Phenylacetonitrile derivative as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of chemical medicine preparation, and particularly relates to a phenylacetonitrile derivative as well as a preparation method and application thereof. The structural formula of the phenylacetonitrile derivative is # imgabs0, the derivative has a remarkable inhibition effect on various bacteria, the bacteriostasis rate is increased along with increase of concentration, and powerful support is provided for development of novel antibacterial drugs. Meanwhile, a cell compatibility experiment proves that the derivative is safe to human fibroblasts, and a safety guarantee is provided for application of the derivative in the field of medicines. According to the preparation method, a green chemical concept is adopted, the use of toxic solvents is avoided, and the pollution to the environment is reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical drug preparation, and particularly relates to a phenylacetonitrile derivative, a preparation method thereof, and an application thereof. Background Art

[0002] Phenylacetonitrile (C 8 H 7 N), as an important organic compound, has extensive applications in multiple fields such as fine chemical industry, pharmaceutical synthesis, and perfume industry. Especially in the pharmaceutical field, phenylacetonitrile is a key intermediate for synthesizing antibiotics, anticancer drugs, and antidepressants. Its derivatives, such as phenylacetic acid and ethyl phenylacetate, also play important roles in the pharmaceutical, pesticide, and dye industries. In recent years, with the increasing demand for new antibacterial drugs, the application of phenylacetonitrile and its derivatives in antibacterial aspects has gradually attracted attention.

[0003] Traditionally, the preparation of phenylacetonitrile derivatives mostly relies on complex chemical reaction conditions, such as high temperature, high pressure, or the use of toxic catalysts, which not only increases production costs but also may cause environmental pollution. In recent years, with the popularization of the concept of green chemistry, developing an environmentally friendly, energy-efficient, and high-yield preparation method for phenylacetonitrile derivatives has become a research hotspot. In this context, the present invention proposes a new phenylacetonitrile derivative and a preparation method thereof. The preparation method adopts the concept of green chemistry, reduces environmental pollution, and the prepared phenylacetonitrile derivative shows significant inhibitory effects on Escherichia coli, Staphylococcus aureus, and Staphylococcus epidermidis. This discovery provides a new idea for the development of new antibacterial drugs. Summary of the Invention

[0004] In order to overcome the deficiencies of the prior art, the purpose of the present invention is to provide a phenylacetonitrile derivative, a preparation method thereof, and an application thereof. By adopting the concept of green reaction, the use of toxic solvents is avoided, and a phenylacetonitrile derivative that shows inhibitory effects on Escherichia coli, Staphylococcus aureus, and Staphylococcus epidermidis is prepared, providing a new idea for the development of new antibacterial drugs.

[0005] The present invention is achieved by the following technical solutions:

[0006] A phenylacetonitrile derivative, the structural formula of the phenylacetonitrile derivative is as follows:

[0007]

[0008] The preparation method of the above-mentioned phenylacetonitrile derivative includes the following steps:

[0009]

[0010] (1) 2-Amino-5-bromonicotinaldehyde is added to phenylacetonitrile, and then KOH solution is added, followed by microwave irradiation reaction. The reaction solution is separated and purified to obtain Intermediate 1;

[0011] (2) Intermediate 1 is added to HCl solution, and then NaNO 2 is added. Stir the reaction, pour the reaction mixture into ice water, filter, collect the precipitate and wash it. The obtained crude product is recrystallized to obtain Intermediate 2;

[0012] (3) Intermediate 2 is added to POCl 3 and subjected to microwave irradiation reaction. The reaction solution is separated and purified to obtain Intermediate 3;

[0013] (4) Intermediate 3, 4-aminophenylboronic acid, anhydrous N-methylpyrrolidone and K 2 CO 3 are mixed and heated for reaction. The reaction solution is post-treated to obtain Intermediate 4;

[0014] (5) Intermediate 4 and 3,4-dihydroxybenzeneacetonitrile are added to a solvent, stirred for reaction. The reaction solution is dried, concentrated and purified by column chromatography to obtain the phenylacetonitrile derivative.

[0015] As a preferred technical solution of the present invention, in step (1), the molar ratio of 2-amino-5-bromonicotinaldehyde to phenylacetonitrile is 1:(1 - 1.2); the concentration of the KOH solution is 10 - 12 wt%; the operation of the microwave irradiation reaction is: under 400 W microwave irradiation, intermittently irradiate for 3 - 4 min at intervals of 25 - 30 seconds.

[0016] As a preferred technical solution of the present invention, in step (2), the molar ratio of Intermediate 1 to NaNO 2 is 1:(1 - 1.5); the concentration of the HCl solution is 1.5 - 2 mol / L.

[0017] As a preferred technical solution of the present invention, in step (2), the stirring reaction time is 0.5 - 1 h; the solvent for recrystallization is ethanol.

[0018] As a preferred technical solution of the present invention, in step (3), the dosage ratio of Intermediate 2 to POCl 3 is 1 mmol:(14 - 16) mL; the operation of the microwave irradiation reaction is: under 200 W microwave irradiation, intermittently irradiate for 2.5 - 3 min at intervals of 25 - 30 seconds.

[0019] As a preferred technical solution of the present invention, in step (4), the Intermediate 3, 4-aminophenylboronic acid, anhydrous N-methylpyrrolidone and K 2 CO 3The molar ratio is 1:(1 - 1.2):(10 - 11):(3.4 - 3.6); the temperature of the heating reaction is 70 - 80 °C, and the time is 15 - 25 min.

[0020] As a preferred technical solution of the present invention, in step (5), the molar ratio of intermediate 4 to 3,4-dihydroxybenzyl cyanide is 1:(1 - 1.2); the time of the stirring reaction is 16 - 20 h.

[0021] Use of the above-mentioned benzyl cyanide derivative in the preparation of antibacterial drugs.

[0022] The principle of preparing the benzyl cyanide derivative of the present invention is as follows: First, under microwave radiation conditions and in the presence of KOH, 2-amino-5-bromonicotinaldehyde reacts with benzyl cyanide in a solvent-free cyclization reaction to obtain intermediate 1; then, through the reaction of intermediate 1 with NaNO 2 it is converted into intermediate 2; intermediate 2 and POCl 3 can be converted into intermediate 3 in only 2 - 3 min under microwave radiation conditions; then, in the presence of NMP and K 2 CO 3 intermediate 3 undergoes a substitution reaction to introduce a phenylboronic acid group to obtain intermediate 4; finally, the phenylboronic acid group of intermediate 4 forms a borate ester with 3,4-dihydroxybenzyl cyanide to obtain the benzyl cyanide derivative.

[0023] The present invention has the following effects compared with the prior art:

[0024] 1. The present invention provides a benzyl cyanide derivative, which has a significant inhibitory effect on a variety of bacteria, and the antibacterial rate increases with the increase of concentration, providing strong support for the development of new antibacterial drugs. At the same time, the cell compatibility experiment proves that the derivative is safe for human fibroblasts, providing a safety guarantee for its application in the medical field.

[0025] 2. The present invention also provides a preparation method of the above-mentioned benzyl cyanide derivative, adopting the concept of green chemistry, avoiding the use of toxic solvents, and reducing environmental pollution.

[0026] 3. The benzyl cyanide derivative of the present invention can not only be used in the medical field, but also be applied to multiple fields such as food preservation and daily chemical products, with broad market prospects and economic benefits. Description of the Drawings

[0027] Figure 1 It is the antibacterial activity result of the benzyl cyanide derivative of the present invention at different concentrations;

[0028] Figure 2 It is the result of the influence of the benzyl cyanide derivative of the present invention on the survival rate of human fibroblasts at different concentrations. Detailed Embodiments

[0029] The technical solutions of the present invention will be further described below in conjunction with specific embodiments. However, those skilled in the art should understand that the following examples are only used to illustrate the present invention and should not be regarded as a limitation of the present invention. The specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, unless otherwise specified, are all conventional products obtained through commercial channels.

[0030] Example 1

[0031] A phenylacetonitrile derivative has the following structural formula:

[0032]

[0033] The preparation method of the above phenylacetonitrile derivative includes the following steps:

[0034]

[0035] (1) 2-Amino-5-bromonicotinaldehyde (CAS: 206997-15-1, 10 mmol) was added to phenylacetonitrile (11 mmol), and then 1 mL of a 10% KOH solution was added dropwise. Under microwave irradiation at 400 W, it was intermittently irradiated at intervals of 30 seconds for 3 min. After the reaction was completed, the reaction mixture was cooled and then added to ice water. It was filtered, the precipitated solid was collected and washed with water. The obtained crude product was recrystallized with ethanol to obtain Intermediate 1; 1 HNMR:(C 14 H 10 BrN 3 , 400 MHz, DMSO-d6) δ: 8.63 - 8.60 (m, 2H), 8.21 (s, 1H), 7.53 - 7.40 (m, 5H), 6.41 (s, 2H); HRMS(ESI + ): [M + H] + Calculated to be 300.01, found to be 300.02; the above results confirm that the obtained product is the target product.

[0036] (2) Intermediate 1 (10 mmol) was added to HCl solution (2 M, 100 mL). After cooling to 3 °C, 50 mL of NaNO 2 (12 mmol) solution was added. The reaction mixture was stirred at room temperature for 1 h, and the reaction was monitored by TLC. After the reaction was completed, the mixture was poured into ice water, filtered, the precipitate was collected and washed with water. The obtained crude product was recrystallized with ethanol to obtain Intermediate 2; HRMS(ESI + ): [M + H] +Calculated to be 300.99, found 300.99; the above results confirm that the obtained product is the target product.

[0037] (3) Add intermediate 2 (5 mmol) to 75 mL of POCl 3 and irradiate intermittently at 30 - second intervals for 3 min under microwave radiation of 200 W. After the reaction is completed, cool the reaction mixture to room temperature, and then extract and wash successively with ice water (2×100 mL) and saturated sodium carbonate solution. Collect and combine the organic phases. After evaporating the organic phases to dryness, obtain the crude product. Recrystallize the crude product with n - pentane to obtain intermediate 3; HRMS (ESI + ): [M + H] + Calculated to be 318.96, found 318.95; the above results confirm that the obtained product is the target product.

[0038] (4) Mix intermediate 3 (5 mmol), 4 - aminophenylboronic acid (5.5 mmol), anhydrous N - methylpyrrolidone (52 mmol) and K 2 CO 3 (17.5 mmol), stir at 75 °C for 20 min, monitor the reaction by TLC. After the reaction is completed, cool the reaction solution to room temperature, then pour it into ethyl acetate, filter to collect the precipitated solid, wash with water, and finally recrystallize with methanol to obtain intermediate 4; 1 HNMR: (C 20 H 15 BBrN 3 O 2 , 400 MHz, DMSO - d6) δ: 9.21 (s, 1H), 8.63 - 8.60 (m, 2H), 8.21 (s, 1H), 7.72 (d, 2H), 7.53 - 7.40 (m, 5H), 7.19 (d, 2H); HRMS (ESI + ): [M + H] + Calculated to be 420.04, found 420.04; the above results confirm that the obtained product is the target product.

[0039] (5) Add intermediate 4 (5 mmol) and 3,4 - dihydroxybenzyl cyanide (5.5 mmol) to 25 mL of dichloromethane, then dropwise add ethyl acetate until dissolved, stir at room temperature for 18 h. Dry the reaction solution with anhydrous magnesium sulfate, concentrate and purify by column chromatography to obtain the phenylacetonitrile derivative. 1 HNMR: (C 28 H 18 BBrN 4 O 2, 400 MHz, DMSO-d6) δ: 9.21 (s, 1H), 8.63 - 8.60 (m, 2H), 8.21 (s, 1H), 7.72 (d, 2H), 7.53 - 7.40 (m, 5H), 7.19 (d, 2H), 6.60 - 6.55 (m, 3H), 4.32 (s, 2H); HRMS(ESI + ):[M + H] + Calculated: 533.07, Found: 533.07; The above results confirm that the obtained product is the target product.

[0040] Example 2

[0041] A phenylacetonitrile derivative, the structural formula of which is the same as that in Example 1.

[0042] The preparation method of the above phenylacetonitrile derivative, the reaction process is the same as that in Example 1, including the following steps:

[0043] (1) Add 2-amino-5-bromonicotinaldehyde (10 mmol) to phenylacetonitrile (10 mmol), then dropwise add 1 mL of 10% KOH solution, and irradiate intermittently at intervals of 25 seconds for 3 min under microwave radiation of 400 W. After the reaction is completed, cool the reaction mixture and add it to ice water, filter, collect the precipitated solid and wash it with water. The obtained crude product is recrystallized with ethanol to obtain Intermediate 1; the 1 1H NMR, HRMS(ESI + ) are consistent with the results of Example 1.

[0044] (2) Add Intermediate 1 (10 mmol) to HCl solution (1.5 M, 100 mL), cool to 0 °C and then add 50 mL of NaNO 2 (10 mmol) solution. Stir the reaction mixture at room temperature for 0.5 h, monitor the reaction by TLC. After the reaction is completed, pour the mixture into ice water, filter, collect the precipitate and wash it with water. The obtained crude product is recrystallized with ethanol to obtain Intermediate 2; the HRMS(ESI + ) is consistent with the results of Example 1.

[0045] (3) Add Intermediate 2 (5 mmol) to 70 mL of POCl 3 , and irradiate intermittently at intervals of 30 seconds for 2.5 min under microwave radiation of 200 W. After the reaction is completed, cool the reaction mixture to room temperature, and then extract and wash it successively with ice water (2 × 100 mL) and saturated sodium carbonate solution. Collect and combine the organic phases. After the organic phases are evaporated to dryness, the crude product is obtained. The crude product is recrystallized with n-pentane to obtain Intermediate 3; the HRMS(ESI + ) is consistent with the results of Example 1.

[0046] (4) Mix intermediate 3 (5 mmol), 4-aminophenylboronic acid (5 mmol), anhydrous N-methylpyrrolidone (50 mmol) and K 2 CO 3 (17 mmol), stir at 80 °C for 15 min, monitor the reaction by TLC. After the reaction is completed, cool the reaction solution to room temperature, then pour it into ethyl acetate, filter to collect the precipitated solid, wash with water, and finally recrystallize with methanol to obtain intermediate 4; the 1 HNMR and HRMS (ESI + ) are consistent with the results of Example 1.

[0047] (5) Add intermediate 4 (5 mmol) and 3,4-dihydroxybenzyl cyanide (5 mmol) to 20 mL of dichloromethane, then dropwise add ethyl acetate until dissolved, stir at room temperature for 16 h, dry the reaction solution with anhydrous magnesium sulfate, concentrate and purify by column chromatography to obtain the phenylacetonitrile derivative. The 1 HNMR and HRMS (ESI + ) are consistent with the results of Example 1.

[0048] Example 3

[0049] A phenylacetonitrile derivative, the structural formula of which is the same as that in Example 1.

[0050] The preparation method of the above-mentioned phenylacetonitrile derivative includes the following steps:

[0051] (1) Add 2-amino-5-bromonicotinaldehyde (10 mmol) to phenylacetonitrile (12 mmol), then dropwise add 1 mL of a 12% KOH solution, and irradiate intermittently at intervals of 30 seconds for 4 min under microwave radiation of 400 W. After the reaction is completed, cool the reaction mixture, add it to ice water, filter, collect the precipitated solid and wash with water, and recrystallize the obtained crude product with ethanol to obtain intermediate 1; the 1 HNMR and HRMS (ESI + ) are consistent with the results of Example 1.

[0052] (2) Add intermediate 1 (10 mmol) to HCl solution (2 M, 100 mL), cool to 5 °C and then add 50 mL of NaNO 2 (15 mmol) solution, stir the reaction mixture at room temperature for 1 h, monitor the reaction by TLC. After the reaction is completed, pour the mixture into ice water, filter, collect the precipitate and wash with water, and recrystallize the obtained crude product with ethanol to obtain intermediate 2; the HRMS (ESI + ) is consistent with the results of Example 1.

[0053] (3) Add intermediate 2 (5 mmol) to 80 mL of POCl 3 , and irradiate intermittently at intervals of 25 seconds for 3.0 min under microwave radiation of 200 W. After the reaction is completed, cool the reaction mixture to room temperature, and then extract and wash it successively with ice water (2 × 100 mL) and saturated sodium carbonate solution. Collect and combine the organic phases. After evaporating the organic phases to dryness, obtain the crude product. Recrystallize the crude product with n-pentane to obtain intermediate 3; the HRMS (ESI + ) is consistent with the result of Example 1.

[0054] (4) Mix intermediate 3 (5 mmol), 4-aminophenylboronic acid (6 mmol), anhydrous N-methylpyrrolidone (55 mmol), and K 2 CO 3 (18 mmol), stir at 70 °C for 25 min, monitor the reaction by TLC. After the reaction is completed, cool the reaction solution to room temperature, then pour it into ethyl acetate, filter and collect the precipitated solid, wash it with water, and finally recrystallize it with methanol to obtain intermediate 4; the 1 HNMR, HRMS (ESI + ) are consistent with the result of Example 1.

[0055] (5) Add intermediate 4 (5 mmol) and 3,4-dihydroxybenzonitrile (6 mmol) to 30 mL of dichloromethane, and then dropwise add ethyl acetate until dissolved. Stir at room temperature for 20 h. Dry the reaction solution with anhydrous magnesium sulfate, concentrate it, and purify it by column chromatography to obtain the phenylacetonitrile derivative. The 1 HNMR, HRMS (ESI + ) are consistent with the result of Example 1.

[0056] Test Example 1 Test the antibacterial activity of the phenylacetonitrile derivative prepared in the present invention

[0057] Select Gram-negative bacteria (Escherichia coli) and Gram-positive bacteria (Staphylococcus epidermidis, Staphylococcus aureus), and evaluate the antibacterial activity of the phenylacetonitrile derivative by the microbroth dilution method. The specific method is as follows: Inoculate Escherichia coli, Staphylococcus epidermidis, and Staphylococcus aureus into 10 mL of NB culture medium, and culture them in a constant temperature shaker at 37 °C and 220 rpm for 8 h. When the bacteria enter the logarithmic growth phase, dilute them with NB broth to a McFarland turbidity of 0.5. At this time, the number of bacteria is 10 8CFU / mL, and then inoculated into a 96-well plate after dilution at a ratio of 1:1000. It was divided into a negative control group and an experimental group. The negative control group was inoculated only with the bacterial solution, and after the experimental group was inoculated with the bacterial solution, phenylacetonitrile derivative solutions with concentrations of 2.5 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, 160 μg / mL, and 320 μg / mL were added (the phenylacetonitrile derivative was dissolved in a medium containing 0.2% DMSO to prepare a 320 μg / mL stock solution, and then the stock solution was diluted with the medium to the required different concentrations of phenylacetonitrile derivative solutions). At the same time, a blank control group (without adding the bacterial solution and phenylacetonitrile derivative) was set, and each group had 6 replicate wells. After culturing in a constant temperature incubator at 37°C for 24 h, a microplate reader was used to detect the OD value at a wavelength of 600 nm. After repeating the experiment 3 times, the average value was taken. The results are as Figure 1 shown.

[0058] From Figure 1 it can be seen that within the range of 2.5 - 320 μg / mL, the phenylacetonitrile derivative prepared by the present invention has a certain inhibitory effect on Escherichia coli, Staphylococcus aureus, and Staphylococcus epidermidis, and the higher the concentration, the gradually increasing antibacterial rate. When the concentration is 160 μg / mL, the antibacterial rates against Escherichia coli, Staphylococcus aureus, and Staphylococcus epidermidis are 83.33%, 86.57%, and 87.69% respectively. The above results indicate that the phenylacetonitrile derivative prepared by the present invention is expected to be used as an antibacterial drug.

[0059] Test Example 2: Perform a cell compatibility test on the phenylacetonitrile derivative prepared by the present invention

[0060] Add 100 μL of human fibroblast L929 (1×10 5 cells / mL) in the logarithmic growth phase to a 96-well plate. The medium in the 96-well plate is DMEM complete medium. Place the 96-well plate in a cell incubator at 37°C and 5% CO 2 . After culturing for 12 h, gently aspirate the cell culture medium in each well, and add 200 μL of DMEM complete medium containing 320 μg / mL of the phenylacetonitrile derivative to the 96-well plate and continue to culture in the cell incubator for 24 h. DMEM serum-free medium is used as the blank control, and DMEM complete medium supplemented with 5% phenol is used as the positive control, and place it in a 37°C, 5% CO 2 incubator for culturing; After culturing for 48 h, take out the 96-well plate and observe the cell morphology under a microscope. The results are as Figure 2 shown.

[0061] It is Figure 2 known that there is no obvious difference in the fluorescence intensity of the cells in the field of view. The above results indicate that: the phenylacetonitrile compound of the present invention is safe for human fibroblasts.

[0062] In summary, the present invention prepares a phenylacetonitrile derivative by means of a green chemical reaction. This derivative exhibits inhibitory effects against Escherichia coli, Staphylococcus aureus, and Staphylococcus epidermidis, providing strong support for the development of new antibacterial drugs. Meanwhile, the cytotoxicity experiment proves that this derivative is safe for human fibroblasts, providing safety assurance for its application in the medical field.

[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them. The basic principles and main features of the present invention have been described with specific implementation schemes above. On the basis of the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the present invention claimed.

Claims

1. A benzyl cyanide derivative, characterized in that The structural formula of the benzyl cyanide derivative is as follows:

2. The method for preparing the benzyl cyanide derivative according to claim 1, characterized in that: The following steps are involved: (1) adding 2-amino-5-bromonicotinaldehyde to benzyl cyanide, then adding KOH solution, and performing microwave irradiation reaction. The reaction solution is separated and purified to obtain intermediate 1; (2) Adding intermediate 1 to HCl solution, then adding NaNO2, stirring the reaction, pouring the reaction mixture into ice water, filtering, collecting and washing the precipitate, and then recrystallizing the obtained crude product to obtain intermediate 2; (3) adding intermediate 2 to POCl3 and subjecting to microwave irradiation reaction, and separating and purifying the reaction solution to obtain intermediate 3; (4) mixing intermediate 3, p-aminophenylboronic acid, anhydrous N-methylpyrrolidone and K2CO3, heating for reaction, and post-treating the reaction solution to obtain intermediate 4; (5) The intermediate 4,3,4-dihydroxybenzeneacetonitrile is added to a solvent and stirred for reaction. The reaction solution is dried, concentrated, and purified by column chromatography to obtain a benzeneacetonitrile derivative.

3. The method for preparing a benzyl cyanide derivative according to claim 2, characterized in that: In step (1), the molar ratio of 2-amino-5-bromonicotinaldehyde to benzyl cyanide is 1:(1-1.2); the concentration of the KOH solution is 10-12wt%; and the microwave irradiation reaction is performed by intermittently irradiating for 3-4 minutes at intervals of 25-30 seconds under 400W microwave irradiation.

4. The method for preparing a benzyl cyanide derivative according to claim 2, characterized in that: In step (2), the molar ratio of intermediate 1 to NaNO2 is 1:(1-1.5); the concentration of the HCl solution is 1.5-2 mol / L.

5. The method for preparing a benzyl cyanide derivative according to claim 2, characterized in that: The stirring reaction time in step (2) is 0.5-1h; the solvent for the recrystallization is ethanol.

6. The method for preparing a benzyl cyanide derivative according to claim 2, characterized in that: In step (3), the ratio of intermediate 2 to POCl3 is 1 mmol: (14-16) mL; the operation of the microwave irradiation reaction is: under 200 W microwave irradiation, intermittent irradiation is performed for 2.5-3 min at intervals of 25-30 seconds.

7. The method for preparing a benzyl cyanide derivative according to claim 2, characterized in that: In step (4), the molar ratio of intermediate 3, p-aminophenylboronic acid, anhydrous N-methylpyrrolidone and K2CO3 is 1:(1-1.2):(10-11):(3.4-3.6); the temperature of the heating reaction is 70-80°C and the time is 15-25min.

8. The method for preparing a benzyl cyanide derivative according to claim 2, characterized in that: In step (5), the molar ratio of intermediate 4 to 3,4-dihydroxybenzeneacetonitrile is 1:(1-1.2); and the stirring reaction time is 16-20 hours.

9. Use of the benzyl cyanide derivative according to claim 1 in the preparation of antibacterial drugs.

Images