CN111848537B - Synthesis method of chlorogenic acid derivative and antibacterial activity determination method - Google Patents
Synthesis method of chlorogenic acid derivative and antibacterial activity determination method Download PDFInfo
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Abstract
The invention discloses a method for synthesizing chlorogenic acid derivatives, which comprises the following steps: step 1, protecting alcoholic hydroxyl groups of chlorogenic acid; step 2, protecting phenolic hydroxyl groups of chlorogenic acid; step 3, synthesizing chlorogenic acid derivatives containing oxazole or pyrazole rings; step 4, removing a hydroxyl protecting group; the chlorogenic acid high-efficiency antibacterial agent can be prepared, has a remarkable antibacterial effect, and can enhance the stability of unsaturated double bonds and improve the in-vivo biological activity of chlorogenic acid.
Description
Technical Field
The invention relates to the technical field of chlorogenic acid, in particular to a synthetic method of a chlorogenic acid derivative and an antibacterial activity determination method.
Background
Chlorogenic acid (Chlorogenic acid) is dephenolic acid formed by condensing caffeic acid (caffeic acid) and quinic acid (quinic acid, 1-hydroxyhexahydro gallic acid), is allo-caffeoylquinic acid (3-O-caffeoylquinic acid) in chemical name, and is a phenylpropanoid table substance produced by plant bodies through a shikimic acid pathway in an aerobic respiration process. Chlorogenic acid is a main effective component of various medicinal materials, such as honeysuckle, oriental wormwood, eucommia ulmoides and the like, and Chinese patent medicines, such as Fuganni, honeysuckle injection, acne oral liquid and the like, for resisting bacteria, removing toxicity, diminishing inflammation and benefiting gallbladder, and is also an important index for quality control of certain Chinese medicinal preparations. Chlorogenic acid has wide bioactivity, and has antibacterial, antiviral, leukocyte increasing, liver protecting, gallbladder function promoting, antitumor, blood pressure lowering, blood lipid reducing, free radical scavenging, and central nervous system exciting effects. The research on the biological activity of chlorogenic acid by modern science is deeply carried out in a plurality of fields of food, health care, medicine, daily chemical industry and the like.
Due to the existence of ester bonds, unsaturated double bonds and three unstable groups of dihydric phenol in the molecular structure of the chlorogenic acid, the in vivo bioactivity of the chlorogenic acid is reduced to a great extent. Considerable literature and experimental data have been available over the years for chemical modification or remodeling of chlorogenic acid based on its unfavorable physicochemical and biological properties, wherein simple modification of the chlorogenic acid skeleton is an effective way to obtain highly active chlorogenic acid derivatives, and most of the modifications are focused on multiple hydroxyl substituents, carboxyl groups and ester groups in the molecular structure of chlorogenic acid according to the modification sites (Chinese herbal medicine, 2020, 51 (4), 937), while modification of unsaturated carbon-carbon double bonds is rare.
Most of the compounds containing oxazole and pyrazole structures have wide biological activity, have obvious curative effect on resisting fungi, bacteria and malignant tumors, and are used as important medical intermediates and lead compounds. For example, muscial is a compound containing an oxazoline ring skeleton, and has been confirmed to have natural anticancer, antibacterial and tranquilizing functions (eur.j.med.chem.1995, 30, 839); in 2003, barbachyn et al designed and synthesized a series of compounds containing oxazoline parent nucleus, and found that part of the substances have better antibacterial activity and good pharmacokinetic properties than linezolid through activity screening (J.Med.chem.2003, 46, 284); water, etc. reported in 2012 to have 5-phosphoryl-3-aryl isoxazoline, which has a certain inhibitory activity on neuraminidase (chinese.j.org.chem.2012, 32, 1336); in 2013, the compound containing oxazole ring synthesized by Wangshi et al shows strong inhibitory action on escherichia coli and bacillus subtilis (Chin.J.org.chem.2013, 33, 2196); since the 70's of the 20 th century, ankhiwala M.D. et al reported that certain substituted pyrazoline compounds have excellent fungicidal activity (Journal of the Indian Chemical Society,1990, 67 (6), 514); subsequently, antibacterial activity of pyrazoline derivatives was sequentially reported by Turan-Zitounia G and Yangjinmei (Archiv Der Pharmazie,2005, 338, 96; proceedings of the national university of Yunnan (Nature science edition), 2007, 16 (1), 33); in 2020, serial pyrazoline derivatives are combined by the cinnabar subject, the bacteriostatic effects of the series pyrazoline derivatives on gram-positive bacteria, gram-negative bacteria and fungi are analyzed, and the pyrazoline compounds are found to have different degrees of inhibitory action on various bacteria (industrial catalysis, 2020, 28 (2) and 53).
Disclosure of Invention
In order to solve the above problems, the present invention aims to provide a method for synthesizing chlorogenic acid derivatives, and also discloses a method for measuring antibacterial activity of chlorogenic acid derivatives prepared by the above synthesis method.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
a method for synthesizing chlorogenic acid derivatives comprises the following steps:
step 1, protecting alcoholic hydroxyl groups of chlorogenic acid: dissolving chlorogenic acid in acetone, adding p-toluenesulfonic acid into a reaction system, and stirring at room temperature for 3-5 hours; 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 a catalyst 4-Dimethylaminopyridine (DMAP) into a solvent Dimethylformamide (DMF), and stirring and reacting for 2-3 h at room temperature; 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:
further, 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.
Further, in the step 2, the molar ratio of the compound a, acetic anhydride and 4-dimethylaminopyridine 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.
Further, in the step 3, the 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.
Further, in the step 3, the copper salt as the catalyst is cupric chloride, cupric bromide, cupric acetate, cuprous acetate, cupric trifluoroacetate or cupric trifluoromethanesulfonate.
Further, in the step 3, the oxidant is tert-butyl peroxy-butyl alcohol (TBHP), m-chloroperoxybenzoic acid (m-CPBA), N-bromosuccinimideAmines (NBS), 2, 6-Tetramethylpiperidinoammonium tetrafluoroborate (T) + BF 4 ) Or iodobenzene acetate PhI (OAc) 2 。
Further, in the step 3, the organic solvent is DMF, DMA, DMSO, 1,4-dioxane, 1,2-dichloroethane, CH 3 CN or THF.
Further, in the step 4, the molar ratio of the compound D, trifluoroacetic acid and dichloromethane is 1:2 to 3:1, the volume ratio of dichloromethane to methanol is 20:1 to 2.
Further, the extractant used in the above steps 2,3 and 4 is dichloromethane, ethyl acetate, n-butanol or chloroform.
The structural formula of the chlorogenic acid derivative prepared by the method is shown as the 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 method for measuring the antibacterial activity of the chlorogenic acid derivative 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 Uniformly coating the bacteria liquid on the surface of a culture medium plate, and sticking the bacteria liquid to the surface, wherein the bacteria used by the bacteria liquid are 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.
The invention also protects the application of the chlorogenic acid derivative in preparing antibacterial drugs.
Due to the adoption of the technical scheme, the invention has the following advantages:
according to the synthesis method of the chlorogenic acid derivative, an oxazole or pyrazole structure with antibacterial activity is introduced into the chlorogenic acid derivative through a cycloaddition reaction of an unstable group carbon-carbon double bond in the chlorogenic acid structure and a1, 3-dipole according to an activity superposition principle to prepare the chlorogenic acid efficient antibacterial agent, so that the stability of an unsaturated double bond can be enhanced, and the in-vivo biological activity of the chlorogenic acid can be improved; the synthesis route is simple and efficient, the raw material source is wide, the cost is low, the operation is easy, and the yield is high.
The chlorogenic acid derivative prepared by the method has the advantages of novel structure, obvious antibacterial effect and high yield, and is suitable for industrial production.
Detailed Description
The present invention will be further described in detail with reference to the following examples; however, the following examples are merely illustrative, and the present invention is not limited to these examples.
Example 1
A method for synthesizing chlorogenic acid derivatives comprises the following steps:
step 1, protecting alcoholic hydroxyl groups of chlorogenic acid: dissolving 10mmol of chlorogenic acid in 25mL of acetone, adding 10mmol of p-toluenesulfonic acid into a reaction system, and stirring at room temperature for 3 hours; TLC monitoring reaction progress until reactant chlorogenic acid disappears; after the reaction is finished, adding a saturated sodium carbonate solution, neutralizing the residual p-toluenesulfonic acid until the pH value is 7, performing suction filtration, and concentrating the filtrate to obtain a compound A1;
step 2, protecting phenolic hydroxyl of chlorogenic acid: adding 9.2mmol of compound A1, 13.8mmol of acetic anhydride and 9.2mmol of DMAP into a 100mL round-bottom flask, dissolving with 30mL of DMF, and stirring at room temperature for reaction for 2h; the reaction was monitored for completion, and the reaction was washed with 20mL of saturated brine, extracted three times with dichloromethane (3X 10 mL), and the organic phases were combined and purified by silica gel chromatography using dichloromethane and methanol as eluents at a dichloromethane to methanol volume ratio of 30:1, separating out a compound B1;
step 3, synthesis of chlorogenic acid derivative containing oxazole ring: dissolving 8.6mmol of compound B1 in 20mL of DMF, adding 8.6mmol of nitrone dipole C1, 0.86mmol of copper acetate and 1.7mmol of m-CPBA into a reaction system, and reacting at 50 ℃ for 5 hours; the reaction was stopped by TLC monitoring the progress of the reaction until compound B1 completely disappeared, 20mL of saturated brine was added to the reaction system and washed, followed by three times of extraction with dichloromethane (3 × 10 mL), and the organic phases were combined and purified by silica gel chromatography using dichloromethane and methanol as eluents at a volume ratio of dichloromethane to methanol of 10:1, separating out a compound D1;
and 4, removing a hydroxyl protecting group: adding 7.6mmol of compound D1 into a mixture of 15.2mmol of trifluoroacetic acid and 7.6mmol of dichloromethane, reacting at room temperature for 2h, monitoring the reaction progress by TLC, washing twice with 20mL of saturated saline solution after the reaction is finished, extracting three times with dichloromethane (3X 10 mL), combining organic phases, and purifying the organic phases by silica gel chromatography with dichloromethane and methanol as eluent and the volume ratio of dichloromethane to methanol of 20:1, obtaining a compound E1, namely a chlorogenic acid derivative, with the yield of 80 percent, and the nuclear magnetic hydrogen 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
Dissolving 1mmol of compound E1 in acetonitrile with different volumes respectively to prepare stock solutions with mass percent concentrations of 20%, 40%, 60%, 80% and 100%; dipping with sterile cotton swab at 1.0 × 10 3 cfu·m L -1 Uniformly coating the bacterial solution onThe surface of the soybean casein agar medium plate is pasted firmly, and the strain used by the bacterial liquid is staphylococcus aureus; taking out the filter paper pieces soaked by the stock solutions with different concentrations by using a sterile forceps, throwing off the floating liquid on the surfaces of the filter paper pieces, and spreading the filter paper pieces on the surface of a soybean casein agar culture medium; placing the culture medium coated with the bacterial liquid into a culture dish, inverting the culture dish, placing the culture dish in a thermostat at 35 ℃ for culturing for 20 hours, and observing the phenomenon; the transparent circular ring-bacteriostasis ring appears on the culture medium, when the concentration ratio of the compound E1 is 60%, the bacteriostasis effect on staphylococcus aureus is obvious, the diameter of the bacteriostasis ring is 25.28mm, and the diameter of the bacteriostasis ring of chlorogenic acid under the same concentration is only 13.17mm.
Example 2
A method for synthesizing chlorogenic acid derivatives comprises the following steps:
step 1, protecting alcoholic hydroxyl groups of chlorogenic acid: dissolving 10mmol of chlorogenic acid in 30mL of acetone, adding 11mmol of p-toluenesulfonic acid into a reaction system, and stirring at room temperature for 4 hours; TLC monitoring the reaction progress until the reactant chlorogenic acid disappears; after the reaction is finished, adding a saturated sodium carbonate solution, neutralizing the residual p-toluenesulfonic acid until the pH value is 7, performing suction filtration, and concentrating the filtrate to obtain a compound A2;
step 2, protecting phenolic hydroxyl of chlorogenic acid: adding 9.4mmol of compound A2, 18.8mmol of acetic anhydride and 10mmol of DMAP into a 100mL round-bottom flask, dissolving with 30mL of DMF, and stirring for reaction at room temperature for 2 hours; the reaction was monitored for completion, and the reaction was washed with 20mL of saturated brine, extracted three times with dichloromethane (3X 10 mL), and the organic phases were combined and purified by silica gel chromatography using dichloromethane and methanol as eluents at a dichloromethane to methanol volume ratio of 30:2, separating out a compound B2;
step 3, synthesis of chlorogenic acid derivatives containing oxazole ring: dissolving 8.5mmol of compound B2 in 20mL of DMA, adding 10mmol of nitrone dipole C2, 0.85mmol of cupric bromide and 1.7mmol of m-CPBA into a reaction system, and reacting at the temperature of 60 ℃ for 7h; the reaction was stopped by TLC monitoring the progress of the reaction until compound B2 completely disappeared, 20mL of saturated brine was added to the reaction system and washed with dichloromethane three times (3 × 10 mL), the organic phases were combined and purified by silica gel chromatography using dichloromethane and methanol as eluents at a dichloromethane to methanol volume ratio of 10:1.5, separating out a compound D2;
and 4, removing a hydroxyl protecting group: adding 7.4mmol of compound D2 into a mixture of 17mmol of trifluoroacetic acid and 7.6mmol of dichloromethane, reacting at room temperature for 3h, monitoring the reaction progress by TLC, washing twice with 20mL of saturated saline solution after the reaction is finished, extracting five times (5X 10 mL) with dichloromethane, combining organic phases, and purifying the organic phases by silica gel chromatography with dichloromethane and methanol as eluent and the volume ratio of dichloromethane to methanol of 20:1.5, obtaining a compound E2, namely a chlorogenic acid derivative, with the yield of 82.5 percent and the nuclear magnetic hydrogen spectrum data 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
Dissolving 2mmol of compound E2 in acetonitrile with different volumes respectively to prepare stock solutions with mass percentage concentrations of 20%, 40%, 60%, 80% and 100%; dipping with sterile cotton swab at 1.5X 10 3 cfu·m L -1 Uniformly coating the bacterial liquid on the surface of an eosin methylene blue agar culture medium plate, and sticking the bacterial liquid to the surface, wherein the strain used by the bacterial liquid is escherichia coli; taking out the filter paper pieces soaked by the stock solutions with different concentrations by using a sterile forceps, throwing off the floating liquid on the surfaces of the filter paper pieces, and spreading the filter paper pieces on the surface of an eosin methylene blue agar culture medium; putting the culture medium coated with the bacterial liquid into a culture dish, inverting the culture dish, culturing for 20 hours in a 37 ℃ thermostat, and observing the phenomenon; the transparent circular ring-bacteriostasis zone appears on the culture medium, when the concentration ratio of the compound E2 is selected to be 40 percent, the bacteriostasis effect on escherichia coli is obvious, and the diameter of the bacteriostasis zone is 27.33mm, and the diameter of the bacteriostatic circle of chlorogenic acid under the same concentration is 10.26mm.
Example 3
A method for synthesizing chlorogenic acid derivatives comprises the following steps:
step 1, protection of alcoholic hydroxyl group of chlorogenic acid: dissolving 10mmol of chlorogenic acid in 28mL of acetone, adding 11mmol of p-toluenesulfonic acid into a reaction system, and stirring at room temperature for 3.5h; TLC monitoring reaction progress until reactant chlorogenic acid disappears; after the reaction is finished, adding a saturated sodium bicarbonate solution, neutralizing the residual p-toluenesulfonic acid until the pH value is 6.8, performing suction filtration, and concentrating the filtrate to obtain a compound A3;
step 2, protecting phenolic hydroxyl of chlorogenic acid: adding 9.4mmol of compound A3, 15.8mmol of acetic anhydride and 12mmol of DMAP into a 100mL round-bottom flask, dissolving with 32mL of DMF, and stirring at room temperature for reaction for 2.5h; the reaction was monitored for completion, and the reaction was washed with 20mL of saturated brine, extracted four times with ethyl acetate (4X 10 mL), and the organic phases were combined and purified by silica gel chromatography using dichloromethane and methanol as eluents at a dichloromethane to methanol volume ratio of 30:2, separating out a compound B3;
step 3, synthesis of chlorogenic acid derivatives containing oxazole ring: dissolving 8.9mmol of compound B3 in 25mL1, 4-dioxane, adding 9.8mmol of nitrone dipole C3, 0.86mmol of copper trifluoromethanesulfonate and 2.7mmol of NBS into a reaction system, and reacting at 70 ℃ for 6h; the reaction was stopped by TLC monitoring the progress of the reaction until compound B3 completely disappeared, 20mL of saturated brine was added to the reaction system and washed, extracted three times with ethyl acetate (3 × 10 mL), the organic phases were combined and purified by silica gel chromatography using dichloromethane and methanol as eluent at a volume ratio of dichloromethane to methanol of 10:2, separating out a compound D3;
and 4, removing a hydroxyl protecting group: 7.7mmol of compound D3 was added to a mixture of 19.2mmol of trifluoroacetic acid and 7.7mmol of dichloromethane and reacted at room temperature for 2.8h, the progress of the reaction was monitored by TLC, after completion of the reaction, it was washed twice with 20mL of saturated brine, extracted four times with ethyl acetate (4X 10 mL), the organic phases were combined and purified by chromatography on silica gel using dichloromethane and methanol as eluents at a volume ratio of dichloromethane to methanol of 20:1.5, obtaining a compound E3, namely a chlorogenic acid derivative, with the yield of 75.9 percent and the nuclear magnetic hydrogen spectrum data 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
Dissolving 1mmol of compound E3 in acetonitrile with different volumes respectively to prepare stock solutions with mass percentage concentrations of 20%, 40%, 60%, 80% and 100%; dipping with sterile cotton swab at 1.3X 10 3 cfu·m L -1 Uniformly coating the bacterial liquid on the surface of a soybean casein agar culture medium plate, and sticking the bacterial liquid to the surface, wherein the strain used by the bacterial liquid is staphylococcus aureus; taking out the filter paper pieces soaked by the stock solutions with different concentrations by using sterile forceps, throwing off the floating liquid on the surfaces of the filter paper pieces, and spreading the filter paper pieces on the surface of a soybean casein agar culture medium; putting the culture medium coated with the bacterial liquid into a culture dish, inverting the culture dish, culturing for 22 hours in a 37 ℃ thermostat, and observing the phenomenon; the culture medium is found to have a transparent circular-bacteriostatic circle, when the concentration ratio of the compound E3 is 60%, the bacteriostatic effect on staphylococcus aureus is remarkable, the diameter of the bacteriostatic circle is 24.11mm, and is 13.17mm higher than that of chlorogenic acid at the same concentration.
Example 4
A method for synthesizing chlorogenic acid derivatives comprises the following steps:
step 1, protection of alcoholic hydroxyl group of chlorogenic acid: dissolving 10mmol of chlorogenic acid in 30mL of acetone, adding 12mmol of p-toluenesulfonic acid into a reaction system, and stirring at room temperature for 4 hours; TLC monitoring the reaction progress until the reactant chlorogenic acid disappears; after the reaction is finished, adding a saturated sodium bicarbonate solution, neutralizing the residual p-toluenesulfonic acid until the pH value is 7.3, performing suction filtration, and concentrating the filtrate to obtain a compound A4;
step 2, protecting phenolic hydroxyl of chlorogenic acid: adding 9.5mmol of compound A4, 17.1mmol of acetic anhydride and 10.5mmol of DMAP into a 100mL round-bottom flask, dissolving with 25mL of DMF, and stirring for reaction at room temperature for 3h; monitoring the reaction completion, adding 20mL of saturated brine to the reaction system, washing, extracting with n-butanol three times (3X 10 mL), combining the organic phases, purifying the organic phases by silica gel chromatography with dichloromethane and methanol as eluent at a volume ratio of 30:2.5, separating out a compound B4;
step 3, synthesis of chlorogenic acid derivative containing pyrazole ring: dissolving 8.9mmol of compound B4 in 25mL of DMSO, adding 10.7mmol of azomethine imine dipole C4, 0.89mmol of copper chloride and 3.6mmol of TBHP into a reaction system, and reacting at 60 ℃ for 6 hours; the reaction was stopped by TLC monitoring the progress of the reaction until compound B4 completely disappeared, 20mL of saturated brine was added to the reaction system and washed, followed by three times of n-butanol extraction (3 × 10 mL), and the organic phases were combined and purified by silica gel chromatography using dichloromethane and methanol as eluents at a volume ratio of 10:1.2, separating out a compound D4;
and 4, removing a hydroxyl protecting group: adding 7.8mmol of compound D4 into a mixture of 17.2mmol of trifluoroacetic acid and 7.8mmol of dichloromethane, reacting at room temperature for 2.5h, monitoring the reaction progress by TLC, washing the mixture after the reaction is finished with 20mL of saturated saline solution for three times, extracting the mixture with n-butanol for three times (3X 10 mL), combining organic phases, and purifying the organic phases by silica gel chromatography with dichloromethane and methanol as eluent and the volume ratio of dichloromethane to methanol of 20:1.8, compound E4 is obtained in 83.2% yield with the following nuclear magnetic hydrogen spectroscopy data:
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, determination of antibacterial Activity of Compound E4
Dissolving 1mmol of compound E4 in acetonitrile with different volumes respectively to prepare stock solutions with mass percentage concentrations of 20%, 40%, 60%, 80% and 100%; dipping with sterile cotton swab (1.8 × 10) 3 cfu·m L -1 Uniformly coating the bacterial liquid on the surface of a soybean casein agar culture medium plate, and sticking the bacterial liquid to the surface, wherein the strain used by the bacterial liquid is staphylococcus aureus; taking out the filter paper pieces soaked by the stock solutions with different concentrations by using a sterile forceps, throwing off the floating liquid on the surfaces of the filter paper pieces, and spreading the filter paper pieces on the surface of a soybean casein agar culture medium; putting the culture medium coated with the bacterial liquid into a culture dish, inverting the culture dish, culturing for 24 hours in a thermostat at 36 ℃, and observing the phenomenon; the culture medium is found to have a transparent circular ring-bacteriostatic circle, when the concentration ratio of the compound E4 is 60%, the bacteriostatic effect on staphylococcus aureus is remarkable, the diameter of the bacteriostatic circle is 28.04mm, which is far higher than the diameter of the bacteriostatic circle of chlorogenic acid 13.17mm under the same concentration.
Example 5
A method for synthesizing chlorogenic acid derivatives comprises the following steps:
step 1, protection of alcoholic hydroxyl group of chlorogenic acid: dissolving 10mmol of chlorogenic acid in 24mL of acetone, adding 12mmol of p-toluenesulfonic acid into a reaction system, and stirring at room temperature for 4.5h; TLC monitoring reaction progress until reactant chlorogenic acid disappears; after the reaction is finished, adding a saturated sodium bicarbonate solution, neutralizing the residual p-toluenesulfonic acid until the pH value is 7, performing suction filtration, and concentrating the filtrate to obtain a compound A5;
step 2, protecting phenolic hydroxyl of chlorogenic acid: adding 9.3mmol of compound A5, 14.9mmol of acetic anhydride and 11.2mmol of DMAP into a 100mL round-bottom flask, dissolving with 27mL of DMF, and stirring at room temperature for reaction for 3h; the reaction was monitored for completion, and the reaction was washed with 20mL of saturated brine, extracted three times with dichloromethane (3X 10 mL), and the organic phases were combined and purified by silica gel chromatography using dichloromethane and methanol as eluents at a dichloromethane to methanol volume ratio of 30:3, separating out a compound B5;
step 3, synthesis of chlorogenic acid derivative containing pyrazole ring: dissolving 8.9mmol of compound B5 in 30mL of acetonitrile, adding 9.8mmol of azomethine imine dipole C5, 1.3mmol of cuprous acetate and 4.5mmol of iodobenzene acetate into a reaction system, and reacting at 60 ℃ for 8h; the reaction was stopped by TLC monitoring the progress of the reaction until compound B5 completely disappeared, 20mL of saturated brine was added to the reaction system, and the mixture was extracted three times with dichloromethane (3 × 10 mL), the organic phases were combined and purified by silica gel chromatography using dichloromethane and methanol as eluents at a dichloromethane to methanol volume ratio of 10:1.6, isolating compound D5;
and 4, removing a hydroxyl protecting group: taking 7.7mmol of compound D5, adding the compound D5 into a mixture of 16.2mmol of trifluoroacetic acid and 7.7mmol of dichloromethane, reacting at room temperature for 3h, monitoring the progress of the reaction by TLC, washing the reaction product after the reaction is finished with 20mL of saturated saline twice, extracting the reaction product with dichloromethane three times (3X 10 mL), combining organic phases, and purifying the organic phases by silica gel chromatography by taking dichloromethane and methanol as eluent and the volume ratio of dichloromethane to methanol is 20:1.4, compound E5 is obtained in 84.3% yield with the following nuclear magnetic hydrogen spectroscopy data:
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, determination of antibacterial Activity of Compound E5
Dissolving 1.5mmol of compound E5 in acetonitrile of different volumes respectively to obtain 20%, 40%, 60%, 80%, and 100% acetonitrile solutionPreparing a liquid; dipping with sterile cotton swab at 1.6X 10 3 cfu·m L -1 Uniformly coating the bacterial liquid on the surface of an eosin methylene blue agar culture medium plate, and sticking the bacterial liquid to the surface, wherein the strain used by the bacterial liquid is candida albicans; taking out the filter paper pieces soaked by the stock solutions with different concentrations by using a sterile forceps, throwing off the floating liquid on the surfaces of the filter paper pieces, and spreading the filter paper pieces on the surface of an eosin methylene blue agar culture medium; placing the culture medium coated with the bacterial liquid into a culture dish, inverting the culture dish, placing the culture dish in a thermostat at 36 ℃ for culturing for 24 hours, and observing the phenomenon; the culture medium is found to have a transparent circular-bacteriostatic circle, when the concentration ratio of the compound E5 is 80%, the bacteriostatic effect on the Candida albicans is remarkable, the diameter of the bacteriostatic circle is 26.32mm, which is far higher than that of chlorogenic acid at the same concentration by 9.98mm.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
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.
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