Preparation and application of a histidine-derived pillar aromatic bonded silica gel chiral filler

Abstract

The invention discloses a preparation method of a histidine-derived pillar aromatic hydrocarbon bonded silica chiral filler. 1,4-dibromoethoxybenzene is used as a structural unit module, polyformaldehyde is used as a bridging condensation agent, and boron trifluoride etherate is used as a catalyst. The bromine-substituted pillar aromatic hydrocarbon is prepared by condensation at room temperature in a dichloromethane solvent. Then, an L-histidine chiral selector containing an imidazole conjugated ring structure is introduced, so that the chiral filler introduces hydrogen bonding forces on the basis of a large number of π-π conjugated groups in the pillar aromatic hydrocarbon, and the chiral separation performance is enhanced under the driving force of multiple forces. The chiral column is further filled into a chiral chromatographic column by a high-pressure homogenization method and applied to the chiral separation of non-steroidal anti-inflammatory drugs. The chiral liquid chromatographic column provided by the invention has strong selectivity for non-steroidal anti-inflammatory drugs ketoprofen, ibuprofen and carprofen, and exhibits good specificity and repeatability. The standard deviation RSD values ​​of the retention factor, the separation factor and the separation degree are all less than 1.98%.

Description

Technical Field

[0001] The invention relates to the efficient preparation of a histidine-derived columnar aromatics bonded silica gel chiral filler and the separation and evaluation of non-steroidal chiral anti-inflammatory drugs, belonging to the field of high performance liquid chromatography chiral filler enantiomer chromatographic separation. Background Art

[0002] Chirality is closely related to many fields such as life sciences, new drug development, human health, chiral new materials and national economy, and is widely present in the macroscopic and microscopic worlds that are highly related to our lives and nature. Chiral enantiomers are mirror images and cannot be completely overlapped. There are significant differences in pharmacological activity, toxicity and physiological effects, which leads to huge risks in the clinical application of chiral drugs. At present, the main methods for obtaining single enantiomers include natural sources, asymmetric synthesis, crystallization resolution, membrane resolution, enzymatic hydrolysis and chromatographic resolution. Chiral chromatography technology has obvious advantages in resolving chiral racemates with high boiling point, poor thermal stability, large molar mass and strong polarity, and it does not destroy the sample and has been recovered. It is currently one of the most effective methods for separating and obtaining single enantiomers. In chiral chromatography resolution technology, chiral fillers are the key and core of chiral resolution technology to achieve high selective separation. Therefore, it is of practical significance and application prospects to develop new chiral chromatographic fillers, establish chiral separation technology or analytical methods to study chiral drugs at the enantiomer level, and apply them to actual production to more efficiently and environmentally friendly resolve chiral drugs.

[0003] Non-steroidal anti-inflammatory drugs are widely used in clinical practice to relieve a variety of fever, pain, arthritis and other diseases because of their anti-inflammatory, analgesic, antipyretic and anti-rheumatic effects. Among the drugs on the market, most non-steroidal anti-inflammatory drugs have chiral structures, and their enantiomers have one active, two different pharmacological activities or even opposite pharmacological activities. Among them, the S-body of ibuprofen mainly plays a role, the R-body has low activity, but has anti-inflammatory related pharmacology; the S-body of ketoprofen has analgesic and anti-inflammatory effects compared to the R-body; the S-body in flurbiprofen is used to treat arthritis, while the R-body has a certain pharmacological effect on Alzheimer's disease. Therefore, it may be more accurate to perform chiral separation of non-steroidal anti-inflammatory drugs and analyze the pharmacology, efficacy, toxicity, etc. at the enantiomeric level, which is of great development significance while improving efficacy, reducing the burden on human organs, benefiting people's health and reducing costs.

[0004] Based on this, the present invention uses L-histidine containing an imidazole conjugated ring structure as a chiral selector, so that the chiral filler introduces the L-histidine chiral center on the basis of the π-π conjugated structure of the electron-rich benzene ring of the columnar aromatics, and the imidazole conjugated ring structure, and enhances the chiral separation performance driven by multiple forces such as the coordination effect, the π-π "host-guest" effect, and the hydrogen bond force, and then prepares a histidine-derived columnar aromatics bonded silica gel chiral filler in a one-pot method, and applies it to the study of the chiral separation performance of non-steroidal anti-inflammatory drugs. The effects of different flow rates, different isopropanol volume fractions, temperatures, etc. on the separation degree are explored, and the repeatability of multiple injections is examined. It is hoped that these CSPs and methods can be efficiently applied to the chirality evaluation of non-steroidal anti-inflammatory drugs and promote chirality research. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing a histidine-derived pillar aromatic hydrocarbon bonded silica gel chiral filler;

[0006] Another object of the present invention is to provide application of the chiral filler in chiral separation of non-steroidal anti-inflammatory drugs.

[0007] 1. Preparation of Histidine-derived Pillar Arene Bonded Silica Chiral Packing

[0008] (1) Synthesis of bromoethoxy-substituted pillar aromatics: dissolve p-bromoethoxybenzene in dichloromethane solution, add paraformaldehyde as a bridging agent, boron trifluoride etherate as a catalyst, stir at 25-30°C for 1.5-2.0 hours, quench the reaction with methanol after the reaction is completed, wash the reaction mixture with water and saturated sodium bicarbonate solution, dry the organic layer with anhydrous magnesium sulfate, remove the organic solvent in vacuo, and purify by column chromatography to obtain bromoethoxy-substituted pillar aromatics. The mass ratio of p-bromoethoxybenzene to paraformaldehyde is 2.5:1-3:1; the mass ratio of p-bromoethoxybenzene to boron trifluoride is 1:1-1.5:1.

[0009] The synthetic route is as follows:

[0010]

[0011] (2) Preparation of histidine-derived columnar aromatic hydrocarbon bonded silica chiral filler: Disperse aminopropyl silica gel in anhydrous toluene, add bromoethoxy-substituted columnar aromatic hydrocarbon, L-histidine and diethylamine, and reflux at 85-90°C for 20-24 hours under a nitrogen atmosphere. After the reaction is completed, filter out a light yellow solid powder, wash with acetonitrile, ethanol and methanol, and dry in vacuum to obtain a histidine-derived columnar aromatic hydrocarbon bonded silica chiral filler.

[0012] Among them, the mass ratio of bromoethoxy-substituted pillar aromatic hydrocarbons to L-histidine is 1:1~2:1; the mass ratio of bromoethoxy-substituted pillar aromatic hydrocarbons to diethylamine is 8:1~12:1; the mass ratio of bromoethoxy-substituted pillar aromatic hydrocarbons to aminopropyl silica gel is 1:5~1:6.

[0013] The synthetic route is as follows:

[0014] .

[0015] II. Characterization of Histidine-Derived Pillararene-Bonded Silica Chiral Packing

[0016] 1. Infrared Spectroscopy Analysis

[0017] The infrared spectra of aminopropyl silica and histidine-derived pillararene-bonded silica chiral packing are as shown in Figure 2 . The absorption peak at 1710 wavenumbers indicates the characteristic peak of the carbonyl group, the strong absorption peak at 1632 wavenumbers shows the imine bond group, the sharp peaks at 1494 and 1405 are due to the bending vibration of the benzene ring, and the absorption peak at 685 wavenumbers is the skeletal stretching vibration of the benzene ring. The infrared results indicate the successful preparation of histidine-derived pillararene-bonded silica chiral packing.

[0018] 2. Elemental Analysis

[0019] The results of elemental analysis show that the percentage contents of nitrogen, carbon and hydrogen in aminopropyl silica are 1.45%, 4.04% and 1.02% respectively, while those in histidine-derived pillararene-bonded silica chiral packing are 2.30%, 15.39% and 1.78% respectively. The results of elemental analysis prove the successful preparation of histidine-derived pillararene-bonded silica chiral packing. The specific experimental data are shown in Table 1.

[0020]

[0021] 3. Nitrogen Adsorption-Desorption

[0022] Aminopropyl silica and histidine-derived pillararene-bonded silica chiral packing were analyzed by nitrogen adsorption / desorption isotherms. The experimental results all showed type IV isotherms ( Figure 3 ). At the low-pressure section where P / P0 < 0.4, the adsorption amount increased gently, and at this time, nitrogen molecules were adsorbed on the inner surface of the mesopores from monolayer to multilayer. It should be noted that when 0.4 < P / P0 < 0.85, the nitrogen capture rate increased rapidly, indicating that the prepared material has a mesoporous structure. The test data showed that the specific surface area of aminopropyl silica was 260.13 cm 3 / g, and that of histidine-derived pillararene-bonded silica chiral packing was 2221.33 cm 3 / g, indicating that L-histidine and bromoethoxy-substituted pillararene were successfully bonded, that is, the successful preparation of histidine-derived pillararene-bonded silica chiral packing.

[0023] III. Evaluation of Chiral Resolution of Non-Steroidal Anti-Inflammatory Drugs

[0024] 1. Packing of chiral chromatographic columns

[0025] Weigh 1.5-2.0 g of histidine-derived column aromatic hydrocarbon bonded silica chiral filler, evenly disperse it with 40-45 mL of chromatographic methanol as a homogenate, use 180-200 mL of chromatographic methanol as a displacement liquid, and fill it at a pressure of 45-50 MPa for 25-30 minutes to obtain an L-histidine-modified column aromatic hydrocarbon bonded mesoporous silica chiral column.

[0026] 2. Evaluation of chiral separation of non-steroidal anti-inflammatory drugs

[0027] The chiral separation of nonsteroidal anti-inflammatory drugs was evaluated under the conditions of n-hexane and isopropanol as mobile phase, detection wavelength of 254nm, and flow rate of 1.0mL / min. The experimental results showed that the mobile phase was n-hexane: isopropanol = 95:5~90:10 (volume ratio) and good chiral separation performance was achieved for ketoprofen (a), ibuprofen (b) and carlofen (c). Ketoprofen obtained baseline separation results. The specific chromatogram is shown in the figure below. Figure 4 It is shown that this type of stationary phase has a good chiral separation effect on non-steroidal anti-inflammatory drugs.

[0028] 3. Investigation of different mobile phase ratios

[0029] In normal phase mode, the effect of isopropanol volume fraction in the mobile phase (n-hexane / isopropanol) on the chiral separation performance of ketoprofen was investigated. The experimental results showed that with the increase of isopropanol volume fraction, the retention time, separation factor and resolution of ketoprofen enantiomers gradually decreased. When n-hexane / isopropanol = 95:5, v / v, the maximum resolution reached 3.7. The separation chromatograms affected by different isopropanol volume fractions are shown in Figure 2. Figure 5 shown.

[0030] 4. Temperature responsiveness

[0031] The effect of temperature on the separation of phenoxyethanol was studied in the temperature range of 20°C to 50°C when n-hexane / isopropanol (95 / 5, v / v) was used as the mobile phase. Figure 6 As shown, the experimental results show that with the increase of column temperature, the retention time decreases and the resolution decreases, indicating that the chiral separation of ketoprofen enantiomers on the histidine-derived aromatic bonded silica chiral column is an exothermic process.

[0032] 5. Repeatability test

[0033] Ketoprofen, a nonsteroidal anti-inflammatory drug, was selected as a test substance to detect the repeatability of the histidine-derived column aromatic bonded silica chiral column. Ketoprofen was injected six times in the mobile phase of n-hexane / isopropanol (95 / 5, v / v), and the elution time remained almost unchanged, showing good repeatability. The standard deviation Rs value of the separation degree for six injections was less than 1.98%, showing good stability. The results are shown in Figure 2. Figure 7 shown.

[0034] In summary, the present invention provides a new strategy and new use for the efficient preparation of histidine-derived columnar aromatic hydrocarbon bonded silica chiral fillers. 1,4-dibromoethoxybenzene is used as a structural unit module, polyformaldehyde is used as a condensation agent, and boron trifluoride etherate is used as a catalyst. The bromine-substituted columnar aromatic hydrocarbon is prepared by condensation at room temperature in a dichloromethane solvent, and then L-histidine chiral selector containing an imidazole conjugated ring structure is introduced, so that the chiral filler introduces hydrogen bonding forces based on a large number of π-π conjugated groups in the columnar aromatic hydrocarbon, and the chiral separation performance is enhanced under the driving force of multiple forces. This type of chiral filler has high selectivity for non-steroidal anti-inflammatory drugs ketoprofen, ibuprofen and carprofen. Under the conditions of mobile phase conditions of n-hexane: isopropanol = 95:5 (volume ratio), detection wavelength 254nm, and flow rate of 1mL / min, the enantiomeric separation of ketoprofen is as high as 3.7. The temperature responsiveness experiment confirms that the separation process of ketoprofen on the histidine-derived columnar aromatic hydrocarbon bonded silica chiral column is an exothermic process. The standard deviation values ​​of retention time, separation factor and resolution in the repeatability study of ketoprofen enantiomers were all less than 1.98%. This proves that the chiral column formed by the histidine-derived column aromatic hydrocarbon bonded silica gel chiral filler has strong specificity, selectivity and stability for the evaluation of non-steroidal anti-inflammatory drug enantiomer separation. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the H NMR spectrum of bromoethoxy-substituted pillar aromatics;

[0036] Figure 2 The infrared spectrum of aminopropyl silica gel and histidine-derived pillar aromatic bonded silica gel chiral packing;

[0037] Figure 3 Nitrogen adsorption-desorption spectra of aminopropyl silica and histidine-derived columnar aromatic bonded silica chiral packing;

[0038] Figure 4 Chiral separation of nonsteroidal anti-inflammatory drugs: (a) ketoprofen, (b) ibuprofen, (c) carlofen;

[0039] Figure 5 The effect of different isopropanol volume fractions on the chiral separation performance of ketoprofen;

[0040] Figure 6 The effect of temperature change on the chiral separation performance of ketoprofen;

[0041] Figure 7 To investigate the repeatability of chiral separation performance of ketoprofen; DETAILED DESCRIPTION

[0042] The preparation and application of the histidine-derived pillar aromatic hydrocarbon bonded silica gel chiral filler of the present invention are further described below through specific examples.

[0043] Example 1 Preparation of Histidine-derived Pillar Arene Bonded Silica Chiral Filler

[0044] (1) Synthesis of bromoethoxy-substituted pillar aromatics: At room temperature, weigh 2.5 g of p-bromoethoxybenzene and dissolve it in 50 mL of dichloromethane solution, add 1.0 g of paraformaldehyde as a bridging agent, 2.0 mL of boron trifluoride etherate as a catalyst, stir magnetically for 1.5 hours, pour into 80 mL of methanol to quench the reaction, wash the reaction mixture with 30 mL of water and 30 mL of saturated sodium bicarbonate solution, then dry the organic layer with 20 g of anhydrous magnesium sulfate, and remove the organic solvent in vacuo. Purify by column chromatography using dichloromethane:n-hexane = 1:5~1:3 volume ratio to obtain bromoethoxy-substituted pillar aromatics. The characteristic peak at 6.96 ppm is the hydrogen atom of the benzene ring, the characteristic peak at 4.28 ppm is the hydrogen atom of the methylene group on the alkyl chain adjacent to oxygen, the characteristic peak at 3.86 ppm is the chemical shift value of the methylene peak bridging the benzene ring, and the characteristic peak at 3.66 ppm is the hydrogen atom on the carbon atom of the alkyl chain adjacent to bromine, indicating the successful synthesis of bromoethoxy columnar aromatics. The NMR hydrogen spectrum of bromoethoxy substituted columnar aromatics is shown in Figure 1. Figure 1 . 1 H NMR (400 MHz, CDCl3, rt) δ (ppm): 6.96 (s, 10H), 4.28 (t, J = 6.0 Hz, 20H), 3.86 (s, 10H), 3.66 (t, J =4.0 Hz, 20H).

[0045] (2) Preparation of histidine-derived columnar aromatic hydrocarbon bonded silica chiral filler: Weigh 2.0 g of aminopropyl silica gel and disperse it in 100 mL of anhydrous toluene, add 0.4 g of bromoethoxy-substituted columnar aromatic hydrocarbon obtained in the above step, 0.3 g of L-histidine, and 0.05 g of diethylamine (catalyst), and reflux at 85 °C for 20 hours under nitrogen atmosphere. After the reaction is completed, filter and obtain a light yellow solid powder. Wash with 50-60 mL of acetonitrile, 50-60 mL of ethanol, and 50-60 mL of methanol, and then dry under vacuum at 45-50 °C for 20-24 hours to obtain a histidine-derived columnar aromatic hydrocarbon bonded silica chiral filler.

[0046] Example 2 Chiral separation of nonsteroidal anti-inflammatory drugs

[0047] (1) Packing of chiral chromatographic columns

[0048] Weigh 1.5 g of L-histidine modified column aromatic hydrocarbon bonded mesoporous silica chiral filler, use 40 mL of chromatographic methanol as a homogenate to evenly disperse it, use 180 mL of chromatographic methanol as a displacement liquid, and fill it at a pressure of 45 MPa for 25 minutes to prepare a histidine-derivatized column aromatic hydrocarbon bonded silica chiral chromatographic column;

[0049] (2) Chiral separation: Chiral separation of nonsteroidal anti-inflammatory drugs ketoprofen, ibuprofen, and carlofen was performed under the following chromatographic conditions: the chromatographic column was a histidine-derived column aromatic bonded silica gel chiral chromatographic column, the mobile phase was n-hexane and isopropanol in a volume ratio of 95:5, the detection wavelength was 254nm, the flow rate was 1mL / min, and the column temperature was 20℃. The chromatogram of chiral separation of nonsteroidal anti-inflammatory drugs is shown in the figure. Figure 4 , proving that histidine-derived columnar aromatics bonded silica chiral filler has a good chiral separation effect for non-steroidal anti-inflammatory drugs.

Claims

1. A method for preparing a histidine-derived pillar aromatic bonded silica gel chiral filler, comprising the following steps: (1) Synthesis of bromoethoxy-substituted pillar aromatics: dissolve p-bromoethoxybenzene in dichloromethane solution, add paraformaldehyde as a bridging agent, boron trifluoride etherate as a catalyst, stir at 25-30°C for 1.5-2.0 hours, quench the reaction with methanol after the reaction is completed, wash the reaction mixture with water and saturated sodium bicarbonate solution, dry the organic layer with anhydrous magnesium sulfate, remove the organic solvent in vacuo, and purify by column chromatography to obtain bromoethoxy-substituted pillar aromatics; (2) Preparation of histidine-derived columnar aromatic hydrocarbon bonded silica chiral filler: Disperse aminopropyl silica gel in anhydrous toluene, add bromoethoxy-substituted columnar aromatic hydrocarbon, L-histidine and diethylamine, and reflux at 85-90°C for 20-24 hours under a nitrogen atmosphere. After the reaction is completed, filter out a light yellow solid powder, wash with acetonitrile, ethanol and methanol, and dry in vacuum to obtain a histidine-derived columnar aromatic hydrocarbon bonded silica chiral filler.

2. The method for preparing the histidine-derived pillar aromatic bonded silica gel chiral filler according to claim 1, characterized in that: In step (1), the mass ratio of p-bromoethoxybenzene to paraformaldehyde is 2.5:1 to 3:

1.

3. The method for preparing the histidine-derived pillar aromatic hydrocarbon bonded silica gel chiral filler according to claim 1, characterized in that: In step (1), the mass ratio of p-bromoethoxybenzene to boron trifluoride etherate is 1:1 to 1.5:

1.

4. The method for preparing the histidine-derived pillar aromatic hydrocarbon bonded silica gel chiral filler according to claim 1, characterized in that: In step (2), the mass ratio of the bromoethoxy-substituted pillar aromatic hydrocarbon to L-histidine is 1:1 to 2:

1.

5. The method for preparing the histidine-derived pillar aromatic hydrocarbon bonded silica gel chiral filler according to claim 1, characterized in that: In step (2), the mass ratio of the bromoethoxy-substituted pillar aromatic hydrocarbon to diethylamine is 8:1 to 12:

1.

6. The method for preparing the histidine-derived pillar aromatic bonded silica gel chiral filler according to claim 1, characterized in that: In step (2), the mass ratio of the bromoethoxy-substituted columnar aromatic hydrocarbon to the aminopropyl silica gel is 1:5 to 1:

6.

7. Use of the histidine-derived pillar aromatic hydrocarbon bonded silica gel chiral filler prepared according to the method of claim 1 in the chiral separation of non-steroidal anti-inflammatory drugs.

8. The use of the histidine-derived pillar arene bonded silica gel chiral filler according to claim 7 in the chiral separation of non-steroidal anti-inflammatory drugs, characterized in that: The following steps are included: (1) Filling of chiral chromatographic column: Weigh 1.5-2.0 g of histidine-derived column aromatic hydrocarbon bonded silica chiral filler, evenly disperse it with 40-45 mL of chromatographic methanol as a homogenate, use 180-200 mL of chromatographic methanol as a displacement liquid, and fill it at a pressure of 45-50 MPa for 25-30 minutes to prepare a histidine-derived column aromatic hydrocarbon bonded silica chiral chromatographic column; (2) Chiral separation: Chiral separation of non-steroidal anti-inflammatory drugs was performed under the following chromatographic conditions: the chromatographic column was a histidine-derived aromatic bonded silica gel chiral chromatographic column, the mobile phase was n-hexane and isopropanol in a volume ratio of 95:5, the detection wavelength was 254 nm, the flow rate was 1 mL / min, and the column temperature was 20-50 °C; non-steroidal anti-inflammatory drugs included ketoprofen, ibuprofen, and carprofen.

Images