A polysubstituted pyrazoline compound and a synthesis method thereof

Abstract

The present invention provides a polysubstituted pyrazoline compound and a synthesis method thereof, mainly relating to the technical field of pyrazoline compounds, in an organic solvent, after adding a substrate hydrazone, vinyl isoxazole and a base at room temperature to react completely, a condensed ring 1,3,4,5-tetrasubstituted pyrazoline compound is obtained through separation and purification. The invention is scientifically designed, ingeniously conceived, simple in method, and easy to operate. The method of the present invention can specifically synthesize a new pyrazoline compound with multiple substituents, and has fewer by-products than the traditional method, is easy to purify, and has a very high yield. The limitation of synthesizing such pyrazoline compounds in the known production method is solved, and the practicality of this method is proved.

Description

Technical Field

[0001] The present invention mainly relates to the technical field of pyrazoline compounds, and in particular to a polysubstituted pyrazoline compound and a synthesis method thereof. Background Art

[0002] Pyrazoline compounds are a class of heterocyclic compounds with diverse structures. They exist in many drug intermediates and are also the main structural units of many drugs. It is reported that this class of compounds has a wide range of biological activities, such as anti-inflammatory activity, anti-tumor activity, monoamine oxidase selective inhibitory activity, antibacterial and bactericidal activity, insecticide, potential cytotoxic agent, cholesterol phthalyl transferase or cholesterol absorption inhibitory activity, inhibition of low-concentration lipoprotein oxidation and anti-amoeba activity, etc. Some polysubstituted pyrazoline derivatives have good optical properties, such as being used for fluorescent switches; Among them, the most important representative of pyrazoline compounds as drugs is 3-amino-1-(m-trifluoromethylphenyl)-2-pyrazoline (BW755C), which is a dual inhibitor of cyclooxygenase and lipoxygenase and has good anti-inflammatory effect. In addition, the anti-inflammatory drug celecoxib is also one of the representatives; 9-methoxyacridine (PZA) is also a new type of anti-tumor drug currently in the second phase of clinical trials. In terms of agricultural applications, since the 1970s, this type of compound has been continuously developed into pesticides such as PH60-41 and RH4321.

[0003] Considering the therapeutic potential of this structure, the development of new pyrazoline compounds and new methods for their efficient synthesis has aroused great interest. At present, several typical methods for the synthesis of pyrazoline have been studied and discovered, including 1,3-dipolar cycloaddition, direct cyclization of α,β-unsaturated carbonyl compounds with hydrazine, and direct heating of 1,5-diaminotetrazole with α,β-unsaturated ketone in DMF.

[0004] In addition, Nilo Zanatta et al. found that trifluoromethyl-substituted pyrazoline compounds can be obtained by the [3+2] cyclization concentration method using β-alkoxyvinyl 1,1,1-trifluoromethyl ketone and hydrazine; aminoguanidine can also undergo a [3+2] cyclization reaction with dibenzylideneacetone to obtain pyrazoline derivatives; Werner Seebacher also used ammonium thiocyanate and α,β-unsaturated ketones to obtain 1-aminothiocarbonyl-2-pyrazoline compounds.

[0005] However, these synthetic methods often have shortcomings such as limited substrate range and narrow applicability, which make it difficult to meet the needs of synthesizing pyrazoline derivatives with diverse structures. Therefore, developing new synthetic methods that effectively construct structurally diverse pyrazole derivatives based on simple and readily available raw materials is still a topic worth studying in organic synthetic chemistry. Summary of the invention

[0006] The purpose of the present invention is to provide a polysubstituted pyrazoline compound and a synthesis method thereof, so as to solve the technical problem that the synthesis method in the prior art often has the disadvantages of limited substrate range, narrow application range, etc., and is difficult to meet the synthesis needs of pyrazoline derivatives with diverse structures.

[0007] The invention discloses a method for synthesizing a polysubstituted pyrazoline compound. In an organic solvent, a substrate hydrazone, vinyl isoxazole and a base are added at room temperature for complete reaction, and then separated and purified to obtain a condensed ring 1,3,4,5-tetrasubstituted pyrazoline compound.

[0008] Further, the hydrazone structural formula is

[0009] Wherein, R1 is a benzene ring, a naphthalene ring or a thiophene ring substituted with chlorine, fluorine, bromine and methoxy at different positions;

[0010] R2 is a benzene ring substituted with chlorine, fluorine, methyl and methoxy at different positions.

[0011] Further, the structural formula of the vinyl isoxazole is

[0012] R3 is a benzene ring substituted with fluorine, bromine, chlorine, methyl or methoxy at different positions.

[0013] Furthermore, the organic solvent is selected from the following solvents or combinations of solvents: tetrahydrofuran, ethyl acetate, acetonitrile, dichloromethane, ethanol, preferably dichloromethane, which is low in toxicity and easy to handle.

[0014] Furthermore, the base is selected from Et3N, Cs2CO3, K2CO3, DBU or DIPEA.

[0015] Furthermore, the base is Et3N.

[0016] Furthermore, the molar ratio of the hydrazone, vinyl isoxazole and base is 1:1.2:0.5-1:1.2:2.5.

[0017] Furthermore, the reaction temperature in the synthesis method is 20-40 degrees Celsius; preferably the reaction temperature is room temperature.

[0018] The room temperature described in the present invention is 20-30°C.

[0019] Furthermore, the synthesis method also includes a purification step. Preferably, the purification step includes column chromatography and recrystallization.

[0020] The reaction process of the present invention is shown in equation (A):

[0021] Equation (A):

[0022]

[0023] The second object of the present invention is to protect a polysubstituted pyrazoline compound, which is prepared by the above method and has the structural formula

[0024] Wherein, R1 is a benzene ring, a naphthalene ring or a thiophene ring substituted with chlorine, fluorine, bromine and methoxy at different positions;

[0025] R2 is a benzene ring substituted with chlorine, fluorine, methyl and methoxy at different positions;

[0026] R3 is a benzene ring substituted with fluorine, bromine, chlorine, methyl or methoxy at different positions.

[0027] Further, selected from the following compounds:

[0028]

[0029] The third object of the present invention is to protect the application of a polysubstituted pyrazoline compound for the preparation of antitumor drugs.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1. The invention has a scientific design, ingenious conception, simple method, and easy operation. The method of the invention can specifically synthesize a new pyrazoline compound with multiple substituents, and has fewer by-products than the traditional method, is easy to purify, and has a very high yield. It solves the limitations of the known production methods in synthesizing such pyrazoline compounds, and proves the practicality of this method;

[0032] 2. For the synthetic product 1,3,4,5-tetrasubstituted pyrazoline compounds, the nitro group on the 5-position isoxazolidinone ring can be structurally modified to introduce a target functional group to have different biological activities; 1,3,4,5-tetrasubstituted pyrazole compounds can also be obtained by MnO2 oxidation to have more different pharmacological activities; and another 1,3,4-triaryl pyrazole compound can also be obtained by eliminating the 5-position isoxazolidinone ring by SnCl2. Therefore, the polysubstituted pyrazoline compounds of the present invention have great application prospects in the field of drug molecules;

[0033] 3. The raw materials of the present invention are cheap and easily available, which effectively reduces the reaction cost. The solvent is non-toxic and harmless, does not require metal catalysts, is green and environmentally friendly, does not require the participation of transition metals, and has a wide range of substrate applications. The reaction conditions are simple, and no heating or inert gas protection is required. The target compound can be obtained in high yield in air at room temperature, providing a new route option for the synthesis of active molecules containing such a skeleton, and has a good prospect for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is the hydrogen spectrum of the compound of formula III-1 prepared in Example 1;

[0035] Figure 2 The carbon spectrum of the compound of formula III-1 prepared in Example 1;

[0036] Figure 3 is the hydrogen spectrum of the compound of formula III-2 prepared in Example 2;

[0037] Figure 4 The carbon spectrum of the compound of formula III-2 prepared in Example 2;

[0038] Figure 5 The specific pharmacological activities of compounds III-1, III-2 and III-3 in Example 11. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments.

[0040] Example 1

[0041] Using N-phenylbenzoylhydrazone chloride and styrylisoxazole as standard substrates, the base-catalyzed

[0042] The reaction conditions for the synthesis of polysubstituted pyrazole compounds were studied:

[0043]

[0044]

[0045] Wherein, "molar ratio" means I:II; "equiv" means equivalent; temperature "rt" means room temperature; a means anhydrous and oxygen-free conditions;

[0046] Preparation of 3-methyl-4-nitro-5-(1,3,4-triphenyl-4,5-dihydro-1H-pyrazol-5-yl)isoxazole (Ⅲ-1)

[0047]

[0048] A 10 mL reaction tube and a magnetic stirrer were dried in advance, and then 100 mg N-phenylbenzohydrazone chloride (0.435 mmol) and 120.5 ul Et3N (0.869 mmol) were added to the reaction tube respectively, and then 2.2 mL of dichloromethane solvent was added, and finally 120.0 mg of styrylisoxazole (0.522 mmol) was added, and the whole reaction was reacted at room temperature, and the whole reaction process was monitored by TLC. After the reaction was completed, the solvent was concentrated, and the crude product was separated by column chromatography using a mixed solution of petroleum ether and ethyl acetate (30 / 1, v / v) as an eluent and 300-400 mesh silica gel as a separation resin to obtain 3-methyl-4-nitro-5-(1,3,4-triphenyl-4,5-dihydro-1H-pyrazol-5-yl)isoxazole (92.0 mg, yellow solid), with a yield of 99.80%;

[0049] Ⅲ-1 Compound: 1 H NMR (600MHz, CDCl3) δ7.67 (dd, J=7.9, 1.8Hz, 2H), 7.33 (d, J=6.8Hz, 2H), 7.32–7.21 (m, 8H) ,7.14–7.09(m,2H),6.88(s,1H),6.07(d,J=3.2Hz,1H),4.76(d,J=3.3Hz,1H),2.54(s,3H). 13 C NMR (151MHz, CDCl3) δ169.84,153.48,147.22,141.08,135.50,128.65,127.23,127 .15,126.74,126.25,126.12,125.14,124.20,118.32,111.09,62.93,56.80,9.25.

[0050] Example 2

[0051] Preparation of 5-(4-(4-bromophenyl)-1,3-diphenyl-4,5-dihydro-1H-pyrazol-5-yl)-3-methyl-4-nitroisoxazole (III-2):

[0052]

[0053] A 10 mL reaction tube and a magnetic stirrer were dried in advance, and then 100 mg N-phenylbenzohydrazone chloride (0.435 mmol) and 120.5 ul Et3N (0.869 mmol) were added to the reaction tube respectively, and then 2.2 mL of dichloromethane solvent was added, and finally 160.7 mg of p-bromophenylvinylisoxazole (0.522 mmol) was added. The whole reaction was allowed to react at room temperature, and the whole reaction process was monitored by TLC. After the reaction was completed, the solvent was concentrated, and the crude product was separated by column chromatography using a mixed solution of petroleum ether and ethyl acetate (30 / 1, v / v) as an eluent and 300-400 mesh silica gel as a separation resin to obtain 5-(4-(4-bromophenyl)-1,3-diphenyl-4,5-dihydro-1H-pyrazol-5-yl)-3-methyl-4-nitroisoxazole (109.11 mg, yellow solid), with a yield of 98.80%;

[0054] III-2 Compounds: 1 H NMR(600MHz, CDCl3)δ7.63(dd,J=7.6,2.2Hz,2H),7.50–7.44(m,2H),7.31–7.27(m,3H),7.27–7.23(m,2H),7.20– 7.15(m,2H),7.13–7.07(m,2H),6.89(d,J=1.0Hz,1H),6.03(d,J=3.1Hz,1H),4.72(d,J=3.1Hz,1H),2.54(s,3H). 13 C NMR (151MHz, CDCl3) δ171.9,155.8,149.2,143.2,136.7,132.7,130.7,130. 1,129.5,129.3,129.2,128.7,126.4,122.6,120.9,113.5,65.1,58.4,11.6.

[0055] Example 3

[0056] Preparation of 5-(1,4-diphenyl-3-(thiophene-2-yl)-4,5-dihydro-1H-pyrazol-5-yl)-3-methyl-4-nitroisoxazole (III-3):

[0057]

[0058] A 10 mL reaction tube and a magnetic stirrer were dried in advance, and then 100 mg of N-phenylthiophene-2-carbazone chloride (0.42 mmol) and 117.46 ul of Et3N (0.847 mmol) were added to the reaction tube respectively, and then 2.1 mL of dichloromethane solvent was added, and finally 115.96 mg of styrylisoxazole (0.504 mmol) was added, and the entire reaction was reacted at room temperature, and the entire reaction process was monitored by TLC. After the reaction was completed, the solvent was concentrated, and the crude product was separated by column chromatography using a mixed solution of petroleum ether and ethyl acetate (20 / 1, v / v) as an eluent and 300-400 mesh silica gel as a separation resin to obtain 5-(1,4-diphenyl-3-(thiophene-2-yl)-4,5-dihydro-1H-pyrazol-5-yl)-3-methyl-4-nitroisoxazole (90.51 mg, brown solid), with a yield of 99.32%;

[0059] Ⅲ-3 compounds: 1 H NMR(600MHz, CDCl3)δ7.37–7.31(m,3H),7.31–7.28(m,2H),7.28–7.21(m,3H),7.11–7.05 (m,2H),6.86(d,J=4.4Hz,3H),6.05(d,J=4.0Hz,1H),4.72(d,J=3.9Hz,1H),2.55(s,3H). 13 C NMR (151MHz, CDCl3) δ171.86,155.87,145.84,143.33,137.77,134.68,129.56,129 .47,128.62,127.53,127.41,127.25,127.22,120.78,113.53,65.47,60.19,11.60.

[0060] Example 4

[0061] Preparation of 5-(1-(4-methoxyphenyl)-3-(naphthalen-2-yl)-4-phenyl-4,5-dihydro-1H-pyrazol-5-yl)-3-methyl-4-nitroisoxazole (Ⅲ-4)

[0062]

[0063] A 10 mL reaction tube and a magnetic stirrer were dried in advance, and then 100 mg of N-(4-methoxyphenyl)-2-naphthylhydrazone chloride (0.323 mmol) and 89.40 ul of Et3N (0.645mmol), then 2.1mL of dichloromethane solvent was added, and finally 89.03mg of styryl isoxazole (0.39mmol) was added, and the whole reaction was allowed to react at room temperature. TLC monitored the whole reaction process. After the reaction was completed, the solvent was concentrated, and the crude product was separated by column chromatography using a mixed solution of petroleum ether and ethyl acetate (10 / 1, v / v) as an eluent and 300-400 mesh silica gel as a separation resin to obtain 5-(1-(4-methoxyphenyl)-3-(naphthalen-2-yl)-4-phenyl-4,5-dihydro-1H-pyrazol-5-yl)-3-methyl-4-nitroisoxazole (79.44mg, red solid), with a yield of 97.66%;

[0064] III-4: 1 H NMR(600MHz, CDCl3)δ8.11(dd,J=8.8,1.6Hz,1H),7.83–7.72(m,3H),7.66–7.62(m,1H),7.44–7.32(m,6H),7.28(s,1H ),7.14–7.09(m,2H),6.83(d,J=8.8Hz,2H),6.09(dd,J=3.3,1.2Hz,1H),4.95–4.91(m,1H),3.74(s,3H),2.53(s,3H). 13 C NMR (151MHz, CDCl3) δ172.29,155.76,154.43,149.32,138.03,137.74,133.44,133.07,130.25,129.57,128.72,128 .42,128.34,128.25,127.76,127.55,126.61,126.44,125.99,123.98,115.41,114.87,66.23,59.02,55.64,11.60.

[0065] Example 5

[0066] Preparation of 5-(3-(4-fluorophenyl)-1-mesityl-4-phenyl-4,5-dihydro-1H-pyrazol-5-yl)-3-methyl-4-nitroisoxazole (Ⅲ-5)

[0067]

[0068] A 10 mL reaction tube and a magnetic stirrer were dried in advance, and then 100 mg 3-fluoro-N-mesitylphenylbenzohydrazone chloride (0.344 mmol) and 95.57 ul Et3N (0.689 mmol) were added to the reaction tube respectively, and then 1.7 mL dichloromethane solvent was added, and finally 95.17 mg styrylisoxazole (0.414 mmol) was added, and the whole reaction was reacted at room temperature, and the whole reaction process was monitored by TLC. After the reaction was completed, the solvent was concentrated, and the crude product was separated by column chromatography using a mixed solution of petroleum ether and ethyl acetate (10 / 1, v / v) as an eluent and 300-400 mesh silica gel as a separation resin to obtain 5-(3-(4-fluorophenyl)-1-mesityl-4-phenyl-4,5-dihydro-1H-pyrazol-5-yl)-3-methyl-4-nitroisoxazole (73.23 mg, yellow solid), with a yield of 87.48%;

[0069] Ⅲ-5 Compound: 1 H NMR (600MHz, CDCl3) δ7.56 (dd, J=8.9, 5.4Hz, 2H), 7.41–7.30 (m, 5H), 6.95 (s, 2H), 6.78 (s ,2H),5.93(d,J=6.5Hz,1H),5.19(d,J=6.5Hz,1H),2.49(s,3H),2.22(s,3H),2.14(s,6H). 13 C NMR (151MHz, CDCl3) δ169.51, 162.68 (d, J = 248.7Hz), 155.94, 145.89, 138.22, 137.22, 136.28, 130.8 3,129.84,129.58,128.29,127.86,127.80,115.57(d,J=21.9Hz),67.38,56.32,20.89,18.39,11.55. 19 F NMR (565MHz,CDCl3)δ-112.53.

[0070] Example 6

[0071] Preparation of 5-(3-(3-chlorophenyl)-1,4-bis(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-5-yl)-3-methyl-4-nitroisoxazole (Ⅲ-6)

[0072]

[0073] A 10 mL reaction tube and a magnetic stirrer were dried in advance, and then 100 mg of 3-chloro-N-(4-methoxyphenyl)benzohydrazone chloride (0.340 mmol) and 94.29 ul of Et3N (0.680mmol), then 1.7mL of dichloromethane solvent was added, and finally 106.14mg of p-methoxyphenylphenylisoxazole (0.41mmol) was added, and the whole reaction was allowed to react at room temperature. TLC monitored the whole reaction process. After the reaction was completed, the solvent was concentrated, and the crude product was separated by column chromatography using a mixed solution of petroleum ether and ethyl acetate (10 / 1, v / v) as an eluent and 300-400 mesh silica gel as a separation resin to obtain 5-(3-(3-chlorophenyl)-1,4-bis(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-5-yl)-3-methyl-4-nitroisoxazole (74.30mg, red solid), with a yield of 94.77%;

[0074] III-6: 1 H NMR(600MHz, CDCl3)δ7.74(t,J=1.82Hz,1H),7.44–7.39(m,1H),7.29–7.23(m,1H),7.23–7.16(m,2H),7.09–7.0 3(m,2H),6.88–6.78(m,5H),6.06(d,J=3.52Hz,1H),4.71(d,J=3.51Hz,1H),3.74(d,J=8.42Hz,6H),2.52(s,3H). 13 C NMR (151MHz, CDCl3) δ171.9,160.4,155.8,154.6,147.6,139.0,137.4,134.6,133.0,130.7,130.2, 129.8,128.7,126.2,124.4,119.7,115.5,114.8,113.6,113.3,66.1,58.7,55.6,55.6,55.3,11.6.

[0075] Example 7

[0076] Preparation of 5-(3,4-diphenyl-1-(3-(trifluoromethyl)phenyl)-4,5-dihydro-1H-pyrazol-5-yl)-3-methyl-4-nitroisoxazole (Ⅲ-7)

[0077]

[0078] A 10 mL reaction tube and a magnetic stirrer were dried in advance, and then 100 mg of N-(3-(trifluoromethyl)phenyl)benzohydrazone chloride (0.340 mmol) and 93.02 ul of Et3N (0.680 mmol) were added to the reaction tube respectively, and then 1.7 mL of dichloromethane solvent was added, and finally 92.63 mg of styrylisoxazole (0.40 mmol) was added, and the whole reaction was reacted at room temperature, and the whole reaction process was monitored by TLC. After the reaction was completed, the solvent was concentrated, and the crude product was separated by column chromatography using a mixed solution of petroleum ether and ethyl acetate (30 / 1, v / v) as an eluent and 300-400 mesh silica gel as a separation resin to obtain 5-(3,4-diphenyl-1-(3-(trifluoromethyl)phenyl)-4,5-dihydro-1H-pyrazol-5-yl)-3-methyl-4-nitroisoxazole (71.77 mg, red solid), with a yield of 86.85%;

[0079] III-7 Compound: 1 H NMR(600MHz, CDCl3)δ7.69(dd,J=6.7,3.0Hz,2H),7.53(s,1H),7.38–7.34(m,2H),7.34–7 .27(m,7H),7.14–7.06(m,2H),6.08(d,J=2.9Hz,1H),4.80(d,J=3.0Hz,1H),2.56(s,3H). 13 C NMR (151MHz, CDCl3) δ171.36,155.95,150.87,143.63,137.39,131.93(q,J=32.2Hz),130.49,130.24,129.96,129.68,129.55,1 28.67,128.65,127.41,126.76,124.05(q,J=272.5Hz),116.95(q,J=3.9Hz),115.69,110.52(q,J=4.1Hz),64.97,59.31,11.56. 19 F NMR (565MHz,CDCl3)δ-62.78.

[0080] Example 8

[0081] Preparation of 3-methyl-4-nitro-5-(3-(3-nitrophenyl)-1,4-diphenyl-4,5-dihydro-1H-pyrazol-5-yl)isoxazole (Ⅲ-8)

[0082]

[0083] A 10 mL reaction tube and a magnetic stirrer were dried in advance, and then 100 mg 3-nitro-N-phenylbenzohydrazone chloride (0.364 mmol) and 100.80 ul Et3N (0.727 mmol) were added to the reaction tube respectively, and then 1.8 mL dichloromethane solvent was added, and finally 100.38 mg styrylisoxazole (0.44 mmol) was added, and the whole reaction was reacted at room temperature, and the whole reaction process was monitored by TLC. After the reaction was completed, the solvent was concentrated, and the crude product was separated by column chromatography using a mixed solution of petroleum ether and ethyl acetate (20 / 1, v / v) as an eluent and 300-400 mesh silica gel as a separation resin to obtain 3-methyl-4-nitro-5-(3-(3-nitrophenyl)-1,4-diphenyl-4,5-dihydro-1H-pyrazol-5-yl)isoxazole (76.91 mg, red solid), with a yield of 90.17%;

[0084] III-8 Compound: 1 H NMR (600MHz, CDCl3) δ8.48(s,1H),8.08(ddd,J=8.2,2.2,1.0Hz,1H),7.96(dt,J=7.9,1.3Hz,1H),7.45(t,J=8.0Hz,1H),7.3 9–7.32(m,3H),7.31–7.26(m,4H),7.17–7.12(m,2H),6.93(s,1H),6.15(d,J=3.5Hz,1H),4.82(d,J=3.5Hz,1H),2.56(s,3H). 13 C NMR (151MHz, CDCl3) δ170.60,155.17,147.68,146.39,141.89,136.26,132.01,130.97,129.30, 129.03,128.81,128.80,128.08,126.55,122.41,120.61,120.24,112.84,64.70,57.89,10.76.

[0085] Example 9

[0086] Preparation of 5-(1,3-diphenyl-4-(p-tolyl)-4,5-dihydro-1H-pyrazol-5-yl)-3-methyl-4-nitroisoxazole (Ⅲ-9)

[0087]

[0088] A 10 mL reaction tube and a magnetic stirrer were dried in advance, and then 100 mg N-phenylbenzohydrazone chloride (0.435 mmol) and 120.51 ul Et3N (0.870 mmol) were added to the reaction tube respectively, and then 2.2 mL of dichloromethane solvent was added, and finally 127.32 mg of p-methylphenylisoxazole (0.52 mmol) was added, and the whole reaction was allowed to react at room temperature. TLC monitored the whole reaction process. After the reaction was completed, the solvent was concentrated, and the crude product was separated by column chromatography using a mixed solution of petroleum ether and ethyl acetate (30 / 1, v / v) as an eluent and 300-400 mesh silica gel as a separation resin to obtain 5-(1,3-diphenyl-4-(p-tolyl)-4,5-dihydro-1H-pyrazol-5-yl)-3-methyl-4-nitroisoxazole (95.23 mg, red solid), with a yield of 82.17%;

[0089] III-9 compound: 1 H NMR (600MHz, CDCl3) δ7.69–7.64(m,2H),7.32–7.21(m,5H),7.20–7.01(m,6H),6. 87(s,1H),6.04(d,J=3.3Hz,1H),4.73(d,J=3.2Hz,1H),2.54(s,3H),2.30(s,3H). 13 C NMR (151MHz, CDCl3) δ172.3,155.8,149.7,143.5,138.3,134.9,131.1,130.2,1 30.0,129.5,129.0,128.6,127.4,126.6,120.6,113.4,65.4,58.9,21.2,11.6.

[0090] Example 10

[0091] Preparation of 3-methyl-4-nitro-5-(4-(4-nitrophenyl)-1,3-diphenyl-4,5-dihydro-1H-pyrazol-5-yl)isoxazole (Ⅲ-10)

[0092]

[0093] A 10 mL reaction tube and a magnetic stirrer were dried in advance, and then 100 mg N-phenylbenzohydrazone chloride (0.435 mmol) and 120.51 ul Et3N (0.870 mmol) were added to the reaction tube respectively, and then 2.2 mL of dichloromethane solvent was added, and finally 143.47 mg of p-nitrostyrylisoxazole (0.52 mmol) was added. The whole reaction was allowed to react at room temperature, and the whole reaction process was monitored by TLC. After the reaction was completed, the solvent was concentrated, and the crude product was separated by column chromatography using a mixed solution of petroleum ether and ethyl acetate (20 / 1, v / v) as an eluent and 300-400 mesh silica gel as a separation resin to obtain 3-methyl-4-nitro-5-(4-(4-nitrophenyl)-1,3-diphenyl-4,5-dihydro-1H-pyrazol-5-yl)isoxazole (101.96 mg, red solid), with a yield of 90.23%;

[0094] III-10 Compound: 1 H NMR (600MHz, CDCl3) δ8.24–8.19(m,2H),7.66–7.58(m,2H),7.53–7.47(m,2H),7.32–7.24(m,4H),7 .14–7.09(m,2H),6.92(d,J=1.2Hz,1H),6.07(d,J=3.0Hz,1H),4.86(d,J=3.0Hz,1H),2.56(s,3H). 13 C NMR (151MHz, CDCl3) δ171.4,155.9,148.6,147.9,144.6,143.0,130.3,129.6,129.5,128.8,128.6,126.3,124.9,121.2,113.6,64.9,58.3,11.5.

[0095] Experimental Example 11

[0096]

[0097]

[0098] When the active double bond substrate II-1 was replaced by IV-1, the reaction could not proceed and the expected compound could not be obtained.

[0099] Experimental Example 12 In vitro activity experiment

[0100] 12.1 In vitro antitumor activity of pyrazoline compounds

[0101] Material

[0102] 3-Methyl-4-nitro-5-(1,3,4-triphenyl-4,5-dihydro-1H-pyrazol-5-yl)isoxazole (III-1), 5-(4-(4-bromophenyl)-1,3-diphenyl-4,5-dihydro-1H-pyrazol-5-yl)-3-methyl-4-nitroisoxazole (III-2), and 5-(1,4-diphenyl-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-5-yl)-3-methyl-4-nitroisoxazole (III-3) were prepared from Example 1, Example 2, and Example 3, respectively. Trypsin, MTT (Biofroxx, Germany); dimethyl sulfoxide (DMSO) (MPBiomedicals, France); fetal bovine serum (FBS) (Newzerum, New Zealand); DMEM medium, RPMI1640 medium, culture dishes, 96-well plates (Corning, USA); colon cancer cells HCT116, breast cancer cells MDA-MB-231, non-small cell lung cancer cells A549, and liver cancer cells HepG2 were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences and preserved in our laboratory.

[0103] Cell recovery

[0104] Take out the tumor cell lines HCT116, MDA-MB-231, A549, and HepG2 from the liquid nitrogen tank, place them in a 37°C water bath, and after the frozen cells are thawed into a cell suspension, centrifuge at 1000r / min for 3 minutes, discard the supernatant, add 1ml of fresh complete culture medium containing 10% fetal bovine serum and 1% double antibody to mix the cells, then add 5ml of culture medium, gently blow the cells repeatedly with a pipette to mix them, and finally transfer the cell suspension to a 10mm culture dish, mark the cell name, cell generation, recovery date and operator's name, and place it in a cell culture incubator for culture. (HCT116 and HepG2 cell lines are cultured in DMEM culture medium, and MDA-MB-231 cell lines and A549 cell lines are cultured in RPMI1640 culture medium)

[0105] Cell passaging

[0106] HCT116, MDA-MB-231, A549, and HepG2 cells with good growth status were taken out, the culture medium was removed, 1 ml of sterile PBS was added to wash, 1.5 ml of trypsin was added to digest the cells, and the cells became round and fell off after about 2 minutes. 1.5 ml of culture medium was added to terminate the digestion, and the cells were transferred to a centrifuge tube, centrifuged at 1000 r / min for 3 minutes, the supernatant was discarded, 1 ml of fresh culture medium was added to resuspend, and fresh culture medium was added to divide the cells into other culture dishes at a ratio of 1:3, and the passage date and generation number were marked, and the cells were placed in a cell culture incubator for culture.

[0107] Determination of the antitumor activity of pyrazoline compounds in vitro by MTT assay

[0108] Take HCT116, MDA-MB-231, A549, and HepG2 cells with good growth status and perform the same cell passage experiment. After resuspending the cells, count the cells with a hemocytometer and plant them in a 96-well plate at 2500 cells / well. After culturing the cells for 24 hours, remove the original culture medium and add culture medium containing different concentrations of drugs to continue culturing for 48 hours. After the drug effect ends, remove the original culture medium, add 100μl of fresh culture medium, add 10μl of MTT solution, place incubator and incubate for 2 hours, add 150ml DMSO, incubate on a shaker for 15 minutes, measure the OD value at 490nm with an enzyme marker, and calculate the cell proliferation inhibition rate. Repeat the above steps three times.

[0109] Experimental Results

[0110] The IC50 values ​​of Ⅲ-1 against colon cancer cells HCT116, breast cancer cells MDA-MB-231, lung adenocarcinoma cells A549, and liver cancer cells HepG2 were 37.67±0.34μM, 46.00±0.52μM, 25.67±0.31μM, and 26.22±0.51μM, respectively; the IC50 values ​​of Ⅲ-2 against colon cancer cells HC T116, breast cancer cells MDA-MB-231, lung adenocarcinoma cells A549, and liver cancer cells Hep The IC50 of G2 were 29.33±0.24μM, 51.67±0.56μM, 32.33±0.39μM and 22.67±0.17μM respectively; the IC50 of Ⅲ-3 for colon cancer cells HCT116, breast cancer cells MDA-MB-231, lung adenocarcinoma cells A549 and liver cancer cells HepG2 were 17.01±0.32μM, 21.03±0.44μM, 29.67±0.11μM and 5.33±0.18μM respectively.

[0111] At all experimental concentrations, the three compounds had significant growth inhibition effects on various tumor cells. The above data prove that pyrazoline compounds have good anti-tumor effects in vitro.

[0112] The above are the implementation methods listed in this embodiment, but this embodiment is not limited to the above optional implementation methods. Those skilled in the art can arbitrarily combine the above methods to obtain other various implementation methods. Anyone can derive other various forms of implementation methods under the inspiration of this embodiment. The above specific implementation methods should not be understood as limiting the scope of protection of this embodiment. The scope of protection of this embodiment shall be based on the definition in the claims, and the description can be used to interpret the claims.

Claims

1. A method for synthesizing a polysubstituted pyrazoline compound, characterized in that: In an organic solvent, a substrate hydrazone, vinyl isoxazole and a base are added at room temperature and reacted completely, followed by separation and purification to obtain a 1,3,4,5-tetrasubstituted pyrazoline compound having the structural formula: ; The hydrazone structural formula is ; Wherein, R1 is a benzene ring, a naphthalene ring or a thiophene ring substituted with chlorine, fluorine, bromine or methoxy at different positions; R2 is a benzene ring substituted with chlorine, fluorine, methyl or methoxy at different positions; The structural formula of the vinyl isoxazole is ; R3 is a benzene ring substituted with fluorine, bromine, chlorine, methyl or methoxy at different positions.

2. A method for synthesizing a polysubstituted pyrazoline compound according to claim 1, characterized in that : The organic solvent is selected from the following solvents or a combination of solvents: tetrahydrofuran, ethyl acetate, acetonitrile, dichloromethane, and ethanol.

3. A method for synthesizing a polysubstituted pyrazoline compound according to claim 2, characterized in that : The organic solvent is selected from the following solvents: dichloromethane.

4. A method for synthesizing a polysubstituted pyrazoline compound according to claim 1, characterized in that : The base is selected from Et3N, Cs2CO3, K2CO3, DBU or DIPEA.

5. A method for synthesizing a polysubstituted pyrazoline compound according to claim 4, characterized in that : The base is selected from Et3N.

6. A method for synthesizing a polysubstituted pyrazoline compound according to claim 1, characterized in that :The molar ratio of the hydrazone, vinyl isoxazole and base is 1:1.2:0.5-1:1.2:2.

5.

7. A method for synthesizing a polysubstituted pyrazoline compound according to claim 1, characterized in that : The reaction temperature in the synthesis method is 20-40 degrees Celsius.

8. A method for synthesizing a polysubstituted pyrazoline compound according to claim 7, characterized in that : The reaction temperature in the synthesis method is room temperature.

9. A method for synthesizing a polysubstituted pyrazoline compound according to claim 1, characterized in that : The synthetic method also includes a refining step.

10. A method for synthesizing a polysubstituted pyrazoline compound according to claim 9, characterized in that : The refining step includes column chromatography and recrystallization.

11. A polysubstituted pyrazoline compound, characterized in that : Obtained by the synthesis method of a polysubstituted pyrazoline compound according to any one of claims 1 to 10, the structural formula of which is ; Wherein, R1 is a benzene ring, a naphthalene ring or a thiophene ring substituted with chlorine, fluorine, bromine or methoxy at different positions; R2 is a benzene ring substituted with chlorine, fluorine, methyl or methoxy at different positions; R3 is a benzene ring substituted with fluorine, bromine, chlorine, methyl or methoxy at different positions.

12. An application of a polysubstituted pyrazoline compound, characterized in that : The compound obtained by the synthesis method of a polysubstituted pyrazoline compound according to any one of claims 1 to 10 and the compound according to claim 11 are used for the preparation of drugs for treating colon cancer, breast cancer, lung adenocarcinoma or liver cancer.

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