A BINAM-cellulose derivative composite chiral stationary phase and its preparation method and application

Abstract

The present invention relates to the technical field of chiral stationary phases, and specifically to a BINAM-cellulose derivative composite chiral stationary phase, a preparation method and application thereof. The preparation method comprises the following steps: using BINAM as a raw material to synthesize a BINAM derivative containing a thiourea group spacer arm, and bonding it to the surface of mercapto silica gel through "click chemistry" to obtain BINAM-SiO2; using acryloyl chloride and isocyanate to derivatize the hydroxyl groups on cellulose to obtain a cellulose derivative; and then bonding the cellulose derivative to BINAM-SiO2 to obtain a BINAM-cellulose derivative composite chiral stationary phase. The BINAM-cellulose derivative composite chiral stationary phase prepared by the present invention has stable chiral recognition ability, can achieve baseline separation for chiral solutes of various structural types, and has good stability, and is suitable for use as a high performance liquid chromatography filler.

Description

Technical Field

[0001] The invention relates to the technical field of chiral stationary phases, and in particular to a BINAM-cellulose derivative composite chiral stationary phase and a preparation method and application thereof. Background Art

[0002] Because of the great differences in the biological activity, pharmacological effects and toxicity of chiral drugs, the enantiomer separation of chiral drugs has received more and more attention. High performance liquid chromatography chiral stationary phase (CSP) has become the preferred method for the separation of chiral drug enantiomers and the in vivo and in vitro detection of chiral drugs due to its high efficiency, high sensitivity, wide range of applications, and the ability to be combined with other instruments. It can also achieve the preparation and semi-preparation of enantiomers.

[0003] Commonly used HPLC CSPs include brush-type, polysaccharide derivatives, cyclodextrin and derivatives, crown ethers and derivatives, macrocyclic antibiotics, proteins, etc. These CSPs are widely used, but they also have shortcomings: (1) The aging of chiral selectors limits the separation effect, and it is difficult to quickly discover new and efficient chiral selectors; (2) Except for Pirkle-type chiral stationary phases, the chiral separation mechanism of most CSPs is unclear and needs further in-depth research. The development of composite chiral stationary phases and the study of chiral recognition mechanisms have important academic value and practical significance.

[0004] Polysaccharide derivative chiral stationary phases are mostly prepared from polysaccharides derived from benzoate, substituted benzoate, phenylcarbamate and substituted phenylcarbamate. The structure of polysaccharide macromolecules has a certain influence on their chiral recognition ability. Polysaccharide derivative CSPs with different structures have certain complementarity in the separation of chiral solutes. At present, about 90% of chiral compounds can be separated on polysaccharide derivative CSPs.

[0005] 1,1'-BINAM has a C2 symmetry axis, and its axial chirality is both rigid and flexible. By structural modification and group addition and subtraction, the axial angle of this type of compound can be changed to obtain good asymmetric induction. In recent years, chiral thiourea catalysts derived from 1,1'-BINAM have been applied to asymmetric Michael addition reactions, asymmetric Diels-Alder reactions, etc.

[0006] BINAM and cellulose derivatives are both traditional and highly efficient chiral selectors, but there is no report on the combined use of the two in the prior art. Summary of the invention

[0007] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a BINAM-cellulose derivative composite chiral stationary phase and a preparation method and application thereof.

[0008] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0009] In a first aspect, a method for preparing a BINAM-cellulose derivative composite chiral stationary phase is provided, comprising the following steps:

[0010] Step 1, using BINAM as a raw material to synthesize a BINAM derivative containing a thiourea group spacer arm;

[0011] Step 2: Bond the BINAM derivative to the surface of mercaptosilane by “click chemistry” to obtain BINAM-SiO 2 ;

[0012] Step 3, using acryloyl chloride and isocyanate to derivatize the hydroxyl groups on cellulose to obtain a cellulose derivative;

[0013] Step 4: Bonding cellulose derivatives to BINAM-SiO 2 A BINAM-cellulose derivative composite chiral stationary phase was obtained.

[0014] Preferably, the method of step 1 specifically includes the following sub-steps:

[0015] Step 1-1, one of the amino groups in BINAM is acetylated and protected, and two methyl groups are introduced into the other amino group through formaldehyde solution to obtain a tertiary amine group, and then the acetylamino group is deacetylated and reacted with thiophosgene to obtain an isothiocyanate-containing compound (R)-1, whose structure is shown in Formula VI:

[0016]

[0017] (2) Compound (R)-1 is dissolved in ultra-dry dichloromethane and reacted with spacer arms 1,6-hexanediamine and p-phenylenediamine respectively, and then reacted with allyl isothiocyanate to obtain compounds (R)-1a and (R)-1b respectively, whose structures are shown in Formulas VII and VIII; Compound (R)-1 is dissolved in ultra-dry dichloromethane and reacted with spacer arm 2-methylallylamine to obtain compound (R)-1c, whose structure is shown in Formula IX;

[0018]

[0019]

[0020]

[0021] Preferably, the method of step 2 is as follows: dispersing compounds (R)-1a, (R)-1b, and (R)-1c in methanol, adding a crosslinking agent, and bonding them to the mercapto silica gel carrier through a "click chemistry" reaction to obtain BINAM-SiO 2 (Formula II, Formula III, Formula IV);

[0022]

[0023]

[0024]

[0025] In the structures shown in formula II, III, and IV, Stands for silica gel carrier.

[0026] In the present invention, the configuration of BINAM is R -or S -Configuration.

[0027] Preferably, in step 1-1, the protecting reagent for BINAM acetylation protection is acetic anhydride, the reaction molar ratio is: 1:1.0~2.0, and the reaction temperature is 25°C; the concentration of formaldehyde solution is 35~40%, and the reaction temperature is 40°C; the reagent for deacetylation is hydrochloric acid, the concentration is 2~6 M, the temperature is 85°C, and the reaction time is 24h; the molar ratio of the reaction with thiophosgene is: 1:1.0~3.0, the reaction temperature is 25°C, and the reaction time is 12h.

[0028] Preferably, in step 1-2, the reaction molar ratio of compound (R)-1 to the spacer arm is 1:1.0-4.0, the reaction temperature is 25° C., and the reaction time is 18 h.

[0029] Preferably, in step 2, the mercapto silica gel is obtained by bonding (3-mercaptopropyl)triethoxysilane to chromatographic spherical silica gel; the mass ratio of the reaction of compound (R)-1a and mercapto silica gel is 1:8.0~12.0 (g / g); the cross-linking agent is azobisisobutyronitrile; the bonding reaction temperature is 90°C and the reaction time is 48h.

[0030] Preferably, the acrylate-cellulose derivative in step 3 has a structure shown in Formula V:

[0031]

[0032] R is selected from: , , , , ;

[0033] The preparation method comprises the following steps:

[0034] The microcrystalline cellulose is dissolved in an ultra-dry organic solvent, acryloyl chloride is added for reaction, and then a derivatization reagent isocyanate is added to derivatize all hydroxyl groups on the cellulose. After the reaction is completed, a precipitant is added to precipitate the product, and then the product is dissolved in an organic solvent, subjected to secondary precipitation, filtered, and vacuum dried to obtain an acrylate-cellulose derivative (Formula V);

[0035] Preferably, the microcrystalline cellulose is vacuum dried; the ultra-dry organic solvent is N,N-dimethylacetamide and anhydrous lithium chloride, the ratio of microcrystalline cellulose to the ultra-dry organic solvent is 1g: (20-40)ml: (1.0-2.0)g, the dissolution temperature is 80°C, and the dissolution time is 8h.

[0036] Preferably, the molar ratio of microcrystalline cellulose to acryloyl chloride is 3.0:1.0-2.5, the reaction temperature is 80° C., and the reaction time is 8 hours.

[0037] Preferably, the derivatization agent is any one of 3,5-dimethylphenylisocyanate, 4-methylphenylisocyanate, phenylisocyanate, 3,4-dimethylphenylisocyanate, and 3-methylphenylisocyanate; the reaction molar ratio of microcrystalline cellulose to isocyanate is 1:4.0~8.0, the reaction temperature is 80°C, and the reaction time is 24h.

[0038] Preferably, the precipitant is methanol; the organic solvent for dissolving the precipitate is dimethyl sulfoxide or tetrahydrofuran; the secondary precipitant is methanol; the vacuum drying temperature is 60° C. and the time is 12 h.

[0039] Preferably, the method of step 4 is specifically:

[0040] The acrylate-cellulose derivative was dissolved in an organic solvent and coated on BINAM-SiO 2 The surface of the cellulose is dispersed in methanol, a cross-linking agent is added for reaction, and after the reaction is completed, the cellulose is filtered and washed with methanol and anhydrous ethanol to obtain a BINAM-cellulose derivative composite chiral stationary phase filler;

[0041] Preferably, the acrylate-cellulose derivative is any one of acrylate-3,5-dimethylphenylcarbamate cellulose derivative, acrylate-4-methylphenylcarbamate cellulose derivative, acrylate-phenylcarbamate cellulose derivative, acrylate-3,4-dimethylphenylcarbamate cellulose derivative, acrylate-3-methylphenylcarbamate cellulose derivative;

[0042] Preferably, the configuration of BINAM is R -or S -Configuration.

[0043] Preferably, the organic solvent for dissolving the acrylate-cellulose derivative is tetrahydrofuran, and the ratio of the acrylate-cellulose derivative to tetrahydrofuran is 0.4-0.6 g: 10-20 ml.

[0044] Preferably, the acrylate-cellulose derivative and BINAM-SiO 2 The coating ratio is 1:3.0~5.0 (g / g); the coating method is rotary evaporation.

[0045] Preferably, the cross-linking agent is azobisisobutyronitrile, and the amount added is 1.0-3.0 g; the reaction temperature is 90° C., and the reaction time is 24 h.

[0046] In a second aspect, a BINAM-cellulose derivative composite chiral stationary phase is provided, which has a structure shown in Formula I:

[0047]

[0048] R 1 Selected from:

[0049] ,

[0050] ,

[0051] R 2 Selected from:

[0052] , , , ,

[0053] In the structure shown in Formula I, Stands for silica gel carrier.

[0054] In a third aspect, a method is provided for using a BINAM-cellulose derivative composite chiral stationary phase in the separation of racemic chiral compounds.

[0055] The beneficial effects of the present invention are:

[0056] The BINAM-cellulose derivative composite chiral stationary phase of the present invention combines two traditional chiral selectors BINAM and cellulose derivatives for the first time to prepare a composite CSP. Experimental results show that the introduction of BINAM can enhance the separation effect of CSP on racemates. The composite CSP has excellent chiral separation effect, good stability and repeatability, can separate chiral compounds of various structural types, and is suitable for use as a high performance liquid chromatography filler. The experimental results of the composite CSP provide a new idea for the design and synthesis of a new chiral stationary phase. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 The synthetic route diagram of the preparation method of the embodiment of the present invention;

[0058] Figure 2 BINAM-SiO in Example 1 2 (A) and SEM images of composite CSP (B);

[0059] Figure 3 This is the chromatogram of enantiomeric separation of four chiral compounds in Example 1;

[0060] Figure 4 This is the chromatogram of the enantiomeric separation of three chiral compounds in Example 2;

[0061] Figure 5 This is the chromatogram of enantiomeric separation of four chiral compounds in Example 3. DETAILED DESCRIPTION

[0062] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0063] Example 1

[0064] Reference Figure 1 This embodiment provides a method for preparing a BINAM-cellulose derivative composite chiral stationary phase, which comprises the following steps:

[0065] (1) Synthesis of BINAM derivatives

[0066] Will( R )-(+)-1,1'-bi-2-naphthylamine (2.84 g, 10 mmol) was dissolved in 80 mL of anhydrous dichloromethane. At 0°C, under nitrogen protection, 1.2 mL (12 mmol) of acetic anhydride was added. After 5 minutes, 6 mL (100 mmol) of acetic acid was added. The reaction was continued for 10 minutes. The reaction was continued for 12 hours at room temperature. The mixture was neutralized with saturated sodium bicarbonate solution to pH ≈ 7. The organic solvent was separated. The aqueous solution was extracted with dichloromethane 3 times, 50 mL each time. The organic phases were combined, washed with 50 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. The crude product was purified by normal phase silica gel column chromatography with petroleum ether-ethyl acetate (5:1) to obtain a white solid product R-1.

[0067] R-1 (1.63 g, 5 mmol) was dissolved in 30 mL of tetrahydrofuran, and 5.0 mL (60 mmol) of 37% formaldehyde solution was added. The mixture was stirred under magnetic stirring for 15 min, and 1.32 g (35 mmol) of sodium cyanoborohydride was added. After 15 min, 6.5 mL of glacial acetic acid solution was added, and the mixture was reacted at room temperature for 6 h. After the reaction, the organic solvent was removed by distillation under reduced pressure, and a saturated sodium bicarbonate solution was added to adjust the pH to 7. The mixture was extracted with dichloromethane 3 times, 50 mL each time. The organic phases were combined, washed with 50 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The crude product was subjected to normal phase silica gel column chromatography, and eluted with petroleum ether-ethyl acetate (8:1) to obtain a light yellow solid product R-2.

[0068] R-2 (1.6 g, 4.5 mmol) was dissolved in 150 mL of anhydrous ethanol, 4 M HCl (54 mL) was added, and the reaction was heated under reflux for 24 h. The mixture was cooled to room temperature, and the ethanol was removed by distillation under reduced pressure. Saturated sodium bicarbonate solution was added to neutralize the mixture to pH ≈ 7. The mixture was extracted with dichloromethane for 3 times, 50 mL each time. The organic phases were combined, washed with 50 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and the organic solvent was removed by distillation under reduced pressure. The product R-3 was purified by normal phase silica gel column chromatography and eluted with petroleum ether-ethyl acetate (15:1) to obtain a light yellow solid product.

[0069] R-3 (1.09 g, 3.5 mmol) was dissolved in 30 mL of dichloromethane, 3 mL of saturated sodium bicarbonate solution was added, magnetic stirring was carried out at 0 °C for 5 min, 540 µL (7 mmol) of thiophosgene was slowly added dropwise, the reaction was continued for 1 h, and the temperature was naturally raised to room temperature for 12 h. After the reaction, 60 mL of distilled water was added, and dichloromethane was extracted 3 times, 30 mL each time. The organic phases were combined, washed with 50 mL of saturated sodium chloride solution, dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. The crude product was purified by normal phase silica gel column chromatography, and eluted with petroleum ether-ethyl acetate (30:1) to obtain a yellow powder product R-4.

[0070] The spacer arm 1,6-hexanediamine (1.04 g, 9 mmol) was dissolved in 50 mL of anhydrous dichloromethane, and then R-4 (1.06 g, 3 mmol) dissolved in 50 mL of anhydrous dichloromethane was slowly added dropwise. After the addition was complete, the reaction was carried out at room temperature for 4 hours, and then an excess of allyl isothiocyanate (1.78 g) was added. After continuing the reaction for 12 hours, the organic solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography and eluted with petroleum ether-ethyl acetate (3:1) to obtain a yellow oily product R-4a.

[0071] R-4a NMR and high resolution mass spectrometry data: 1 H NMR (600 MHz, CDCl 3 ) δ 8.01 (d, J= 8.7Hz, 1H), 7.97 (d, J = 8.9 Hz, 1H), 7.94 (d, J = 8.2 Hz, 1H), 7.84 (d, J = 8.1 Hz,1H), 7.58-7.48 (m, 3H), 7.31-7.25 (m, 3H), 7.11 (t, J = 7.1 Hz, 1H), 6.86 (d, J =7.6 Hz, 1H), 5.84 (ddd, J = 22.5, 10.6, 5.5 Hz, 1H), 5.22 (d, J = 17.2 Hz, 1H),5.15 (d, J = 10.3 Hz, 1H), 4.11 (q, J = 7.1 Hz, 2H), 3.50-3.25 (m, 4H), 2.55 (s,6H), 1.48-1.34 (m, 2H), 1.29-1.26 (m, 2H), 1.22-1.13 (m, 2H), 1.11-0.98 (m,2H). 13 C NMR (150 MHz, CDCl 3 ) δ 182.1, 180.1, 149.8, 134.1, 133.6, 133.3,132.4, 132.1, 130.3, 129.9, 129.7, 128.4, 128.3, 127.2, 127.1, 126.6, 126.3,124.9, 124.4, 124.1, 123.2, 118.8, 117.1, 46.83, 44.72, 43.98, 28.50, 25.86.HRMS (ESI) calculated for C 33 H 39 N 5 S 2 [M+H] + 570.2732, found 570.2759.

[0072] (2) BINAM-SiO 2 Preparation of silica gel

[0073] Preparation of thiolated silica gel: Add 20.0g silica gel and 100mL 10% hydrochloric acid solution to a 250mL round-bottom flask, heat and reflux for 24h, cool to room temperature, wash with distilled water until pH ≈ 7, and vacuum dry at 120℃ for 12h. Transfer the dried silica gel to a 250mL round-bottom flask, add 200mL anhydrous toluene, 4mL pyridine, 2.0mL triethylamine, 20.0mL (3-mercaptopropyl) triethoxysilane in sequence, and heat and reflux at 120℃ for 24h under nitrogen protection. After the reaction, wash with toluene, acetone, methanol, and dichloromethane, respectively, and vacuum dry at 60℃ to obtain thiolated silica gel.

[0074] It was determined that the elemental analysis of the mercapto silica gel synthesized in this example contained 2.67% carbon, 0.76% hydrogen and 0.0% nitrogen; and the weight loss rate determined by thermogravimetric analysis was 8.88%.

[0075] The BINAM derivative R-4a was bonded to the surface of thiolated silica gel by "click chemistry". 10.0 g of thiolated silica gel was weighed and dispersed in 60 mL of methanol, and then 200.0 mg of azobisisobutyronitrile and 1.0 g of BINAM derivative R-4a were added. The mixture was reacted at 90 °C for 48 h under nitrogen protection. After the reaction, the mixture was cooled to room temperature, filtered, washed with methanol, anhydrous ethanol, and dichloromethane, respectively, and dried under vacuum at 60 °C to obtain BINAM-SiO 2 Silica gel, named RM569-SiO 2 .

[0076] It was determined that the RM569-SiO synthesized in this embodiment 2 The elemental analysis showed that the carbon content was 8.65%, the hydrogen content was 1.34%, and the nitrogen content was 1.12%; the weight loss rate of thermogravimetric analysis was 16.92%.

[0077] (3) Synthesis of cellulose derivatives

[0078] Weigh 1.0 g of dry microcrystalline cellulose to a 100 mL round-bottom flask, add 30 mL of ultra-dry N, N-dimethylacetamide, stir and swell at 80 ° C for 8 h under nitrogen protection, cool to room temperature, add 1.5 g of anhydrous lithium chloride, continue stirring for 2 h, add 30.0 mg of 4-dimethylaminopyridine, 185.0 mg of acryloyl chloride, 207.0 mg of triethylamine, heat to 80 ° C for reaction for 8 h, then add excess cellulose derivatization reagent 3,5-dimethylphenylisocyanate (5.4 g), and continue to react for 24 h. After the reaction, the solution is yellow-brown, cool to room temperature, slowly drop into 500 mL of methanol for precipitation, stand for 2 h, filter, dissolve the filter cake with a small amount of dimethyl sulfoxide, and perform secondary precipitation with methanol, filter, wash with methanol, and vacuum dry at 60 ° C to obtain acrylate-3,5-dimethylphenylcarbamate cellulose derivatives.

[0079] (4) Preparation of BINAM-cellulose derivative composite chiral stationary phase

[0080] 0.55 g of acrylate-3,5-dimethylphenylcarbamate cellulose derivative was added to a 50 mL round-bottom flask and dissolved in 15 mL of tetrahydrofuran to form a viscous liquid. The viscous liquid was coated onto 2.2 g of RM569-SiO by rotary evaporation. 2 The silica gel surface was decompressed and distilled to remove the organic solvent. The silica gel coated with cellulose derivatives was dispersed in 30 mL of methanol solution, and 2.0 g of azobisisobutyronitrile was added. The reaction was carried out at 90 ° C under nitrogen protection for 24 hours. After the reaction was completed, it was filtered, washed with methanol and anhydrous ethanol, and dried in vacuum at 60 ° C to obtain RM569-acrylate-3,5-dimethylphenylcarbamate cellulose derivative chiral stationary phase.

[0081] It was determined that the elemental analysis of the chiral stationary phase of the RM569-acrylate-3,5-dimethylphenylcarbamate cellulose derivative synthesized in this example contained 18.95% carbon, 2.09% hydrogen and 2.23% nitrogen. The weight loss rate of the thermogravimetric analysis was 31.93%.

[0082] (5) Filling of chiral chromatography columns

[0083] The chiral stationary phase was loaded into the empty column by homogenization method. First, the empty column (150×4.6 mm) was connected to the lower end of the homogenization tank, 2.1 g of RM569-acrylate-3,5-dimethylphenylcarbamate cellulose derivative chiral stationary phase was weighed into a 50 mL beaker, 30 mL of homogenate (n-hexane / isopropanol 90:10) was added, and it was ultrasonicated for 3 min to make it a suspension, and then transferred to the homogenization tank, and n-hexane / isopropanol (90:10) was used as the displacement liquid, and it was quickly filled into the empty column at a pressure of 40 MPa, and maintained for 30 min to prepare a chiral chromatographic column.

[0084] Example 2

[0085] The steps and methods of this example are basically the same as those of Example 1, except that the spacer arm of the BINAM derivative is p-phenylenediamine to obtain R-4b.

[0086] R-4b NMR and high-resolution mass spectrometry data: 1 H NMR (600 MHz, CDCl 3 ) δ 7.95 (t, J = 8.8Hz, 2H), 7.90 (d, J = 8.2 Hz, 1H), 7.85 (d, J= 8.1 Hz, 1H), 7.65-7.41 (m, 3H),7.31 (t, J = 7.4 Hz, 1H), 7.29-7.21 (m, 2H), 7.06 (t, J = 7.6 Hz, 1H), 7.00 (d, J =7.9 Hz, 2H), 6.88-6.77 (m, 3H), 5.76 (ddd, J = 22.4, 10.6, 5.4 Hz, 1H), 5.13(d, J = 17.1 Hz, 1H), 5.08 (d, J = 10.2 Hz, 1H), 4.50 (s, 4H), 3.97 (s, 2H), 2.52(s, 6H). 13 C NMR (150 MHz, CDCl 3 ) δ 182.2, 180.6, 149.8, 136.7, 134.1, 133.3,132.4, 131.9, 130.3, 130.1, 129.9, 129.7, 128.4, 128.3, 127.9, 127.5, 127.3,127.1, 126.7, 126.4, 124.9, 124.4, 124.2, 123.3, 118.8, 117.0, 48.5, 48.1,46.8, 43.9.HRMS (ESI) calculated for C 35 H 35 N 5 S 2 [M+H] + 590.2467, found 590.2442.

[0087] It was determined that the elemental analysis of the BINAM composite chiral stationary phase synthesized in this example contained 17.63% carbon, 2.63% hydrogen and 2.32% nitrogen. The weight loss rate of the thermogravimetric analysis was 28.46%.

[0088] Example 3

[0089] The steps and methods of this example are basically the same as those of Example 1, except that the spacer arm of the BINAM derivative is 2-methylallylamine, to obtain R-4c.

[0090] R-4c NMR and high resolution mass spectrometry data: 1 H NMR (600 MHz, CDCl3 ) δ 7.99 (d, J = 8.7Hz, 1H), 7.95 (d, J = 9.0 Hz, 1H), 7.92 (d, J = 8.2 Hz, 1H), 7.83 (d, J = 8.1 Hz,1H), 7.77-7.60 (m, 1H), 7.52 (d, J = 8.9 Hz, 1H), 7.47 (t, J = 6.2 Hz, 1H), 7.31-7.24 (m, 3H), 7.10 (t, J = 7.7 Hz, 1H), 6.87 (d, J = 7.6 Hz, 1H), 4.62 (s, 1H), 4.49 (s, 1H), 3.98 (s, 2H), 2.55 (s, 6H), 1.53 (s, 3H). 13 C NMR (150 MHz, CDCl 3 ) δ 181.0, 149.9, 141.2, 134.2, 133.4, 132.3, 130.2, 130.1, 129.5,128.4, 128.3, 127.1, 126.6, 126.3, 124.9, 124.5, 124.1, 123.4, 118.9, 111.2,50.8, 43.9, 20.3.HRMS (ESI) calculated for C 27 H 27 N 3 S[M+H] + 426.1962, found426.2040.

[0091] It was determined that the elemental analysis of the BINAM composite chiral stationary phase synthesized in this example contained 18.28% carbon, 2.71% hydrogen and 2.10% nitrogen. The weight loss rate of the thermogravimetric analysis was 29.43%.

[0092] Example 4

[0093] The steps and methods of this embodiment are basically the same as those of Embodiment 1, except that the cellulose derivatization reagent is 4-methylphenyl isocyanate.

[0094] It was determined that the elemental analysis of the BINAM composite chiral stationary phase synthesized in this example contained 18.80% carbon, 1.95% hydrogen and 2.23% nitrogen. The weight loss rate of the thermogravimetric analysis was 29.12%.

[0095] Example 5

[0096] The steps and methods of this example are basically the same as those of Example 1, except that the spacer arm of the BINAM derivative is p-phenylenediamine, and the cellulose derivatization agent is 4-methylphenylisocyanate.

[0097] It was determined that the elemental analysis of the BINAM composite chiral stationary phase synthesized in this example contained 17.95% carbon, 2.50% hydrogen and 2.55% nitrogen. The weight loss rate of the thermogravimetric analysis was 28.68%.

[0098] Example 6

[0099] The steps and methods of this example are basically the same as those of Example 1, except that the spacer arm of the BINAM derivative is 2-methylallylamine, and the cellulose derivatization agent is 4-methylphenylisocyanate.

[0100] It was determined that the elemental analysis of the BINAM composite chiral stationary phase synthesized in this example contained 18.69% carbon, 2.59% hydrogen and 2.38% nitrogen. The weight loss rate of the thermogravimetric analysis was 29.51%.

[0101] Example 7

[0102] The steps and methods of this embodiment are basically the same as those of embodiment 1, except that the cellulose derivatization reagent is phenyl isocyanate.

[0103] It was determined that the elemental analysis of the BINAM composite chiral stationary phase synthesized in this example contained 18.81% carbon, 1.89% hydrogen and 2.32% nitrogen. The weight loss rate of the thermogravimetric analysis was 30.26%.

[0104] Example 8

[0105] The steps and methods of this example are basically the same as those of Example 1, except that the spacer arm of the BINAM derivative is p-phenylenediamine, and the cellulose derivatization agent is phenylisocyanate.

[0106] It was determined that the elemental analysis of the BINAM composite chiral stationary phase synthesized in this example contained 18.77% carbon, 2.51% hydrogen and 2.75% nitrogen. The weight loss rate of the thermogravimetric analysis was 29.87%.

[0107] Example 9

[0108] The steps and methods of this example are basically the same as those of Example 1, except that the spacer arm of the BINAM derivative is 2-methylallylamine, and the cellulose derivatization agent is phenylisocyanate.

[0109] It was determined that the elemental analysis of the BINAM composite chiral stationary phase synthesized in this example contained 18.49% carbon, 2.50% hydrogen and 2.54% nitrogen. The weight loss rate of the thermogravimetric analysis was 30.86%.

[0110] Example 10

[0111] The steps and methods of this embodiment are basically the same as those of Embodiment 1, except that the cellulose derivatization reagent is 3,4-dimethylphenyl isocyanate.

[0112] It was determined that the elemental analysis of the BINAM composite chiral stationary phase synthesized in this example contained 17.85% carbon, 1.97% hydrogen and 2.12% nitrogen. The weight loss rate of the thermogravimetric analysis was 28.17%.

[0113] Embodiment 11

[0114] The steps and methods of this example are basically the same as those of Example 1, except that the spacer arm of the BINAM derivative is p-phenylenediamine, and the cellulose derivatization agent is 3,4-dimethylphenylisocyanate.

[0115] It was determined that the elemental analysis of the BINAM composite chiral stationary phase synthesized in this example contained 19.28% carbon, 2.68% hydrogen and 2.53% nitrogen. The weight loss rate of the thermogravimetric analysis was 30.00%.

[0116] Example 12

[0117] The steps and methods of this example are basically the same as those of Example 1, except that the spacer arm of the BINAM derivative is 2-methylallylamine, and the cellulose derivatization agent is 3,4-dimethylphenylisocyanate.

[0118] It was determined that the elemental analysis of the BINAM composite chiral stationary phase synthesized in this example contained 19.85% carbon, 2.89% hydrogen and 2.25% nitrogen. The weight loss rate of the thermogravimetric analysis was 30.66%.

[0119] Embodiment 13

[0120] The steps and methods of this embodiment are basically the same as those of Embodiment 1, except that the cellulose derivatization reagent is 3-methylphenyl isocyanate.

[0121] It was determined that the elemental analysis of the BINAM composite chiral stationary phase synthesized in this example contained 19.48% carbon, 1.99% hydrogen and 2.43% nitrogen. The weight loss rate of the thermogravimetric analysis was 31.18%.

[0122] Embodiment 14

[0123] The steps and methods of this example are basically the same as those of Example 1, except that the spacer arm of the BINAM derivative is p-phenylenediamine, and the cellulose derivatization agent is 3-methylphenylisocyanate.

[0124] It was determined that the elemental analysis of the BINAM composite chiral stationary phase synthesized in this example contained 18.65% carbon, 2.65% hydrogen and 2.52% nitrogen. The weight loss rate of the thermogravimetric analysis was 29.78%.

[0125] Embodiment 15

[0126] The steps and methods of this example are basically the same as those of Example 1, except that the spacer arm of the BINAM derivative is 2-methylallylamine, and the cellulose derivatization agent is 3-methylphenylisocyanate.

[0127] It was determined that the elemental analysis of the BINAM composite chiral stationary phase synthesized in this example contained 18.82% carbon, 2.61% hydrogen and 2.35% nitrogen. The weight loss rate of the thermogravimetric analysis was 30.19%.

[0128] Comparative Example 1

[0129] (1) Synthesis of cellulose derivatives

[0130] Weigh 1.0 g of dry microcrystalline cellulose to a 100 mL round-bottom flask, add 30 mL of ultra-dry N, N-dimethylacetamide, stir and swell at 80 ° C for 8 h under nitrogen protection, cool to room temperature, add 1.5 g of anhydrous lithium chloride, continue stirring for 2 h, add 30.0 mg of 4-dimethylaminopyridine, 185.0 mg of acryloyl chloride, 207.0 mg of triethylamine, heat to 80 ° C for reaction for 8 h, then add excess cellulose derivatization reagent 3,5-dimethylphenylisocyanate (5.4 g), and continue to react for 24 h. After the reaction, the solution is yellow-brown, cool to room temperature, slowly drop into 500 mL of methanol for precipitation, stand for 2 h, filter, dissolve the filter cake with a small amount of dimethyl sulfoxide, and perform secondary precipitation with methanol, filter, wash with methanol, and vacuum dry at 60 ° C to obtain acrylate-3,5-dimethylphenylcarbamate cellulose derivatives.

[0131] (2) Preparation of cellulose derivative chiral stationary phase

[0132] Add 0.55g of acrylate-3,5-dimethylphenylcarbamate cellulose derivative to a 50mL round-bottom flask, dissolve it in 15mL of tetrahydrofuran, and coat it on the surface of 2.2g of mercapto silica gel by rotary evaporation. Remove the organic solvent by vacuum distillation. Disperse the silica gel coated with cellulose derivative in 30mL of methanol solution, add 2.0g of azobisisobutyronitrile, and react at 90℃ under nitrogen protection for 24h. After the reaction is completed, filter, wash with methanol and anhydrous ethanol, and vacuum dry at 60℃ to obtain acrylate-3,5-dimethylphenylcarbamate cellulose derivative chiral stationary phase.

[0133] It was determined that the chiral stationary phase of the acrylate-3,5-dimethylphenylcarbamate cellulose derivative synthesized in this comparative example had a carbon content of 14.89%, a hydrogen content of 2.20%, and a nitrogen content of 1.85% according to elemental analysis; and a weight loss rate of 25.86% according to thermogravimetric analysis.

[0134] (3) Filling of chiral chromatographic columns

[0135] The chiral stationary phase was loaded into the empty column by homogenization method. First, the empty column (150×4.6 mm) was connected to the lower end of the homogenization tank, 2.1 g of acrylate-3,5-dimethylphenylcarbamate cellulose derivative chiral stationary phase was weighed into a 50 mL beaker, 30 mL of homogenate (n-hexane / isopropanol 90:10) was added, and it was ultrasonicated for 3 min to make it a suspension, and then transferred to the homogenization tank, and n-hexane / isopropanol (90:10) was used as the displacement liquid, and it was quickly filled into the empty column at a pressure of 40 MPa, and maintained for 30 min to prepare a chiral chromatographic column.

[0136] Comparative Example 2

[0137] The steps and methods of this comparative example are basically the same as those of comparative example 1, except that the cellulose derivatization reagent is 4-methylphenyl isocyanate.

[0138] It was determined that the chiral stationary phase of the acrylate-4-methylphenylcarbamate cellulose derivative synthesized in this comparative example contained 14.38% carbon, 2.21% hydrogen and 1.74% nitrogen in elemental analysis; and a weight loss rate of 25.43% in thermogravimetric analysis.

[0139] Comparative Example 3

[0140] The steps and methods of this comparative example are basically the same as those of comparative example 1, except that the cellulose derivatization reagent is phenyl isocyanate.

[0141] It was determined that the chiral stationary phase of the acrylate-phenylcarbamate cellulose derivative synthesized in this comparative example had a carbon content of 14.81%, a hydrogen content of 2.01%, and a nitrogen content of 2.08% according to elemental analysis; and a weight loss rate of 26.60% according to thermogravimetric analysis.

[0142] Comparative Example 4

[0143] The steps and methods of this comparative example are basically the same as those of comparative example 1, except that the cellulose derivatization reagent is 3,4-dimethylphenyl isocyanate.

[0144] It was determined that the chiral stationary phase of the acrylate-3,4-dimethylphenylcarbamate cellulose derivative synthesized in this comparative example had a carbon content of 17.91%, a hydrogen content of 2.59%, and a nitrogen content of 2.21% according to elemental analysis; and a weight loss rate of 25.47% according to thermogravimetric analysis.

[0145] Comparative Example 5

[0146] The steps and methods of this comparative example are basically the same as those of comparative example 1, except that the cellulose derivatization reagent is 3-methylphenyl isocyanate.

[0147] It was determined that the chiral stationary phase of the acrylate-3-methylphenylcarbamate cellulose derivative synthesized in this comparative example had a carbon content of 14.93%, a hydrogen content of 2.51%, and a nitrogen content of 1.38% according to elemental analysis; and a weight loss rate of 25.26% according to thermogravimetric analysis.

[0148] Application Example 1

[0149] Chiral chromatographic columns were prepared using Example 1, Example 2, Example 3, and Control Example 1, and were used to separate trans-1,2-diphenylethylene oxide, 4,4'-dimethylbenzoin, benzoin, benzoin methyl ether, 2,2,2-trifluoro-1-(p-tolyl)ethanol, and 1-(4-chlorophenyl)-2,2,2-trifluoroethanol chiral compounds under normal phase conditions. The detection conditions were as follows: the mobile phase was n-hexane / isopropanol (90 / 10, v / v); the flow rate was 0.5 mL / min; the column temperature was 25°C, and the wavelength of the ultraviolet detector was 254 nm. The separation effect of the chiral chromatographic columns obtained in the examples and control examples on chiral compounds was tested under these chromatographic conditions. The retention factor ( k' 1 ) and the separation factor ( α ) to evaluate the chiral recognition ability of the chiral stationary phase; the separation results of chiral compounds in Application Example 1 are shown in Tables 1 and 2. Figure 3 The chromatogram of enantiomer separation of four chiral solutes separated under normal phase conditions in Example 1; Figure 4 The chromatogram of the enantiomer separation of three chiral solutes separated under normal phase conditions in Example 2; Figure 5 The enantiomer separation chromatogram of four chiral solutes separated under normal phase conditions in Example 3 is shown.

[0150] Table 1 Enantiomer separation results of chiral solutes in Example 1 and Comparative Example 1

[0151]

[0152] Table 2 Enantiomer separation results of chiral solutes in Example 2 and Comparative Example 1

[0153]

[0154] Table 3 Enantiomer separation results of chiral solutes in Example 3 and Comparative Example 1

[0155]

[0156] Application Example 2

[0157] Chiral chromatographic columns were prepared using Example 4, Example 5, Example 6, and Control Example 2, and were used to separate four chiral compounds, trans-1,2-diphenylethylene oxide, benzoin methyl ether, trogol base, and 2,2,2-trifluoro-1-(naphthalene-1-yl)ethanol, under normal phase conditions. The detection conditions were as follows: the mobile phase was n-hexane / isopropanol (90 / 10, v / v); the flow rate was 0.5 mL / min; the column temperature was 25°C, and the wavelength of the UV detector was 254 nm. The separation effects of the chiral chromatographic columns obtained in the Examples and Control Examples on the four chiral compounds were tested under these chromatographic conditions. The retention factor ( k' 1 ) and the separation factor ( α ) to evaluate the chiral recognition ability of the chiral stationary phase; the separation results of four chiral compounds in Application Example 2 are shown in Table 4.

[0158] Table 4 Enantiomer separation results of four chiral solutes in Application Example 2

[0159]

[0160] Application Example 3

[0161] Chiral chromatographic columns were prepared using Example 7, Example 8, Example 9, and Control Example 3, and were used to separate four chiral compounds, trans-1,2-diphenyloxirane, trogol base, 2,2,2-trifluoro-1-(9-anthryl)ethanol, and 4-chromanol, under normal phase conditions. The detection conditions were as follows: the mobile phase was n-hexane / isopropanol (90 / 10, v / v); the flow rate was 0.5 mL / min; the column temperature was 25°C, and the wavelength of the UV detector was 254 nm. The separation effects of the chiral chromatographic columns obtained in the Examples and Control Examples on the four chiral compounds were tested under these chromatographic conditions. The retention factor ( k' 1 ) and the separation factor ( α ) to evaluate the chiral recognition ability of the chiral stationary phase; the separation results of four chiral compounds in Application Example 3 are shown in Table 5.

[0162] Table 5 Enantiomer separation results of four chiral solutes in Application Example 3

[0163]

[0164] Application Example 4

[0165] Chiral chromatographic columns were prepared using Example 10, Example 11, Example 12, and Control Example 4, and were used to separate four chiral compounds, 1-(1-naphthyl)ethanol, 2,2,2-trifluoro-1-(naphthyl-1-yl)ethanol, 2,2,2-trifluoro-1-(naphthyl-2-yl)ethanol, and flavanone, under normal phase conditions. The detection conditions were as follows: the mobile phase was n-hexane / isopropanol (90 / 10, v / v); the flow rate was 0.5 mL / min; the column temperature was 25°C, and the wavelength of the UV detector was 254 nm. The separation effects of the chiral chromatographic columns obtained in the Examples and Control Examples on the four chiral compounds were tested under these chromatographic conditions. The retention factor ( k' 1 ) and the separation factor ( α ) to evaluate the chiral recognition ability of the chiral stationary phase; the separation results of 4 chiral compounds in Application Example 4 are shown in Table 6.

[0166] Table 6 Enantiomer separation results of four chiral solutes in Application Example 4

[0167]

[0168] Application Example 5

[0169] Chiral chromatographic columns were prepared using Example 13, Example 14, Example 15, and Control Example 5, and were used to separate four chiral compounds, namely, benzoin methyl ether, 2,2,2-trifluoro-1-(9-anthryl)ethanol, 1-(1-naphthalene)ethanol, and 2,2,2-trifluoro-1-(naphthalene-1-yl)ethanol, under normal phase conditions. The detection conditions were as follows: the mobile phase was n-hexane / isopropanol (90 / 10, v / v); the flow rate was 0.5 mL / min; the column temperature was 25°C, and the wavelength of the ultraviolet detector was 254 nm. The separation effects of the chiral chromatographic columns obtained in the Examples and Control Examples on the four chiral compounds were tested under these chromatographic conditions. The retention factor ( k' 1 ) and the separation factor ( α ) to evaluate the chiral recognition ability of the chiral stationary phase; the separation results of four chiral compounds in Application Example 5 are shown in Table 7.

[0170] Table 7 Enantiomer separation results of four chiral solutes in Application Example 5

[0171]

[0172] The BINAM-cellulose derivative composite chiral stationary phase of the present invention combines two traditional chiral selectors BINAM and cellulose derivatives for the first time to prepare a composite CSP. Experimental results show that the introduction of BINAM can enhance the separation effect of CSP on racemates. The composite CSP has excellent chiral separation effect, good stability and repeatability, can separate chiral compounds of various structural types, and is suitable for use as a high performance liquid chromatography filler. The experimental results of the composite CSP provide a new idea for the design and synthesis of a new chiral stationary phase.

[0173] 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 implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0174] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description 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 may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for preparing a BINAM-cellulose derivative composite chiral stationary phase, characterized in that: The following steps are involved: Step 1, using BINAM as a raw material to synthesize a BINAM derivative containing a thiourea group spacer arm; Step 2: Bonding the BINAM derivative to the surface of mercapto silica gel by "click chemistry" to obtain BINAM-SiO2; wherein the reaction ratio of the BINAM derivative to the mercapto silica gel is 1:10; Step 3, using acryloyl chloride and isocyanate to derivatize the hydroxyl groups on cellulose to obtain a cellulose derivative; Step 4, bonding the cellulose derivative to BINAM-SiO2 to obtain a BINAM-cellulose derivative composite chiral stationary phase; The method of step 1 specifically includes the following sub-steps: Step 1-1, one amino group in BINAM is acetylated and protected, and two methyl groups are introduced into the other amino group through formaldehyde solution to obtain a tertiary amine group, and then the acetylamino group is deacetylated and reacted with thiophosgene to obtain an isothiocyanate compound; Step 1-2, dissolving the isothiocyanate compound in ultra-dry dichloromethane and reacting with the spacer arm to obtain a BINAM derivative; The specific method of step 3 is: The microcrystalline cellulose is dissolved in an ultra-dry organic solvent, acryloyl chloride is added for reaction, and then a derivatization reagent isocyanate is added to derivatize all hydroxyl groups on the cellulose. After the reaction is completed, a precipitant is added to precipitate the product, and then the product is dissolved in an organic solvent, subjected to secondary precipitation, filtered, and vacuum dried to obtain an acrylate-cellulose derivative; wherein the molar ratio of the microcrystalline cellulose to the acryloyl chloride is 3.0:1.0-2.5, and the reaction molar ratio of the microcrystalline cellulose to the derivatization reagent isocyanate is 1:4.0-8.0; The specific method of step 4 is: The acrylate-cellulose derivative is dissolved in an organic solvent, coated on the surface of BINAM-SiO2, dispersed in methanol, and a cross-linking agent is added to react. After the reaction is completed, the mixture is filtered and washed with methanol and anhydrous ethanol to obtain a BINAM-cellulose derivative composite chiral stationary phase filler. The coating ratio of acrylate-cellulose derivative to BINAM-SiO2 is 1:3.0~5.0 (g / g).

2. The preparation method according to claim 1, characterized in that: In step 1-1, the protecting reagent for acetylation protection of BINAM amino group is acetic anhydride, the reaction molar ratio is 1:1.0~2.0, and the reaction temperature is 25°C; the concentration of formaldehyde solution is 30~45%, and the reaction temperature is 40°C; the reagent for deacetylation of acetylamino group is hydrochloric acid, the concentration is 2~6 M, the temperature is 85°C, and the reaction time is 24h; the molar ratio of reaction with thiophosgene is 1:1.0~3.0, the reaction temperature is 25°C, and the reaction time is 12h; In step 1-2, the reaction molar ratio of the isothiocyanate compound to the spacer arm is 1:1.0-4.0, the reaction temperature is 25° C., and the reaction time is 18 h.

3. The preparation method according to claim 1, characterized in that: In step 2, the mercapto silica gel is obtained by bonding (3-mercaptopropyl)triethoxysilane to chromatographic spherical silica gel; the bonding reaction temperature is 90° C. and the reaction time is 48 hours.

4. The preparation method according to claim 1, characterized in that: The ultra-dry organic solvent is N, N-dimethylacetamide and anhydrous lithium chloride, the ratio of microcrystalline cellulose to ultra-dry organic solvent is 1g: (20-40)ml, the dissolution temperature is 80°C, and the dissolution time is 8h; The reaction temperature of microcrystalline cellulose and acryloyl chloride is 80°C and the reaction time is 8h; The reaction temperature of microcrystalline cellulose and the derivatization reagent isocyanate is 80°C and the reaction time is 24h; The precipitant is methanol; the organic solvent for dissolving the precipitate is dimethyl sulfoxide or tetrahydrofuran; the vacuum drying temperature is 60° C. and the time is 12 hours.

5. The preparation method according to claim 1, characterized in that: The organic solvent for dissolving the acrylate-cellulose derivative is tetrahydrofuran, and the ratio of the acrylate-cellulose derivative to tetrahydrofuran is 0.4-0.6 g: 10-20 ml; The coating method adopts rotary evaporation; The cross-linking agent is azobisisobutyronitrile, and the amount added is 1.0~3.0g; the reaction temperature is 90℃ and the time is 24h.

6. A BINAM-cellulose derivative composite chiral stationary phase, characterized in that: Obtained according to any one of the preparation methods of claims 1-5, having a structure shown in formula I: ； R1 is selected from: 、 、 ； R2 is selected from: 、 、 、 、 ； In the structure shown in Formula I, Stands for silica gel carrier.

7. Use of the BINAM-cellulose derivative composite chiral stationary phase as claimed in claim 6 in the separation of racemic chiral compounds.

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