A BINAM-based chiral stationary phase of β-cyclodextrin derivative, its preparation method and application

Abstract

The present invention relates to the technical field of chiral stationary phase packing materials, and particularly relates to a BINAM-based β -cyclodextrin derivative chiral stationary phase and its preparation method and application. The structure of the BINAM-based β -cyclodextrin derivative chiral stationary phase is: β A BINAM derivative is connected to the 6-position of the cyclodextrin and is bonded to the silica gel surface through a carbamate bond, and the structure is as shown in Formula I. The novel β -cyclodextrin CSP packing material provided by the present invention can separate chiral compounds of various structural types, is applicable to different mobile phase systems of high performance liquid chromatography (HPLC), and also has good stability in strongly polar and high-water-content mobile phases. The CSP packing material provided by the present invention can resolve various types of chiral drugs such as imidazole antibacterial drugs and β receptor blockers, as well as various triazole pesticides. It can meet the needs of daily analysis, quality control in the production process and use process of these chiral drugs and chiral pesticides.

Description

Technical Field

[0001] The present invention relates to the technical field of chiral stationary phase packing materials, and particularly relates to a BINAM-based β -cyclodextrin derivative chiral stationary phase and its preparation method and application. Background Art

[0002] Chirality is one of the basic properties of the living system. Biomacromolecules such as enzymes, receptors, plasma proteins, and ion channels all have chirality, and the living body itself is a chiral environment ( J. Am. Chem. Soc. 2024, 146, 26,17765–17772). When chiral drugs enter the human body, there may be stereoselective differences in the chiral recognition between enantiomers and macromolecules in the body, resulting in different pharmacodynamics, pharmacokinetics, and toxicology between enantiomers, and thus different pharmacological effects and adverse reactions. Clinically, nearly half of the drugs are chiral, but most drugs are used clinically as racemates. Usually, only one of the enantiomers exerts the drug effect. For example, β -receptor blockers are an important class of drugs widely used clinically. Chiral β -receptor blockers contain at least one chiral center, and there are significant differences in pharmacodynamics between different enantiomers. S The activity of the R -enantiomer is β 50-500 times that of the R -enantiomer. Except for timolol, most chiral Molecules -receptor blockers are used clinically as racemates, and side effects such as dizziness and hallucinations caused by drug use are considered to be related to their

[0003] -isomers ( Environ. Monit. Assess . 2021, 26: 468).

[0004] The research on chiral pesticides has also begun to receive extensive attention. Among racemic pesticides, half of them may be inactive. Sprayed on farmland, they not only pollute the environment but also cause waste of resources. According to statistics, among more than 650 pesticides on the market, 173 are chiral, and only dozens of them are single enantiomer pesticides. With the increasing requirements for environmental safety, the development of single enantiomer chiral pesticides has been put on the agenda ( Environ. Monit. Assess . 2024, 196, 153.).

[0004] Therefore, establishing separation and analysis methods for enantiomers of chiral drugs and chiral pesticides is a key prerequisite for the research on pharmacodynamics, pharmacokinetics, and interactions of enantiomers of chiral drugs and chiral pesticides and quality control during use, and has important practical significance for the research, evaluation, and use of highly efficient, low-toxic, and specific drugs / pesticides.

[0005] Due to its high efficiency, high sensitivity, wide application range, and the ability to be combined with other instruments, high-performance liquid chromatography with chiral stationary phases can not only be used for analysis but also for the preparation and semi-preparation of enantiomers. It has become the preferred method for the separation of chiral drug enantiomers and the in vitro and in vivo detection of chiral drugs.

[0006] β -Cyclodextrin ( β -cyclodextrins, β -CD), composed of 7 glucopyranose units, forms a conical structure with a hydrophobic cavity inside and a hydrophilic surface outside. This unique cavity structure can form inclusion complexes with chiral molecules and plays a decisive role in chiral separation. Chemical modification of the hydroxyl groups of cyclodextrin can not only change the shape and size of its cavity to meet various spatial requirements but also the introduced substituents can provide different binding sites. This is beneficial for expanding the chiral separation throughput of the stationary phase and improving its chiral selectivity. Currently, the vast majority of cyclodextrin-based chiral columns on the market are fully derivatized with carbamate on the hydroxyl groups of cyclodextrin, and the separation effect is limited, only applicable to the separation of some chiral drugs. 1,1'-Binaphthalene-2,2'-diamine (BINAM) is a typical axially chiral compound with a C2 symmetry axis and contains two identical naphthalene units. The two naphthalene rings hinder the free rotation of the 1,1'-bond, so the chiral binaphthalene molecule has a stable chiral configuration. In recent years, it has been widely used as a hot molecule in asymmetric catalysis and chiral synthesis research ( Tetrahedron 2024, 167, 134274.). Summary of the Invention

[0007] The purpose of the present invention is to provide a chiral stationary phase (CSP) based on BINAM β -cyclodextrin derivative and its preparation method and application. The present invention combines two traditional chiral selectors, BINAM derivative and β -cyclodextrin, to prepare a novel chiral stationary phase and enhance its chiral recognition ability and application range.

[0008] To achieve the above invention purpose, the technical solution adopted by the present invention is as follows:

[0009] In the first aspect, a chiral stationary phase based on 1,1'-binaphthalene-2,2'-diamine (BINAM) β -cyclodextrin derivative is provided, β A BINAM derivative is connected to the 6-position of

[0010]

[0011] Formula I

[0012] R is selected from:

[0013]

[0014] The configuration of BINAM is R - or S - configuration.

[0015] In a second aspect, a method for preparing a chiral stationary phase based on 1,1'-binaphthalene-2,2'-diamine (BINAM)-cyclodextrin derivative is provided, which comprises the following steps: β S1. Dissolve dry

[0016] -cyclodextrin in an aqueous sodium hydroxide solution, add an acetonitrile solution of p-toluenesulfonyl chloride at room temperature or in an ice bath for reaction, filter impurities after the reaction, adjust the filtrate to pH≈7 with hydrochloric acid (HCl, 30~38%), refrigerate overnight at 4°C to precipitate, filter and recrystallize the solid twice with a small amount of hot water, filter and dry to obtain mono-6-p-toluenesulfonylated- β -cyclodextrin (TsO- β -cyclodextrin (TsO- β CD) as shown in formula II.

[0017]

[0018] Formula II

[0019] S2. Take the TsO- β CD obtained in S1 and add it to ammonia water, stir and react at 40~70°C for 10~24h, then remove most of the solvent under reduced pressure, drop it into acetone solution to form a precipitate, filter and dry to obtain 6-amino-6-deoxy- β -cyclodextrin (NH 2 - β CD) as shown in formula III.

[0020]

[0021] Formula III

[0022] S3. Dissolve BINAM in anhydrous dichloromethane, add 4-NitrophenylIsocyanate for reaction, and purify by silica gel column chromatography to obtain compound AM-1 as shown in formula IV.

[0023]

[0024] Formula IV

[0025] S4. Dissolve compound AM-1 in anhydrous dichloromethane, add phenyl isocyanate with different substituents for reaction, and purify by silica gel column chromatography to obtain compound AM-2 as shown in Formula V.

[0026]

[0027] Formula V

[0028] R is selected from:

[0029]

[0030] S5. Put compound AM-2 into a round-bottom flask, add iron, ethanol and hydrochloric acid, heat and react for 5 - 12 h. After the reaction is completed, adjust the pH to about 7 with saturated sodium bicarbonate (NaHCO 3 ), extract with ethyl acetate (EtOAc) three times, combine the EtOAc layers, distill off the solvent under reduced pressure, and purify by silica gel column chromatography to obtain compound AM-3 as shown in Formula VI.

[0031]

[0032] Formula VI

[0033] R is selected from:

[0034]

[0035] S6. Dissolve AM-3 in ethyl acetate or dichloromethane, add thiophosgene and saturated NaHCO 3 aqueous solution. After the reaction is completed, separate the ethyl acetate layer with a separatory funnel, add anhydrous sodium sulfate for drying, distill the solution under reduced pressure, and purify by silica gel column chromatography to obtain compound AM-4 as shown in Formula VII:

[0036]

[0037] Formula VII

[0038] R is selected from:

[0039]

[0040] S7. Add compound AM-4 (Formula VII), NH 2 -CD (Formula III) and a catalytic amount of 4-dimethylaminopyridine (DMAP) to N,N-dimethylformamide (DMF) for reaction for 15 - 30 h, then drop into acetone to form a precipitate and filter. Wash the solid with acetone multiple times and dry it under vacuum at 60 °C to obtain the β -cyclodextrin-BINAM derivative (AM-CD) as shown in Formula VIII.

[0041]

[0042] Formula VIII

[0043] R is selected from:

[0044]

[0045] S8. Add AM - CD and isocyanatopropyltriethoxysilane to DMF and react for a period of time, then add activated silica gel and continue the reaction. After cooling to room temperature, filter. The obtained solid is washed repeatedly with DMF and methanol in sequence, and after vacuum drying, the BINAM - based β -cyclodextrin derivative CSP packing material as shown in Formula I is obtained.

[0046] Furthermore, in S1 β The molar mass ratio of -cyclodextrin to p - toluenesulfonyl chloride is 1:0.8 - 1.5; the concentration of the sodium hydroxide aqueous solution is 5 - 10 mol / L, and the dosage of the acetonitrile solution is 2 - 5 mL of acetonitrile per gram of p - toluenesulfonyl chloride dissolved; the reaction is carried out at room temperature, and the reaction time is 5 - 12 h.

[0047] Furthermore, in S2, the dosage of ammonia water is 20 - 40 mL of ammonia water per gram of TsO - β CD.

[0048] Furthermore, in S3, the configuration of BINAM is R - or S - configuration, and its molar mass ratio to p - nitrophenyl isocyanate is 1:1 - 2; the reaction temperature is room temperature, and the reaction time is 2 - 10 h.

[0049] Further, the phenyl isocyanates with different substituents in S4 are: 4-Nitrophenyl Isocyanate, 4-Chlorophenyl Isocyanate, p-Tolyl Isocyanate, Phenyl isocyanate, 4-Methoxyphenylisocyanate, 4-(Trifluoromethyl)PhenylIsocyanate, 4-Dimethlaminophenylisocyanate, 1-(Tert-Butyl)-4-Isocyanatobenzene, 3,5-Dimethylphenyl Isocyanate, 3,5-DichlorophenylIsocyanate. The molar mass ratio of AM-1 to the phenyl isocyanates with different substituents is 1:1 - 2; the reaction temperature is room temperature, and the reaction time is 2 - 10 h.

[0050] Further, in S5, the molar mass ratio of AM-2 to iron is 1:5 - 20; the ratio of ethanol to hydrochloric acid is 4:3 (v / v); the reaction temperature is 60 - 80 °C; the reaction time is 10 - 24 h.

[0051] Further, in S6, the molar mass ratio of AM-3 to phosgene is 1:1 - 2; the amount of saturated NaHCO 3 aqueous solution is 10 mL per gram of AM-3; the reaction is carried out at room temperature or in an ice bath, and the reaction time is 0.5 - 2 h.

[0052] Further, in S7, the molar mass ratio of NH 2 - β CD, AM-4, and DMAP is 1:1 - 2:0.1; the reaction is carried out under nitrogen protection; the amount of the solvent DMF is 10 - 20 mL per gram of NH 2 - β CD. The reaction time is 20 - 24 h.

[0053] Further, in S8, all reactions are carried out under nitrogen protection; the molar mass ratio of AM-CD to isocyanatopropyltriethoxysilane 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.

[0054] Further, the preparation method of the activated silica gel in S8 is as follows: The chromatographic stationary phase silica gel is heated under reflux with 10% hydrochloric acid solution, cooled and washed with distilled water until the pH is approximately 7, and then dried in vacuo at 120 °C for 12 h. The amount of the hydrochloric acid solution is determined according to 10 - 20 mL per gram of silica gel. The reaction temperature is 100 - 120 °C, and the reaction time is 10 - 20 h.

[0055] Thirdly, the application of the BINAM-based β -cyclodextrin derivative chiral stationary phase shown in Formula I in the resolution of chiral compounds is provided. The chiral compounds that can be resolved include: imidazole antibacterial drugs, β receptor blockers, nimodipine, aminoglutethimide, equol, and triazole chiral pesticides.

[0056] The beneficial effects of the present invention are as follows:

[0057] 1. The raw materials of the products of the present invention are widely sourced, the reaction conditions are mild, and the cost is relatively low.

[0058] 2. The CSP filler prepared by combining the BINAM derivative with β -cyclodextrin can, while exerting the β inclusion effect of -cyclodextrin, provide more active sites by the BINAM derivative. Compared with the undervivatized β -cyclodextrin CSP, the chiral resolution effect and resolution range of the cyclodextrin-based stationary phase are greatly enhanced.

[0059] 3. The CSP filler prepared in the present invention has a definite structure compared with the traditional polymer CSP. The structure of the BINAM derivative is determined by nuclear magnetic resonance 1 H NMR, 13 C NMR combined with mass spectrometry (MS). The structure of the β -cyclodextrin derivative and the CSP filler are jointly characterized by elemental analysis (EA), thermogravimetric analysis (TG), etc.

[0060] 4. In the products of the present invention, the cyclodextrin derivative is bonded to the silica gel through a urea bond, with good stability. It is applicable to normal phase, reverse phase, and polar mobile phase HPLC, resistant to acids and alkalis (pH = 4 - 10), resistant to high pressure (40 MPa), and can still maintain good chiral separation performance in strongly polar and high water content mobile phases.

[0061] 5. The CSP filler prepared in the present invention is applicable to the resolution of chiral compounds of various structural types, and can resolve imidazole antibacterial drugs, β receptor blockers, nimodipine, aminoglutethimide, equol, and various triazole chiral pesticides, etc., and can meet the quality control in the daily analysis, production, and clinical use of these chiral drugs and chiral pesticides. Description of the Drawings

[0062] Figure 1 It is the synthetic route diagram of the embodiment of the present invention;

[0063] Figure 2 It is the enantiomeric resolution chromatogram of 9 chiral drugs in Example 1;

[0064] Figure 3 It is the enantiomeric resolution chromatogram of 6 chiral pesticides in Example 1. Detailed Embodiments

[0065] The following describes the detailed embodiments of the present invention to facilitate those skilled in the art of this technology to understand the present invention. However, it should be clear that the present invention is not limited to the scope of the detailed embodiments. For those of ordinary skill in the art of this technology, as long as various changes are within the spirit and scope of the present invention defined and determined by the appended claims, these changes are obvious, and all inventions made using the concept of the present invention are within the scope of protection.

[0066] Example 1

[0067] Refer to Figure 1 , a preparation method of a chiral stationary phase based on ( S )-BINAM β -cyclodextrin derivative, specifically including the following steps:

[0068] (1) Preparation of activated silica gel

[0069] 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 under reflux for 24 h, cool to room temperature, wash with distilled water until pH≈7, and vacuum dry at 120 °C for 12 h to obtain activated silica gel.

[0070] The chromatographic stationary phase silica gel used is produced by Qingdao Meigao Chemical Co., Ltd., China, with a particle size of 5 μ m and a specific surface area of 214 m 2 / g.

[0071] (2) Preparation of 6-amino-6-deoxy- β -cyclodextrin

[0072] Disperse 50 g of β -cyclodextrin in 100 ml of pure water, add 16 ml of 8.25 mol / L NaOH aqueous solution to dissolve it, add 8 g of p-toluenesulfonyl chloride acetonitrile solution under ice bath conditions, react at room temperature for 12 h, filter impurities, adjust the filtrate to pH≈7 with HCl, refrigerate overnight at 4 °C to precipitate, filter and recrystallize the precipitate twice with a small amount of hot water, and vacuum dry to obtain mono-6-p-toluenesulfonylated-β -Cyclodextrin (TsO- β CD), with a yield of 10%. The structure is shown in Formula II.

[0073]

[0074] Formula II

[0075] Take 5 g of dry mono-6-p-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 it into acetone to form a white flocculent precipitate, filter and dry to obtain 6-amino-6-deoxy- β -cyclodextrin (NH 2 - β CD), with a yield of 95%. The structure is shown in Formula III.

[0076]

[0077] Formula III

[0078] (3) Preparation of BINAM derivatives

[0079] Dissolve 2 g of ( S )-BINAM in 20 mL of dichloromethane, add 1.15 g of p-nitrophenyl isocyanate, stir at room temperature for 4 h, purify the crude product by normal-phase silica gel column chromatography, and elute with petroleum ether-ethyl acetate (3:1) to obtain a yellow solid product AM-1, with a yield of 70%, and the structure is shown in Formula IV.

[0080]

[0081] Formula IV

[0082] Dissolve 2 g of compound AM-1 in 20 mL of dichloromethane, add 1.1 g of p-nitrophenyl isocyanate, stir at room temperature for 4 h, purify the crude product by normal-phase silica gel column chromatography, and elute with petroleum ether-ethyl acetate (3:1) to obtain a yellow solid product AM-2a, with a yield of 90%, and the structure is shown in Formula V-a.

[0083]

[0084] Formula V-a

[0085] Put 2 g of compound AM-2a (Formula V-a) and 1.8 g of iron into a round-bottom flask, add 20 ml of EtOH and 15 ml of HCl, react in an oil bath at 75 °C for 12 h, and then use NaHCO 3Adjust to pH≈7, extract 3 times with EtOAc, remove the solvent by distillation under reduced pressure, purify the crude product by normal-phase silica gel column chromatography, and elute with petroleum ether - ethyl acetate (1:1) to obtain the yellow solid product AM-3a with a yield of 20%, and the structure is shown in Formula VI-a.

[0086]

[0087] Formula VI-a

[0088] Dissolve 0.5 g of compound AM-3a in 5 mL of ethyl acetate, add 0.12 mL of thiophosgene and 5 mL of saturated NaHCO 3 aqueous solution, react for 0.5 h in an ice bath, separate the ethyl acetate layer with a separatory funnel, add anhydrous sodium sulfate for drying, distill the solution under reduced pressure, purify the crude product by normal-phase silica gel column chromatography, and elute with petroleum ether - ethyl acetate (8:1) to obtain the brown solid product AM-4a with a yield of 95%, and the structure is shown in Formula VII-a.

[0089]

[0090] Formula VII-a

[0091] NMR and mass spectrometry data of AM-4a: 1 H NMR (600 MHz, Chloroform- d ) δ 8.14 (d, J = 9.0Hz, 1H), 7.92 (d, J = 11.1 Hz, 2H), 7.88 (m, 1H), 7.83 (m, 2H), 7.78 (m, 3H),7.36 (q, J = 7.5 Hz, 2H), 7.19 (m, 2H), 7.14 (m, 1H), 7.09 (m, 3H), 7.01 (d, J =8.5 Hz, 1H), 6.95 (d, J = 8.5 Hz, 1H), 6.82 (s, 1H), 6.79 (s, 4H). 13 C NMR (150MHz, CDCl 3 ) δ154.10, 152.75, 144.93, 142.07, 136.48, 135.54, 134.97, 134.85, 132.94, 132.84, 131.38, 131.11, 129.84, 129.81, 128.47, 128.40, 127.50, 127.46, 126.86, 126.40, 125.93, 125.69, 125.40, 125.14, 125.12, 123.09, 122.87, 122.09, 121.51, 118.10. MS (ESI) calculated for [M+H] + 625。

[0092] (4)Based on BINAM β -cyclodextrin derivative preparation

[0093] Take 0.4 g of compound AM-4a and 0.5 g of NH 2 - β -CD and dissolve it in 20 mL of DMF. Add 10 mg of DMAP and react at 85 °C for 24 h under nitrogen protection. After the reaction is completed, drop it into 200 mL of acetone to precipitate. Filter it with a sintered glass funnel (3-4 μ m) and wash it with acetone multiple times. Dry it in vacuo at 60 °C for 24 h to obtain a white solid product AM-CDa with a yield of 70%. The structure is shown in Formula VIII-a.

[0094]

[0095] Formula VIII-a

[0096] (5)Based on BINAM β -cyclodextrin derivative CSP packing preparation

[0097] Take 0.6 g of AM-CDa and dissolve it in DMF. Add 0.22 mL of 3-isocyanatopropyltriethoxysilane and stir and react at 65 °C for 6 h under nitrogen protection. Then add 2.2 g of activated silica gel, raise the temperature to 115 °C and keep the temperature constant for 24 h. The obtained solid is filtered and washed repeatedly with DMF, methanol, and acetone. After drying in vacuo at 100 °C, the CSP packing is obtained. The structure is shown in Formula I-a. Elemental analysis shows that its carbon content is 3.06%, hydrogen content is 1.27%, and nitrogen content is 0.19%; the weight loss rate of thermogravimetric analysis is 8.76%.

[0098]

[0099] Formula I-a

[0100] (6) Packing of Chiral Chromatographic Column

[0101] The chiral stationary phase was packed into an empty column by the slurry packing method. First, connect the empty column (150×4.6 mm) to the lower end of the slurry tank. Weigh 2.1 g of CSP packing into a 50 mL beaker, add 30 mL of slurry solution (methanol), and ultrasonicate for 1 min to make it a suspension. Transfer it to the slurry tank, use methanol as the displacement liquid, and quickly fill it into the empty column under a pressure of 40 MPa and keep it for 30 min to prepare a chiral chromatographic column.

[0102] Example 2

[0103] A preparation method of a chiral stationary phase based on ( S )-BINAM β -cyclodextrin derivative is basically the same as that in Example 1, except that the reagent reacting with AM-1 is p-chlorophenyl isocyanate.

[0104] The NMR and MS data of AM-4b are as follows: 1 H NMR (600 MHz, Chloroform- d ) δ 7.89 (d, J =9.1 Hz, 1H), 7.85 (m, 3H), 7.81 (t, J = 7.9 Hz, 2H), 7.37 (m, 2H), 7.20 (s,1H), 7.13 (q, J = 6.9 Hz, 2H), 7.06 (s, 1H), 6.95 (d, J = 8.5 Hz, 1H), 6.92 (d, J =8.5 Hz, 1H), 6.87 (d, J = 8.5 Hz, 2H), 6.84 (s, 1H), 6.79 (s, 1H), 6.75 (d, J =8.5 Hz, 2H), 6.62 (d, J = 8.3 Hz, 2H), 6.56 (d, J = 8.3 Hz, 2H). 13 C NMR (150 MHz,CDCl 3 ) δ154.12, 153.61, 136.71, 135.82, 135.20, 135.11, 132.78, 132.74, 131.19, 131.08, 129.57, 129.10, 128.42, 128.40, 127.32, 126.41, 126.32, 125.71, 125.60, 125.23, 125.10, 122.99, 122.62, 122.46, 122.21, 121.69, 121.24. MS (ESI) calculated for [M+H] + 614.

[0105] The BINAM synthesized in this example β -cyclodextrin CSP has a structure as shown in Formula I-b. Elemental analysis shows that its carbon content is 3.52%, hydrogen content is 1.63%, and nitrogen content is 0.43%; the weight loss rate of thermogravimetric analysis is 6.26%.

[0106]

[0107] Formula I-b

[0108] Example 3

[0109] A preparation method of a chiral stationary phase based on ([[]] S )-BINAM β -cyclodextrin derivative is basically the same as that in Example 1, except that the reagent reacting with AM-1 is p-tolyl isocyanate.

[0110] The nuclear magnetic resonance and mass spectrometry data of AM-4c are as follows: 1 H NMR (600 MHz, Chloroform- d ) δ 8.44 (d, J = 9.1 Hz, 1H), 8.13 (d, J = 8.9 Hz, 1H), 7.98 (d, J = 8.8 Hz, 1H), 7.95 (d, J = 9.1 Hz, 1H), 7.89 (d, J = 8.2 Hz, 1H), 7.87 (d, J = 8.1 Hz, 1H), 7.44 – 7.41 (m,1H), 7.36 (d, J= 8.0 Hz, 2H), 7.25 – 7.22 (m, 1H), 7.18 – 7.15 (m, 1H), 7.03(t, J = 9.7 Hz, 3H), 6.92 (d, J = 8.3 Hz, 3H), 6.78 (d, J = 7.0 Hz, 3H), 6.68 (d, J =7.9 Hz, 2H), 6.53 (s, 1H), 6.46 (s, 1H), 2.21 (s, 3H). 13 C NMR (150 MHz, CDCl 3 ) δ 154.98, 153.05, 137.37, 135.56, 134.93, 132.79, 132.63, 131.19, 130.55,129.75, 129.60, 129.50, 128.22, 127.34, 127.18, 126.41, 126.20, 125.70,125.62, 125.30, 124.98, 124.64, 122.92, 122.40, 121.26, 120.55, 20.84. MS(ESI)calculated for [M+H] + 594.

[0111] The BINAM- β -cyclodextrin CSP synthesized in this example has the structure shown in Formula I-c. Elemental analysis shows that its carbon content is 2.61%, hydrogen content is 1.87%, and nitrogen content is 0.32%. The weight loss rate of thermogravimetric analysis is 6.58%.

[0112]

[0113] Formula I-c

[0114] Example 4

[0115] A method for preparing a chiral stationary phase based on ( S )-BINAM β -cyclodextrin derivative is basically the same as that in Example 1, except that the reagent reacting with AM-1 is phenyl isocyanate.

[0116] The nuclear magnetic resonance and mass spectrometry data of AM-4d are as follows: 1 H NMR (600 MHz, Chloroform- d ) δ8.28 (d, J J = 9.0 Hz, 1H), 8.05 (d, J J = 9.0 Hz, 1H), 7.93 (m, 2H), 7.86 (m, 2H), 7.39 (m, 3H), 7.22 (d, J J = 7.8 Hz, 1H), 7.16 (t, J J = 7.7 Hz, 1H), 7.00 (t, J J = 8.3 Hz, 4H), 6.93 (m, 6H), 6.86 (m, 2H), 6.80 (d, J J = 7.7 Hz, 2H). 13 C NMR (150 MHz, CDCl 3 ) δ 154.50, 153.20, 137.10, 136.80, 135.36, 134.91, 134.52, 132.77, 132.70, 131.19, 130.66, 129.59, 129.09, 128.26, 128.21, 127.27, 127.18, 126.41, 126.19, 125.79, 125.66, 125.27, 125.14, 124.81, 124.62, 123.02, 121.54, 121.41, 120.73. MS (ESI) calculated for [M+H] + 580.

[0117] The BINAM- β -cyclodextrin CSP synthesized in this example has the structure shown in Formula I-d. Elemental analysis shows that its carbon content is 2.54%, hydrogen content is 2.62%, and nitrogen content is 0.50%; the weight loss rate of thermogravimetric analysis is 6.09%.

[0118]

[0119] Formula I-d

[0120] Example 5

[0121] A method for preparing a chiral stationary phase of a ([[]]-BINAM-based S )-cyclodextrin derivative is basically the same as that in Example 1, except that the reagent reacting with AM-1 is 4-methoxyphenyl isocyanate. β The nuclear magnetic resonance and mass spectrometry data of AM-4e are as follows:

[0122] The nuclear magnetic resonance and mass spectrometry data of AM-4e are as follows:1 1H NMR (600 MHz, Chloroform- d ) δ 8.13 (m,1H), 7.94 (m, 1H), 7.84 (m, 2H), 7.79 (t, J J = 8.3 Hz, 2H), 7.36 (m, 3H), 7.17(t, J J = 7.7 Hz, 1H), 7.09 (t, J J = 8.2 Hz, 1H), 6.98 (d, J J = 8.5 Hz, 1H), 6.85 (d, J J =8.4 Hz, 2H), 6.76 (m, 4H), 6.69 (m, 1H), 6.57 (m, 1H), 6.49 (d, J J = 8.4 Hz,2H), 6.40 (d, J J = 8.7 Hz, 2H), 3.70 (s, 3H). 13 13C NMR (150 MHz, CDCl 3 ) δ 153.99,152.02, 136.05, 134.35, 134.22, 133.65, 131.64, 131.48, 130.04, 129.61,128.44, 128.38, 127.19, 126.12, 126.02, 125.11, 124.76, 124.44, 124.10,124.01, 123.67, 121.81, 120.89, 120.52, 119.85, 113.24, 54.30. MS(ESI)calculated for [M+H] + 610.

[0123] The BINAM- β -cyclodextrin CSP synthesized in this example has the structure shown in Formula I-e. Elemental analysis shows that its carbon content is 4.08%, hydrogen content is 1.18%, and nitrogen content is 0.78%. The weight loss rate of thermogravimetric analysis is 7.95%.

[0124]

[0125] Formula I-e

[0126] Example 6

[0127] A kind based on ( S) - Preparation method of chiral stationary phase of BINAM β - cyclodextrin derivative, which is basically the same as that in Example 1, except that the reagent reacting with AM-1 is 4-trifluoromethylphenyl isocyanate.

[0128] The nuclear magnetic resonance and mass spectrometry data of AM-4f are as follows: 1 H NMR (600 MHz, Chloroform- d ) δ 8.55 (d, J =9.0 Hz, 1H), 8.49 (d, J = 8.9 Hz, 1H), 8.37 (t, J = 10.6 Hz, 2H), 8.29 (m, 2H),7.92 (m, 1H), 7.83 (q, J = 7.0 Hz, 2H), 7.70 (d, J = 7.9 Hz, 3H), 7.63 (q, J = 7.0Hz, 2H), 7.43 (m, 5H), 7.35 (m, 1H), 7.26 (m, 4H). 13 C NMR (150 MHz, CDCl 3 ) δ 152.68, 152.43, 139.96, 135.51, 134.04, 133.95, 133.89, 131.77, 131.74,130.00, 129.97, 128.61, 128.59, 127.26, 126.29, 125.40, 125.20, 125.06,125.03, 125.01, 124.98, 124.56, 124.54, 124.22, 124.12, 124.06, 124.00,123.91, 122.11, 121.47, 121.43, 120.73, 120.55, 120.07, 118.23. MS (ESI) calculated for [M+H] + 648.

[0129] The BINAM- β - cyclodextrin CSP synthesized in this example has the structure shown in Formula I-f. Elemental analysis shows that its carbon content is 2.61%, hydrogen content is 1.87%, and nitrogen content is 0.71%; the weight loss rate of thermogravimetric analysis is 5.58%.

[0130]

[0131] Formula Ⅰ-f

[0132] Example 7

[0133] A preparation method of a chiral stationary phase based on ( S )-BINAM and β -cyclodextrin derivative is basically the same as that in Example 1, except that the reagent reacting with AM-1 is 4-(dimethylamino)phenyl isocyanate.

[0134] The nuclear magnetic resonance and mass spectrometry data of AM-4g are as follows: 1 H NMR (600 MHz, Chloroform- d ) δ 8.34 (d, J J = 9.1 Hz, 1H), 8.20 (d, J J = 4.6 Hz, 1H), 7.95 (d, J J = 8.9 Hz, 1H), 7.90 (t, J J = 9.0 Hz, 2H), 7.84 (d, J J = 8.2 Hz, 1H), 7.66 (m, 1H), 7.53 (m, 1H), 7.40 (m, 2H), 7.26 (m, 1H), 7.25 (m, 1H), 7.15 (d, J J = 8.3 Hz, 1H), 7.09 (m, 3H), 7.03 (d, J J = 8.5 Hz, 1H), 6.97 (d, J J = 8.6 Hz, 2H), 6.86 (d, J J = 8.8 Hz, 2H), 6.81 (m, 1H), 6.39 (m, 2H), 2.93 (s, 6H). 13 C NMR (150 MHz, CDCl 3 ) δ152.84, 152.53, 137.63, 135.95, 135.07, 134.26, 132.78, 132.55, 131.72, 130.34, 129.79, 129.77, 128.60, 128.40, 128.32, 127.29, 127.22, 126.40, 126.25, 126.22, 125.88, 125.58, 125.34, 124.86, 124.81, 123.38, 123.25, 120.36, 120.22, 119.43, 118.30, 110.97, 39.98. MS (ESI) calculated for [M+H] + 623.

[0135] The BINAM synthesized in this example - β -cyclodextrin CSP structure is shown in Formula I-g. Elemental analysis shows that its carbon content is 3.88%, hydrogen content is 1.94%, and nitrogen content is 0.85%; the weight loss rate of thermogravimetric analysis is 7.76%.

[0136]

[0137] Formula I-g

[0138] Example 8

[0139] A preparation method of a chiral stationary phase based on ( S )-BINAM β -cyclodextrin derivative is basically the same as that of Example 1, except that the reagent reacting with AM-1 is 1-(tert-butyl)-4-isocyanatobenzene.

[0140] The nuclear magnetic resonance and mass spectrometry data of AM-4h are as follows: 1 H NMR (600 MHz, Chloroform- d ) δ 8.15 (d, J = 9.1 Hz, 1H), 8.11 (d, J = 8.9 Hz, 1H), 7.80 (m, 4H), 7.64 (m, 2H), 7.58 (s,1H), 7.33 (t, J = 5.6 Hz, 1H), 7.28 (t, J = 7.4 Hz, 1H), 7.15 (t, J= 7.4 Hz, 2H), 7.09 (m, 3H), 7.02 (s, 1H), 6.95 (m, 3H), 6.74 (d, J = 8.8 Hz, 2H), 6.71 (d, J = 8.9 Hz, 2H), 1.14 (s, 9H). 13 C NMR (150 MHz, CDCl 3 ) δ 166.81, 155.89, 152.76, 137.35, 135.77, 134.50, 134.36, 132.55, 132.30, 131.77, 130.48, 130.46, 129.75, 129.71, 128.39, 128.25, 127.34, 127.28, 126.70, 126.67, 126.09, 125.94, 125.65, 125.43, 125.40, 125.33, 125.01, 124.53, 123.81, 122.75, 121.06, 120.36, 119.08, 34.87, 30.96. MS (ESI) calculated for [M+H] + 636.

[0141] The BINAM- β -cyclodextrin CSP synthesized in this example has the structure shown in Formula I-h. Elemental analysis shows that its carbon content is 3.43%, hydrogen content is 1.11%, and nitrogen content is 0.62%; the weight loss rate of thermogravimetric analysis is 6.36%.

[0142]

[0143] Formula I-h

[0144] Example 9

[0145] A preparation method of a chiral stationary phase based on ( S )-BINAM β -cyclodextrin derivative is basically the same as that in Example 1, except that the reagent reacting with AM-1 is 3,5-dimethylphenyl isocyanate.

[0146] The nuclear magnetic resonance and mass spectrometry data of AM-4i are as follows: 1 H NMR (600 MHz, Chloroform- d ) δ 8.27 (d, J= 9.0 Hz, 1H), 8.01 (d, J = 8.9 Hz, 1H), 7.92 (d, J = 8.9 Hz, 1H), 7.88 (d, J = 9.0 Hz, 1H), 7.83 (s, 1H), 7.80 (d, J = 8.2 Hz, 1H), 7.55 (s, 1H), 7.38 (t, J = 7.9 Hz, 1H), 7.32 (t, J = 8.0 Hz, 1H), 7.17 (t, J = 8.3 Hz, 1H), 7.11 (t, J = 7.7 Hz, 1H), 6.98 (d, J = 8.4 Hz, 2H), 6.92 (d, J = 8.6 Hz, 2H), 6.89 (d, J = 8.6 Hz, 1H), 6.83 (d, J = 8.8 Hz, 3H), 6.69 (m, 1H), 6.58 (m, 1H), 6.42 (m, 2H), 2.02 (s, 6H). 13 C NMR (150 MHz, CDCl 3 ) δ 154.83, 153.15, 138.97, 138.77, 137.29, 136.59, 135.37, 135.01, 134.37, 132.81, 132.70, 131.22, 130.53, 130.50, 129.52, 128.32, 128.19, 128.16, 127.14, 127.04, 126.99, 126.54, 126.34, 126.07, 125.73, 125.59, 125.49, 125.35, 125.02, 124.97, 124.88, 123.36, 122.84, 121.37, 120.44, 120.04, 119.43, 119.34, 21.08. MS (ESI) calculated for [M+H] + 608.

[0147] The BINAM synthesized in this example βThe structure of the β-cyclodextrin CSP is shown in Formula I-i. Elemental analysis shows that its carbon content is 2.48%, hydrogen content is 2.93%, and nitrogen content is 0.78%. The weight loss rate of thermogravimetric analysis is 6.81%.

[0148]

[0149] Formula I-i

[0150] Example 10

[0151] A preparation method of a chiral stationary phase based on ( S )-BINAM-β-cyclodextrin derivative is basically the same as that in Example 1, except that the reagent reacting with AM-1 is 3,5-dichlorophenyl isocyanate. β

[0152] The nuclear magnetic resonance and mass spectrometry data of AM-4g are as follows: 1 H NMR (600 MHz, Chloroform- d ) δ 8.09 (d, J J = 9.0 Hz, 1H), 8.04 (d, J J = 9.0 Hz, 1H), 7.95 (d, J J = 9.0 Hz, 1H), 7.85 (t, J J =8.2 Hz, 2H), 7.76 (d, J J = 8.2 Hz, 1H), 7.46 (d, J J = 11.8 Hz, 2H), 7.38 (t, J J = 7.5Hz, 1H), 7.33 (t, J J = 7.5 Hz, 1H), 7.14 (t, J J = 7.7 Hz, 1H), 7.10 (t, J J = 8.0 Hz,1H), 7.01 (m, 2H), 6.88 (m, 9H). 13 C NMR (150 MHz, CDCl 3 ) δ ​153.73, 153.44, 140.12, 136.80, 135.04, 134.97, 134.75, 134.63, 132.94, 132.89, 131.03, 130.85, 129.53, 129.51, 128.16, 128.15, 127.08, 127.03, 126.17, 125.95, 125.46, 125.38, 125.33, 125.30, 122.87, 122.73, 122.48, 122.17, 121.80, 120.69, 117.30. MS (ESI) calculated for [M+H]+ 648.

[0153] The BINAM- β -cyclodextrin CSP synthesized in this example has the structure shown in Formula I-j. Elemental analysis shows that its carbon content is 3.37%, hydrogen content is 1.15%, and nitrogen content is 0.33%. The weight loss rate of thermogravimetric analysis is 6.23%.

[0154]

[0155] Formula I-j

[0156] Application Example 1: Application of β -cyclodextrin CSP based on BINAM in high performance liquid chromatography

[0157] The chiral stationary phase described in Formula I of the present invention has good stability and wide chiral selectivity, and can be used to resolve a series of enantiomeric compounds in high performance liquid chromatography. For convenience of description, the high performance liquid chromatography used in this example is Agilent 1260, but the present invention is not limited thereto.

[0158] Prepare a chiral chromatographic column with the chiral stationary phase described in Example 1. Most β β-blockers (Table 1) can be resolved in polar mobile phases, and chiral drugs such as imidazole antibacterial drugs, nimodipine, aminoglutethimide, equol, ketoprofen, thalidomide, etc. (Table 1) and most triazole pesticides (Table 2) can be resolved in reverse mobile phases.

[0159] Chromatographic conditions: flow rate 0.8 mL / min, room temperature.

[0160] Wherein k 1 is the retention factor of the first eluted enantiomer, and is calculated by the formula k 1 = (t 1 -t 0 ) / t 0Calculated, where t 1 and t 2 are the elution times of the first peak and the second peak respectively, and t 0 is the dead time, which is taken as 2.34 min here;

[0161] α is the selectivity factor, and the calculation formula is α = k 2 / k 1

[0162] R s is the resolution, and the calculation formula is R s = 2(t 2 -t 1 ) / (W 1 +W 2 ), where W 1 and W 2 are the peak widths of the first and second eluted enantiomers respectively.

[0163] Table 1 Resolution results of chiral drugs by BINAM- β -cyclodextrin CSP synthesized in Example 1

[0164]

[0165] a. Mobile phase: ACN / 0.3% FA (15 / 85)

[0166] b. Mobile phase: ACN / 0.1% FA (10 / 90)

[0167] c. Mobile phase: ACN / MeOH / TEA / AcOH (95 / 5 / 1.2 / 0.8, v / v / v / v)

[0168] d. Mobile phase: MeOH / 0.1% FA (40 / 60)

[0169] e. Mobile phase: ACN / H 2 O (10 / 90)

[0170] f. Mobile phase: MeOH / H 2 O (30 / 70)

[0171] g. Mobile phase: MeOH / H 2 O (50 / 50)

[0172] Table 2 Resolution results of chiral pesticides by BINAM- β -cyclodextrin CSP synthesized in Example 1

[0173]

[0174] a mobile phase: MeOH / H 2 O (35 / 65)

[0175] b mobile phase: MeOH / H 2 O (45 / 55)

[0176] c mobile phase: MeOH / H 2 O (50 / 50)

[0177] d mobile phase: ACN / 0.1% FA (10 / 90)

[0178] In summary, the novel β -cyclodextrin CSP packing material provided by the present invention can separate chiral compounds of various structural types, is applicable to different mobile phase systems of high performance liquid chromatography (HPLC), and also has good stability in strongly polar and high water content mobile phases. The CSP packing material provided by the present invention can resolve various chiral drugs such as imidazole antibacterial drugs, β receptor blockers, etc., and various triazole pesticides. It can meet the needs of daily analysis, quality control in the production process and use process of these chiral drugs and chiral pesticides.

[0179] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and without departing from the spirit or basic characteristics of the present invention, the present invention can be implemented in other specific forms. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present invention.

[0180] In addition, it should be understood that although this specification is described according to embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A BINAM-based β -Cyclodextrin derivative chiral stationary phase, characterized in that β -A BINAM derivative is connected to the 6-position of cyclodextrin and bonded to the silica surface via carbamate. The structure is shown in Formula I: Formula I R is selected from: The configuration of BINAM is R -or S -Configuration.

2. The BINAM-based method of claim 1 β - A method for preparing a cyclodextrin derivative chiral stationary phase, characterized in that: The steps include: S1. β -cyclodextrin reacts with p-toluenesulfonyl chloride to obtain mono-6-p-toluenesulfonyl- β -Cyclodextrin TsO- β CD; Formula II S2. Take the TsO- β CD is added to ammonia water, and the mixture is stirred at 40-70°C for 10-24 hours to obtain 6-amino-6-deoxy- β -Cyclodextrin NH2- β CD; Formula III S3. BINAM was dissolved in anhydrous dichloromethane, p-nitrobenzene isocyanate was added to react, and the compound AM-1 as shown in Formula Ⅳ was obtained by purification by silica gel column chromatography; Formula IV S4. Compound AM-1 is dissolved in anhydrous dichloromethane, phenyl isocyanate containing different substituents is added to react, and purified by silica gel column chromatography to obtain compound AM-2 as shown in formula V; Formula V R is selected from: S5. Add iron, ethanol and hydrochloric acid to compound AM-2, heat the reaction for 5 to 12 hours, and purify by silica gel column chromatography to obtain compound AM-3 as shown in formula VI; Formula VI R is selected from: S6. AM-3 was dissolved in ethyl acetate or dichloromethane, thiophosgene and saturated aqueous NaHCO3 were added, and the mixture was purified by silica gel column chromatography to obtain compound AM-4 as shown in formula VII; Formula VII R is selected from: S7. Compound AM-4 of formula VII and compound NH2- β CD and a catalytic amount of DMAP are added to DMF and reacted for 15 to 30 hours to obtain the product shown in Formula VIII. β - Cyclodextrin-BINAM derivative AM-CD; Formula VIII R is selected from: S8. AM-CD and isocyanatepropyltriethoxysilane were added to DMF to react for a period of time, and activated silica gel was added to continue the reaction. After cooling to room temperature, the solid was filtered, and the obtained solid was washed repeatedly with DMF and methanol for several times, and then vacuum dried to obtain a BINAM-based compound as shown in Formula I. β - Cyclodextrin derivative CSP filler.

3. The method according to claim 2 based on BINAM β - A method for preparing a cyclodextrin derivative chiral stationary phase, characterized in that: The method of S1 specifically comprises the following steps: Will β -cyclodextrin is dissolved in an aqueous sodium hydroxide solution, and a solution of p-toluenesulfonyl chloride in acetonitrile is added to react at room temperature or under ice bath conditions. After the reaction is completed, impurities are filtered out, the filtrate is adjusted to pH with hydrochloric acid, and refrigerated overnight to precipitate. The solid is filtered and recrystallized several times with a small amount of hot water, filtered and dried to obtain a mono-6-p-toluenesulfonyl- β -Cyclodextrin TsO- β CD; β The molar mass ratio of -cyclodextrin and p-toluenesulfonyl chloride is 1:0.8~1.5; the concentration of the sodium hydroxide aqueous solution is 5~10 mol / L, and the amount of acetonitrile solution is 1 gram of p-toluenesulfonyl chloride dissolved in 2~5 mL of acetonitrile.

4. The method according to claim 2 based on BINAM β - A method for preparing a cyclodextrin derivative chiral stationary phase, characterized in that: The configuration of BINAM in S3 is R -or S -configuration, the molar mass ratio of BINAM to p-nitrobenzene isocyanate is 1:1~2; the reaction temperature is room temperature, and the reaction time is 2~10 h.

5. The BINAM-based method according to claim 2 β - A method for preparing a cyclodextrin derivative chiral stationary phase, characterized in that: The phenyl isocyanates containing different substituents in S4 include: p-nitrobenzene isocyanate, p-chlorophenyl isocyanate, p-toluene isocyanate, phenyl isocyanate, 4-methoxyphenyl isocyanate, 4-trifluoromethylphenyl isocyanate, 4-(dimethylamino)phenyl isocyanate, 1-(tert-butyl)-4-isocyanatebenzene, 3,5-dimethylphenyl isocyanate, and 3,5-dichlorophenyl isocyanate. The molar mass ratio of AM-1 used to the phenyl isocyanate containing different substituents is 1:1~2; the reaction temperature is room temperature, and the reaction time is 2~10 h.

6. The BINAM-based method according to claim 2 β - A method for preparing a cyclodextrin derivative chiral stationary phase, characterized in that: The molar mass ratio of AM-2 to iron in S5 is 1:5~20; the ratio of ethanol to hydrochloric acid is 4:3 (v / v); the reaction temperature is 60~80 ℃; and the reaction time is 10~24 h.

7. The BINAM-based method according to claim 2 β - A method for preparing a cyclodextrin derivative chiral stationary phase, characterized in that: The molar mass ratio of AM-3 to thiophosgene in S6 is 1:1~2; the amount of saturated NaHCO3 aqueous solution used is 10 mL per gram of AM-3; the reaction is carried out at room temperature or in an ice bath, and the reaction time is 0.5~2 h.

8. The BINAM-based method according to claim 2 β - A method for preparing a cyclodextrin derivative chiral stationary phase, characterized in that: NH2- in S7 β The molar mass ratio of CD, AM-4 and DMAP is 1:1~2:0.1; the reaction is carried out under nitrogen protection; the amount of solvent DMF is based on 1:1 per gram of NH2- β Add 10~20 mL of CD; the reaction time is 20~24 h.

9. The BINAM-based method according to claim 2 β - A method for preparing a cyclodextrin derivative chiral stationary phase, characterized in that: All reactions in S8 were carried out under nitrogen protection; the molar mass ratio of AM-CD to triethoxypropyl isocyanate was 1:2~5; the reaction time was 4~10 h; the reaction temperature after adding activated silica gel was 110~120°C, and the reaction time was 20~36 h.

10. The BINAM-based method of claim 1 β -Application of cyclodextrin derivative chiral stationary phase in separation of chiral compounds, characterized in that: The chiral compounds that can be separated include: imidazole antibiotics, β Beta-blockers, nimodipine, aminoglutethimide, equol and triazole chiral pesticides.

Images