CN111848537B - A kind of synthetic method and antibacterial activity determination method of chlorogenic acid derivative - Google Patents

Abstract

The invention discloses a method for synthesizing chlorogenic acid derivatives, which comprises the following steps: step 1, protection of chlorogenic acid alcohol hydroxyl group; step 2, protection of chlorogenic acid phenolic hydroxyl group; step 3, containing oxazole or pyrazole Synthesis of cyclic chlorogenic acid derivatives; Step 4, removal of hydroxyl protective group; the present invention can prepare high-efficiency antibacterial agents of chlorogenic acids, with remarkable antibacterial effect, and can also enhance the stability of unsaturated double bonds and improve green In vivo biological activity of ortho acids.

Description

Synthesis method and antibacterial activity determination method of a chlorogenic acid derivative

technical field

The invention relates to the technical field of chlorogenic acid, in particular to a synthesis method and antibacterial activity determination method of a chlorogenic acid derivative.

Background technique

Chlorogenic acid is a depsipolic acid formed by the condensation of caffeic acid and quinic acid (1-hydroxyhexahydrogallic acid). -Caffeoylquinic acid (3-O-caffeoylquinic acid) is a phenylpropanoid compound produced by plants through the shikimic acid pathway during aerobic respiration. Chlorogenic acid is the main active ingredient of antibacterial and detoxifying, anti-inflammatory and choleretic of many medicinal materials, such as honeysuckle, Yinchen, Eucommia, etc. An important indicator of preparation quality control. Chlorogenic acid has a wide range of biological activities, such as antibacterial, antiviral, increasing white blood cells, protecting liver and gallbladder, antitumor, lowering blood pressure, lowering blood fat, scavenging free radicals and stimulating the central nervous system. Modern scientific research on the biological activity of chlorogenic acid has penetrated into many fields such as food, health care, medicine and daily chemical industry.

Due to the presence of ester bond, unsaturated double bond and dihydric phenol three unstable groups in the molecular structure of chlorogenic acid, the biological activity of chlorogenic acid in vivo is greatly reduced. For many years, there have been a considerable amount of literature or experimental data on the chemical modification or modification of the unfavorable physical and chemical properties and biological activities of chlorogenic acid. Among them, simple modification based on the chlorogenic acid skeleton is a way to obtain highly active chlorogenic acid derivatives. According to its modification sites, most of them focus on the modification of multiple hydroxyl substituents, carboxyl groups and ester groups in the molecular structure of chlorogenic acid (Chinese herbal medicine, 2020, 51(4), 937), while for The modification of unsaturated carbon-carbon double bonds is relatively rare.

Most compounds containing oxazole and pyrazole structures have a wide range of biological activities, and have significant curative effects in antifungal, antibacterial and anti-malignant tumors, and have been used as an important class of pharmaceutical intermediates and lead compounds. For example, muscarinol is a compound containing an oxazoline ring skeleton, which has been proven to have natural anticancer, antibacterial and calming functions (Eur.J.Med.Chem.1995,30,839); in 2003, Barbachyn et al. designed and synthesized a compound containing A series of compounds based on the oxazoline core, some substances have better antibacterial activity and good pharmacokinetic properties than linezolid through activity screening (J.Med.Chem.2003, 46, 284); in 2012, Zhang Changshui et al reported 5 -Phosphoryl-3-arylisoxazoline has certain inhibitory activity on neuraminidase (Chin.J.Org.Chem.2012,32,1336); in 2013, Wang Shifa et al. Compounds with azole rings have shown strong inhibitory effects on Escherichia coli and Bacillus subtilis (Chin.J.Org.Chem.2013,33,2196); since the 1970s, Ankhiwala M.D. et al. have reported certain substituted Pyrazoline compounds have excellent bactericidal activity (Journal of the Indian Chemical Society, 1990,67 (6), 514); Subsequently, Turan-Zitounia G and Yang Jinmei have reported the antibacterial activity of pyrazoline derivatives (Archiv Der Pharmazie , 2005, 338, 96; Journal of Yunnan University for Nationalities (Natural Science Edition), 2007, 16(1), 33); in 2020, Zhu Hong's research group formed a series of pyrazoline derivatives and analyzed their effect on Gram-positive According to the antibacterial effect of bacteria, Gram-negative bacteria and fungi, this kind of pyrazoline compounds have different degrees of inhibitory effects on various bacteria (Industrial Catalysis, 2020, 28(2), 53).

SUMMARY OF THE INVENTION

In order to solve the above problems, the object of the present invention is to provide a synthesis method of chlorogenic acid derivatives, and at the same time, disclose an antibacterial activity assay method of the chlorogenic acid derivatives prepared by the above synthesis method.

In order to realize the above-mentioned purpose of the invention, the present invention adopts following technical scheme:

A synthetic method for chlorogenic acid derivatives, comprising the following steps:

Step 1. Protection of chlorogenic acid alcoholic hydroxyl group: Dissolve chlorogenic acid in acetone, then add p-toluenesulfonic acid to the reaction system, stir at room temperature for 3-5 hours; TLC monitors the reaction process until the reactant chlorogenic acid Disappeared; after the reaction, add alkaline substances to neutralize the remaining p-toluenesulfonic acid to a pH value of 6-7.5, filter with suction, and concentrate the filtrate to obtain Compound A;

Step 2, the protection of chlorogenic acid phenolic hydroxyl group: the compound A obtained in step 1, the phenolic hydroxyl protecting group acetic anhydride and the catalyst 4-dimethylaminopyridine (DMAP) are added in the solvent dimethylformamide (DMF), Stir the reaction at room temperature for 2 to 3 hours; monitor the completion of the reaction, add saturated brine to the reaction system to wash, then extract with an extractant, combine the organic phases, and purify the organic phases by silica gel chromatography, using dichloromethane and methanol as eluents , Compound B is isolated;

Step 3, synthesis of chlorogenic acid derivatives containing oxazole or pyrazole ring: the compound B obtained in step 2 is dissolved in an organic solvent, and then 1,3-dipole C, catalyst copper salt, and oxidizing agent are added to In the reaction system, react at a temperature of 50-70°C for 5-8 hours; TLC monitors the reaction process until the compound B completely disappears, stop the reaction, add saturated brine to the reaction system to wash, then extract with an extractant, and combine the organic phases , the organic phase was purified by silica gel chromatography, using dichloromethane and methanol as eluents to isolate compound D;

The 1,3-dipole C is formula (I)

Wherein, R is H, Bn, Me, Ph, 4-MePh, 4-MeOPh, 4-ClPh or CH ₂ CH ₂ CO; Ar is Ph, 4-MePh, 4-MeOPh, 4-ClPh, 2-MePh or 2-ClPh; X is O or N;

Step 4. Removal of hydroxyl protecting group: Add the compound D obtained in step 3 into the mixture of trifluoroacetic acid and dichloromethane, react at room temperature for 1.5-3 hours, monitor the reaction progress by TLC, after the reaction, add Wash with saturated brine, extract with extractant, combine the organic phases, and purify the organic phases with silica gel chromatography, using dichloromethane and methanol as eluents to obtain the final product E, namely chlorogenic acid derivatives, with a yield of 70-85% %;

The synthetic route is:

Further, in the above step 1, the dosage ratio of chlorogenic acid to acetone is 10 mmol: 20-30 mL, the molar ratio of chlorogenic acid to p-toluenesulfonic acid is 1: 1.0-1.2, and the alkaline substance is saturated sodium carbonate or Saturated sodium bicarbonate.

Further, in the above step 2, the molar ratio of compound A, acetic anhydride and 4-dimethylaminopyridine is 1:1.5～2.0:1～1.5, and the dosage ratio of compound A and dimethylformamide is 10mmol:25 ~35mL, the volume ratio of dichloromethane to methanol is 30:1~3.

Further, in the above step 3, the dosage ratio of compound B to organic solvent is 10 mmol: 20-30 mL, and the molar ratio of compound B to 1,3-dipole C, catalyst copper salt, and oxidant is 1: 1.0-1.2 : 0.1～0.2: 0.2～0.5, the volume ratio of dichloromethane to methanol is 10:1～2.

Further, in the above step 3, the catalyst copper salt is copper chloride, copper bromide, copper acetate, cuprous acetate, copper trifluoroacetate or copper trifluoromethanesulfonate.

Further, in the above step 3, the oxidant is tert-butanol peroxy (TBHP), m-chloroperoxybenzoic acid (m-CPBA), N-bromosuccinimide (NBS), 2,2,6 , 6-tetramethylpiperidinium ammonium tetrafluoroborate (T ⁺ BF ⁴ ) or iodophenyl acetate PhI(OAc) ₂ .

Further, in the above step 3, the organic solvent is DMF, DMA, DMSO, 1,4-dioxane, 1,2-dichloroethane, CH ₃ CN or THF.

Further, in the above step 4, the molar ratio of compound D, trifluoroacetic acid and dichloromethane is 1:2-3:1, and the volume ratio of dichloromethane and methanol is 20:1-2.

Further, the extractant used in the above steps 2, 3, 4 is dichloromethane, ethyl acetate, n-butanol or chloroform.

The above-mentioned synthetic method of chlorogenic acid derivatives, the structural formula of the chlorogenic acid derivatives prepared by it is formula (II):

Wherein, R is H, Bn, Me, Ph, 4-MePh, 4-MeOPh, 4-ClPh or CH ₂ CH ₂ CO; Ar is Ph, 4-MePh, 4-MeOPh, 4-ClPh, 2-MePh or 2-ClPh; X is O or N.

The antibacterial activity assay method of the above-mentioned chlorogenic acid derivatives comprises the following steps: taking the chlorogenic acid derivatives and dissolving them in acetonitrile as a solvent, and respectively configuring the mass percentage concentrations to be 20%, 40%, 60%, 80%, 100% % stock solution; dip 1.0×10 ³ to 2.0×10 ³ cfu·mL ^-1 bacterial solution with a sterile cotton swab, spread it evenly on the surface of the medium plate, and stick it firmly. The strain used for the bacterial solution is large coliform Bacillus, Staphylococcus aureus, or Candida albicans; use sterile tweezers to take out the filter paper soaked with the stock solution of different concentrations above, and spread it on the surface of different culture media; put the culture medium coated with bacterial liquid into a petri dish In the method, the culture dish was inverted, and placed in a 35-37°C incubator for 20-24 hours to observe the phenomenon; transparent circles of different sizes appeared on the culture medium-inhibition zones, and the green circles could be obtained by measuring the diameter of the inhibition zone. Antibacterial activity of ortho acid derivatives.

The present invention also protects the application of the above-mentioned chlorogenic acid derivatives in the preparation of antibacterial drugs.

Owing to adopting above-mentioned technical scheme, the present invention has following advantage:

The synthesis method of the chlorogenic acid derivatives of the present invention is based on the principle of active superposition, through the cycloaddition reaction of the unstable group carbon-carbon double bond and 1,3-dipole in the structure of chlorogenic acid, which will have antibacterial activity The oxazole or pyrazole structure is introduced into it to prepare high-efficiency chlorogenic acid antibacterial agents, which can also enhance the stability of unsaturated double bonds and enhance the biological activity of chlorogenic acid in vivo; the synthesis route is simple and efficient, the source of raw materials is wide, and the cost is low , easy to operate and high yield.

The synthesis method of the chlorogenic acid derivative of the present invention has the novel structure of the prepared chlorogenic acid derivative, remarkable antibacterial effect and high yield, and is suitable for industrialized production.

Detailed ways

The present invention can be further described in detail with reference to the following examples; however, the following examples are merely illustrations, and the present invention is not limited to these examples.

Example 1

A synthetic method for chlorogenic acid derivatives, comprising the following steps:

Step 1. Protection of chlorogenic acid alcoholic hydroxyl group: Dissolve 10 mmol chlorogenic acid in 25 mL of acetone, then add 10 mmol p-toluenesulfonic acid to the reaction system, stir at room temperature for 3 h; TLC monitors the reaction process until the reactant is green The original acid disappeared; after the reaction was completed, a saturated sodium carbonate solution was added to neutralize the remaining p-toluenesulfonic acid to a pH value of 7, suction filtered, and the filtrate was concentrated to obtain compound A1;

Step 2, protection of chlorogenic acid phenolic hydroxyl group: join 9.2mmol compound A1, 13.8mmol acetic anhydride and 9.2mmol DMAP in a 100mL round-bottomed flask, dissolve with 30mL DMF, stir and react at room temperature for 2h; Add 20 mL of saturated brine to the reaction system to wash, then extract three times with dichloromethane (3 × 10 mL), combine the organic phase, and purify the organic phase by silica gel chromatography, using dichloromethane and methanol as eluents, dichloromethane and methanol The volume ratio is 30:1, and the compound B1 is isolated;

Step 3, Synthesis of chlorogenic acid derivatives containing oxazole ring: Dissolve 8.6mmol of compound B1 in 20mL of DMF, then add 8.6mmol of nitrone dipole C1, 0.86mmol of copper acetate, and 1.7mmol of m-CPBA to the reaction In the system, react at a temperature of 50° C. for 5 h; monitor the progress of the reaction by TLC until the compound B1 disappears completely, stop the reaction, add 20 mL of saturated brine to the reaction system for washing, and then extract three times with dichloromethane (3×10 mL) , combined the organic phase, the organic phase was purified by silica gel chromatography, using dichloromethane and methanol as eluents, the volume ratio of dichloromethane to methanol was 10:1, and compound D1 was isolated;

Step 4. Removal of hydroxyl protecting group: take 7.6 mmol of compound D1 and add it to a mixture of 15.2 mmol of trifluoroacetic acid and 7.6 mmol of dichloromethane, react at room temperature for 2 hours, monitor the reaction progress by TLC, wash with 20 mL of saturated saline after the reaction Twice, and then extracted three times with dichloromethane (3 × 10mL), combined organic phase, the organic phase was purified by silica gel chromatography, with dichloromethane and methanol as eluent, the volume ratio of dichloromethane to methanol was 20:1 , to obtain compound E1, i.e. chlorogenic acid derivatives, with a yield of 80%, and the H NMR spectrum data are as follows:

¹ HNMR (CDCl ₃ , 400MHz) δ: 1.55-1.61 (m, 2H), 2.01 (s, 1H), 2.06 (s, 1H), 2.23-2.34 (m, 2H), 3.31 (s, 3H), 3.87 (q,J=8.0Hz,1H),4.03(m,1H),4.17(t,J=6.5,1H),4.42(s,1H),5.0(s,1H),6.08(s,1H), 6.79(s,1H),7.13(t,J＝6.0Hz,2H),7.28-7.36(m,2H),7.48-7.63(m,3H),8.13(s,1H),10.50(s,1H) .

Step 5, Determination of Antibacterial Activity of Compound E1

Dissolve 1 mmol of compound E1 in different volumes of acetonitrile to prepare stock solutions with mass percent concentrations of 20%, 40%, 60%, 80%, and 100%; dip 1.0×10 ³ cfu·m L ^-1 bacteria solution, spread it evenly on the surface of the soybean casein agar medium plate, stick it firmly, the bacteria used in the bacteria solution is Staphylococcus aureus; Shake off the floating liquid on the surface of the filter paper, and spread it on the surface of the soybean casein agar medium; put the culture medium coated with the bacterial solution into a petri dish, turn the petri dish upside down, and place it in a 35°C incubator for cultivation 20h, observe phenomenon; Find that transparent ring-inhibition zone appears on the culture medium, compound E1 is when selecting the concentration ratio of 60%, has remarkable antibacterial effect to Staphylococcus aureus, and the diameter of the inhibition zone is 25.28mm, while the same concentration The diameter of the inhibition zone of lower chlorogenic acid is only 13.17mm.

Example 2

A synthetic method for chlorogenic acid derivatives, comprising the following steps:

Step 1. Protection of chlorogenic acid alcohol hydroxyl group: Dissolve 10mmol chlorogenic acid in 30mL of acetone, then add 11mmol p-toluenesulfonic acid into the reaction system, stir at room temperature for 4h; TLC monitors the reaction process until the reactant is green The original acid disappeared; after the reaction was completed, a saturated sodium carbonate solution was added to neutralize the remaining p-toluenesulfonic acid to a pH value of 7, suction filtered, and the filtrate was concentrated to obtain compound A2;

Step 2. Protection of phenolic hydroxyl group of chlorogenic acid: Add 9.4mmol compound A2, 18.8mmol acetic anhydride and 10mmol DMAP into a 100mL round bottom flask, dissolve with 30mL DMF, stir and react at room temperature for 2h; monitor the completion of the reaction, and report to the reaction Add 20 mL of saturated brine to the system to wash, then extract three times with dichloromethane (3 × 10 mL), combine the organic phases, and purify the organic phases by silica gel chromatography, using dichloromethane and methanol as eluents, the separation of dichloromethane and methanol The volume ratio is 30:2, and the compound B2 is isolated;

Step 3. Synthesis of chlorogenic acid derivatives containing an oxazole ring: Dissolve 8.5mmol of compound B2 in 20mL of DMA, then add 10mmol of nitrone dipole C2, 0.85mmol of copper bromide, and 1.7mmol of m-CPBA to the reaction In the system, react at a temperature of 60°C for 7h; monitor the progress of the reaction by TLC until the compound B2 disappears completely, stop the reaction, add 20mL saturated brine to the reaction system to wash, and then extract three times with dichloromethane (3×10mL) , combined the organic phase, the organic phase was purified by silica gel chromatography, using dichloromethane and methanol as eluents, the volume ratio of dichloromethane to methanol was 10:1.5, and compound D2 was isolated;

Step 4, removal of hydroxyl protecting group: Take 7.4mmol of compound D2 and add it to a mixture of 17mmol of trifluoroacetic acid and 7.6mmol of dichloromethane, react at room temperature for 3h, monitor the progress of the reaction by TLC, after the reaction, wash the mixture with 20mL of saturated saline times, extracted five times with dichloromethane (5×10 mL), combined the organic phases, and purified the organic phases by silica gel chromatography, using dichloromethane and methanol as eluents, the volume ratio of dichloromethane to methanol was 20:1.5, Obtain compound E2, i.e. chlorogenic acid derivative, the yield is 82.5%, and the H NMR spectrum data are as follows:

¹ HNMR (CDCl ₃ , 400MHz) δ: 1.72-1.76 (m, 2H), 2.12 (s, 1H), 2.26-2.34 (m, 2H), 2.49 (s, 1H), 3.00 (s, 1H), 3.99 (t,J=8.5Hz,1H),4.05(q,J=8.5Hz,1H),4.24(m,1H),4.60(s,1H),6.54(s,1H),6.89(s,1H) , 7.32-7.55 (m, 5H), 7.61-7.78 (m, 4H), 7.84-7.89 (m, 3H), 8.70 (s, 1H), 11.03 (s, 1H).

Step 5, Determination of Antibacterial Activity of Compound E2

Dissolve 2 mmol of compound E2 in different volumes of acetonitrile to prepare stock solutions with mass percent concentrations of 20%, 40%, 60%, 80%, and 100%; dip 1.5×10 ³ cfu·m L ^-1 bacteria solution, spread it evenly on the surface of the eosin methylene blue agar medium plate, stick it firmly, the bacteria used in the bacteria solution is E. Filter paper, shake off the floating liquid on the surface of the filter paper, spread it on the surface of eosin methylene blue agar medium; put the culture medium coated with bacterial liquid into a petri dish, put the petri dish upside down, and place it in a 37°C incubator for cultivation 20h, observe phenomenon; Find that transparent circular ring-inhibition zone appears on the culture medium, compound E2 is when selecting the concentration ratio of 40%, all antibacterial effect is remarkable to E. The diameter of the inhibition zone of chlorogenic acid is 10.26mm.

Example 3

A synthetic method for chlorogenic acid derivatives, comprising the following steps:

Step 1. Protection of chlorogenic acid alcohol hydroxyl group: Dissolve 10mmol chlorogenic acid in 28mL of acetone, then add 11mmol p-toluenesulfonic acid to the reaction system, stir at room temperature for 3.5h; TLC monitors the reaction process until the reactant Chlorogenic acid disappears; after the reaction is completed, add saturated sodium bicarbonate solution to neutralize the remaining p-toluenesulfonic acid to a pH value of 6.8, filter with suction, and concentrate the filtrate to obtain compound A3;

Step 2, protection of chlorogenic acid phenolic hydroxyl group: join 9.4mmol compound A3, 15.8mmol acetic anhydride and 12mmol DMAP in a 100mL round-bottomed flask, dissolve with 32mL DMF, stir and react at room temperature for 2.5h; The reaction system was washed with 20 mL of saturated brine, extracted four times with ethyl acetate (4×10 mL), the organic phases were combined, and the organic phase was purified by silica gel chromatography, using dichloromethane and methanol as eluents, dichloromethane and The volume ratio of methanol is 30:2, and compound B3 is isolated;

Step 3. Synthesis of chlorogenic acid derivatives containing oxazole ring: Dissolve 8.9 mmol of compound B3 in 25 mL of 1,4-dioxane, then add 9.8 mmol of nitrone dipole C3 and 0.86 mmol of trifluoromethanesulfonate Copper acid and 2.7 mmol NBS were added to the reaction system, and reacted at a temperature of 70 ° C for 6 h; TLC monitored the progress of the reaction until the compound B3 disappeared completely, and stopped the reaction. The ester was extracted three times (3×10 mL), the organic phases were combined, and the organic phase was purified by silica gel chromatography, using dichloromethane and methanol as eluents, the volume ratio of dichloromethane and methanol was 10:2, and compound D3 was isolated;

Step 4. Removal of hydroxyl protecting group: take 7.7mmol of compound D3 and add it to a mixture of 19.2mmol of trifluoroacetic acid and 7.7mmol of dichloromethane, react at room temperature for 2.8h, monitor the progress of the reaction by TLC, after the reaction, use 20mL of saturated saline Wash twice, extract four times with ethyl acetate (4×10 mL), combine the organic phases, and purify the organic phases by silica gel chromatography, using dichloromethane and methanol as eluents, and the volume ratio of dichloromethane to methanol is 20: 1.5, to obtain compound E3, namely chlorogenic acid derivatives, with a yield of 75.9%, and the H NMR spectrum data are as follows:

¹ HNMR (CDCl ₃ , 400MHz) δ: 1.57-1.61 (m, 2H), 2.04 (s, 1H), 2.33-2.38 (m, 2H), 2.47 (s, 1H), 3.83 (t, J = 8.5Hz ,1H),3.96(q,J=8.5Hz,1H),4.26-4.29(m,1H),4.41(s,2H),4.57(t,J=8.5Hz,1H),4.78(s,1H) ,5.45(s,1H),6.76(s,1H),7.10-7.26(m,6H),7.31-7.48(m,5H),7.50-7.52(m,1H),8.59(s,1H),10.16 (s,1H).

Step 5, Determination of Antibacterial Activity of Compound E3

Dissolve 1 mmol of compound E3 in different volumes of acetonitrile to prepare stock solutions with mass percent concentrations of 20%, 40%, 60%, 80%, and 100%; dip 1.3×10 ³ cfu·m L ^-1 bacteria solution, spread it evenly on the surface of the soybean casein agar medium plate, stick it firmly, the bacteria used in the bacteria solution is Staphylococcus aureus; Shake off the floating liquid on the surface of the filter paper, and spread it on the surface of the soybean casein agar medium; put the culture medium coated with the bacterial solution into a petri dish, turn the petri dish upside down, and place it in a 37°C incubator for cultivation 22h, observe the phenomenon; find that a transparent ring-inhibition zone appears on the culture medium, and compound E3 has a significant antibacterial effect on Staphylococcus aureus when the concentration ratio of 60% is selected, and the diameter of the inhibition zone is 24.11mm, which is higher than that of the same The diameter of the inhibition zone of chlorogenic acid under the concentration is 13.17mm.

Example 4

A synthetic method for chlorogenic acid derivatives, comprising the following steps:

Step 1. Protection of chlorogenic acid alcohol hydroxyl group: Dissolve 10mmol chlorogenic acid in 30mL acetone, then add 12mmol p-toluenesulfonic acid into the reaction system, stir at room temperature for 4h; TLC monitors the reaction process until the reactant is green The original acid disappeared; after the reaction was completed, a saturated sodium bicarbonate solution was added to neutralize the remaining p-toluenesulfonic acid to a pH value of 7.3, suction filtered, and the filtrate was concentrated to obtain compound A4;

Step 2, protection of chlorogenic acid phenolic hydroxyl group: join 9.5mmol compound A4, 17.1mmol acetic anhydride and 10.5mmol DMAP in a 100mL round-bottomed flask, dissolve with 25mL DMF, stir and react at room temperature for 3h; Add 20 mL of saturated brine to the reaction system to wash, then extract three times with n-butanol (3 × 10 mL), combine the organic phases, and purify the organic phases by silica gel chromatography, using dichloromethane and methanol as eluents, dichloromethane and methanol The volume ratio is 30:2.5, and the compound B4 is isolated;

Step 3. Synthesis of chlorogenic acid derivatives containing pyrazole rings: Dissolve 8.9 mmol of compound B4 in 25 mL of DMSO, then add 10.7 mmol of azomethene imine dipole C4, 0.89 mmol of copper chloride, Add 3.6mmol TBHP into the reaction system and react at 60°C for 6h; TLC monitors the progress of the reaction until the compound B4 disappears completely, then stop the reaction, add 20mL saturated saline to the reaction system to wash, and then extract three times with n-butanol (3×10mL), the organic phases were combined, and the organic phase was purified by silica gel chromatography, using dichloromethane and methanol as eluents, the volume ratio of dichloromethane to methanol was 10:1.2, and compound D4 was isolated;

Step 4, removal of hydroxyl protecting group: take 7.8mmol of compound D4 and add it to a mixture of 17.2mmol of trifluoroacetic acid and 7.8mmol of dichloromethane, react at room temperature for 2.5h, monitor the progress of the reaction by TLC, after the reaction, use 20mL of saturated saline Wash three times, extract three times with n-butanol (3 × 10mL), combine the organic phase, and purify the organic phase by silica gel chromatography, using dichloromethane and methanol as eluents, the volume ratio of dichloromethane and methanol is 20:1.8, Compound E4 was obtained with a yield of 83.2%, and the H NMR spectrum data were as follows:

¹ HNMR (CDCl ₃ , 400MHz) δ: 1.53-1.57(m, 2H), 2.00(s, 1H), 2.09(s, 3H), 2.16-2.22(m, 2H), 2.19(t, J=6.5Hz ,2H),2.33(s,1H),3.10(s,1H),3.57(t,J=6.5,2H),3.91(t,J=8.5Hz,1H),4.16(q,t=8.5Hz, 1H),4.20(m,1H),4.48(s,1H),5.25(s,1H),6.81(s,1H),7.06-7.10(m,3H),7.26-7.36(m,3H),8.61 (s,1H),10.27(s,1H).

Step 5, the antibacterial activity assay of compound E4

Dissolve 1 mmol of compound E4 in different volumes of acetonitrile to prepare stock solutions with mass percent concentrations of 20%, 40%, 60%, 80%, and 100%; dip 1.8×10 ³ cfu·m L ^-1 bacteria solution, spread it evenly on the surface of the soybean casein agar medium plate, stick it firmly, the bacteria used in the bacteria solution is Staphylococcus aureus; Shake off the floating liquid on the surface of the filter paper, and spread it on the surface of the soybean casein agar medium; put the culture medium coated with the bacterial solution into a petri dish, turn the petri dish upside down, and place it in a 36°C incubator for cultivation 24h, observe phenomenon; Find that transparent ring-inhibition zone appears on the culture medium, compound E4 is when selecting the concentration ratio of 60%, has remarkable antibacterial effect to Staphylococcus aureus, and the diameter of inhibition zone is 28.04mm, far higher than The diameter of the inhibition zone of chlorogenic acid at the same concentration was 13.17mm.

Example 5

A synthetic method for chlorogenic acid derivatives, comprising the following steps:

Step 1. Protection of chlorogenic acid alcohol hydroxyl group: Dissolve 10mmol chlorogenic acid in 24mL acetone, then add 12mmol p-toluenesulfonic acid to the reaction system, stir at room temperature for 4.5h; TLC monitors the reaction process until the reactant Chlorogenic acid disappears; after the reaction is completed, add saturated sodium bicarbonate solution to neutralize the remaining p-toluenesulfonic acid to a pH value of 7, filter with suction, and concentrate the filtrate to obtain compound A5;

Step 2, protection of chlorogenic acid phenolic hydroxyl group: join 9.3mmol compound A5, 14.9mmol acetic anhydride and 11.2mmol DMAP in a 100mL round bottom flask, dissolve with 27mL DMF, stir and react at room temperature for 3h; Add 20 mL of saturated brine to the reaction system to wash, then extract three times with dichloromethane (3 × 10 mL), combine the organic phase, and purify the organic phase by silica gel chromatography, using dichloromethane and methanol as eluents, dichloromethane and methanol The volume ratio is 30:3, and the compound B5 is isolated;

Step 3. Synthesis of chlorogenic acid derivatives containing pyrazole rings: Dissolve 8.9 mmol of compound B5 in 30 mL of acetonitrile, then add 9.8 mmol of azomethene imine dipole C5, 1.3 mmol of cuprous acetate, Add 4.5mmol of iodobenzene acetate to the reaction system, and react at a temperature of 60°C for 8h; TLC monitors the progress of the reaction until compound B5 disappears completely, then stops the reaction, adds 20mL of saturated saline to the reaction system to wash, and then washes with dichloromethane Extract three times (3×10mL), combine the organic phase, and purify the organic phase by silica gel chromatography, using dichloromethane and methanol as eluents, the volume ratio of dichloromethane and methanol is 10:1.6, and isolate compound D5;

Step 4. Removal of hydroxyl protecting group: take 7.7 mmol of compound D5 and add it to a mixture of 16.2 mmol of trifluoroacetic acid and 7.7 mmol of dichloromethane, react at room temperature for 3 hours, monitor the reaction progress by TLC, wash with 20 mL of saturated saline after the reaction is completed Twice, extracted three times with dichloromethane (3×10mL), combined the organic phase, and purified the organic phase with silica gel chromatography, using dichloromethane and methanol as eluents, the volume ratio of dichloromethane to methanol was 20:1.4, Compound E5 was obtained with a yield of 84.3%, and the H NMR spectrum data were as follows:

¹ HNMR (CDCl ₃ , 400MHz) δ: 1.81-1.84(m, 2H), 2.07(s, 1H), 2.47-2.49(m, 2H), 2.23(t, J=6.5Hz, 2H), 2.31(s ,1H),2.64(s,1H),3.63(t,J=6.5Hz,2H),3.79(s,1H),4.03(t,J=8.5Hz,1H),4.16(q,t=8.5Hz ,1H),4.22(m,1H),5.61(s,1H),6.95(s,1H),7.16-7.22(m,3H),7.28-7.35(m,2H),7.43(m,1H), 8.25(s,1H), 11.13(s,1H).

Step 5, the antibacterial activity assay of compound E5

Dissolve 1.5mmol of compound E5 in different volumes of acetonitrile to prepare stock solutions with concentrations of 20%, 40%, 60%, 80%, and 100% by mass percentage; dip 1.6×10 ³ cfu· m L ^-1 bacterial solution, spread it evenly on the surface of the eosin methylene blue agar medium plate, and stick it firmly. The strain used in the bacterial solution is Candida albicans; take it out with sterile tweezers and soak it in the above-mentioned stock solutions of different concentrations filter paper, shake off the floating liquid on the surface of the filter paper, and spread it on the surface of the eosin methylene blue agar medium; put the culture medium coated with the bacterial solution into a petri dish, put the petri dish upside down, and place it in a 36°C incubator Cultured in medium for 24h, observe the phenomenon; found that a transparent ring-inhibition zone appeared on the culture medium, compound E5 had a significant antibacterial effect on Candida albicans when the concentration ratio of 80% was selected, and the diameter of the inhibition zone was 26.32mm, which was much higher than The diameter of the inhibition zone of chlorogenic acid at the same concentration is 9.98mm.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention should be included in the protection of the present invention. within range.

Claims (10)

1. A method for synthesizing chlorogenic acid derivatives is characterized by comprising the following steps: which comprises the following steps:

step 1, protection of alcoholic hydroxyl group of chlorogenic acid: dissolving chlorogenic acid in acetone, adding p-toluenesulfonic acid into a reaction system, and stirring at room temperature for 3-5 h; TLC monitoring reaction progress until reactant chlorogenic acid disappears; after the reaction is finished, adding an alkaline substance, neutralizing the residual p-toluenesulfonic acid until the pH value is 6-7.5, performing suction filtration, and concentrating the filtrate to obtain a compound A;

step 2, protecting phenolic hydroxyl of chlorogenic acid: adding the compound A obtained in the step 1, acetic anhydride with a phenolic hydroxyl protecting group and 4-dimethylaminopyridine serving as a catalyst into dimethylformamide serving as a solvent, and stirring and reacting at room temperature for 2-3 hours; monitoring the reaction, adding saturated saline solution into the reaction system for washing, then extracting by using an extracting agent, combining organic phases, purifying the organic phases by using silica gel chromatography, and separating out a compound B by using dichloromethane and methanol as eluent;

and 3, synthesizing chlorogenic acid derivatives containing oxazole or pyrazole rings: dissolving the compound B obtained in the step 2 in an organic solvent, adding 1, 3-dipole C, a catalyst copper salt and an oxidant into a reaction system, and reacting for 5-8 h at the temperature of 50-70 ℃; TLC monitoring reaction progress until compound B disappears completely, stopping reaction, adding saturated salt solution into reaction system, washing, extracting with extractant, combining organic phase, purifying organic phase with silica gel chromatography, eluting with dichloromethane and methanol to separate out compound D;

the 1, 3-dipole C is of the formula (I)

Wherein R is H, bn, me, ph, 4-MePh, 4-MeOPh, 4-ClPh or CH ₂ CH ₂ CO; ar is Ph, 4-MePh, 4-MeOPh, 4-ClPh, 2-MePh or 2-ClPh; x is O or N;

and 4, removing a hydroxyl protecting group: adding the compound D obtained in the step 3 into a mixture of trifluoroacetic acid and dichloromethane, reacting at room temperature for 1.5-3 h, monitoring the reaction process by TLC, after the reaction is finished, adding saturated salt solution into a reaction system for washing, extracting by using an extracting agent, combining organic phases, purifying the organic phases by using silica gel chromatography, and taking dichloromethane and methanol as eluent to obtain a final product E, namely a chlorogenic acid derivative, wherein the yield is 70-85%;

the synthetic route is as follows:

2. the method for synthesizing a chlorogenic acid derivative as claimed in claim 1, wherein: in the step 1, the dosage ratio of chlorogenic acid to acetone is 10mmol: 20-30 mL, wherein the molar ratio of chlorogenic acid to p-toluenesulfonic acid is 1:1.0 to 1.2, and the alkaline substance is saturated sodium carbonate or saturated sodium bicarbonate.

3. The method for synthesizing a chlorogenic acid derivative as claimed in claim 1, wherein: in the step 2, the molar ratio of the compound A, acetic anhydride and 4-dimethylamino pyridine is 1: 1.5-2.0: 1-1.5, the dosage ratio of the compound A to the dimethylformamide is 10mmol: 25-35 mL, wherein the volume ratio of dichloromethane to methanol is 30:1 to 3.

4. The method for synthesizing a chlorogenic acid derivative as claimed in claim 1, wherein: in the step 3, the dosage ratio of the compound B to the organic solvent is 10mmol: 20-30 mL, wherein the molar ratio of the compound B to the 1, 3-dipole C to the catalyst copper salt to the oxidant is 1:1.0 to 1.2:0.1 to 0.2: 0.2-0.5, wherein the volume ratio of dichloromethane to methanol is 10:1 to 2.

5. The method for synthesizing a chlorogenic acid derivative according to claim 1 or 4, characterized in that: in the step 3, the copper salt of the catalyst is cupric chloride, cupric bromide, cupric acetate, cuprous acetate, cupric trifluoroacetate or cupric trifluoromethanesulfonate.

6. The method for synthesizing a chlorogenic acid derivative according to claim 1 or 4, characterized in that: in the step 3, the oxidant is tert-butyl peroxy, m-chloroperoxybenzoic acid, N-bromosuccinimide, 2, 6-tetramethyl piperidinyloxy ammonium tetrafluoroborate or iodobenzene acetate; the organic solvent is DMF, DMA, DMSO, 1,4-dioxane, 1,2-dichloroethane, or CH ₃ CN or THF.

7. The method for synthesizing a chlorogenic acid derivative as claimed in claim 1, wherein: in the step 4, the molar ratio of the compound D, trifluoroacetic acid and dichloromethane is 1:2 to 3:1, wherein the volume ratio of dichloromethane to methanol is 20:1 to 2.

8. A method for synthesizing a chlorogenic acid derivative according to any of claims 1 to 7, characterized in that: the structural formula of the chlorogenic acid derivative prepared by the method is shown as formula (II):

wherein R is H, bn, me, ph, 4-MePh, 4-MeOPh, 4-ClPh or CH ₂ CH ₂ CO; ar is Ph, 4-MePh, 4-MeOPh, 4-ClPh, 2-MePh or 2-ClPh; x is O or N.

9. A method for measuring antibacterial activity of chlorogenic acid derivative prepared by the synthesis method of claim 8, which comprises the following steps: which comprises the following steps: dissolving chlorogenic acid derivatives with acetonitrile as solvent, and preparing into stock solutions with mass percentage concentrations of 20%, 40%, 60%, 80%, and 100%, respectively; dipping with sterile cotton swab at 1.0 × 10 ³ ～2.0×10 ³ cfu·mL ^-1 The bacterial liquid is uniformly coated on the surface of a culture medium plate and is pasted firmly, and the used strain of the bacterial liquid is escherichia coli, staphylococcus aureus or candida albicans; taking out the filter paper sheets soaked by the stock solutions with different concentrations by using sterile forceps, and spreading the filter paper sheets on the surfaces of different culture media; putting the culture medium coated with the bacterial liquid into a culture dish, inverting the culture dish, culturing for 20-24 h in a thermostat at 35-37 ℃, and observing the phenomenon; transparent circular rings-bacteriostatic rings with different sizes appear on the culture medium respectively, and the bacteriostatic activity of the chlorogenic acid derivative can be obtained by measuring the diameters of the bacteriostatic rings.

10. Use of chlorogenic acid derivative prepared by the synthesis method of claim 8 in preparing antibacterial drugs.