minutes at 4°C, and the supernatant was collected and analyzed. The concentrations of the compounds in plasma were determined by high-performance liquid chromatography, and non-compartmental analysis was performed using WinNonlin software.

[0115] 2. Experimental Results

[0116] The results showed that after the three derivatives were injected into rats via the peritoneal cavity and tail vein, the peak concentration, area under the drug-time curve and bioavailability of group 1a were higher than those of derivatives 1c and 1e ( Figure 3and Table 2). Furthermore, compared to intravenous administration, intraperitoneal administration can increase the half-life of the three derivatives. We also used oral administration to study the pharmacokinetic properties of derivatives 1a, 1c, and 1e, but the results showed that the three derivatives were metabolized very rapidly after oral administration, and the compounds were almost undetectable in blood samples (data not shown). Based on the superior pharmacokinetic properties and significant anti-hydatid activity of compound 1a, we selected compound 1a as the preferred compound for subsequent studies.

[0117] Table 2 Pharmacokinetic parameters of compounds 1a, 1c, and 1e

[0118]

[0119] Note: Absolute bioavailability = AUC 0→∞(ip) ×D iv AUC 0→∞(iv) ×D iP ×100%; * Compared with group 1a, P<0.05, ** Compared with group 1a, P < 0.01 (bioavailability = AUC 0→∞(ip) ×D iv AUC 0→∞(iv) ×D iP ×100%; * compared with 1a group,P<0.05, ** compared with HBN5 group, P<0.01)

[0120] Example 28: In vivo anti-hydatid disease activity study of compound 1a

[0121] 1. Experimental Methods

[0122] For infection of mice, PSCs were pre-cultured in vitro to produce small cysts (microcysts, 200-300 μm in diameter). 25 microcysts were transplanted into the peritoneal cavity of each mouse and suspended in 0.4 mL RPMI 1640 medium. Approximately 180 days after infection, the modeling was detected by B-ultrasound, and the modeling mice were randomly divided into 10 groups, with 6 mice in each group. (1) The model was given 0.1 mL / 10 g / day normal saline; (2) ABZ (high, medium, and low) dose groups were given 50, 25, and 12.5 mg / kg / day ABZ suspension, respectively; (3) HM (high, medium, and low) dose groups were given 50, 25, and 12.5 mg / kg / day HM solution, respectively; (4) 1a (high, medium, and low) dose groups were given 50, 25, and 12.5 mg / kg / day 1a solution, respectively. Intraperitoneal administration was continued for 14 and 28 days. During treatment, the mice were monitored daily for weight changes and survival. After the end of administration, the mice were sacrificed, and the peritoneal cysts were isolated and weighed. The reduction in cyst wet weight was calculated as (average cyst weight of the model control group - average cyst weight of the treatment group) / average cyst weight of the model control group × 100%. The therapeutic effect was evaluated based on the ultrastructural changes of the cysts. Brain tissues of the mice were stained with HE and observed under a light microscope after 28 days of treatment, and hippocampal tissues were observed under a transmission electron microscope.

[0123] 2. Experimental Results

[0124] The mice were randomly divided into 10 groups after modeling. After 14 and 28 days of intraperitoneal administration, the animals were euthanized for visual inspection, and the vesicle tissue was isolated from each experimental mouse and weighed. The infected group mice had a large number of protosegments in their bodies, which were transparent, with high tension and clear cystic fluid. The mice in the compound 1a group had a small number of vesicles in their bodies, with low cystic wall tension, mostly translucent or hard calcified nodules, and the cystic fluid was light yellow. The effect was more obvious after 28 days of treatment ( Figure 4 B, D in the figure). The results of the cyst inhibition rate are shown in Table 3. The cyst inhibition rate of the compound 1a group was as high as 76.87%, which was about 1.4 times higher than that of the ABZ group and the HM group. In addition, the inhibitory effect of compound 1a on the growth of the protosegment in mice showed a dose-dependent trend. The therapeutic effect of the drug was analyzed after 14 and 28 days of treatment. Compared with the model group, the average wet weight of the cyst in each drug group after treatment was reduced to varying degrees, which was statistically significant ( Figure 4 A, C in the figure). Compared with the ABZ dose groups, the average cystic wet weight of mice in the corresponding dose groups of compound 1a after 14 days of treatment was significantly decreased, which was statistically significant. However, only the HM high dose group and the 1a high dose group showed a statistically significant decrease in the average cystic wet weight ( Figure 4 In addition, compared with the ABZ and HM dose groups, the corresponding dose groups of compound 1a significantly reduced the average cyst wet weight of mice after 28 days of treatment ( Figure 4 C in the above table). Therefore, the preferred compound 1a can effectively inhibit the growth of Echinococcus granulosus in mice, outperforming the positive drugs ABZ and HM. Even at a dose of 25 mg / kg, the in vivo cyst inhibition rate of compound 1a is higher than that of compound DH-330 (32.91% cyst inhibition rate). In summary, compound 1a has excellent anti-echinococcosis activity and is promising as a potential anti-echinococcosis drug candidate.

[0125] Table 3 Effects of compound 1a on mice infected with Echinococcus granulosus 14 and 28 days later (n=6)

[0126]

[0127] Note: Data were indicated as tH&E mean±standard error(SEM) *** P<0.001, ** P<0.01, * P<0.05.

[0128] On the basis of the in vivo therapeutic activity of 1a, TEM ( Figure 5 ) further confirmed the optimal efficacy of 1a against hydatid cysts at the ultrastructural level. In the model group, the cyst wall stratum corneum and germinal layer had normal and clear structures, with numerous and neatly arranged microvilli between the stratum corneum and the germinal layer. The germinal layer cell structure was normal, with clear nucleoli. In the ABZ (50 mg / kg) and HM (50 mg / kg) dose groups, microvilli were reduced, the germinal layer structure was unclear, and the cells were reduced, but the nucleoli were clear. At the same dose, in the 1a group, in addition to the disappearance of microvilli, the reduction of germinal layer cells, and the loosening of the structure, vacuolar structures were also present, with disrupted cell structure and the disappearance of nucleoli.

[0129] To study the brain tissue toxicity of compound 1a, Kunming mice were administered compound 1a for 28 days. H&E staining results showed that ( Figure 6 A in the figure shows that the number of pyramidal cells in the brain tissue of the control group, ABZ group, and 1a group was large and tightly arranged, with clearly visible nuclei and cytoplasm. However, the number of pyramidal cell layers in the brain tissue of the HM group decreased, the cells showed obvious degeneration, the cells were loosely arranged, and they were swollen and deformed. Similarly, transmission electron microscopy was used to observe the hippocampal tissue of mice. The structure of hippocampal neurons in the control group, ABZ group, and 1a group was normal, with neatly arranged cristae, little heterochromatin, and no damage to organelles or nuclei. However, the hippocampal neurons in the HM group showed widening of the perinuclear system, incomplete mitochondrial membranes and cristae, and synaptic damage manifested as blurred synaptic clefts and increased postsynaptic density ( Figure 6Therefore, by modifying HM, its derivative 1a significantly reduced its toxicity to mouse brain tissue, avoiding the neurotoxicity and other problems associated with HM. Compound 1a is promising for further research as a potential anti-echinococcosis drug candidate.

Claims

1. Use of a compound in the preparation of a medicament for preventing and / or treating echinococcosis, characterized in that: The compound is a compound represented by Formula I or II, or a pharmaceutically acceptable salt thereof: Among them, R 1 Selected from or -CH3; R 2 Selected from R 3 Selected from R 4 Selected from 2. Use of the compound according to claim 1 in the preparation of a medicament for preventing and / or treating echinococcosis, characterized in that: The compound represented by formula I is selected from: 1a: 4-((4-Hydroxypiperidin-1-yl)methyl)-N-(1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl)benzamide 1c: 4-([1,4'-bipiperidinyl]-1'-ylmethyl)-N-(1-(3,4-dimethoxyphenyl)-9H-pyridinyl[3,4-b]indol-3-yl)benzamide 1d: N-(1-(3,4-dimethoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl)-4-((4-methyl-1,4-diazepin-1-yl)methyl)benzamide 1e: 4-([1,4'-bipiperidinyl]-1'-ylmethyl)-N-(1-methyl-9H-pyridinyl[3,4-b]indol-3-yl)benzamide 1f: 4-([1,4'-bipiperidinyl]-1'-ylmethyl)-N-(1-(p-tolyl)-9H-pyridinyl[3,4-b]indol-3-yl)benzamide 1g: 4-((4-methyl-1,4-diazepin-1-yl)methyl)-N-(1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl)benzamide 3. Use of the compound according to claim 1 in the preparation of a medicament for preventing and / or treating echinococcosis, characterized in that: The compound represented by formula II is selected from: 2a: N-(4-([1,4'-bipiperidinyl]-1'-ylmethyl)phenyl)-1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxamide 2c: N-(4-([1,4'-bipiperidinyl]-1'-ylmethyl)phenyl)-1-(3,4-dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxamide 2d: N-(4-([1,4'-bipiperidinyl]-1'-ylmethyl)phenyl)-1-(2-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxamide 2e: N-(4-([1,4'-bipiperidinyl]-1'-ylmethyl)phenyl)-1-(2,3-dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxamide 2f: N-(4-([1,4'-bipiperidinyl]-1'-ylmethyl)phenyl)-1-(2,4-dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxamide 2g: N-(4-([1,4'-bipiperidinyl]-1'-ylmethyl)phenyl)-1-(3,5-dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxamide 4. Use of a compound in the preparation of a medicament for preventing and / or treating echinococcosis, characterized in that: Compound selected from: 1b: N-(1-(3-methoxyphenyl)-9Hpyridin[3,4-b]indol-3-yl)-4-((4-methyl-1,4-diazepin-1-yl)methyl)benzamide 5. Use of a compound in the preparation of a medicament for preventing and / or treating echinococcosis, characterized in that: Compound selected from: 2b: N-(4-([1,4'-bipiperidinyl]-1'-ylmethyl)phenyl)-1-(3-methoxyphenyl)-9Hpyridinyl[3,4-b]indole-3-carboxamide 6. A compound characterized in that Selected from: 1g: 4-((4-methyl-1,4-diazepin-1-yl)methyl)-N-(1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl)benzamide 7. A compound, characterized in that Selected from: 2b: N-(4-([1,4'-bipiperidinyl]-1'-ylmethyl)phenyl)-1-(3-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxamide 8. A compound, characterized in that The compound represented by formula II, or a pharmaceutically acceptable salt thereof: Among them, R 3 Selected from R 4 Selected from 9. The compound according to claim 8, characterized in that The compound represented by formula II is selected from: 2a: N-(4-([1,4'-bipiperidinyl]-1'-ylmethyl)phenyl)-1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxamide 2c: N-(4-([1,4'-bipiperidinyl]-1'-ylmethyl)phenyl)-1-(3,4-dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxamide 2d: N-(4-([1,4'-bipiperidinyl]-1'-ylmethyl)phenyl)-1-(2-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxamide 2e: N-(4-([1,4'-bipiperidinyl]-1'-ylmethyl)phenyl)-1-(2,3-dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxamide 2f: N-(4-([1,4'-bipiperidinyl]-1'-ylmethyl)phenyl)-1-(2,4-dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxamide 2g: N-(4-([1,4'-bipiperidinyl]-1'-ylmethyl)phenyl)-1-(3,5-dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxamide 10. The method for preparing the compound according to claim 8, characterized in that: The preparation method of compound II comprises: Compound 2-1R 3 CHO and L-tryptophan undergo Pictet-Spengler reaction under acidic conditions to obtain intermediate 2-2. Intermediate 2-2 is oxidized to give intermediate 2-3; 4-Nitrobenzyl bromide and compound 2-4R 4 H undergoes a nucleophilic substitution reaction to give intermediate 2-5, and the nitro group of intermediate 2-5 is reduced to an amino group to give intermediate 2-6; Intermediate 2-3 and intermediate 2-6 undergo amide condensation reaction to generate target compound II; 11. The method for preparing the compound according to claim 10, characterized in that: The synthetic route of compound II includes:

12. A pharmaceutical composition, characterized in that The invention comprises a therapeutically effective amount of the compound according to any one of claims 6 to 9 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

13. Use of the pharmaceutical composition according to claim 12 in the preparation of a medicament for preventing and / or treating echinococcosis.

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