Quinoxaline compound, preparation method and application

Abstract

The invention discloses a quinoxaline compound as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The structural formula of the compound is # imgabs0, in the formula, R1 is alkyl or allyl, and R2 is alkyl; or R1 is alkyl, and R2 is carboxyl. According to the invention, a quinoxaline skeleton is subjected to structural modification and transformation, a series of novel compounds with antiviral activity are provided, or powerful candidate substances are provided for new drug research and development of topoisomerase inhibitors. The method is suitable for preparing antiviral, antibacterial, anti-inflammatory or anti-tumor preparations.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to a novel quinoxaline compound, specifically a quinoxaline compound, a preparation method and an application thereof. Background Art

[0002] Cancer is a serious threat to human health. Early anti-cancer drugs were mostly cytotoxic, unable to effectively distinguish cancer cells from normal cells and resulting in severe toxic side effects. Consequently, in recent decades, research on anti-cancer drugs has gradually shifted towards drugs that target specific cancer cell targets. Targeted drugs can specifically bind to targets on cancer cells, increasing selectivity for tumor cells and thus effectively reducing the toxic side effects of chemotherapy. Topoisomerase (Topo) is one of the most widely studied anti-cancer drug targets. Topoisomerase is involved in regulating the topological structure of DNA during replication and transcription, maintaining the homeostasis of DNA within the cell. Studies have found that the content and activity of Topo in tumor cells are much higher than in normal somatic cells. Topoisomerase inhibitors are a class of compounds that inhibit the activity of DNA topoisomerase and are cytotoxic drugs. By inhibiting Topo activity, they can prevent the rapid proliferation of tumor cells and induce apoptosis. Quinoxaline-based topoisomerase inhibitors are currently a hot topic in anti-cancer drug research. Quinoxaline topoisomerase inhibitors have the advantages of good efficacy and broad anti-tumor spectrum. Practice has shown that they have high activity and good selectivity against various solid tumors. Therefore, the development of new quinoxaline topoisomerase inhibitors is of great significance.

[0003] Quinoxaline structures possess a wide range of biological activities and serve as the core framework for the development of numerous innovative drugs. Quinoxaline and its derivatives possess diverse pharmacological activities, including antibacterial, anticancer, anti-inflammatory, and antiviral properties, and are widely used in biopharmaceuticals, industrial chemistry, and organic chemistry. Therefore, the development of novel quinoxaline compounds is of great significance and may also provide opportunities for the development of novel topoisomerase inhibitors. Summary of the Invention

[0004] The purpose of the present invention is to provide a quinoxaline compound, preparation method and application, and to provide a compound with better efficacy by structurally modifying and transforming the quinoxaline skeleton, which may provide a new candidate substance for the development of new anti-tumor drugs.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] A quinoxaline compound having the structural formula

[0007] In the formula, R1 is an alkyl group or an allyl group; R2 is an alkyl group.

[0008] Another quinoxaline compound has the structural formula

[0009] In the formula, R1 is an alkyl group; R2 is a carboxyl group.

[0010] As a limitation, the alkyl group is methyl.

[0011] The present invention also provides a method for preparing the above-mentioned quinoxaline compound, comprising the following steps:

[0012] S1. o-phenylenediamine and diethyl oxalate are condensed to obtain intermediate A1;

[0013] S2. Intermediate A1 is subjected to bromination reaction with phosphorus oxybromide to obtain intermediate A2;

[0014] S3. Intermediate A2 and an allylamine compound undergo nucleophilic substitution reaction to obtain a third intermediate;

[0015] S4. The third intermediate and tris(dibenzylideneacetone)dipalladium, tri(o-methylphenyl)phosphine and triethylamine are subjected to an intramolecular Heck cyclization reaction to obtain the quinoxaline compound;

[0016] The structural formula of the intermediate A1 is The structural formula of intermediate A2 is

[0017]

[0018] The allylamine compound includes N-allylmethylamine or diallylamine.

[0019] As a limitation, the condensation reaction is carried out at reflux temperature for 1.5 to 2 hours;

[0020] The bromination reaction is carried out under the following conditions: acetonitrile solvent, N,N-dimethylformamide as catalyst, reflux temperature, and time of 1.5 to 2.5 hours.

[0021] As another limitation, the molar ratio of the intermediate A2 and N-allylmethylamine is 1:2, and the conditions for the nucleophilic substitution reaction are: in tetrahydrofuran solvent, at reflux temperature, for 4.0 to 4.5 hours;

[0022] The molar ratio of the intermediate A2 and diallylamine is 1:6, and the conditions for the nucleophilic substitution reaction are: in 1,4-dioxane solvent, the temperature is reflux temperature, and the time is 15.0 to 15.5 hours.

[0023] As a further limitation, the intramolecular Heck cyclization reaction is carried out in N-methylpyrrolidone as solvent, at a temperature of 110 to 120° C., for 4.0 to 4.5 hours.

[0024] The present invention also provides another method for preparing a quinoxaline compound, wherein the quinoxaline compound is It is Obtained by oxidation reaction.

[0025] As a limitation, in the oxidation reaction, the oxidant is potassium permanganate, the phase transfer catalyst is dicyclohexyl-18-crown-6, the solvent is water, and the temperature is room temperature.

[0026] The present invention also provides a use of the above-mentioned quinoxaline compound, wherein the quinoxaline compound is used to prepare an antiviral, antibacterial, anti-inflammatory or antitumor preparation.

[0027] Due to the adoption of the above technical solution, the present invention has the following technical advancements compared with the prior art:

[0028] ① The present invention provides a quinoxaline compound, which, by structurally modifying and remodeling the quinoxaline skeleton, provides a series of novel compounds with antiviral activity, which may provide strong candidate substances for the development of new drugs for topoisomerase inhibitors;

[0029] ② The present invention provides a method for preparing quinoxaline compounds, which achieves efficient synthesis of the target compound through specific catalyst and reaction condition control, mild reaction conditions and simple operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the structural detection result of product B in Example 3, wherein Figure 1 A is the hydrogen nuclear magnetic spectrum, Figure 1 B is the carbon NMR spectrum, Figure 1 C is the high-resolution mass spectrum;

[0031] Figure 2 is the structural detection result of product C in Example 4, wherein Figure 2 A is the hydrogen nuclear magnetic spectrum, Figure 2 B is the carbon NMR spectrum, Figure 2 C is a high-resolution mass spectrum;

[0032] Figure 3 is the structural detection result of product G in Example 5, wherein Figure 3 A is the hydrogen nuclear magnetic spectrum, Figure 3 B is the carbon NMR spectrum, Figure 3 C is the high-resolution mass spectrum;

[0033] Figure 4is the structural detection result of product D in comparative example 1, wherein Figure 4 A is the hydrogen nuclear magnetic spectrum, Figure 4 B is the carbon NMR spectrum, Figure 4 C is the high-resolution mass spectrum;

[0034] Figure 5 The structural test results of product E in Comparative Example 2 are shown in FIG. Figure 5 A is the hydrogen nuclear magnetic spectrum, Figure 5 B is the carbon NMR spectrum, Figure 5 C is the high-resolution mass spectrum;

[0035] Figure 6 The structural test results of product F in comparative example 3 are shown in FIG. Figure 6 A is the hydrogen nuclear magnetic spectrum, Figure 6 B is the carbon NMR spectrum, Figure 6 C is the high-resolution mass spectrum;

[0036] Figure 7 Graph showing the antiviral test results of product B at different concentrations;

[0037] Figure 8 Figure 2 is the antiviral test result of product C at different concentrations;

[0038] Figure 9 Figure 2 is the antiviral test result of product G at different concentrations;

[0039] Figure 10 Figure 2 shows the cytotoxicity test results of products D, E and F. DETAILED DESCRIPTION

[0040] The present invention will be further described in detail below by way of specific examples. It should be understood that the described examples are only used to illustrate the present invention and are not intended to limit the present invention.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods in the art.

[0042] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0043] Example 1

[0044] This embodiment provides a quinoxaline compound, the structural formula of which is

[0045] In the formula, R1 can be optionally an alkyl group (such as methyl, ethyl or propyl, etc.) or an allyl group (-CH2-CH=CH2); R2 is an alkyl group, which can be optionally a methyl group. The quinoxaline compound can be used to prepare antiviral, antibacterial, anti-inflammatory or anti-tumor preparations.

[0046] Example 2

[0047] This embodiment provides a quinoxaline compound, the structural formula of which is

[0048] In the formula, R1 is an alkyl group, which can be methyl, ethyl or propyl, etc.; R2 is a carboxyl group (-COOH). The quinoxaline compound can be used to prepare antiviral, antibacterial, anti-inflammatory or anti-tumor preparations.

[0049] Example 3

[0050] This example prepares a quinoxaline compound, whose structural formula is Specifically, the following steps are performed in sequence:

[0051] S1. o-phenylenediamine and diethyl oxalate undergo condensation reaction to obtain intermediate A1

[0052] To a 500 mL single-necked flask, 12 g of o-phenylenediamine, 16.2 g of diethyl oxalate, and 2 mol / L HCl (324 mL) were added in sequence. The mixture was stirred magnetically and heated to maintain reflux for 1.5 h, resulting in the precipitation of a large amount of white flocculent solids. (Experimental results show that the condensation reaction time is 1.5 to 2 h, for example, 1.5 h, 1.6 h, 1.7 h, 1.8 h, 1.9 h, 2 h, etc., which can all react completely to form a white flocculent solid. This example shows the results of the reaction for 1.5 h.) The reaction progress was monitored by TLC, indicating that the raw materials reacted completely and product was generated. The reaction solution was cooled to room temperature, filtered, and the filter cake was washed with water. The filter cake was dried in a vacuum drying oven at 50° C. to obtain 16.14 g of light green flocculent crystals, i.e., intermediate A1.

[0053] Calculations show that the yield of intermediate A1 in the condensation reaction is 90%. Testing shows that the melting range of intermediate A1 is greater than 300°C.

[0054] The molecular structure of intermediate A1 was determined by H NMR spectroscopy, and the H NMR characterization data were as follows: 1 HNMR (500MHz, DMSO-d6) δ11.89 (s, 2H), 7.13–7.11 (m, 2H), 7.09–7.06 (m, 2H). Therefore, the structural formula of intermediate A1 is

[0055] S2. Intermediate A1 and phosphorus oxybromide undergo bromination reaction to obtain intermediate A2

[0056] Under nitrogen protection, 10g intermediate A1, 200mL acetonitrile and 53.8g tribromide phosphorus oxychloride were added to a 1000mL four-necked flask in sequence. After magnetic stirring for 10min, 14.7mL of N,N-dimethylformamide was dissolved in 5mL liquid acetonitrile solution and added dropwise to the reaction solution using a constant pressure dropping funnel. Heat and keep the reflux temperature for 2h (experimentally verified, the bromination reaction time is 1.5 to 2.5h, such as 1.5h, 1.7h, 1.9h, 2h, 2.3h, 2.5h, etc., and all can react to obtain a product. The present embodiment provides the result of the reaction for 2h). TLC was used to monitor the reaction progress. The raw material reaction was complete. After the reaction solution was cooled to room temperature, an ice-water mixture was slowly added, stirred at room temperature for 1h, and a large amount of white solid was separated out. The filter cake obtained was a white solid, and the filter cake was dried at room temperature for 3h in a vacuum drying oven to obtain 15.23g of white solid, i.e., intermediate A2.

[0057] Calculations show that the yield of intermediate A2 in the bromination reaction is 86%. Testing shows that the melting range of intermediate A2 is 160-161°C.

[0058] The molecular structure of intermediate A2 was determined by H NMR spectroscopy, and the H NMR characterization data were as follows: 1 HNMR (500MHz,CDCl3)δ8.04(q,J=3.5Hz,2H),7.82(q,J=3.5Hz,2H). Therefore, the structural formula of intermediate A2 is

[0059] S3. Intermediate A2 and N-allylmethylamine undergo nucleophilic substitution reaction to obtain intermediate A3

[0060] Under nitrogen protection, 15 g of intermediate A2, 7.41 g of N-allylmethylamine (the molar ratio of intermediate A2 to N-allylmethylamine is 1:2) and 170 mL of tetrahydrofuran were added to a 250 mL four-necked flask in sequence, and stirring was started. The mixture was heated and maintained at reflux for 4 h (experimental verification shows that the nucleophilic substitution reaction time is 4 to 4.5 h, for example, 4 h, 4.1 h, 4.2 h, 4.3 h, 4.4 h, 4.5 h, etc., which can complete the reaction and generate the product. This example shows the result of 4 h of reaction). The reaction progress was monitored by TLC. After the reaction, the reaction solution was cooled to room temperature and rotary evaporated to dryness to obtain a crude product of intermediate A3. The crude product was preliminarily purified by a 300-400 mesh silica gel chromatography column to obtain a light yellow-green liquid, i.e., intermediate A3.

[0061] According to calculation, the yield of intermediate A3 in the nucleophilic substitution reaction is 95%.

[0062] The molecular structure of intermediate A3 was determined by H NMR spectroscopy, and the H NMR characterization data were as follows: 1HNMR (400MHz,CDCl3)δ7.88(d,J=8.0Hz,1H),7.80(d,J=8.0Hz,1H),7.63(t,J=8.0Hz,1H),7.49(t,J=8.4Hz,1H),6.10–6.00(m,1H),5.38–5.27(m,2H),4.11(d,J=6.0Hz,2H),3.08(s,3H). Therefore, the structural formula of intermediate A3 is

[0063] S4. Intermediate A3 and tris(dibenzylideneacetone)dipalladium, tri(o-methylphenyl)phosphine and triethylamine undergo intramolecular Heck cyclization to obtain

[0064] Under nitrogen protection, intermediate A3 (1.8 mmol), tris(dibenzylideneacetone)dipalladium (0.09 mmol), tri(o-methylphenyl)phosphine (0.36 mmol), triethylamine (1.98 mmol) and N-methylpyrrolidone were added sequentially to a 50 mL four-necked flask, and heated at 110 ° C for 4 h (experimentally verified that the intramolecular Heck cyclization reaction can react completely to form a product at a temperature of 110-120 ° C and a time of 4-4.5 h, for example, heating at 113 ° C for 4 h, heating at 110 ° C for 4.1 h, heating at 115 ° C for 4.2 h, heating at 120 ° C for 4.3 h, heating at 117 ° C for 4.4 h, heating at 110 ° C for 4.5 h, etc. This example gives the result of heating at 110 ° C for 4 h). The reaction process was monitored by TLC, and it was seen that the product was generated and the raw material was reacted completely. The reaction solution was cooled to room temperature and transferred to a separatory funnel. Chloroform and water were added and the mixture was separated after shaking. The organic phase was a yellow oily liquid. The aqueous phase was washed with chloroform, and the organic phases were combined. The combined organic phases were washed twice with saturated brine. TLC monitoring showed that the organic phase was the product. The organic phase was dried over anhydrous magnesium sulfate and filtered to obtain a clear orange-red liquid. The crude product was obtained by rotary evaporation to dryness and purified by column chromatography to obtain product B.

[0065] Calculation showed that the yield of product B in the intramolecular Heck cyclization reaction was 81%.

[0066] The molecular structure of product B was determined by hydrogen nuclear magnetic resonance (H-NMR), carbon nuclear magnetic resonance (C-NMR) and high-resolution mass spectrometry. Figure 1 As shown;

[0067] Depend on Figure 1 A shows that the hydrogen nuclear magnetic characterization data of product B is: 1H NMR (500MHz, DMSO-d6) δ8.17(d,J=8.5Hz,1H),8.08(d,J=8.0Hz,1H),8.01(s,1H),7.73–7.66(m,2H),3.85(s,3H),2.38(s,3H);

[0068] Depend on Figure 1 B shows that the carbon NMR characterization data of product B is: 13 C NMR (101MHz, CDCl3) δ142.9,142.5,140.0,139.3,136.8,129.3,128.2,127.6,126.2,109.2,31.2,8.8;

[0069] Depend on Figure 1 C shows that the high-resolution mass spectrometry data of product B is: 198.1018 [M+H] + .

[0070] Therefore, the structural formula of product B is

[0071] Example 4

[0072] This example prepares a quinoxaline compound, whose structural formula is Specifically, the following steps are performed in sequence:

[0073] S1. o-phenylenediamine and diethyl oxalate undergo condensation reaction to obtain intermediate A1

[0074] The intermediate A1 was prepared by the method in Example 3, and its structural formula is

[0075] S2. Intermediate A1 and phosphorus oxybromide undergo bromination reaction to obtain intermediate A2

[0076] The intermediate A2 was prepared by the method in Example 3, and its structural formula is

[0077] S3. Intermediate A2 and diallylamine undergo nucleophilic substitution reaction to obtain intermediate A4

[0078] Under nitrogen protection, 2 g of intermediate A2, 3.38 g of diallylamine (the molar ratio of intermediate A2 to diallylamine is 1:6) and 40 mL of 1,4-dioxane were added sequentially to a 50 mL four-necked flask, and stirring was started. The mixture was heated and maintained at reflux for 15 h (experimental verification shows that the nucleophilic substitution reaction time is 15 to 15.5 h, for example, 15 h, 15.1 h, 15.2 h, 15.3 h, 15.4 h, 15.5 h, etc., which can complete the reaction and generate the product. This example shows the result of the reaction for 15 h). The reaction progress was monitored by TLC. After the reaction, the reaction solution was cooled to room temperature and rotary evaporated to dryness to obtain a crude product of intermediate A4. The crude product was preliminarily purified by passing it through a 300-400 mesh silica gel chromatography column to obtain an orange-yellow liquid, namely, intermediate A4.

[0079] According to calculation, the yield of intermediate A4 in the nucleophilic substitution reaction is 73%.

[0080] The molecular structure of intermediate A4 was determined by H NMR spectroscopy, and the H NMR characterization data were as follows: 1 HNMR (400MHz,CDCl3)δ7.88(d,J=8.0Hz,1H),7.80(d,J=8.4Hz,1H),7.63(t,J=8.4Hz,1H),7.50(t,J=8.4Hz,1H),6.07–5.97(m,2H),5.30(dd,J=17.2,1.2Hz,2H),5.24(dd,J=10.0,1.2Hz,2H),4.16(d,J=5.6Hz,4H). Therefore, the structural formula of intermediate A4 is

[0081]

[0082] S4. Intermediate A4 reacts with tris(dibenzylideneacetone)dipalladium, tri(o-methylphenyl)phosphine and triethylamine via intramolecular Heck cyclization to obtain

[0083] Under nitrogen protection, intermediate A4 (1.8 mmol), tris(dibenzylideneacetone)dipalladium (0.09 mmol), tri(o-methylphenyl)phosphine (0.36 mmol), triethylamine (1.98 mmol) and N-methylpyrrolidone were added to a 50 mL four-necked flask in sequence and heated at 113 ° C for 4.5 h. The reaction process was monitored by TLC, and the product was formed and the raw materials reacted completely. The reaction solution was cooled to room temperature and transferred to a separatory funnel. Chloroform and water were added and shaken for separation. The organic phase was a yellow oily liquid. The aqueous phase was washed with chloroform, the organic phases were combined, and the combined organic phases were washed twice with saturated brine. TLC monitoring showed that the organic phase was the product. The organic phase was dried over anhydrous magnesium sulfate and filtered to obtain a clear orange-red liquid. The crude product was rotary evaporated to dryness and purified by column chromatography to obtain product C.

[0084] Calculation showed that the yield of product C in the intramolecular Heck cyclization reaction was 86%.

[0085] The molecular structure of product C was determined by H NMR, C NMR and high resolution mass spectrometry. Figure 2 As shown;

[0086] Depend on Figure 2 A shows that the hydrogen nuclear magnetic characterization data of product C is: 1 H NMR(400MHz, CDCl3)δ8.29(d,J=7.6Hz,1H),8.10(d,J=7.6Hz,1H),7.70–7.63(m,2H), 7.55(s,1H),6.10–6.01(m,1H),5.28–5.18(m,2H),4.93(d,J=5.6Hz,2H),2.50(s,3H);

[0087] Depend on Figure 2 B shows that the carbon NMR characterization data of product C are: 13 C NMR (101MHz, CDCl3) δ143.0,142.0,140.3,139.3,135.3,133.4,129.3,128.3,127.6,126.2,118.1,109.8,46.4,8.9;

[0088] Depend on Figure 2 C shows that the high-resolution mass spectrometry data of product C is: 224.1172[M+H] + .

[0089] Therefore, the structural formula of product C is

[0090] Example 5

[0091] This example prepares a quinoxaline compound, whose structural formula is Specifically, the following steps are performed in sequence:

[0092] S1. o-phenylenediamine and diethyl oxalate undergo condensation reaction to obtain intermediate A1

[0093] The intermediate A1 was prepared by the method in Example 3, and its structural formula is

[0094] S2. Intermediate A1 and phosphorus oxybromide undergo bromination reaction to obtain intermediate A2

[0095] The intermediate A2 was prepared by the method in Example 3, and its structural formula is

[0096] S3. Intermediate A2 and N-allylmethylamine undergo nucleophilic substitution reaction to obtain intermediate A3

[0097] The intermediate A3 was prepared by the method in Example 3, and its structural formula is

[0098] S4. Intermediate A3 and tris(dibenzylideneacetone)dipalladium, tri(o-methylphenyl)phosphine and triethylamine undergo intramolecular Heck cyclization to obtain

[0099] The method in Example 3 was used to prepare product B, whose structural formula is

[0100] S5. After oxidation reaction,

[0101] To a 250mL round-bottom flask, 2g of product B, 7.65g of potassium permanganate, 3.1g of dicyclohexyl-18-crown ether-6, and 40mL of water were added in sequence. Stirring was started at room temperature and the reaction was allowed to proceed for 8h. TLC monitoring showed that the raw materials had reacted completely and product was produced. Filtered with suction, the filtrate was a light yellow solution. After adding 1mol / L dilute hydrochloric acid to adjust the pH to 1, the filtrate was a dark yellow liquid. 150mL of ethyl acetate was added for extraction. The organic phase was a clear yellow-green liquid and the aqueous phase was a clear yellow liquid. The aqueous phase was washed with 120mL of ethyl acetate (×3). The organic phases were combined and washed with 50mL of water (×1). The organic phase was a clear yellow-green liquid and the aqueous phase was a clear yellow liquid. TLC monitoring showed that the organic phase was the product. The organic phase was dried over anhydrous magnesium sulfate for 1h and filtered with suction to obtain a clear yellow filtrate. Rotary evaporation to dryness gave 3.42g of a yellow liquid. Column chromatography was used to preliminarily purify the target substance, yielding 0.05g of a white solid, i.e., product G.

[0102] Calculation showed that the yield of product G in the intramolecular Heck cyclization reaction was 86%.

[0103] The molecular structure of product G was determined by H NMR, C NMR and high resolution mass spectrometry. Figure 3 As shown;

[0104] Depend on Figure 3 As can be seen from A, the hydrogen nuclear magnetic characterization data of product G is: 1 H NMR (400MHz, DMSO-d6) δ12.67(s,1H),9.55(s,1H),7.66(d,J=8.0Hz,1H),7.46(t,J=7.3Hz,1H),7.30(t,J=8.4Hz,2H),3.29(s,3H);

[0105] Depend on Figure 3 B shows that the carbon NMR characterization data of product G are: 13 C NMR (101MHz, DMSO-d6) δ163.9,152.0,148.5,130.6,130.4,128.6,127.2,123.6,115.0,40.2,39.9,39.7,39.5,39.3,39.1,38.9,29.0;

[0106] Depend on Figure 3 C shows that the high-resolution mass spectrometry data of product G is: 228.1485[M+H] + .

[0107] Therefore, the structural formula of product G is

[0108] Comparative Example 1

[0109] This comparative example prepared a quinoxaline compound, whose structural formula is Specifically, the following steps are performed in sequence:

[0110] S1. o-phenylenediamine and diethyl oxalate undergo condensation reaction to obtain intermediate A1

[0111] The intermediate A1 was prepared by the method in Example 3, and its structural formula is

[0112] S2. Intermediate A1 and phosphorus oxybromide undergo bromination reaction to obtain intermediate A2

[0113] The intermediate A2 was prepared by the method in Example 3, and its structural formula is

[0114] S3. Intermediate A2 and N-allylbenzylamine undergo nucleophilic substitution reaction to obtain intermediate A5

[0115] Under nitrogen protection, 0.39 g of intermediate A2, 1 g of N-allylbenzylamine and 30 mL of 1,4-dioxane were added sequentially to a 50 mL four-necked flask. The mixture was stirred and heated to maintain reflux for 15 h. The reaction progress was monitored by TLC. After the reaction was completed, the reaction solution was cooled to room temperature and rotary evaporated to dryness to obtain a crude intermediate A5 product. The crude product was initially purified by a 300-400 mesh silica gel chromatography column. To further improve the purity of the product, a secondary purification was performed to obtain a brown-yellow liquid, namely, intermediate A5.

[0116] According to calculation, the yield of intermediate A5 in the nucleophilic substitution reaction is 83%.

[0117] The molecular structure of intermediate A5 was determined by H NMR spectroscopy, and the H NMR characterization data were as follows: 1 HNMR (400MHz,CDCl3)δ7.89(d,J=8.4Hz,1H),7.79(d,J=8.4Hz,1H),7.63(t,J=8.4Hz,1H),7.51(t,J=8.4Hz,1H),7.42(d,J=7.2Hz,2H),7.31(t,J=7.2Hz,2H),7.25–7.21(m,1H),6.07–5.99(m,1H),5.23(t,J=10.4Hz,2H),4.75(s,2H),4.11(d,J=6Hz,2H). Therefore, the structural formula of intermediate A5 is

[0118] S4. Intermediate A5 is reacted with tris(dibenzylideneacetone)dipalladium, tri(o-methylphenyl)phosphine and triethylamine via intramolecular Heck cyclization to obtain

[0119] Under nitrogen protection, intermediate A5 (1.8 mmol), tris(dibenzylideneacetone)dipalladium (0.09 mmol), tris(o-methylphenyl)phosphine (0.36 mmol), triethylamine (1.98 mmol) and N-methylpyrrolidone were added to a 50 mL four-necked flask in sequence and heated at 110 ° C for 4 h. The reaction process was monitored by TLC, and the product was formed and the raw materials reacted completely. The reaction solution was cooled to room temperature and transferred to a separatory funnel. Chloroform and water were added and shaken for separation. The organic phase was a yellow oily liquid. The aqueous phase was washed with chloroform, the organic phases were combined, and the combined organic phases were washed twice with saturated brine. TLC monitoring showed that the organic phase was the product. The organic phase was dried over anhydrous magnesium sulfate and filtered to obtain a clear orange-red liquid. The crude product was rotary evaporated to dryness and purified by column chromatography to obtain product D.

[0120] According to calculation, the yield of product D in the intramolecular Heck cyclization reaction is 50%.

[0121] The molecular structure of product D was determined by H NMR, C NMR and high resolution mass spectrometry. Figure 4 As shown;

[0122] Depend on Figure 4 A shows that the hydrogen nuclear magnetic characterization data of product D is: 1 H NMR (400MHz, CDCl3) δ8.27(d,J=7.6Hz,1H),8.12(d,J=9.6Hz,1H),7.67(t,J=8.0Hz,2H),7.47(s,1H),7.33–7.26(m,5H),5.50(s,2H),2.46(s,3H);

[0123] Depend on Figure 4 B shows that the carbon NMR characterization data of product D is: 13 C NMR (101MHz, CDCl3) δ143.0,142.4,140.4,139.5,137.3,135.3,129.4,129.0,128.4,128.0,127.9,127.7,126.3,110.1,47.8,8.9;

[0124] Depend on Figure 4 C shows that the high-resolution mass spectrometry data of product D is: 274.1324 [M+H] + .

[0125] Therefore, the structural formula of product D is

[0126] Comparative Example 2

[0127] This comparative example prepared a quinoxaline compound, whose structural formula is Specifically, the following steps are performed in sequence:

[0128] S1. o-phenylenediamine and diethyl oxalate undergo condensation reaction to obtain intermediate A1

[0129] The intermediate A1 was prepared by the method in Example 3, and its structural formula is

[0130] S2. Intermediate A1 and phosphorus oxybromide undergo bromination reaction to obtain intermediate A2

[0131] The intermediate A2 was prepared by the method in Example 3, and its structural formula is

[0132] S3. Intermediate A2 and N-allylcyclohexaneamine undergo nucleophilic substitution reaction to obtain intermediate A6

[0133] Under nitrogen protection, 0.4 g of intermediate A2, 1 g of N-allylcyclohexane, and 30 mL of 1,4-dioxane were added sequentially to a 50 mL four-necked flask. The mixture was stirred and heated to maintain reflux for 12 h. The reaction progress was monitored by TLC. After the reaction, the reaction solution was cooled to room temperature and rotary evaporated to dryness to obtain a crude intermediate A6. The crude product was initially purified by a 300-400 mesh silica gel chromatography column. To further improve the purity of the product, a secondary purification was performed to obtain a yellow-green solid, namely, intermediate A6.

[0134] According to calculation, the yield of intermediate A6 in the nucleophilic substitution reaction is 70%.

[0135] The molecular structure of intermediate A6 was determined by H NMR spectroscopy, and the H NMR characterization data were as follows: 1 HNMR(400MHz, CDCl3)δ7.87(d,J=7.6Hz,1H),7.78(d,J=7.6Hz,1H),7.62(t,J= 8.4Hz,1H),7.50(t,J=8.4Hz,1H),5.87–5.78(m,1H),5.23(d,J=17.2Hz,1H),4. 99(d,J=10.0Hz,1H),4.13(d,J=5.2Hz,2H),3.85(tt,J=11.6,3.6Hz,1H),1.94( d,J=11.2Hz,2H),1.83(d,J=13.2Hz,2H),1.68–1.54(m,3H),1.37–1.26(m,3H). Therefore, the structural formula of intermediate A6 is

[0136] S4. Intermediate A6 is reacted with tris(dibenzylideneacetone)dipalladium, tri(o-methylphenyl)phosphine and triethylamine via intramolecular Heck cyclization to obtain

[0137] Under nitrogen protection, intermediate A6 (1.8 mmol), tris(dibenzylideneacetone)dipalladium (0.09 mmol), tri(o-methylphenyl)phosphine (0.36 mmol), triethylamine (1.98 mmol) and N-methylpyrrolidone were added to a 50 mL four-necked flask in sequence and heated at 110 ° C for 4 h. The reaction process was monitored by TLC, and the product was formed and the raw materials reacted completely. The reaction solution was cooled to room temperature and transferred to a separatory funnel. Chloroform and water were added and shaken for separation. The organic phase was a yellow oily liquid. The aqueous phase was washed with chloroform, the organic phases were combined, and the combined organic phases were washed twice with saturated brine. TLC monitoring showed that the organic phase was the product. The organic phase was dried over anhydrous magnesium sulfate and filtered to obtain a clear orange-red liquid. The crude product was rotary evaporated to dryness and purified by column chromatography to obtain product E.

[0138] According to calculation, the yield of product E in the intramolecular Heck cyclization reaction is 40%.

[0139] The molecular structure of product E was determined by H NMR, C NMR and high resolution mass spectrometry. Figure 5 As shown;

[0140] Depend on Figure 5 A shows that the hydrogen nuclear magnetic characterization data of product E is: 1 H NMR (400MHz, CDCl3) δ8.24(d,J=7.6Hz,1H),8.10(d,J=9.6Hz,1H),7.65(s,1H),7.64–7.61(m,2H),4.82 (tt,J=12.0,4.0Hz,1H),2.49(s,3H),2.12(d,J=10.8Hz,2H),1.94(d,J=13.2Hz,2H),1.96–1.58(m,6H);

[0141] Depend on Figure 5 B shows that the carbon NMR characterization data of product E is: 13 C NMR (101MHz, CDCl3) δ143.2,141.7,140.4,139.2,132.8,129.3,128.3,127.4,126.1,109.1,52.9,33.4,25.9,25.7,9.0;

[0142] Depend on Figure 5 C shows that the high-resolution mass spectrometry data of product E is: 266.1640 [M+H] + .

[0143] Therefore, the structural formula of product E is

[0144] Comparative Example 3

[0145] This comparative example prepared a quinoxaline compound, whose structural formula is Specifically, the following steps are performed in sequence:

[0146] S1. o-phenylenediamine and diethyl oxalate undergo condensation reaction to obtain intermediate A1

[0147] The intermediate A1 was prepared by the method in Example 3, and its structural formula is

[0148] S2. Intermediate A1 and phosphorus oxybromide undergo bromination reaction to obtain intermediate A2

[0149] The intermediate A2 was prepared by the method in Example 3, and its structural formula is

[0150] S3. Intermediate A2 and N-ethylmethylpropenamine undergo nucleophilic substitution reaction to obtain intermediate A7

[0151] Under nitrogen protection, 0.58 g of intermediate A2, 1 g of N-ethylmethylpropenamine and 30 mL of 1,4-dioxane were added to a 50 mL four-necked flask in sequence. The mixture was stirred and heated to maintain reflux for 9 h. The reaction progress was monitored by TLC. After the reaction, the reaction solution was cooled to room temperature and rotary evaporated to dryness to obtain a crude intermediate A7. The crude product was preliminarily purified by a 300-400 mesh silica gel chromatography column to obtain a brown-yellow viscous solid, namely, intermediate A7.

[0152] According to calculation, the yield of intermediate A7 in the nucleophilic substitution reaction is 86%.

[0153] The molecular structure of intermediate A7 was determined by H NMR spectroscopy, and the H NMR characterization data were as follows: 1 HNMR (400MHz,CDCl3)δ7.87(d,J=8.4Hz,1H),7.78(d,J=8.0Hz,1H),7.62(t,J=8.4Hz,1H),7.48(t,J=8.4Hz,1H),5.03(s,1H),4.92(s,1H),4.10(s,2H),3.6(q,J=6.8Hz,2H),1.76(s,3H),1.22(t,J=6.8,3H). Therefore, the structural formula of intermediate A7 is

[0154]

[0155] S4. Intermediate A7 is reacted with tris(dibenzylideneacetone)dipalladium, tri(o-methylphenyl)phosphine and triethylamine via intramolecular Heck cyclization to obtain

[0156] Under nitrogen protection, intermediate A7 (1.8 mmol), tris(dibenzylideneacetone)dipalladium (0.09 mmol), tris(o-methylphenyl)phosphine (0.36 mmol), triethylamine (1.98 mmol) and N-methylpyrrolidone were added to a 50 mL four-necked flask in sequence and heated at 110 ° C for 4 h. The reaction process was monitored by TLC, and the product was formed and the raw materials reacted completely. The reaction solution was cooled to room temperature and transferred to a separatory funnel. Chloroform and water were added and shaken for separation. The organic phase was a yellow oily liquid. The aqueous phase was washed with chloroform, the organic phases were combined, and the combined organic phases were washed twice with saturated brine. TLC monitoring showed that the organic phase was the product. The organic phase was dried over anhydrous magnesium sulfate and filtered to obtain a clear orange-red liquid. The crude product was rotary evaporated to dryness and purified by column chromatography to obtain product F.

[0157] Calculation showed that the yield of product F in the intramolecular Heck cyclization reaction was 29%.

[0158] The molecular structure of product F was determined by H NMR, C NMR and high resolution mass spectrometry. Figure 6 As shown;

[0159] Depend on Figure 6 As can be seen from A, the hydrogen nuclear magnetic characterization data of product F are: 1 H NMR (500MHz, DMSO-d6) δ7.71(d,J=8.0Hz,1H),7.51(d,J=8.0Hz,1H),7.44(t,J=7.0Hz,1H),7 .25(t,J=7.0Hz,1H),3.52(q,J=7.0Hz,2H),3.48(s,2H),1.36(s,6H),1.19(t,J=7.5Hz,3H);

[0160] Depend on Figure 6 B shows that the carbon NMR characterization data of product F are: 13 C NMR (101MHz, CDCl3) δ160.3,153.2,142.2,137.7,128.7,128.5,125.7,123.4,61.2,39.0,38.6,26.6,12.1;

[0161] Depend on Figure 6 C shows that the high-resolution mass spectrum data of product F is: 228.1485[M+H] + .

[0162] Therefore, the structural formula of product F is

[0163] Antiviral activity assay

[0164] To systematically evaluate the potential antiviral activity of six quinoxaline compounds (products B–G), in vitro antiviral activity was assayed in PK15 cells. Untreated PK15 cells served as a negative control (denoted as Mock), and PK15 cells infected with PRV-GFP (pseudorabies virus labeled with green fluorescent protein) served as a positive control (denoted as PRV+) to test the antiviral effects of the quinoxaline compounds against PRV.

[0165] The percentage of GFP+ cells can intuitively reflect the degree of viral infection, and therefore becomes a key indicator for judging the antiviral activity of quinoxaline compounds. If the percentage of GFP+ cells in the quinoxaline compound-treated group is significantly decreased compared with the positive control group, then the corresponding compound has antiviral activity.

[0166] (1) Experimental preparation

[0167] Remove the PK15 cell cryopreservation tube from the liquid nitrogen tank and quickly place it in a 37°C water bath for thawing. Keep the temperature constant during this process and shake it slightly. After all the cells have melted, wipe the outer wall of the tube with alcohol to keep it clean, and then place it in a biosafety cabinet. Use a pipette to slowly transfer the thawed cells to a 15mL centrifuge tube containing 2mL of DMEM medium (supplemented with 10% FBS and 1% penicillin-streptomycin), mix gently, centrifuge at 700r / min for 5min at room temperature, remove the supernatant, and add 1mL of fresh culture medium. Resuspend the cells and transfer them to a cell culture flask. Shake the culture flask slightly and then place the culture flask in a cell culture incubator at 37°C and 5% CO2 for culture.

[0168] When the cell density in the cell culture flask exceeds 90%, perform the subculture operation. Remove the old culture medium, add 1mL of PBS buffer for washing, then carefully aspirate and discard the PBS with a pipette, add 1mL of trypsin digestion solution, and gently shake the cell culture flask left and right to ensure that the digestion solution is evenly covered on the cell surface. Place the culture flask in the incubator for 2 to 5 minutes. During this process, when the cell morphology is observed to become round under the microscope, the digestion process is immediately terminated, 2mL of fresh culture medium is added, and the side wall of the culture flask is tapped to promote the cells to fall off the wall of the flask to form a cell suspension. Then use a pipette to gently blow the cell suspension to evenly disperse the cells. Transfer the cell suspension to a 15mL centrifuge tube and centrifuge at 700r / min at room temperature for 5 minutes. After the end, carefully aspirate the supernatant and retain the cell pellet. Add 2 to 3mL of culture medium again to resuspend the cells. Adjust the cell density according to 2.5×10 5 The cell suspension was plated into 12-well plates at a density of 10 cells / mL.

[0169] (II) Antiviral activity test of products B, C and G

[0170] Product B, product C and product G were taken respectively and prepared into 6 μg / mL, 12.5 μg / mL and 25 μg / mL solutions with deionized water. After the cells attached for 12 hours, they were infected with PRV-GFP (MOI = 0.1). Then different concentrations of products were added and the cells were treated for 24 hours. The samples were harvested and analyzed by flow cytometry. The results were as follows: Figures 7-9 As shown;

[0171] Depend on Figure 7 It can be seen that the percentage of GFP+ cells in the positive control group was 80.7%, and when the concentration of product B was 6 μg / mL, the percentage of GFP+ cells was 79.4%, which showed a lower antiviral effect; as the concentration increased to 12.5 μg / mL, the percentage of GFP+ cells decreased to 62.9%, which indicated that product B began to show a certain antiviral potential; when the concentration was further increased to 25 μg / mL, the percentage of GFP+ cells dropped sharply to 8.14%. This significant change indicated that product B had excellent antiviral activity at a concentration of 25 μg / mL.

[0172] Depend on Figure 8 It can be seen that the percentage of GFP+ cells in the positive control group was 34.8%. When the concentration of product C was 25 μg / mL, its antiviral activity was particularly outstanding, and the percentage of GFP+ cells was only 9.05%, which was a significant decrease compared with the positive control. Therefore, product C has antiviral activity at a concentration of 25 μg / mL.

[0173] Depend on Figure 9 It can be seen that the percentage of GFP+ cells in the positive control group was 80.7%, and the percentage of GFP+ cells of product G at a concentration of 6 μg / mL was 79.3%, which was similar to the performance of product B at the same concentration; when the concentration increased to 12.5 μg / mL, the percentage of GFP+ cells dropped to 66.0%. Although it decreased, the decrease was not significant; however, when the concentration was further increased to 25 μg / mL, the percentage of GFP+ cells was observed to decrease to 48.2%, indicating that product G also exhibited antiviral activity at higher concentrations. Although its antiviral effect was slightly inferior to that of product B, compared with the positive control, product G still exhibited significant antiviral activity at a concentration of 25 μg / mL.

[0174] (III) Cytotoxicity experiments of products D, E, and F

[0175] During the experiment, it was found that products D, E and F were cytotoxic even at low concentrations and would seriously damage cells, so the antiviral activity experiment was not carried out.

[0176] Take product D, product E and product F respectively, and prepare them into 6 μg / mL solution with deionized water. After the cells adhere for 12 hours, they are infected with PRV-GFP (MOI = 0.1), and then the products are added respectively. After 24 hours, the samples are harvested and the cells are observed under a microscope. The results are as follows Figure 10 As shown;

[0177] Depend on Figure 10 As can be seen, the cell morphology of cells treated with products D, E, and F changed significantly, with cells shrinking, becoming rounded, or deformed, losing their original regular shape. The connections between cells became loose or broken, resulting in increased intercellular spaces or cells falling off the cell flask wall. These all indicate that products D, E, and F have a strong toxic effect on cells at low concentrations.

[0178] In summary, products B, C, and G all exhibited excellent antiviral activity at a concentration of 25 μg / mL, while products D, E, and F also caused severe cell damage at lower concentrations. Comparison of chemical structure and activity revealed that compounds exhibited activity when the nitrogen substituents were methyl or allyl, while other substituents exhibited stronger cytotoxicity.

Claims

1. A quinoxaline compound, characterized in that Its structural formula is In the formula, R1 is an alkyl group or an allyl group; R2 is an alkyl group.

2. A quinoxaline compound, characterized in that Its structural formula is In the formula, R1 is an alkyl group; R2 is a carboxyl group.

3. A quinoxaline compound according to claim 1 or 2, characterized in that, The alkyl group is methyl.

4. The method for preparing a quinoxaline compound according to claim 1, wherein The following steps are involved: S1. o-phenylenediamine and diethyl oxalate are condensed to obtain intermediate A1; S2. Intermediate A1 is subjected to bromination reaction with phosphorus oxybromide to obtain intermediate A2; S3. Intermediate A2 and an allylamine compound undergo nucleophilic substitution reaction to obtain a third intermediate; S4. The third intermediate and tris(dibenzylideneacetone)dipalladium, tri(o-methylphenyl)phosphine and triethylamine are subjected to an intramolecular Heck cyclization reaction to obtain the quinoxaline compound; The structural formula of the intermediate A1 is The structural formula of intermediate A2 is The allylamine compound includes N-allylmethylamine or diallylamine.

5. The method for preparing a quinoxaline compound according to claim 4, wherein: The condensation reaction is carried out at reflux temperature for 1.5 to 2 hours; The bromination reaction is carried out under the following conditions: acetonitrile solvent, N,N-dimethylformamide as catalyst, temperature at reflux temperature, and time for 1.5 to 2.5 hours.

6. The method for preparing a quinoxaline compound according to claim 4 or 5, wherein: The molar ratio of the intermediate A2 to N-allylmethylamine is 1:2, and the conditions for the nucleophilic substitution reaction are: in tetrahydrofuran solvent, at reflux temperature, for 4.0 to 4.5 hours; The molar ratio of the intermediate A2 and diallylamine is 1:6, and the conditions for the nucleophilic substitution reaction are: in 1,4-dioxane solvent, the temperature is reflux temperature, and the time is 15.0 to 15.5 hours.

7. The method for preparing a quinoxaline compound according to claim 6, wherein: The intramolecular Heck cyclization reaction is carried out in N-methylpyrrolidone as solvent, at a temperature of 110 to 120° C., for a time of 4.0 to 4.5 hours.

8. The method for preparing a quinoxaline compound according to claim 2, wherein: The quinoxaline compound is It is Obtained by oxidation reaction.

9. The method for preparing a quinoxaline compound according to claim 8, wherein: In the oxidation reaction, the oxidant is potassium permanganate, the phase transfer catalyst is dicyclohexyl-18-crown-6, the solvent is water, and the temperature is room temperature.

10. The use of a quinoxaline compound according to any one of claims 1 to 3, characterized in that: The quinoxaline compounds are used for preparing antiviral, antibacterial, anti-inflammatory or antitumor preparations.

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