Underived beta-cyclodextrin chiral stationary phase as well as preparation method and application thereof

Abstract

The invention relates to the technical field of chiral stationary phase fillers, in particular to an underived beta-cyclodextrin chiral stationary phase as well as a preparation method and application thereof. Underived beta-cyclodextrin is bonded on the surface of silica gel through urea or thiourea groups respectively, and the structure is shown as a formula I in the specification. The preparation process of the underived beta-cyclodextrin chiral stationary phase filler provided by the invention has the advantages of simple and easily-controlled conditions, no need of complex derivatization and good repeatability. The novel chiral stationary phase is suitable for different mobile phase systems of high performance liquid chromatography, can efficiently separate N-containing chiral compounds such as imidazole antibacterial drugs and triazole pesticides in various structure types, and can meet the requirements of daily analysis, production process and use process quality control and the like of the chiral drugs and the chiral pesticides.

Description

Technical Field

[0001] The present invention relates to the technical field of chiral stationary phase fillers, and in particular to a class of underivatized β -Cyclodextrin chiral stationary phase and its preparation method and application. Background Art

[0002] Enantiomers are ubiquitous in nature and are particularly important in the pharmaceutical field. Although enantiomers of chiral drugs share identical chemical structures, they exhibit significant differences in pharmacology, toxicology, pharmacokinetics, metabolism, and other biological activities. To ensure drug safety and efficacy, enantiomer separation is often necessary. Furthermore, chiral separation is also crucial in other fields, such as assessing the ecotoxicity and biodegradability of chiral pollutants in the environment and assessing enantiomer content and safety in the food industry. Therefore, enantiomeric separation of chiral compounds is of vital importance in fields such as medicine, agriculture, and life sciences.

[0003] High-performance liquid chromatography (HPLC) chiral stationary phase (CSP) is considered to be the preferred method for enantiomeric separation analysis and purity detection. It has the advantages of fast separation speed, high sensitivity, convenient operation, and good stability. It can also complete the determination of the enantiomeric purity of drugs in complex substrates.

[0004] Among the many chiral selectors, β -cyclodextrin ( β -CD) has a unique structure. It consists of 7 pyranose glucose units forming a conical structure, forming an inner hydrophobic cavity and an outer hydrophilic surface. This unique cavity structure can form an inclusion complex with chiral molecules and plays a decisive role in chiral separation. β The hydroxyl groups at the -CD end can be introduced by derivatization to provide additional forces (such as hydrogen bonding, π-π conjugation, charge interaction and steric repulsion). β -CD chiral stationary phases have been extensively studied, including single, partial, and complete substitution of hydroxyl groups, as well as dimer substitution. β -CD CSP. Many studies have shown that the secondary hydroxyl groups of cyclodextrin are essential for the separation of most chiral compounds. Excessive derivatization will not only consume β The hydroxyl groups on the edge of the -CD lead to the loss of the key chiral recognition site of hydrogen bonding and blockage of the cyclodextrin port, which is not conducive to the full utilization of the cavity encapsulation by cyclodextrin. β The derivatization of -CD often fails to achieve the expected separation effect and may even lose the original separation ability. Summary of the Invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention aims to provide a class of β-Cyclodextrin chiral stationary phase and its preparation method and application. β -Cyclodextrins are bonded to silica gel via urea or thiourea spacer arms to prepare a new class of underivatized β -CD CSP, compared with traditional natural β -CD CSP (CD CSP), its chiral splitting effect and range have been greatly improved.

[0006] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is: First, a class of underived β -cyclodextrin chiral stationary phase, β -Cyclodextrin is bonded to the silica gel surface via urea or thiourea, and its structure is shown in Formula I:

[0007] Formula I.

[0008] In the second aspect, there is provided an underivatized β - A method for preparing a cyclodextrin chiral stationary phase, comprising the following steps: S1. β -cyclodextrin, p-toluenesulfonyl chloride, and tetrabutylammonium bromide (TBAB) are added to water and stirred for 8-24 hours, and then sodium hydroxide aqueous solution is added and stirred for 2-10 hours. After the reaction is completed, impurities are filtered, and ammonium chloride is added to the filtrate and refrigerated overnight to precipitate. The solid is filtered and recrystallized with a small amount of hot water several times, filtered and dried to obtain the mono-6-p-toluenesulfonyl- β -Cyclodextrin TsO- β CD;

[0009] Formula II S2. Take the TsO- β CD was added to ammonia water and heated to react to obtain 6-amino-6-deoxy- β -cyclodextrinNH2- β CD;

[0010] Formula III S3. Recycle the NH2- β CD and triethoxysilane iso(thio)cyanate are added to dimethylformamide (DMF) and reacted for a period of time, and then activated silica gel is added to continue the reaction. After cooling to room temperature, the reaction is filtered. The obtained solid is repeatedly washed multiple times and vacuum dried to obtain a CSP filler as shown in Formula I.

[0011] Furthermore, the reaction in S1 is carried out at room temperature; β - Add 10-30 ml of water to cyclodextrin; β The molar mass ratio of cyclodextrin, p-toluenesulfonyl chloride, and tetrabutylammonium bromide is 1:0.8~1.5:0.01-0.1; the concentration of the sodium hydroxide aqueous solution is 5~10 mol / L, and the amount used is 10~20 mL of sodium hydroxide aqueous solution per 100 mL of water; the amount of ammonium chloride used is 3~10 g per 100 mL of solution.

[0012] Furthermore, the amount of ammonia in S2 is per gram of TsO- β Add 20-40 mL of aqueous ammonia to CD; the reaction temperature is 30-70°C and the reaction time is 10-24 h.

[0013] Furthermore, the preparation method of activated silica gel in S3 is as follows: the chromatographic stationary phase silica gel is heated to reflux with a 10% hydrochloric acid solution, cooled and washed with distilled water to a pH of ≈ 7, and vacuum dried at 120°C for 12 h; the amount of hydrochloric acid solution is determined at 10-20 mL per gram of silica gel; the reaction temperature is 100-120°C, and the reaction time is 10-20 h.

[0014] Furthermore, all reactions in S3 were carried out under nitrogen protection; NH2- β The molar mass ratio of CD to triethoxysilane is 1:2-5; the reaction time is 4-10 h; the reaction temperature after adding activated silica gel is 110-120°C, and the reaction time is 20-36 h.

[0015] Furthermore, NH2- β 10-50 mg of 4-dimethylaminopyridine (DMAP) can be added to the reaction between CD and triethoxysilane iso(thio)cyanate.

[0016] Furthermore, the iso(thio)cyanate triethoxysilane in S3 can be of different chain lengths, including: triethoxy(isocyanatomethyl)silane; triethoxy(2-isocyanatoethyl)silane; (3-isocyanatopropyl)triethoxysilane; triethoxy(4-isocyanatobutyl)silane; triethoxy(5-isocyanatopentyl)silane; triethoxy(6-isocyanatohexyl)silane; (triethoxysilyl)methyl isothiocyanate; 3-triethoxysilylpropyl isothiocyanate; triethoxy(6-isothiocyanatohexyl)silane.

[0017] In the third aspect, there is provided an underivatized β - The application of cyclodextrin chiral stationary phase in the separation of chiral compounds, the chiral compounds that can be separated include: imidazole antibiotics, βBeta-blockers, aromatic acid drugs, nimodipine, aminoglutethimide, equol, chlorpheniramine and triazole chiral pesticides.

[0018] The beneficial effects of the present invention are: 1. The raw materials of the product of the present invention are widely available and the cost is low.

[0019] 2. The present invention provides β -The preparation method of cyclodextrin CSP is simple, and excellent chiral resolution ability can be obtained without complex derivatization. The reaction conditions are mild and easy to control, and the repeatability and reproducibility are good, which is conducive to mass production.

[0020] 3. The present invention prepares β - Cyclodextrin CSP filler uses urea or thiourea as a spacer arm to bond to silica gel, compared to traditional natural β -cyclodextrin CSP, its chiral resolution ability and scope have been greatly improved, proving the important influence of CSP spacer arm on the chiral resolution effect.

[0021] 4. The present invention prepares β -Cyclodextrin CSP filler has good stability and is suitable for normal phase, reverse phase and polar mobile phase HPLC. It is acid and alkali resistant (pH = 4~10) and high pressure resistant (40 MPa). It can still maintain good chiral separation performance in highly polar and high water content mobile phases.

[0022] 5. The present invention prepares β -Cyclodextrin CSP filler can be used for the separation of chiral compounds of various structural types, and can separate imidazole antibiotics, β Beta-blockers, aromatic acid drugs, nimodipine, aminoglutethimide, equol, chlorpheniramine and other drugs as well as triazole chiral pesticides. It can meet the daily analysis and production of these chiral drugs and chiral pesticides and the quality control during clinical use. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The synthetic route diagram of the embodiment of the present invention; Figure 2 Prepared for Example 1 and Example 2 β -Cyclodextrin CSP (CSP-1, CSP-2) and traditional natural β -Structure diagram of cyclodextrin CSP (CD CSP); Figure 3 Prepared for Example 1 and Example 2 β - Chromatograms of enantiomeric separation of some chiral drugs by cyclodextrin CSP (CSP-1, CSP-2). DETAILED DESCRIPTION

[0024] The specific embodiments of the present invention are described below to facilitate understanding of the present invention by those skilled in the art. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the appended claims, these changes are obvious, and all inventions and creations utilizing the concepts of the present invention are protected.

[0025] Example 1 Reference Figure 1 , a urea spacer-based β - A method for preparing a cyclodextrin chiral stationary phase, which specifically comprises the following steps: (1) Preparation of activated silica gel Add 20.0 g of chromatographic stationary phase silica gel and 200 ml of 10% hydrochloric acid solution into a 500 ml round-bottom flask, heat and reflux for 24 h, cool to room temperature, wash with distilled water to pH ≈ 7, and vacuum dry at 120°C for 12 h to obtain activated silica gel.

[0026] The chromatographic stationary phase silica gel used was produced by Suzhou Nano-Tech Co., Ltd., China, with a particle size of 5.38 μ m, specific surface area 303 m 2 / g.

[0027] (2) 6-amino-6-deoxy- β - Preparation of cyclodextrin Will β -CD (36 g), p-toluenesulfonyl chloride (9.36 g), and tetrabutylammonium bromide (0.312 g) were added to 750 mL of water and stirred at room temperature for 12 hours. Then, 128 mL of 8.25 mol / L sodium hydroxide aqueous solution was added and stirring was continued for 2 hours. The mixture was then filtered, and 38 g of ammonium chloride was added to the filtrate. The mixture was allowed to stand at 4°C for 48 hours to induce precipitation. The precipitate was collected by filtration, recrystallized twice in hot water, and dried in vacuo to obtain mono-6-p-toluenesulfonyl- β -cyclodextrin (TsO- β CD), yield 28%. The structure is shown in Formula II.

[0028]

[0029] Formula II Take 5 g of dry mono-6-toluenesulfonyl- β -cyclodextrin, add 165 mL of concentrated ammonia water, stir and react at 50 ° C for 20 h, then remove most of the solvent under reduced pressure, drop into acetone to form a white flocculent precipitate, filter and dry to obtain 6-amino-6-deoxy- β -cyclodextrin (NH2- βCD), yield 95%. The structure is shown in Formula III.

[0030]

[0031] Formula III (3) β -Cyclodextrin CSP filler preparation Take 0.6 g NH2- β CD was dissolved in DMF, and 0.32 mL of (3-isocyanatopropyl)triethoxysilane was added. The mixture was stirred at 60°C under nitrogen for 6 h. Then, 2.2 g of activated silica gel was added, and the temperature was raised to 115°C and kept constant for 24 h. The filtered solid was washed repeatedly with DMF, methanol, and acetone, and dried under vacuum at 100°C to obtain CSP-1 filler, whose structure is shown in Formula IV. Elemental analysis showed a carbon content of 4.04%, a hydrogen content of 1.07%, and a nitrogen content of 0.49%. Thermogravimetric analysis revealed a weight loss of 14.75%.

[0032]

[0033] Formula IV (4) Filling of chiral chromatography column The chiral stationary phase was loaded onto an empty column using the homogenization method. First, an empty column (150 × 4.6 mm) was connected to the lower end of a homogenization tank. 2.1 g of CSP filler was weighed into a 50 mL beaker, and 30 mL of homogenate (methanol) was added. The suspension was ultrasonicated for 1 minute and transferred to a homogenization tank. Methanol was used as the displacement fluid and the empty column was rapidly filled at 40 MPa pressure for 30 minutes to prepare the chiral chromatographic column.

[0034] Example 2 Reference Figure 1 , a thiourea spacer-based β -cyclodextrin chiral stationary phase preparation method, the steps are basically the same as those in Example 1, except that NH2- β CD reacts with 3-triethoxysilylpropyl isothiocyanate, and the specific steps are as follows: Take 0.6 g NH2- βCD was dissolved in DMF, and 50 mg of DMAP and 0.35 mL of 3-triethoxysilylpropyl isothiocyanate were added. The mixture was stirred at 60°C under nitrogen for 6 h. Then, 2.2 g of activated silica gel was added, and the temperature was raised to 115°C and kept constant for 24 h. The filtered solid was washed repeatedly with DMF, methanol, and acetone, and dried under vacuum at 100°C to obtain CSP-2 filler, the structure of which is shown in Formula V. Elemental analysis showed a carbon content of 5.67%, a hydrogen content of 1.10%, a nitrogen content of 0.80%, and a sulfur content of 0.40%. The weight loss rate by thermogravimetric analysis was 15.49%.

[0035]

[0036] Formula V Application Example 1: β -Application of Cyclodextrin CSP in High Performance Liquid Chromatography The chiral stationary phase described in Formula I of the present invention has good stability and broad chiral selectivity and can be used to resolve multiple enantiomeric compounds in high performance liquid chromatography. For ease of illustration, the high performance liquid chromatograph used in this application example is an Agilent 1260, but this does not limit the present invention.

[0037] The chiral chromatographic columns (CSP-1 and CSP-2) prepared in Example 1 and Example 2 can separate most of the β Receptor blockers / agonists can be separated in reverse phase flow, including imidazole antibiotics, nimodipine, chlorpheniramine, ketoprofen and other chiral drugs (Table 1) and most triazole pesticides (Table 2). β -cyclodextrin CSP (CD CSP) were compared, and their structures are as follows Figure 2 As shown in Tables 1 and 2, the resolution effect and range of CSP-1 and CSP-2 are greatly improved compared with CD CSP, indicating that the spacer arm has a great influence on the chiral resolution ability of CSP. This may be because compared with urea and thiourea spacer arms, carbamate has an additional NH group, which can provide more hydrogen bonding force for CSP, thereby enhancing the chiral recognition ability of CSP.

[0038] Chromatographic conditions: flow rate 0.8 mL / min, room temperature.

[0039] in k 1 is the retention factor of the first eluting enantiomer, which is given by the formula k 1 = (t1-t0) / t0, where t1 and t2 are the elution times of the first and second peaks, respectively, and t0 is the dead time, which is 2.34 min here; α is the selection factor, and the calculation formula isα = k 2 / k 1; R s is the separation degree, and the calculation formula is R s = 2(t2-t1) / (W1+W2), where W1 and W2 are the peak widths of the first and second eluting enantiomers, respectively.

[0040] Table 1 β - Results of separation of chiral drugs by cyclodextrin CSP

[0041] a Mobile phase: ACN / MeOH / TEA / AcOH (95 / 5 / 1.2 / 0.8, v / v / v / v); b Mobile phase: MeOH / 0.1% FA (30 / 70, v / v); c Mobile phase: MeOH / 0.1% FA (50 / 50, v / v); d Mobile phase: ACN / H2O (10 / 90, v / v); e Mobile phase: MeOH / H2O (30 / 70, v / v); f Mobile phase: MeOH / H2O (50 / 50, v / v).

[0042] Table 2 β - Results of the separation of triazole pesticides by cyclodextrin CSP

[0043] a Mobile phase: MeOH / H2O (35 / 65, v / v); b Mobile phase: MeOH / H2O (45 / 55, v / v).

[0044] c Mobile phase: MeOH / 0.1% FA (30 / 70, v / v).

[0045] In summary, the present invention retains β -The hydroxyl group of the CD port only changes β -CD and silica gel spacer arm, maximize the original advantages of cyclodextrin. βCyclodextrin chiral stationary phases (CD CSPs) typically form a carbamate spacer arm. This new system replaces the spacer arm with urea or thiourea, significantly improving the separation efficiency and range compared to traditional CD CSPs. This system demonstrates superior enantiomeric separation capabilities, particularly for N-chiral compounds such as imidazole antibiotics and triazole pesticides. Furthermore, this system eliminates the need for complex derivatization, resulting in a simple and easily controllable preparation process and conditions.

[0046] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the present invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations that come within the meaning and range of equivalents of the claims be embraced therein.

[0047] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A class of underived β -cyclodextrin chiral stationary phase, characterized in that β -Cyclodextrin is bonded to the silica gel surface via urea or thiourea, and its structure is shown in Formula I: ； Formula I.

2. An underivatized form of claim 1 β -A method for preparing a cyclodextrin chiral stationary phase, characterized in that The steps include: S1. β -cyclodextrin, p-toluenesulfonyl chloride, and tetrabutylammonium bromide are added to water and stirred for 8 to 24 hours, and then sodium hydroxide aqueous solution is added and stirred for 2 to 10 hours. After the reaction is completed, impurities are filtered, and ammonium chloride is added to the filtrate and refrigerated overnight to precipitate. The solid is filtered and recrystallized with a small amount of hot water several times, filtered and dried to obtain the mono-6-p-toluenesulfonyl- β -Cyclodextrin TsO- β CD; ； Formula II S2. Take the TsO- β CD was added to ammonia water and heated to react to obtain 6-amino-6-deoxy- β -cyclodextrinNH2- β CD; ； Formula III S3. Recycle the NH2- β CD and different isocyanate triethoxysilanes are added to dimethylformamide to react for a period of time, and then activated silica gel is added to continue the reaction. After cooling to room temperature, it is filtered. The obtained solid is repeatedly washed several times and vacuum dried to obtain the CSP filler shown in Formula I.

3. The underivatized form of claim 2 β -A method for preparing a cyclodextrin chiral stationary phase, characterized in that The reaction in S1 was carried out at room temperature; β - Add 10-30 ml of water to cyclodextrin; β The molar mass ratio of cyclodextrin, p-toluenesulfonyl chloride, and tetrabutylammonium bromide is 1:0.8~1.5:0.01-0.1; the concentration of the sodium hydroxide aqueous solution is 5~10 mol / L, and the amount used is 10~20 mL of sodium hydroxide aqueous solution per 100 mL of water; the amount of ammonium chloride used is 3~10 g per 100 mL of solution.

4. The underivatized form of claim 2 β -A method for preparing a cyclodextrin chiral stationary phase, characterized in that The amount of ammonia in S2 is per gram of TsO- β Add 20-40 mL of aqueous ammonia to CD; the reaction temperature is 30-70°C and the reaction time is 10-24 h.

5. The underivatized form of claim 2 β -A method for preparing a cyclodextrin chiral stationary phase, characterized in that The preparation method of activated silica gel in S3 is as follows: the chromatographic stationary phase silica gel and hydrochloric acid solution are heated to reflux, washed with distilled water after cooling, and vacuum dried at 120°C for 12 hours; the amount of hydrochloric acid solution is determined at 10-20 mL per gram of silica gel; the reaction temperature is 100-120°C, and the reaction time is 10-20 hours.

6. The underivatized form of claim 2 β -A method for preparing a cyclodextrin chiral stationary phase, characterized in that All reactions in S3 were carried out under nitrogen protection; NH2- β The molar mass ratio of CD to triethoxysilane is 1:2-5; the reaction time is 4-10 h; the reaction temperature after adding activated silica gel is 110-120°C, and the reaction time is 20-36 h.

7. The underivatized form of claim 6 β -A method for preparing a cyclodextrin chiral stationary phase, characterized in that NH2- in S3 β The reaction of CD with different isocyanate triethoxysilanes was carried out by adding 10-50 mg of 4-dimethylaminopyridine.

8. The underivatized form of claim 2 β -A method for preparing a cyclodextrin chiral stationary phase, characterized in that The isocyanate triethoxysilanes in S3 are of different chain lengths, including: triethoxy(isocyanatomethyl)silane; triethoxy(2-isocyanatoethyl)silane; (3-isocyanatopropyl)triethoxysilane; triethoxy(4-isocyanatobutyl)silane; triethoxy(5-isocyanatopentyl)silane; triethoxy(6-isocyanatohexyl)silane; (triethoxysilyl)methyl isothiocyanate; 3-triethoxysilylpropyl isothiocyanate; and triethoxy(6-isothiocyanatohexyl)silane.

9. The underivatized β -Application of cyclodextrin chiral stationary phase in separation of chiral compounds, characterized in that, The chiral compounds that can be separated include: imidazole antibiotics, β Beta-blockers, aromatic acid drugs, nimodipine, aminoglutethimide, equol, chlorpheniramine and triazole chiral pesticides.

Images