Preparation method and application of a small molecule compound and derivatives of squaric acid

Abstract

The present invention relates to the technical field of squaric acid therapeutic compounds, and discloses a preparation method and application of a squaric acid small molecule compound and derivatives. The preparation method of the squaric acid small molecule compound and derivatives comprises the following steps: mixing and stirring compound A, ethylenediamine, and tert-butanol, adding Na2CO3 and I2, heating for reaction, purifying the reaction product mixture to obtain a nitro compound; dissolving the nitro compound, adding palladium-carbon and introducing hydrogen for reaction, filtering the reaction mixture, and evaporating to obtain an amine compound; stirring N,N-dimethylformamide, toluene, 3,4-diethoxycyclobutene-3-ene-1,2-dione (1.3 mmol), and zinc trifluoromethanesulfonate at room temperature, then adding the above amine compound, heating for reaction, and purifying the reaction mixture to obtain a squaric acid compound. The present invention provides a class of squaric acid small molecule compounds and derivatives with novel structures and having the effect of killing Babesia.

Description

Technical Field

[0001] The present invention relates to the technical field of a squaric acid therapeutic compound, in particular to a preparation method and application of a squaric acid small molecule compound and a derivative. Background Art

[0002] Babesiosis is a zoonosis caused by Babesia parasiticus in red blood cells. It is transmitted by hard ticks. Its pathogenic mechanism is mainly the lysis of infected red blood cells leading to hemolytic anemia, which can further lead to organ failure or even death under special circumstances. Babesia uses mammals as intermediate hosts and ticks as final hosts. It needs to switch between two hosts to complete its life cycle. This type of pathogen has a wide range of hosts. It can not only parasitize a variety of vertebrates and invertebrates, but some Babesia can also infect humans. The more effective and commonly used drugs for the treatment of babesiosis are mainly imidazopyrimidine, triazoguanidine, azithromycin, atovaquone, clindamycin, quinine, etc. The pathogenic life cycle of babesiosis is complex, and most of them have recurrent infections. Most of the chemical drugs commonly used to treat babesiosis have a strong inhibitory effect on Babesia, but due to the special parasitic mechanism of Babesia, it is resistant to most large molecular, highly charged and low lipid-soluble drugs, and is prone to drug resistance. In addition, genetic variation in Babesia increases the risk of drug resistance.

[0003] Imidazole urea, also known as imidoxacarb and diimidazole urea, is a symmetrical phenylurea compound. It was first introduced in Switzerland in 1969. Imidazole urea is not absorbed orally. Most of the drugs used clinically are dipropionate imidazole urea injections, but the drug has great liver and kidney toxicity and a long withdrawal period. Triazoline, also known as Bernier and Xuechongjing, is an aromatic diamidine drug. my country synthesized the drug in the early 1960s and was subsequently approved for the treatment of animal piroplasmosis. Triazoline has a good insecticidal effect, but it is highly toxic: for example, cattle are severely poisoned at 25 mg / kg, and buffaloes will have toxic reactions or even death if they are continuously used. The drugs commonly used to treat babesiosis currently have obvious toxic and side effects and parasites develop drug resistance, so safe and effective alternative drugs and new treatment plans are urgently needed. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a method for preparing a small molecule compound and a derivative of a square acid, comprising the following steps:

[0005] Step (1) Compound A, ethylenediamine and tert-butyl alcohol are mixed and stirred, and Na 2 CO 3 and I 2 , heating to react, and purifying the reaction product mixture to obtain a nitro compound;

[0006] Step (2) dissolving the nitro compound, adding palladium carbon and introducing hydrogen to react, filtering the reaction product mixture and evaporating to obtain an amine compound;

[0007] Step (3) N,N-dimethylformamide, anhydrous toluene, 3,4-diethoxycyclobutene-3-ene-1,2-dione (1.3 mmol) and zinc trifluoromethanesulfonate solution are stirred at room temperature, and then the above-mentioned amine compound is added and heated for reaction. The reaction product mixture is purified to obtain a derivative of a small molecule compound of a square acid, whose general structural formula is:

[0008]

[0009] Wherein, X includes any one of halogen, hydrogen, and alkoxy.

[0010] excellent

[0011] Preferably, in step (1), the general structural formula of compound A is: The general structural formula of nitro compounds is .

[0012] Preferably, in step (1), the compound A, ethylenediamine, Na 2 CO 3 and I 2 The dosage ratio is (2 mmol):(1.1 equiv):(3 equiv):(1.25 equiv).

[0013] Preferably, in step (1), the mixing and stirring time is 30 min; the heating reaction conditions are: heating to 70° C. and stirring the reaction for 3 h.

[0014] Preferably, in step (1), the reaction product mixture is purified by: first removing the reaction solvent tert-butyl alcohol from the reaction product mixture system under reduced pressure, then adding ethyl acetate, ultrasonicating and filtering to remove insoluble matter, and purifying the organic layer with saturated Na 2 SO 3 The solution was washed with aqueous solution and saline, and then with anhydrous MgSO 4 After drying, it was purified by column chromatography.

[0015] Preferably, in step (2), the solvent for dissolving the nitro compound includes methanol; the amount of palladium carbon added is 5wt%; the reaction conditions are: deoxygenating the reaction system and adding H 2 , react at room temperature overnight; Filtration method: the reaction product mixture is filtered through celite.

[0016] Preferably, in step (3), the structural formula of 3,4-diethoxycyclobutene-3-ene-1,2-dione is .

[0017] Preferably, in step (3), the heating reaction conditions are: heating to 100° C. and stirring for 18 h; the dosage ratio of the amine compound, 3,4-diethoxycyclobutene-3-ene-1,2-dione, and zinc trifluoromethanesulfonate is (1 mmol):(0.480 equivalent):(0.095 equivalent).

[0018] Preferably, in step (3), the reaction product mixture is purified by: detecting the completion of the reaction by thin layer chromatography and observing precipitation, filtering, washing with methanol (3×50 mL) and n-hexane (50 mL) in sequence, and drying under vacuum to obtain the desired product.

[0019] The squaric acid small molecule compound and derivatives are used for treating babesiosis.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention provides a class of novel-structured squaric acid small molecule compounds and derivatives thereof, and the preparation method thereof has the advantages of short synthesis route, mild reaction conditions, abundant and readily available raw materials, simple operation and post-treatment, etc. The squaric acid small molecule compounds and derivatives of the present invention have the effect of killing Babesia.

[0022] The squaric acid small molecule compound and derivatives are used for treating babesiosis.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention provides a class of novel-structured squaric acid small molecule compounds and derivatives thereof, and the preparation method thereof has the advantages of short synthesis route, mild reaction conditions, abundant and readily available raw materials, simple operation and post-treatment, etc. The squaric acid small molecule compounds and derivatives of the present invention have the effect of killing Babesia. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a schematic diagram of the synthesis of the square acid small molecule compounds and derivatives of the present invention. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0027] Example 1: Preparation of N-[3-(4,5-dihydro-3H-imidazol-2-yl)phenyl]-1-[(4-fluorophenyl)amino]methaneamide:

[0028] Step (1) 3-nitrobenzaldehyde (2 mmol) and ethylenediamine (1.1 equivalent) were mixed with tert-butyl alcohol and stirred for 30 minutes, and then Na 2 CO 3 (3 equivalents) and I 2 (1.25 equivalents), and the reaction temperature was raised to 70 ° C and stirred for 3 hours. When the reaction was completed, the reaction solvent tert-butyl alcohol was removed under reduced pressure, ethyl acetate was added to the mixture, and the insoluble matter was removed by ultrasonication and filtration. The organic layer was washed with saturated Na 2 SO 3 The solution was washed with aqueous solution and brine, and then with anhydrous MgSO 4 The mixture was dried and purified by column chromatography to obtain 2-(3-nitrophenyl)-4,5-dihydro-3H-imidazole, the chemical structure of which is as follows:

[0029]

[0030] 1 H NMR (400 MHz, Chloroform- d ) δ 8.51 (t, J = 2.0 Hz, 1H), 8.24 ( J =8.3,2.3, 1.1 Hz, 1H), 8.11 (dt, J =7.8, 1.4 Hz, 1H), 7.54 (t, J =8.0 Hz, 1H), 3.79(s, 4H).

[0031] Step (2) The 2-(3-nitrophenyl)-4,5-dihydro-3H-imidazole obtained in the previous step was dissolved in methanol, and then 5 wt% palladium carbon was added. The reaction vessel was deoxygenated and H was added via a balloon. 2 The reaction was allowed to proceed overnight at room temperature, and the resulting mixture was filtered through diatomaceous earth. The filtrate was evaporated to obtain 5-(4,5-dihydro-3H-imidazol-2-yl)aniline, the chemical structure of which is as follows:

[0032]

[0033] 1 H NMR (400 MHz, DMSO- d 6 ) δ 7.17 (t, J= 7.8 Hz, 1H), 7.09 - 7.00 (m,2H), 6.82 (dd, J = 7.9, 2.2 Hz, 1H), 5.46 (s, 2H), 3.80 (s, 4H).

[0034] Step (3) Anhydrous toluene (19 mL), anhydrous N,N-dimethylformamide (1 mL), 3,4-diethoxycyclobutene-3-ene-1,2-dione (1.3 mmol) and zinc trifluoromethanesulfonate (0.26 mmol) were added to a 100 mL round-bottom flask equipped with a magnetic stirring bar and a reflux condenser. The resulting suspension was stirred at room temperature, followed by the addition of 5-(4,5-dihydro-3H-imidazol-2-yl)aniline (2.73 mmol). The solution was heated to 100°C and stirred for 18 hours, during which time a precipitate was observed to form. After the reaction mixture was cooled to room temperature, it was filtered and the precipitate was washed with methanol (3 times, 50 mL each time) and n-hexane (50 mL) in turn. Finally, it was dried under vacuum conditions to obtain 4-{[3-(4,5-dihydro-1H-imidazol-2-yl)phenyl]amino}-3-{[3-(4,5-dihydro-3H-imidazol-2-yl)phenyl]amino}cyclobut-3-ene-1,2-dione, the chemical structure of which is as follows:

[0035]

[0036] 1 H NMR (400 MHz, DMSO- d 6 ) δ 10.76 (s, 1H), 10.59 (s, 2H), 7.90 (q, J =2.2 Hz, 1H),7.81 (dd, J = 8.0, 2.5 Hz, 1H), 7.68 (td, J = 8.0, 2.5 Hz, 1H), 7.60(d, J = 7.8 Hz, 1H), 4.05 (d, J= 2.5 Hz, 4H). ESI-MS m / z: [M+H] + Calculated for C 22 H 21 N 6 O 2 : 401.1720; found: 401.1731.

[0037] Example 2: Preparation of 4-{[3-(4,5-dihydro-1H-imidazol-2-yl)-5-fluorophenyl]amino}-3-{[5-(4,5-dihydro-3H-imidazol-2-yl)-3-fluorophenyl]amino}cyclobut-3-ene-1,2-dione:

[0038] Step (1) 3-Fluoro-5-nitrobenzaldehyde (2 mmol) and ethylenediamine (1.1 equivalents) were mixed with tert-butyl alcohol and stirred for 30 minutes, and then Na 2 CO 3 (3 equivalents) and I 2 (1.25 equivalents), and the reaction temperature was raised to 70 ° C and stirred for 3 hours. When the reaction was completed, the reaction solvent tert-butyl alcohol was removed under reduced pressure, ethyl acetate was added to the mixture, and the insoluble matter was removed by ultrasonication and filtration. The organic layer was washed with saturated Na 2 SO 3 The solution was washed with aqueous solution and brine, and then with anhydrous MgSO 4 The mixture was dried and purified by column chromatography to obtain 2-(3-fluoro-5-nitrophenyl)-4,5-dihydro-1H-imidazole, the chemical structure of which is as follows:

[0039]

[0040] 1 H NMR (400 MHz, DMSO- d 6 ) δ 8.51 (t, J = 1.7 Hz, 1H), 8.24 (dt, J = 8.5,2.3 Hz, 1H), 8.07 ( J = 9.2, 2.6, 1.4 Hz, 1H), 7.40 (s, 1H), 3.68 (s, 4H).

[0041] Step (2) The 2-(3-fluoro-5-nitrophenyl)-4,5-dihydro-1H-imidazole obtained in the previous step was dissolved in methanol, and then 5 wt% palladium carbon was added. The reaction vessel was deoxygenated and H was added via a balloon. 2 The reaction was allowed to proceed overnight at room temperature, and the resulting mixture was filtered through diatomaceous earth. The filtrate was evaporated to obtain 3-(4,5-dihydro-1H-imidazol-2-yl)-5-fluoroaniline, the chemical structure of which is as follows:

[0042]

[0043] 1 H NMR (400 MHz, DMSO- d 6) δ 10.50 (s, 1H), 6.89 (dd, J = 8.3, 2.1 Hz,2H), 6.66 (dt, J = 11.5, 2.1 Hz, 1H), 5.96 (s, 2H), 3.92 (s, 4H).

[0044] Step (3) Anhydrous toluene (19 mL), anhydrous N,N-dimethylformamide (1 mL), 3,4-diethoxycyclobutene-3-ene-1,2-dione (1.3 mmol) and zinc trifluoromethanesulfonate (0.26 mmol) were added to a 100 mL round-bottom flask equipped with a magnetic stirring bar and a reflux condenser. The resulting suspension was stirred at room temperature, followed by the addition of 5-(4,5-dihydro-3H-imidazole-2-yl)aniline (2.73 mmol). The solution was heated to 100°C and stirred for 18 hours, during which time a precipitate was observed to form. After the reaction mixture was cooled to room temperature, it was filtered and the precipitate was washed with methanol (3 times, 50 mL each time) and n-hexane (50 mL) in turn. Finally, it was dried under vacuum conditions to obtain the chemical structure of 4-{[3-(4,5-dihydro-1H-imidazol-2-yl)-5-fluorophenyl]amino}-3-{[5-(4,5-dihydro-3H-imidazol-2-yl)-3-fluorophenyl]amino}cyclobut-3-ene-1,2-dione as follows:

[0045]

[0046] 1 H NMR (400 MHz, DMSO- d 6 ) δ 10.94 (s, 1H), 10.69 (s, 2H), 7.79 (dt, J =10.4, 2.2 Hz, 1H), 7.68 (t, J = 1.7 Hz, 1H), 7.47 (dt, J = 8.9, 1.9 Hz, 1H), 4.05(s, 4H). ESI-MS m / z: [M+H] + Calculated for C 22 H 19 F 2 N 6 O 2 : 437.1531; found:437.1541.

[0047] Example 3: Preparation of 4-{[3-(4,5-dihydro-1H-imidazol-2-yl)-5-methylphenyl]amino}-3-{[5-(4,5-dihydro-3H-imidazol-2-yl)-3-methylphenyl]amino}cyclobut-3-ene-1,2-dione:

[0048] Step (1) 3-Methyl-5-nitrobenzaldehyde (2 mmol) and ethylenediamine (1.1 equivalent) were mixed with tert-butyl alcohol and stirred for 30 minutes, and then Na 2 CO 3 (3 equivalents) and I 2 (1.25 equivalents), and the reaction temperature was raised to 70 ° C and stirred for 3 hours. When the reaction was completed, the reaction solvent tert-butyl alcohol was removed under reduced pressure, ethyl acetate was added to the mixture, and the insoluble matter was removed by ultrasonication and filtration. The organic layer was washed with saturated Na 2 SO 3 The solution was washed with aqueous solution and brine, and then with anhydrous MgSO 4 The mixture was dried and purified by column chromatography to obtain 2-(3-methyl-5-nitrophenyl)-4,5-dihydro-1H-imidazole, the chemical structure of which is as follows:

[0049]

[0050] 1 H NMR (400 MHz, DMSO- d 6 ) δ 8.43 (t, J = 1.9 Hz, 1H), 8.15 (d, J = 1.9 Hz,1H), 8.08 (s, 1H), 3.65 (s, 4H), 2.47 (s, 3H).

[0051] Step (2) The 2-(3-methyl-5-nitrophenyl)-4,5-dihydro-1H-imidazole obtained in the previous step was dissolved in methanol, and then 5 wt% palladium carbon was added. The reaction vessel was deoxygenated and H was added via a balloon. 2 The reaction was allowed to proceed overnight at room temperature, and the resulting mixture was filtered through diatomaceous earth. The filtrate was evaporated to obtain 3-(4,5-dihydro-1H-imidazol-2-yl)-5-methylaniline, the chemical structure of which is as follows:

[0052]

[0053] 1 H NMR (400 MHz, DMSO- d 6 ) δ 6.85 (d, J= 1.9 Hz, 1H), 6.77 (s, 1H), 6.46(d, J = 1.8 Hz, 1H), 5.04 (s, 2H), 3.53 (s, 4H), 2.17 (s, 3H).

[0054] Step (3) Anhydrous toluene (19 mL), anhydrous N,N-dimethylformamide (1 mL), 3,4-diethoxycyclobutene-3-ene-1,2-dione (1.3 mmol) and zinc trifluoromethanesulfonate (0.26 mmol) were added to a 100 mL round-bottom flask equipped with a magnetic stirring bar and a reflux condenser. The resulting suspension was stirred at room temperature, followed by the addition of 3-(4,5-dihydro-1H-imidazole-2-yl)-5-methylaniline (2.73 mmol). The solution was heated to 100°C and stirred for 18 hours, during which time a precipitate was observed to form. After the reaction mixture was cooled to room temperature, it was filtered and the precipitate was washed with methanol (3 times, 50 mL each) and n-hexane (50 mL) in sequence. Finally, it was dried under vacuum conditions to obtain the chemical structure of 4-{[3-(4,5-dihydro-1H-imidazol-2-yl)-5-methylphenyl]amino}-3-{[5-(4,5-dihydro-3H-imidazol-2-yl)-3-methylphenyl]amino}cyclobut-3-ene-1,2-dione as follows:

[0055]

[0056] 1 H NMR (400 MHz, DMSO- d 6 ) δ 10.67 (s, 1H), 10.55 (s, 2H), 7.70 (d, J =2.0Hz, 1H), 7.61 (s, 1H), 7.43 (s, 1H), 4.03 (s, 4H), 2.39 (s, 3H). ESI-MS m / z: [M+H] + Calculated for C 24 H 25 N 6 O 2 : 429.2033; found: 429.2038.

[0057] Example 4: Preparation of 3-{[3-chloro-5-(4,5-dihydro-3H-imidazol-2-yl)phenyl]amino}-4-{[5-chloro-3-(4,5-dihydro-1H-imidazol-2-yl)phenyl]amino}cyclobut-3-ene-1,2-dione:

[0058] Step (1) 3-Chloro-5-nitrobenzaldehyde (2 mmol) and ethylenediamine (1.1 equivalents) were mixed with tert-butyl alcohol and stirred for 30 minutes, and then Na 2 CO 3 (3 equivalents) and I 2 (1.25 equivalents), and the reaction temperature was raised to 70 ° C and stirred for 3 hours. When the reaction was completed, the reaction solvent tert-butyl alcohol was removed under reduced pressure, ethyl acetate was added to the mixture, and the insoluble matter was removed by ultrasonication and filtration. The organic layer was washed with saturated Na 2 SO 3 The solution was washed with aqueous solution and brine, and then with anhydrous MgSO 4 The mixture was dried and purified by column chromatography to obtain 2-(3-chloro-5-nitrophenyl)-4,5-dihydroimidazole, the chemical structure of which is as follows:

[0059]

[0060] 1 H NMR (400 MHz, DMSO- d 6 ) δ 8.58 (t, J = 1.8 Hz, 1H), 8.37 (t, J = 2.1 Hz,1H), 8.26 (t, J = 1.7 Hz, 1H), 7.35 (s, 1H), 3.67 (s, 4H).

[0061] Step (2) The 2-(3-chloro-5-nitrophenyl)-4,5-dihydroimidazole obtained in the previous step was dissolved in methanol, and then 5 wt% palladium carbon was added. The reaction vessel was deoxygenated and H was added via a balloon. 2 The reaction was allowed to proceed overnight at room temperature. The resulting mixture was filtered through diatomaceous earth and the filtrate was evaporated to obtain 3-chloro-5-(4,5-dihydro-1H-imidazol-2-yl)aniline, the chemical structure of which is as follows:

[0062]

[0063] 1 H NMR (400 MHz, DMSO- d 6 ) δ 7.17 (t, J = 7.8 Hz, 1H), 7.09 - 7.00 (m,2H), 6.82 (dd, J= 7.9, 2.2 Hz, 1H), 5.46 (s, 2H), 3.80 (s, 4H).

[0064] Step (3) Anhydrous toluene (19 mL), anhydrous N,N-dimethylformamide (1 mL), 3,4-diethoxycyclobutene-3-ene-1,2-dione (1.3 mmol) and zinc trifluoromethanesulfonate (0.26 mmol) were added to a 100 mL round-bottom flask equipped with a magnetic stirring bar and a reflux condenser. The resulting suspension was stirred at room temperature, followed by the addition of 3-chloro-5-(4,5-dihydro-1H-imidazol-2-yl)aniline (2.73 mmol). The solution was heated to 100°C and stirred for 18 hours, during which time a precipitate was observed to form. After the reaction mixture was cooled to room temperature, it was filtered and the precipitate was washed with methanol (3 times, 50 mL each) and n-hexane (50 mL) in turn. Finally, it was dried under vacuum conditions to obtain the chemical structure of 3-{[3-chloro-5-(4,5-dihydro-3H-imidazol-2-yl)phenyl]amino}-4-{[5-chloro-3-(4,5-dihydro-1H-imidazol-2-yl)phenyl]amino}cyclobut-3-ene-1,2-dione as follows:

[0065]

[0066] 1 H NMR (400 MHz, DMSO- d 6 ) δ 10.98 (s, 1H), 10.69 (s, 2H), 7.95 (t, J =2.0 Hz, 1H), 7.77 (t, J = 1.8 Hz, 1H), 7.67 (d, J = 1.8 Hz, 1H), 4.04 (s, 4H).ESI-MS m / z: [M+H] + Calculated for C 22 H 19 Cl 2 N 6 O 2 : 469.0940; found: 469.0940.

[0067] Example 5: Preparation of 4-{[3-(4,5-dihydro-1H-imidazol-2-yl)-5-methoxyphenyl]amino}-3-{[5-(4,5-dihydro-3H-imidazol-2-yl)-3-methoxyphenyl]amino}cyclobut-3-ene-1,2-dione:

[0068] Step (1) 3-methoxy-5-nitrobenzaldehyde (2 mmol) and ethylenediamine (1.1 equivalents) were mixed with tert-butyl alcohol and stirred for 30 minutes, and then Na 2 CO 3 (3 equivalents) and I 2 (1.25 equivalents), and the reaction temperature was raised to 70 ° C and stirred for 3 hours. When the reaction was completed, the reaction solvent tert-butyl alcohol was removed under reduced pressure, ethyl acetate was added to the mixture, and the insoluble matter was removed by ultrasonication and filtration. The organic layer was washed with saturated Na 2 SO 3 The solution was washed with aqueous solution and brine, and then with anhydrous MgSO 4 The mixture was dried and purified by column chromatography to obtain 2-(3-methoxy-5-nitrophenyl)-4,5-dihydroimidazole, the chemical structure of which is as follows:

[0069]

[0070] 1 H NMR (400 MHz, DMSO- d 6 ) δ 8.26 (t, J = 1.7 Hz, 1H), 7.81 (q, J = 1.7,1.1 Hz, 2H), 3.92 (s, 3H), 3.68 (s, 4H).

[0071] Step (2) The 2-(3-methoxy-5-nitrophenyl)-4,5-dihydroimidazole obtained in the previous step was dissolved in methanol, and then 5 wt% palladium carbon was added. The reaction vessel was deoxygenated and H was added via a balloon. 2 The reaction was allowed to proceed overnight at room temperature. The resulting mixture was filtered through diatomaceous earth and the filtrate was evaporated to obtain 5-(4,5-dihydro-1H-imidazol-2-yl)-3-methoxyaniline, the chemical structure of which is as follows:

[0072]

[0073] 1 H NMR (400 MHz, DMSO- d 6 ) δ 6.65 (t, J = 1.7 Hz, 1H), 6.61 - 6.51 (m,1H), 6.22 (t, J = 2.2 Hz, 1H), 5.19 (s, 2H), 3.68 (s, 3H), 3.56 (s, 4H).

[0074] Step (3) Anhydrous toluene (19 mL), anhydrous N,N-dimethylformamide (1 mL), 3,4-diethoxycyclobutene-3-ene-1,2-dione (1.3 mmol) and zinc trifluoromethanesulfonate (0.26 mmol) were added to a 100 mL round-bottom flask equipped with a magnetic stirring bar and a reflux condenser. The resulting suspension was stirred at room temperature, followed by the addition of 5-(4,5-dihydro-1H-imidazole-2-yl)-3-methoxyaniline (2.73 mmol). The solution was heated to 100°C and stirred for 18 hours, during which time a precipitate was observed to form. After the reaction mixture was cooled to room temperature, it was filtered and the precipitate was washed with methanol (3 times, 50 mL each) and n-hexane (50 mL) in turn. Finally, it was dried under vacuum conditions, and the chemical structure of the obtained 4-{[3-(4,5-dihydro-1H-imidazol-2-yl)-5-methoxyphenyl]amino}-3-{[5-(4,5-dihydro-3H-imidazol-2-yl)-3-methoxyphenyl]amino}cyclobut-3-ene-1,2-dione was as follows:

[0075]

[0076] 1 H NMR (400 MHz, DMSO- d 6 ) δ 10.69 (s, 1H), 10.60 (s, 2H), 7.52 (s,1H), 7.43 (s, 1H), 7.19 (s, 1H), 4.04 (s, 4H), 3.86 (s, 3H). ESI-MS m / z: [M+H] + Calculated for C 24 H 25 N 6 O 4 : 461.1931; found: 461.1936.

[0077] Experimental example

[0078] Preparation of Babesia strains for testing:

[0079] The parasite strains tested in this experimental example involved Babesia gibsoni.

[0080] Components and preparation of Babesia culture medium:

[0081] Components and preparation method of the basic culture medium for Babesia gibbsii: Prepare 1000 ml of SFM basic culture medium (VP-SFM ATG 12559-019 17.6 g, Albumax I 2 g, L-glutamine 200 mM (Sigma) 20 mL, Antibiotic / Antimycotic 10 mL (100×), dd-H 2 O 970 mL). When using, take 47.5 mL SFM culture medium into a 50 mL centrifuge tube and add 2.5 ml of self-collected canine serum (from beagle dogs). This liquid culture medium is a typical liquid culture medium for Babesia gibbsii. The above operations are all carried out in the sterile environment of a conventional cell room clean bench.

[0082] Preparation of test compounds:

[0083] The compounds of Examples 1-5 were used as solutions. Dimethyl sulfoxide (DMSO), 10× phosphate buffered saline (10× PBS) and SFM culture medium were used to dissolve the inhibitor powder for subsequent experimental use. Specific preparation method: First, a compound concentrate with an original concentration of 10 mM was prepared for storage and then diluted with 10× PBS to an intermediate concentration (200 μM). When determining the half-maximal inhibitory concentration of the compound against Babesia gibbsii, the prepared inhibitor concentrate was diluted with SFM culture medium for a concentration gradient experiment, and the concentration gradients were designed to be: 0.01, 0.1, 1, 10, 50 μM.

[0084] Babesia culture method:

[0085] Babesia gibbsii: Add the conventional culture system of Babesia gibbsii (50 μL red blood cells, 450 μL Babesia gibbsii culture medium (SFM culture medium containing canine serum)) to a 48-well culture plate and place in a 5% CO 2 Culture in an incubator at 37°C. Change the medium every 24 hours (absorb 400 μL of medium and add 400 μL of new medium) and mix thoroughly with the red blood cells at the bottom. Subculture once every 72 hours (mix the old well thoroughly, aspirate 50 μL and add it to the new well, then add 45 μL of fresh canine red blood cells and 405 μL of Babesia gibbsii medium).

[0086] In vitro growth inhibition test of Babesia gibsoni

[0087] Inhibition test on Babesia gibsoni: According to the aseptic operation procedure, 0.3 μL of red blood cell mud was aspirated in the 48-well culture plate of Babesia gibsoni, and a blood smear was made. After Giemsa staining, the infection rate was observed under a microscope. The infection rate was diluted to 0.2% with red blood cells of healthy dogs. Mix thoroughly with SFM culture medium and divide equally into new 96-well culture plates with a volume of 50 μL per well. Take the above candidate inhibitors and perform three replicates for each concentration. Mix the mixture in each well thoroughly and culture it at 37°C for 72 hours. By counting the infection rate, the effect of the compound on the in vitro growth of Babesia gibsoni was observed, and the half-maximal inhibitory concentration (IC) was calculated. 50 )The entire operation was carried out under the aseptic operation procedures of the commonly used cell room clean bench.

[0088] The test results of compound activity data are shown in Table 1:

[0089] Table 1

[0090]

[0091] From the test results in Table 1, it can be seen that the half inhibitory concentration of the compound prepared in Example 2 of the present invention against Babesia gibbsii can reach as low as IC 50 =0.04 μM, the half inhibitory concentration IC of the compounds prepared in Examples 1, 3 and 4 against Babesia gisoni 50 <10 μM, indicating that the small molecule compounds of the present invention and their derivatives have good anti-Babesia activity.

[0092] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a small molecule compound of a square acid type, characterized in that: The following steps are involved: Step (1) Compound A, ethylenediamine and tert-butanol are mixed and stirred, Na2CO3 and I2 are added, and the mixture is heated for reaction, and the reaction product mixture is purified to obtain a nitro compound; Step (2) dissolving the nitro compound, adding palladium carbon and introducing hydrogen to react, filtering the reaction product mixture and evaporating to obtain an amine compound; Step (3) N,N-dimethylformamide, anhydrous toluene, 3,4-diethoxycyclobutene-3-ene-1,2-dione and zinc trifluoromethanesulfonate solution are stirred at room temperature, and then the above-mentioned amine compound is added and heated for reaction. The reaction product mixture is purified to obtain a small molecule square acid compound, whose general structural formula is: ； Wherein, X is any one of halogen, hydrogen, and methyl; located at the meta position of the benzene ring; In the step (1), the general structural formula of compound A is: The general structural formula of nitro compounds is ; In the step (3), the structural formula of 3,4-diethoxycyclobutene-3-ene-1,2-dione is .

2. The method for preparing a small molecule compound of a square acid type according to claim 1, characterized in that: In the step (1), the usage ratio of compound A, ethylenediamine, Na2CO3 and I2 is (2 mmol):(1.1 mmol):(3 mmol):(1.25 mmol).

3. The method for preparing a small molecule compound of a square acid type according to claim 1, characterized in that: In the step (1), the mixing and stirring time is 30 minutes; the heating reaction conditions are: heating to 70° C. and stirring the reaction for 3 hours.

4. The method for preparing a small molecule compound of a square acid type according to claim 1, characterized in that: In the step (1), the reaction product mixture purification method is as follows: the reaction product mixture system is first decompressed to remove the reaction solvent tert-butyl alcohol, then ethyl acetate is added, and insoluble matter is removed by ultrasonication and filtration. The organic layer is washed with saturated Na2SO3 aqueous solution and brine, and dried with anhydrous MgSO4, and then purified by column chromatography.

5. The method for preparing a small molecule compound of a square acid type according to claim 1, characterized in that: In the step (2), the solvent for dissolving the nitro compound is methanol; the amount of palladium carbon added is 5wt%; the reaction conditions are: the reaction system is deoxygenated, H2 is added through a balloon, and the reaction is carried out at room temperature overnight; the filtering method is: the reaction product mixture is filtered through diatomaceous earth.

6. The method for preparing a small molecule compound of a square acid type according to claim 1, characterized in that: In the step (3), the heating reaction conditions are: heating to 100° C. and stirring for 18 hours; the dosage ratio of the amine compound, 3,4-diethoxycyclobutene-3-ene-1,2-dione, and zinc trifluoromethanesulfonate is (1 mmol):(0.480 mmol):(0.095 mmol).

7. The method for preparing a small molecule compound of a square acid type according to claim 1, characterized in that: In the step (3), the reaction product mixture is purified by: using thin layer chromatography to detect the completion of the reaction and observing the precipitation, filtering, washing with methanol and n-hexane in sequence, and drying under vacuum to obtain the product.

8. Use of the squaraine-based small molecule compound prepared by the method for preparing a squaraine-based small molecule compound according to claim 1 for preparing a drug for treating babesiosis.

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