A method for preparing (+)-crispine A by enzymatic resolution

Abstract

The present application relates to the technical field of organic synthesis, and particularly relates to a method for preparing (+)-crispine A by means of biological enzyme resolution. First, 6,7-dimethoxy-3,4-dihydroisoquinoline-2-oxide is subjected to 1,3-dipolar cycloaddition with allyl alcohol to obtain a first compound; then the first compound is added into a sulfonylation reagent and zinc powder to obtain a racemate second compound through a three-step continuous series reaction of primary alcohol sulfonylation, nitrogen-oxygen bond cleavage and ring formation; then the second compound is mixed with vinyl acetate and biological enzyme, and chiral resolution is carried out under the action of the biological enzyme to obtain a third compound with right-handed optical rotation and a fourth compound with left-handed optical rotation; finally, the third compound is mixed with an alkaline reagent and a sulfonylation reagent, and then secondary alcohol sulfonylation is carried out to obtain a fifth compound with right-handed optical rotation; the fifth compound is mixed with a deoxidizing reagent to carry out deoxidation reaction, and (+)-crispine A is prepared. The method is simple, green and environmentally friendly, and can be produced in large quantities.

Description

Technical Field

[0001] This invention relates to the field of organic synthesis technology, and in particular to a method for preparing (+)-crispine A by enzymatic resolution. Background Technology

[0002] Tetrahydroisoquinoline (THIQ) is widely found in natural products and drugs. Due to its complex structural features and various important physiological activities, it has significant clinical medicinal value, and many synthetic methods strive to construct this type of structure. In 2002, Zhao's research group derived this structure from traditional Chinese medicines used to treat stomach pain, colds, and rheumatism. carduus crispua (+)-crispine A was obtained by separation from the middle. Tetrahedron 2002, 58 (6795), its structural formula is shown below. (+)-CrispineA belongs to the tetrahydroisoquinoline structure compound. Activity tests show that it has a significant inhibitory effect on SKOV3, KB and HeLa human cancer cell lines. It is a promising natural anticancer drug molecule that has attracted the interest of many synthetic chemists and medicinal chemists. Currently, the main methods for synthesizing (+)-crispine A are classified into five types: Bischler-Napieralski cyclization, Pictet-Spengler cyclization, N-alkylation / acylation cyclization, oxidative cyclization, and asymmetric hydrogenation. In 2005, the Czarnocki group first reported the asymmetric synthesis of (+)-crispine A, starting from 3,4-dimethoxyphenethylamine, obtaining a key imine intermediate via Bischler-Napieralski cyclization, followed by Ru-catalyzed asymmetric hydrogenation to yield (+)-crispine A (Tetrahedron: Asymmetry 2005). 169 , 3619). In 2007, Turner's group used the monoamine oxidase MAO-N enzyme to catalyze the resolution of the racemic crispine A to obtain chiral nitrogen oxides, which were then reduced with borane to give (+)-crispine A ( Chem. Commun. 2007, 3640). In the same year, Allin's group utilized chiral raw materials ( S )-1-(1-(3,4-dimethoxyphenyl)-3-hydroxypropan-2-yl)Pyrrole Synthesis of (+)-crispine A ( J. Org. Chem. 2007, 72, 8972). In 2013, Yuste's group synthesized (+)-crispine A using an asymmetric Pictet-Spengler reaction controlled by a chiral sulfonyl cofactor. Tetrahedron Lett. 2013, 54 , 1893). In 2014, Hiemstra's group synthesized (+)-crispine A using a chiral phosphoric acid-catalyzed asymmetric Pictet-Spengler reaction. J. Org. Chem. 2014, 79 ,7380).

[0003] In 2006, Chong's group, starting from 6,7-dimethoxy-3,4-dihydroisoquinoline, synthesized (+)-crispine A through an asymmetric addition reaction of allyl boron with an imine. J. Am. Chem. Soc. 2006, 128 , 9646). In 2011, Itoh's group, starting from 6,7-dimethoxy-3,4-dihydroisoquinoline, utilized chiral phosphoric acid to catalyze the asymmetric addition reaction of allylsilane with imine, ultimately completing the synthesis of (+)-crispine A. J. Org. Chem. 2011, 76 , 534). In 2011, Campos' group, starting from Boc-protected pyrrolidine, utilized palladium-catalyzed asymmetric α-arylation of pyrrole to ultimately complete the synthesis of (+)-crispine A. J. Org. Chem. 2011, 76 ,5936). In 2011, Fülöp's group used Lipase PS lipase to selectively hydrolyze the α-side chain of isoquinoline to obtain a chiral compound, and finally completed the synthesis of (+)-crispine A. Tetrahedron: Asymmetry 2011, 22 , 1255). In 2013, the Chittiboyina group started with 3,4-dimethoxybenzaldehyde, and used chiral phosphoric acid to catalyze an asymmetric allylation reaction, followed by several transformations to finally synthesize (+)-crispine A. J. Org. Chem. 2013, 78 , 6355). In 2014, Ma's group, starting from 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline, utilized chiral phosphoric acid to catalyze an asymmetric addition reaction of an imine with an alkynyl group, ultimately completing the synthesis of (+)-crispine A. Org. Chem. Front. 2014, 1, 338). In 2018, the Sun group, using chiral sulfones to control and promote asymmetric allylation with indium metal, finally completed the synthesis of (+)-crispine A. J. Saudi. Chem. Soc. 2018, 22 , 654). In 2009, Zhou's group completed the synthesis of (+)-crispine A using iridium-catalyzed asymmetric hydrogenation of enamines. J. Am. Chem. Soc. 2009, 131 , 1366).

[0004] Existing literature on the preparation of (+)-crispine A mostly employs chemical synthesis methods, which are cumbersome and lengthy. The use of precious metal catalysis further increases the preparation cost, and the demanding reaction conditions significantly limit the large-scale preparation of the target compound. While two existing studies have used enzymatic catalysis to prepare (+)-crispine A, these methods suffer from lengthy synthetic routes. Furthermore, resolution using monoamine oxidases requires borane reduction to obtain the final product, while esterase hydrolysis necessitates the introduction of a large ester side chain onto the primary alcohol for optimal resolution. Moreover, after resolution, alkaline hydrolysis is required to remove the introduced ester side chain to obtain the desired configuration. Therefore, developing a rapid and green method combining chemical and biological approaches for the preparation of (+)-crispine A has significant application value. Summary of the Invention

[0005] To address the aforementioned technical problems, the present invention aims to provide a method for preparing (+)-Crispine A via enzymatic resolution. The main principle of this enzymatic resolution is that the catalytic center of the enzyme has an asymmetric structure. Only the levorotatory second compound can enter the enzyme cavity and undergo esterification to generate the levorotatory fourth compound, while the dextrorotatory second compound (i.e., the "dextrorotatory optically active third compound") does not participate in the reaction. The dextrorotatory second compound and the levorotatory fourth compound have significantly different polarities, allowing them to be separated by conventional column chromatography, thus achieving enzymatic resolution and separating the levorotatory and dextrorotatory optical isomers. In this invention, (+)-crispine A is dextrorotatory optically active crispin A.

[0006] The objective of this invention can be achieved through the following technical solutions: This invention provides a method for preparing (+)-crispine A by enzymatic resolution, comprising the following steps: (S1) The first compound was obtained by reacting 6,7-dimethoxy-3,4-dihydroisoquinoline-2-oxide with allyl alcohol in a 1,3-dipolar cycloaddition reaction. (S2) The first compound prepared in step (S1) is added to a sulfonating agent and zinc powder, and the racemic second compound is obtained through a three-step series reaction of primary alcohol sulfonation, nitrogen-oxygen bond cleavage and cyclization. (S3) The second compound obtained in step (S2) is mixed with vinyl acetate and a biological enzyme and then subjected to chiral resolution under the action of the biological enzyme to obtain a third compound with dextrorotatory optical activity and a fourth compound with levorotatory optical activity. (S4) After mixing the third compound obtained in step (S3) with a basic reagent and a sulfonating reagent, the secondary alcohol is sulfonated to obtain a fifth compound with dextrorotatory optical activity. The fifth compound is mixed with a deoxygenating reducing agent to carry out a deoxygenation reaction to prepare (+)-crispine A. The chemical structural formulas of the first compound, the second compound, the third compound, the fourth compound, the fifth compound, and (+)-crispine A are shown in formulas (I), (II), (III), (IV), (V), and (VI), respectively: Equation (I), Equation (II), Equation (III), Formula (IV), Formula (V), Formula (VI).

[0007] In one embodiment of the present invention, in step (S1), the molar ratio of 6,7-dimethoxy-3,4-dihydroisoquinoline-2-oxide to allyl alcohol is 1:1.5~3.5.

[0008] In one embodiment of the present invention, in step (S1), during the 1,3-dipolar cycloaddition reaction, the temperature is 60°C to 110°C and the reaction time is 3h to 8h.

[0009] In one embodiment of the present invention, in step (S2), the molar ratio of the first compound, the sulfonating agent and the zinc powder is 1:1.5~3.5:3~12.

[0010] In one embodiment of the present invention, in step (S2), the first compound is further mixed with acetic acid.

[0011] In one embodiment of the present invention, the sulfonating agent is selected from one of methanesulfonyl chloride, methanesulfonic anhydride, benzenesulfonyl chloride, benzenesulfonic anhydride, p-toluenesulfonyl chloride, or p-toluenesulfonic anhydride; Preferably, the sulfonating agent is methanesulfonyl chloride.

[0012] In one embodiment of the present invention, in step (S2), during the primary alcohol sulfonation reaction, the temperature is 0~30°C and the time is 1h~3h; During the nitrogen-oxygen bond breaking and cyclization reaction, the temperature is 30℃~100℃ and the time is 2h~24h.

[0013] In one embodiment of the present invention, in step (S3), the bioenzyme is selected from one of immobilized lipase (Novozym 435), Candida antarcticis lipase (CAL-A), Candida antarcticis lipase B (CAL-B), Candida columnaris lipase (CRL), acetate kinase (AK), phosphatidylserine synthase (PS), porcine liver lipase (PLE), and lipase (PLL); preferably, the bioenzyme is Candida antarcticis lipase B (CAL-B) (1800 U / g).

[0014] In one embodiment of the present invention, the second compound is dissolved in an organic reagent selected from tetrahydrofuran, ethyl acetate, toluene, 2-methyltetrahydrofuran (2-Me-THF), methyl tert-butyl ether, diethyl ether, 1,4-dioxane, or acetonitrile; preferably, the organic solvent is 2-methyltetrahydrofuran (2-Me-THF).

[0015] In one embodiment of the present invention, in step (S3), the molar ratio of the second compound to vinyl acetate is 1:4~10; The ratio of the second compound to the biological enzyme is 0.01~1 mmol:1 U.

[0016] In one embodiment of the present invention, in step (S3), the chiral separation process is carried out at a temperature of 10~35°C for 8~72 hours.

[0017] In one embodiment of the present invention, the alkaline reagent is selected from one of LDA, LHMDS, KHMDS, NaHMDS, Et3N, DMAP, Py, DIPEA, Diazabicyclo or NaH; preferably, the alkaline reagent is Et3N; The sulfonating agent is selected from one of p-toluenesulfonyl chloride, p-toluenesulfonic anhydride, benzenesulfonyl chloride, benzenesulfonic anhydride, methanesulfonyl chloride, or methanesulfonic anhydride; preferably, the sulfonating agent is p-toluenesulfonyl chloride; The deoxygenation reducing agent is selected from either LiAlH4 or DIBAL-H; preferably, the deoxygenation reducing agent is LiAlH4.

[0018] In one embodiment of the present invention, in step (S4), the third compound is dissolved in an organic solvent selected from tetrahydrofuran, acetonitrile, or dichloromethane; preferably, the organic solvent is dichloromethane.

[0019] In one embodiment of the present invention, in step (S4), the molar ratio of the third compound, the basic reagent, the sulfonating reagent, and the deoxygenating reducing reagent is 1:2~10:1.2~5:2~10.

[0020] In one embodiment of the present invention, in step (S4), during the secondary alcohol sulfonation process, the temperature is 0~30°C and the time is 3h~5h.

[0021] In one embodiment of the present invention, in step (S4), the temperature during the deoxygenation reaction is 0~80℃ and the time is 3h~7h.

[0022] In this invention, one preparation process of (+)-crispine A is as follows: .

[0023] This invention uses 6,7-dimethoxy-3,4-dihydroisoquinoline-2-oxide as a starting material, which undergoes a 1,3-dipolar cycloaddition reaction with allyl alcohol to obtain a first compound. Subsequently, the primary alcohol is sulfonated, and hydroxysulfonation, nitrogen-oxygen bond cleavage, and intramolecular ring closure are completed in the presence of acetic acid and zinc powder. This "one-pot" method achieves a rapid three-step tandem reaction to prepare a racemic second compound. The second compound is then chirally resolved using a bioenzymatic method to obtain optically pure third and fourth compounds. The optically pure third compound is sulfonated with a secondary alcohol, followed by reduction and deoxygenation with lithium aluminum hydride to prepare optically pure (+)-crispine A. This invention completes a three-step tandem reaction in a one-pot method to obtain the basic skeleton of crispine A; and uses a bioenzymatic method to resolve and prepare the optically pure intermediate third compound, followed by a two-step "one-pot" method for the green and efficient asymmetric synthesis of (+)-crispine A. The method of this invention is simple, environmentally friendly, and can be mass-produced.

[0024] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention uses 6,7-dimethoxy-3,4-dihydroisoquinoline-2-oxide as raw material. After dipole addition, the basic skeleton of crispine A is obtained by completing three tandem reactions in a "one-pot" manner. Finally, the asymmetric synthesis of (+)-crispine A is completed in only four chemical transformations. The route is short and efficient. (2) The present invention reduces the separation and purification process by using a multi-step combined one-pot method, which is beneficial to the industrialization of (+)-crispine A preparation; (3) Most of the intermediates of the present invention are solids and can be recrystallized and purified, which is beneficial to the industrialization of (+)-crispine A preparation; (4) The present invention uses a biological enzymatic method to obtain optically pure intermediate compounds with high optical purity, which can be used for drug development with a skeleton containing a five-membered ring of benzisoquinoline and for asymmetric synthesis. (5) This invention utilizes a combination of biological and chemical methods to complete the asymmetric synthesis of (+)-crispine A intermediates, which is green and environmentally friendly, easy to operate, and can be used for process optimization and large-scale production. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of single-crystal diffraction of the first compound; Figure 2 The HPLC liquid phase spectrum of the second compound (racemic mixture); Figure 3 The HPLC liquid phase spectrum of the third compound (which exhibits dextrorotatory optical rotation); Figure 4 The HPLC liquid phase spectrum of the fourth compound (racemic mixture); Figure 5 The HPLC liquid phase spectrum of the fourth compound (which exhibits levorotatory optical rotation); Figure 6 The 1H NMR spectrum of the natural product (+)-crispine A; Figure 7 The image shows the carbon NMR spectrum of the natural product (+)-crispine A. Detailed Implementation

[0026] This invention provides a method for preparing (+)-crispine A by enzymatic resolution, comprising the following steps: (S1) The first compound was obtained by reacting 6,7-dimethoxy-3,4-dihydroisoquinoline-2-oxide with allyl alcohol in a 1,3-dipolar cycloaddition reaction. (S2) The first compound prepared in step (S1) is added to a sulfonating agent and zinc powder, and the racemic second compound is obtained through a three-step series reaction of primary alcohol sulfonation, nitrogen-oxygen bond cleavage and cyclization. (S3) The second compound obtained in step (S2) is mixed with vinyl acetate and a biological enzyme and then subjected to chiral resolution under the action of the biological enzyme to obtain a third compound with dextrorotatory optical activity and a fourth compound with levorotatory optical activity. (S4) After mixing the third compound obtained in step (S3) with a basic reagent and a sulfonating reagent, the secondary alcohol is sulfonated to obtain a fifth compound with dextrorotatory optical activity. The fifth compound is mixed with a deoxygenating reducing agent to carry out a deoxygenation reaction to prepare (+)-crispine A. The chemical structural formulas of the first compound, the second compound, the third compound, the fourth compound, the fifth compound, and (+)-crispine A are shown in formulas (I), (II), (III), (IV), (V), and (VI), respectively: Equation (I), Equation (II), Equation (III), Formula (IV), Formula (V), Formula (VI).

[0027] Furthermore, in step (S1), the molar ratio of 6,7-dimethoxy-3,4-dihydroisoquinoline-2-oxide to allyl alcohol is 1:1.5~3.5.

[0028] Furthermore, in step (S1), during the 1,3-dipolar cycloaddition reaction, the temperature is 60℃~110℃ and the reaction time is 3h~8h.

[0029] Furthermore, in step (S2), the molar ratio of the first compound, the sulfonating agent, and the zinc powder is 1:1.5~3.5:3~12.

[0030] Furthermore, in step (S2), the first compound is also mixed with acetic acid.

[0031] Furthermore, the sulfonating agent is selected from one of methanesulfonyl chloride, methanesulfonic anhydride, benzenesulfonyl chloride, benzenesulfonic anhydride, p-toluenesulfonyl chloride, or p-toluenesulfonic anhydride; Preferably, the sulfonating agent is methanesulfonyl chloride.

[0032] Furthermore, in step (S2), during the primary alcohol sulfonation reaction, the temperature is 0~30℃ and the time is 1h~3h; During the nitrogen-oxygen bond breaking and cyclization reaction, the temperature is 30℃~100℃ and the time is 2h~24h.

[0033] Further, in step (S3), the bioenzyme is selected from one of immobilized lipase (Novozym 435), Candida antarcticis lipase (CAL-A), Candida antarcticis lipase B (CAL-B), Candida columnaris lipase (CRL), acetate kinase (AK), phosphatidylserine synthase (PS), porcine liver lipase (PLE), and lipase (PLL); preferably, the bioenzyme is Candida antarcticis lipase B (CAL-B) (1800 U / g).

[0034] Furthermore, the second compound is dissolved in an organic reagent selected from tetrahydrofuran, ethyl acetate, toluene, 2-methyltetrahydrofuran (2-Me-THF), methyl tert-butyl ether, diethyl ether, 1,4-dioxane, or acetonitrile; preferably, the organic solvent is 2-methyltetrahydrofuran (2-Me-THF).

[0035] Furthermore, in step (S3), the molar ratio of the second compound to vinyl acetate is 1:4~10; The ratio of the second compound to the biological enzyme is 0.01~1 mmol:1 U.

[0036] Furthermore, in step (S3), during the chiral separation process, the temperature is 10~35℃ and the time is 8~72h.

[0037] Furthermore, the alkaline reagent is selected from one of LDA, LHMDS, KHMDS, NaHMDS, Et3N, DMAP, Py, DIPEA, Diazabicyclo, or NaH; preferably, the alkaline reagent is Et3N. The sulfonating agent is selected from one of p-toluenesulfonyl chloride, p-toluenesulfonic anhydride, benzenesulfonyl chloride, benzenesulfonic anhydride, methanesulfonyl chloride, or methanesulfonic anhydride; preferably, the sulfonating agent is p-toluenesulfonyl chloride; The deoxygenation reducing agent is selected from either LiAlH4 or DIBAL-H; preferably, the deoxygenation reducing agent is LiAlH4.

[0038] Furthermore, in step (S4), the third compound is dissolved in an organic solvent selected from tetrahydrofuran, acetonitrile, or dichloromethane; preferably, the organic solvent is dichloromethane.

[0039] Furthermore, in step (S4), the molar ratio of the third compound, the basic reagent, the sulfonating reagent, and the deoxygenating reducing reagent is 1:2~10:1.2~5:2~10.

[0040] Furthermore, in step (S4), during the secondary alcohol sulfonation process, the temperature is 0~30℃ and the time is 3h~5h.

[0041] Furthermore, in step (S4), the temperature during the deoxygenation reaction is 0~80℃ and the time is 3h~7h.

[0042] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0043] Unless otherwise specified, all reagents used in the following embodiments are commercially available reagents; and all detection methods and techniques used are conventional detection methods and techniques in the art.

[0044] Example 1 This embodiment provides (+)-crispine A and its preparation method, specifically including the following steps: (S1) Preparation of the first compound: Weigh 7.70 g (37.2 mmol) of 6,7-dimethoxy-3,4-dihydroisoquinoline-2-oxide and add toluene (150 mL) as solvent, followed by allyl alcohol (5.1 mL, 74.4 mmol). Transfer the reaction mixture to an oil bath at 80 °C and heat for 5 hours. Cool to room temperature and concentrate under reduced pressure. Recrystallize the crude product from ethyl acetate to give a grayish-white solid, compound I (as shown in formula (I), 7.49 g, 76% yield).

[0045] Structural identification of the first compound: The grayish-white solid obtained by recrystallization (the first compound) was analyzed by X-ray single-crystal diffraction. The X-ray single-crystal diffraction pattern is as follows: Figure 1 As shown, the specific data are as follows. Based on the characterization results of X-ray single-crystal diffraction, it can be determined that the first compound is the compound shown in Formula I, which is also an exo-type (exo-type) racemic compound.

[0046] The single-crystal unit cell data of the compound shown in formula (I) are as follows: a = 7.8532(4) Å; a = 90°. b = 17.2712(8) Å b= 100.678(2)°. c = 9.8998(4) Å g = 90°. Data for the first compound (formula (I)): mp 182-183.5 ℃. 1 H NMR (500 MHz, CDCl3) δ ppm: 6.59 (s, 1H), 6.58 (s, 1H), 4.53 (t, J= 8.4 Hz, 1H), 4.50-4.44 (m, 1H), 3.85 (s, 3H), 3.84 (s, 3H), 3.81 (dd, J =12.1, 2.6 Hz, 1H), 3.62 (dd, J = 12.1, 3.9 Hz, 1H), 3.25 (dt, J = 9.5, 4.4Hz, 1H), 3.12 (td, J = 10.4, 3.8 Hz, 1H), 2.96 (ddd, J = 15.6, 10.4, 4.8 Hz,1H), 2.78 (dt, J = 16.1, 3.9 Hz, 1H), 2.62 (ddd, J = 12.2, 8.0, 4.3 Hz, 1H),2.40-2.31 (m, 1H). 13 C NMR (101 MHz, CDCl3) δ ppm:148.03, 148.00, 127.7, 125.1, 111.1,110.2, 78.1, 64.4, 63.1, 56.2, 56.0, 48.4, 38.4, 27.7. (S2) Preparation of the second compound: (S201) The first compound (7.82 g, 29.5 mmol) obtained in step (S1) was dissolved in dichloromethane (150 mL), cooled in an ice-water bath, and Et3N (8.26 mL, 59 mmol) and methanesulfonyl chloride (2.72 mL, 35.4 mmol) were added sequentially. After the addition was complete, the mixture was stirred at the same temperature until the reaction was finished. Then, dichloromethane was added for dilution, followed by the addition of saturated NH4Cl (20 mL). The mixture was separated, the aqueous phase was extracted with dichloromethane, the organic phases were combined, washed once with saturated NaHCO3, dried over anhydrous Na2SO4, filtered, and the organic phase was concentrated under reduced pressure to obtain the secondary alcohol methanesulfonated product, which was directly added to the next reaction without purification.

[0047] (S202) The residue obtained in step (S201) was dissolved in AcOH / H2O (V / V = 9:1, 300 mL), and zinc powder (15.6 g, 238.4 mmol) was added in three batches. The mixture was heated at 75 °C for 5 h, cooled to room temperature, and filtered to remove the zinc powder. The filtrate was concentrated under reduced pressure to remove most of the acetic acid, and the residue was dissolved in methanol (400 mL). Immediately afterwards, excess Na2CO3 (31.27 g, 295 mmol) was added and stirred at room temperature for 5 h. The solid was removed by diatomaceous earth filtration, and the solid was washed with dichloromethane. The filtrates were combined, concentrated under reduced pressure, and purified by column chromatography (CH2Cl2 / MeOH / NH3 (7 M (in MeOH): 12 / 1 / 0.1), to give a white solid, racemic second compound (as shown in formula (II), 6.61 g, 90% yield).

[0048] Data for the second compound (formula (II)): mp 109.6-111.2 ℃.

[0049] 1 H NMR (500 MHz, CDCl3) δ ppm: 6.62 (s, 1H), 6.52 (s, 1H), 4.48-4.40 (m, 1H), 3.85 (s, 6H), 3.31-3.21 (m, 2H), 3.15-3.08 (m, 1H), 3.05 (d, J =10.3 Hz, 1H), 2.82 (dt, J = 13.2, 7.4 Hz, 1H), 2.73 (dd, J = 16.4, 4.8 Hz, 1H), 2.63 (dd, J = 10.2, 5.7 Hz, 1H), 2.56 (td, J = 11.1, 4.7 Hz, 1H), 2.04(brs, 1H), 1.71 (ddd, J = 13.1, 9.4, 3.6 Hz, 1H). 13 C NMR (101 MHz, CDCl3) δ ppm:147.8, 147.5, 130.4, 126.3, 111.8,108.8, 70.7, 63.0 (2C), 56.2, 56.1, 48.7, 41.8, 28.1. HPLC analysis conditions for the racemic sample of the second compound: Daicel IK3 column, i-PrOH(0.1%HNEt2) / n-Hexane = 30 / 70, flow rate = 0.7 mL / min, T = 25 ℃, λ = 254 nm. The HPLC chromatogram of the second compound is shown below. Figure 2 As shown, by Figure 2 It can be seen that the elution times of the racemic second compound are 15.84 min and 22.41 min, and the peak areas are 49.86% and 50.14%, respectively, indicating that it does not have a chiral environment (enantiomer excess).

[0050] (S3) Preparation of the third and fourth compounds: At room temperature, a suspension of the racemic second compound (2.5 g, 10.5 mmol) prepared in step (S202) in 2-Me-THF (335 mL) was added to the bioenzyme CAL-B (465 mg) and vinyl acetate (7.4 mL, 80.8 mmol). The reaction was carried out at 35 °C for 36 h. The mixture was filtered through diatomaceous earth, eluted with dichloromethane, and the combined filtrates were concentrated under reduced pressure and purified by column chromatography (CH2Cl2 / MeOH (30 / 1)) to obtain a yellow oily liquid: a fourth compound (1.53 g, 51% yield, 93.5%) exhibiting levorotatory optical activity. ee (CH2Cl2 / MeOH / NH3 (7 M in MeOH): 12 / 1 / 0.1) yields a white solid: a third compound with dextrorotatory optical activity (1.22 g, 49% yield, 97.5%). ee ).

[0051] Among them, the third and fourth compounds are easily separated by conventional column chromatography due to their significant difference in polarity.

[0052] HPLC analysis conditions for the third compound: Daicel IK3 column, i-PrOH (0.1% HNEt2) / n-Hexane = 30 / 70, flow rate = 0.7 mL / min, T = 25 ℃, λ = 254 nm. The HPLC chromatogram of the third compound is shown below. Figure 3 As shown, by Figure 3It can be seen that the peak elution times of the third compound are 15.99 min and 22.27 min, and the peak areas are 1.25% and 98.75%, respectively, indicating that it has the properties of [(98.75-1.25) / (98.75+1.25)]. 100% = 97.5% of the ee value.

[0053] HPLC analysis conditions for the racemic sample of the fourth compound: Dassell IK3 column, i-PrOH (0.1% HNEt2) / n-Hexane = 30 / 70, flow rate = 0.7 mL / min, T = 25 ℃, λ = 285 nm. The HPLC chromatogram of the racemic fourth compound is shown below. Figure 4 As shown, by Figure 4 It can be seen that the peak elution times of the racemic fourth compound are 61.23 min and 80.03 min, and the peak areas are 50.27% and 49.73%, respectively, indicating that it does not have a chiral environment (enantiomer excess).

[0054] The HPLC chromatogram of the fourth compound is shown below. Figure 5 As shown, by Figure 5 It can be seen that the peak elution times of the fourth compound are 60.56 min and 80.57 min, and the peak areas are 97.05% and 2.95%, respectively, indicating that it possesses [(97.05-2.95) / (97.05)]. + 2.95)] 100% = 94.1% of the ee value.

[0055] Data for compound three (formula (III)): mp 104.7 -106.8 ℃. = +106.3 ( c = 0.91 in CHCl3); 1 H NMR (500 MHz, CDCl3) δ ppm: 6.62 (s, 1H), 6.52 (s, 1H), 4.48-4.40 (m, 1H), 3.85 (s, 6H), 3.31-3.21 (m, 2H), 3.15-3.08 (m, 1H), 3.05 (d, J =10.3 Hz, 1H), 2.82 (dt, J = 13.2, 7.4 Hz, 1H), 2.73 (dd, J= 16.4, 4.8 Hz, 1H), 2.63 (dd, J = 10.2, 5.7 Hz, 1H), 2.56 (td, J = 11.1, 4.7 Hz, 1H), 2.04(brs, 1H), 1.71 (ddd, J = 13.1, 9.4, 3.6 Hz, 1H). 13 C NMR (101 MHz, CDCl3) δ ppm:147.8, 147.5, 130.4, 126.3, 111.8,108.8, 70.7, 63.0 (2C), 56.2, 56.1, 48.7, 41.8, 28.1. Data for compound four (formula (IV)): = -42.0 ( c = 1.00 in CHCl3); 1 H NMR (500 MHz, CDCl3) δ ppm: 6.62 (s, 1H), 6.50 (s, 1H), 5.28-5.22(m, 1H), 3.85 (s, 3H), 3.83 (s, 3H), 3.32-3.21 (m, 2H), 3.19 (d, J = 11.2 Hz,1H), 3.16-3.06 (m, 1H), 2.92-2.84 (m, 1H), 2.80-2.68 (m, 2H), 2.57-2.49 (m,1H), 2.02 (s, 3H), 1.85-1.77 (m, 1H). 13 C NMR (101 MHz, CDCl3) δ ppm: 171.3, 147.9, 147.5, 129.9, 126.3,111.8, 108.6, 73.6, 63.0, 60.0, 56.2, 56.1, 48.7, 38.6, 28.4, 21.3. Preparation of (S4)(+)-crispine A: (S401) The third compound (1.5 g, 6.00 mmol) obtained in step (S3) was dissolved in dichloromethane (20 mL) and placed in an ice-water bath. Et3N (2.52 mL, 18 mmol) and TsCl (1.72 g, 9 mmol) were added. After the addition was complete, the reaction was carried out at this temperature for 10 min. Then, the temperature was restored to room temperature and the reaction was allowed to proceed for 3 h. The reaction was detected by TLC. Saturated NaHCO3 (20 mL) was added and the mixture was stirred at room temperature for 15 min. The mixture was separated, and the aqueous phase was extracted with dichloromethane (3 × 20 mL). The organic phases were combined and washed once with saturated NaHCO3 and once with saturated NaCl. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude fifth compound with dextrorotatory optical activity. No purification was required before proceeding to the next step of the reaction.

[0056] (S402) The crude fifth compound obtained in step (S401) was dissolved in THF (60 mL) and placed in an ice-water bath. LiAlH4 (1.14 g, 30 mmol) was added in three batches, and the reaction was carried out at this temperature for 10 min after the addition was completed. Then, the mixture was brought back to room temperature and immediately refluxed at 80 °C for 5 h. After cooling to room temperature, it was placed in an ice-water bath, and H2O (1.14 mL), 15% NaOH aq. (1.14 mL), and H2O (3.42 mL) were added sequentially. After stirring at room temperature for 3 h, DCM (50 mL) was added for dilution, followed by the addition of diatomaceous earth (10 g), and stirring was continued for 2 h. The diatomaceous earth was then filtered, and the mixture was washed with dichloromethane. The filtrates were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (CH2Cl2 / MeOH / NH3 (7 M In MeOH): 12 / 1 / 0.1) yielded a white solid (+)-crisipine A (1.12 g, 80% yield); its 1H NMR spectrum is shown below. Figure 6 As shown, the carbon NMR spectrum is as follows: Figure 7 As shown; all are compatible with existing literature ( Org. Lett. 2022, 24 Corresponding to , 6531.).

[0057] Existing literature ( Org. Lett. 2022, 24 , 6531.)(+)-crisipine A NMR data: 1H NMR (400 MHz, CDCl3) δ ppm 6.61 (s, 1 H), 6.57 (s, 1 H), 3.84 - 3.85 (m, 6 H), 3.56 (t, J = 8.13 Hz, 1 H), 3.17 - 3.27 (m, 1 H), 2.98 - 3.16 (m, 2 H), 2.63- 2.85 (m, 3 H), 2.32 - 2.44 (m, 1 H), 1.87 - 2.03 (m, 2 H), 1.72 -1.85 (m, 1H). 13 C NMR (100 MHz, CDCl3) δ ppm 147.5, 147.4, 130.4, 126.0, 111.4,108.9, 62.8, 56.1, 55.9, 53.2, 48.3, 30.7, 27.8, 22.3. Data for (+)-crispine A in this example: mp 55 - 57 ℃. = +79.6 ( c = 0.99 in MeOH); [lit = +91 (MeOH)] (Reference data: Zhang, Q.; Tu, G.; Zhao, Y.; Cheng, T.) Tetrahedron 2002, 58 , 6795); 1 H NMR (400 MHz, CDCl3) δ ppm: 6.60 (s, 1H), 6.56 (s, 1H), 3.84 (s, 3H), 3.83 (s, 3H), 3.52 (t, J = 8.2 Hz, 1H), 3.21-3.12 (m, 1H), 3.11-2.95 (m,2H), 2.81-2.61 (m, 3H), 2.40-2.28 (m, 1H), 2.00-1.82 (m, 2H), 1.80-1.67 (m,1H). 13C NMR (101 MHz, CDCl3) δ ppm: 147.6, 147.5, 130.5, 126.2, 111.5, 109.1, 62.9, 56.2, 56.0, 53.3, 48.4, 30.8, 27.9, 22.4. Example 2 This embodiment provides (+)-crispine A and its preparation method, specifically including the following steps: (S1) Preparation of the first compound: Weigh 15 g (96.6 mmol) of 6,7-dimethoxy-3,4-dihydroisoquinoline-2-oxide and add toluene (300 mL) as solvent, followed by allyl alcohol (13.2 mL, 193.2 mmol). Transfer the reaction mixture to an oil bath at 80 °C and heat for 5 hours. Cool to room temperature and concentrate under reduced pressure. Recrystallize the crude ethyl acetate to give a grayish-white solid, compound I (as shown in formula (I), 20.05 g, 78% yield)).

[0058] (S2) Preparation of the second compound: (S201) The first compound (20 g, 75.47 mmol) obtained in step (S1) was dissolved in dichloromethane (250 mL), cooled in an ice-water bath, and Et3N (21 mL, 150.94 mmol) and methanesulfonyl chloride (6.43 mL, 83.02 mmol) were added sequentially. After the addition was complete, the reaction was carried out at this temperature for half an hour. Then, dichloromethane was added for dilution, followed by the addition of saturated NH4Cl (50 mL). The mixture was separated, and the aqueous phase was extracted with dichloromethane. The organic phases were combined and washed once with saturated NaHCO3. The organic phase was dried with anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the secondary alcohol methanesulfonation product (residue), which was directly added to the next reaction without purification.

[0059] (S202) The residue obtained in step (S201) was dissolved in AcOH / H2O (V / V: 9:1, 300 mL), and zinc powder (49.35 g, 754.7 mmol) was added in three batches. The mixture was heated at 75 °C for 7 h, cooled to room temperature, and filtered to remove the zinc powder. The filtrate was concentrated under reduced pressure to remove most of the acetic acid, and the residue was dissolved in methanol (400 mL). Immediately afterwards, excess NaOH (9.06 g, 226.41 mmol) was added, and the mixture was stirred at room temperature for 5 h. The solid was removed by diatomaceous earth filtration, eluted with dichloromethane, and the combined organic phases were concentrated under reduced pressure and subjected to column chromatography (CH2Cl2 / MeOH / NH3 (7 M (in MeOH): 12 / 1 / 0.1) gives a white solid, racemic second compound (as shown in formula (II), 17.29 g, 92% yield).

[0060] (S3) Preparation of the third and fourth compounds At room temperature, a suspension of the racemic second compound (10 g, 40.16 mmol) prepared in step (S202) in 2-Me-THF (670 mL) was added to the bioenzyme CAL-B (1.87 g) and vinyl acetate (29.61 mL, 312.28 mmol). The reaction was allowed to proceed for 72 h at room temperature. The mixture was then filtered through diatomaceous earth, eluted with dichloromethane, concentrated under reduced pressure, and purified by column chromatography (CH2Cl2 / MeOH 30 / 1) to obtain a yellow oily liquid: a fourth compound exhibiting levorotatory optical activity (6.04 g, 51% yield, 94%). ee (CH2Cl2 / MeOH / NH3 (7 M in MeOH): 12 / 1 / 0.1) yields a white solid: a third compound with dextrorotatory optical activity (4.9 g, 49% yield, 98%). ee ).

[0061] Preparation of (S4)(+)-crispine A (S401) The third compound (5 g, 20.08 mmol) obtained in step (S3) was dissolved in dichloromethane (100 mL) and placed in an ice-water bath. Et3N (8.37 mL, 60.24 mmol) and TsCl (5.74 g, 30.12 mmol) were added, and the mixture was reacted at this temperature for 10 min after the addition was complete. Then, the mixture was brought to room temperature and reacted for 5 h. Saturated NaHCO3 (60 mL) was added and stirred at room temperature for 15 min. The mixture was separated, and the aqueous phase was extracted with dichloromethane (3 × 60 mL). The organic phases were combined and washed once with saturated NaHCO3 and once with saturated NaCl. The mixture was dried over anhydrous sodium sulfate, filtered, and the organic phase was concentrated under reduced pressure to obtain the crude fifth compound with dextrorotatory optical activity. No purification was required for the next reaction.

[0062] (S402) The crude fifth compound obtained in step (S401) was dissolved in THF (200 mL) and placed in an ice-water bath. LiAlH4 (3.82 g, 100.4 mmol) was added in three batches. After the addition was complete, the mixture was reacted at this temperature for 10 min, then brought to room temperature, and then refluxed at 80 °C for 6 h. After cooling to room temperature, it was placed in an ice-water bath, and H2O (3.82 mL), 15% NaOH aq. (3.82 mL), and H2O (11.46 mL) were added sequentially. After stirring at room temperature for 3 h, DCM (150 mL) was added for dilution, followed by diatomaceous earth (40 g), and stirring was continued for 2 h. The diatomaceous earth was then filtered, and the mixture was washed with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and subjected to column chromatography (CH2Cl2 / MeOH / NH3 (7)). M (in MeOH): 12 / 1 / 0.1) yielded a white solid (+)-crisipine A (3.98 g, 85% yield).

[0063] Example 3 This embodiment is the same as Embodiment 1 except for the following differences. At room temperature, vinyl acetate (1.48 mL, 16.08 mmol) was added to a THF (100 mL) suspension of racemic compound II (0.5 g, 2.01 mmol), followed immediately by the addition of the bioenzyme CAL-B (930 mg). The reaction was allowed to proceed for 6 h at room temperature. The mixture was then filtered through diatomaceous earth, eluted with dichloromethane, concentrated under reduced pressure, and purified by column chromatography (CH2Cl2 / MeOH, 30 / 1) to give a yellow oily liquid: a levorotatory optically active compound IV (0.19 g, 32% yield, 96.6%). ee), and (CH2Cl2 / MeOH / NH3 (7 M in MeOH): 12 / 1 / 0.1) yield a white solid: a third compound with dextrorotatory optical activity (0.34 g, 68% yield, 36.6%). ee ).

[0064] Example 4 This embodiment is the same as Embodiment 1 except for the following differences. At room temperature, vinyl acetate (1.48 ml, 16.08 mmol) was added to a suspension of racemic compound II (0.5 g, 2.01 mmol) in EA (100 mL), followed by the addition of the bioenzyme CAL-B (930 mg). The reaction was allowed to proceed for 4 h at room temperature. The mixture was then filtered through diatomaceous earth, eluted with dichloromethane, concentrated under reduced pressure, and purified by column chromatography (CH2Cl2 / MeOH, 30 / 1) to obtain a yellow oily liquid: a levorotatory optically active compound IV (0.26 g, 45% yield, 96.1%). ee (CH2Cl2 / MeOH / NH3 (7 min MeOH): 12 / 1 / 0.1) yields a white solid: a third compound with dextrorotatory optical activity (0.32 g, 65% yield, 76.6%). ee ).

[0065] Example 5 This embodiment is the same as Embodiment 1 except for the following differences. At room temperature, vinyl acetate (1.48 ml, 16.08 mmol) was added to a suspension of racemic compound II (0.5 g, 2.01 mmol) in Et₂O (100 mL), followed by the addition of the enzyme CAL-B (930 mg). The reaction was allowed to proceed for 4 h at room temperature. The mixture was then filtered through diatomaceous earth, eluted with dichloromethane, concentrated under reduced pressure, and purified by column chromatography (CH₂Cl₂ / MeOH, 30 / 1) to give a yellow oily liquid: a levorotatory optically active compound IV (0.31 g, 52% yield, 88.8%). ee (CH2Cl2 / MeOH / NH3 (7 min MeOH): 12 / 1 / 0.1) yields a white solid: a third compound with dextrorotatory optical activity (0.24 g, 48% yield, 96.6%). ee ).

[0066] Example 6 This embodiment is the same as Embodiment 1 except for the following differences. At room temperature, vinyl acetate (1.48 ml, 16.08 mmol) was added to a suspension of racemic compound II (0.5 g, 2.01 mmol) in 2-Me-THF (100 mL), followed by the addition of the enzyme CAL-B (930 mg). The reaction was allowed to proceed for 4 h at room temperature. The mixture was then filtered through diatomaceous earth, eluted with dichloromethane, concentrated under reduced pressure, and purified by column chromatography (CH2Cl2 / MeOH, 30 / 1) to obtain a yellow oily liquid: a levorotatory optically active compound IV (301 mg, 51% yield, 96.4%). ee (CH2Cl2 / MeOH / NH3 (7M in MeOH): 12 / 1 / 0.1) yields a white solid: a third compound with dextrorotatory optical activity (245 mg, 49% yield, 97%). ee ).

[0067] Example 7 This embodiment is the same as Embodiment 1 except for the following differences. At room temperature, vinyl acetate (1.48 ml, 16.08 mmol) was added to a suspension of racemic compound II (0.5 g, 2.01 mmol) in 2-Me-THF (100 mL), followed by the addition of the enzyme CAL-B (465 mg). The reaction was allowed to proceed for 21 h at room temperature. The mixture was then filtered through diatomaceous earth, eluted with dichloromethane, concentrated under reduced pressure, and purified by column chromatography (CH2Cl2 / MeOH, 30 / 1) to obtain a yellow oily liquid: a levorotatory optically active compound IV (301 mg, 51% yield, 96.4%). ee (CH2Cl2 / MeOH / NH3 (7 M in MeOH): 12 / 1 / 0.1) yields a white solid: a third compound with dextrorotatory optical activity (245 mg, 49% yield, 97%). ee ).

[0068] Example 8 This embodiment is the same as Embodiment 1 except for the following differences. At room temperature, vinyl acetate (1.48 ml, 16.08 mmol) was added to a suspension of racemic compound II (0.5 g, 2.01 mmol) in 2-Me-THF (100 mL), followed by the addition of the enzyme CAL-B (232 mg). The reaction was allowed to proceed for 48 h at room temperature. The mixture was then filtered through diatomaceous earth, eluted with dichloromethane, concentrated under reduced pressure, and purified by column chromatography (CH2Cl2 / MeOH, 30 / 1) to obtain a yellow oily liquid: a levorotatory optically active compound IV (301 mg, 51% yield, 96.0%). ee (CH2Cl2 / MeOH / NH3 (7 M in MeOH): 12 / 1 / 0.1) yields a white solid: a third compound with dextrorotatory optical activity (245 mg, 49% yield, 96.1%). ee ).

[0069] Example 9 This embodiment is the same as Embodiment 1 except for the following differences. At room temperature, vinyl acetate (1.48 ml, 16.08 mmol) was added to a suspension of racemic compound II (0.5 g, 2.01 mmol) in 2-Me-THF (100 mL), followed by the addition of the enzyme CAL-B (162 mg). The reaction was allowed to proceed for 72 h at room temperature. The mixture was then filtered through diatomaceous earth, eluted with dichloromethane, concentrated under reduced pressure, and purified by column chromatography (CH2Cl2 / MeOH, 30 / 1) to give a yellow oily liquid: a levorotatory optically active compound IV (284 mg, 48% yield, 98.5%). ee (CH2Cl2 / MeOH / NH3 (7 M in MeOH): 12 / 1 / 0.1) yields a white solid: a third compound with dextrorotatory optical activity (260 mg, 52% yield, 85.5%). ee ).

[0070] Example 10 This embodiment is the same as Embodiment 1 except for the following differences. At room temperature, vinyl acetate (1.48 ml, 16.08 mmol) was added to a suspension of racemic compound II (0.5 g, 2.01 mmol) in 100 mL of 2-Me-THF, followed by the addition of the enzyme CAL-B (162 mg). The reaction was allowed to proceed for 42 h at room temperature. The mixture was then filtered through diatomaceous earth, eluted with dichloromethane, concentrated under reduced pressure, and purified by column chromatography (CH2Cl2 / MeOH, 30 / 1) to give a yellow oily liquid: a levorotatory optically active compound IV (290 mg, 49% yield, 96.7%). ee (CH2Cl2 / MeOH / NH3 (7 M in MeOH): 12 / 1 / 0.1) yields a white solid: a third compound with dextrorotatory optical activity (255 mg, 51% yield, 87.2%). ee ).

[0071] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the interpretation of the present invention, without departing from the scope of the invention, should be within the protection scope of the present invention.

Claims

1. A method for preparing (+)-crispine A by enzymatic resolution, characterized in that, Includes the following steps: (S1) The first compound was obtained by reacting 6,7-dimethoxy-3,4-dihydroisoquinoline-2-oxide with allyl alcohol in a 1,3-dipolar cycloaddition reaction. The chemical structural formula of the 6,7-dimethoxy-3,4-dihydroisoquinoline-2-oxide is shown below: ； (S2) The first compound prepared in step (S1) is added to a sulfonating agent and zinc powder, and the racemic second compound is obtained through a three-step series reaction of primary alcohol sulfonation, nitrogen-oxygen bond cleavage and cyclization. (S3) The second compound obtained in step (S2) is mixed with vinyl acetate and a biological enzyme and then subjected to chiral resolution under the action of the biological enzyme to obtain a third compound with dextrorotatory optical activity and a fourth compound with levorotatory optical activity. The bioenzyme is CAL-B; (S4) After mixing the third compound obtained in step (S3) with a basic reagent and a sulfonating reagent, the secondary alcohol is sulfonated to obtain a fifth compound with dextrorotatory optical activity. The fifth compound is mixed with a deoxygenating reducing agent to carry out a deoxygenation reaction to prepare (+)-crispine A. The chemical structural formulas of the first compound, the second compound, the third compound, the fourth compound, the fifth compound, and (+)-crispine A are shown as formula (I), (±)-formula (II), (+)-formula (III), (-)-formula (IV), (+)-formula (V), and (+)-formula (VI), respectively: 。 2. The method for preparing (+)-crispine A by enzymatic resolution according to claim 1, characterized in that, In step (S1), the molar ratio of 6,7-dimethoxy-3,4-dihydroisoquinoline-2-oxide to allyl alcohol is 1:1.5~3.

5.

3. The method for preparing (+)-Crispine A by enzymatic resolution according to claim 1, characterized in that, In step (S1), the temperature during the 1,3-dipolar cycloaddition reaction is 60℃~110℃ and the reaction time is 3h~8h.

4. The method for preparing (+)-crispine A by enzymatic resolution according to claim 1, characterized in that, In step (S2), the molar ratio of the first compound, the sulfonating agent, and the zinc powder is 1:1.5~3.5:3~12; The sulfonating agent is selected from one of methanesulfonyl chloride, methanesulfonic anhydride, benzenesulfonyl chloride, benzenesulfonic anhydride, p-toluenesulfonyl chloride, or p-toluenesulfonic anhydride.

5. The method for preparing (+)-crispine A by enzymatic resolution according to claim 1, characterized in that, In step (S2), the temperature during the primary alcohol sulfonation reaction is 0~30℃ and the time is 1h~3h; During the nitrogen-oxygen bond breaking and cyclization reaction, the temperature is 30℃~100℃ and the time is 2h~24h.

6. The method for preparing (+)-crispine A by enzymatic resolution according to claim 1, characterized in that, In step (S3), the molar ratio of the second compound to vinyl acetate is 1:4~10; The ratio of the second compound to the biological enzyme is 0.01~1 mmol:1 U.

7. The method for preparing (+)-crispine A by enzymatic resolution according to claim 1, characterized in that, In step (S3), the chiral separation process is carried out at a temperature of 10~35℃ for 8~72h.

8. The method for preparing (+)-crispine A by enzymatic resolution according to claim 1, characterized in that, In step (S4), the molar ratio of the third compound, the basic reagent, the sulfonating reagent, and the deoxygenating reducing reagent is 1:2~10:1.2~5:2~10; The alkaline reagent is selected from one of LDA, LHMDS, KHMDS, NaHMDS, Et3N, DMAP, Py, DIPEA or NaH; The sulfonating agent is selected from one of p-toluenesulfonyl chloride, p-toluenesulfonic anhydride, benzenesulfonyl chloride, benzenesulfonic anhydride, methanesulfonyl chloride, or methanesulfonic anhydride; The deoxygenating reducing agent is selected from either LiAlH4 or DIBAL-H.

9. The method for preparing (+)-crispine A by enzymatic resolution according to claim 1, characterized in that, In step (S4), during the secondary alcohol sulfonation process, the temperature is 0~30℃ and the time is 3h~5h.

10. The method for preparing (+)-crispine A by enzymatic resolution according to claim 1, characterized in that, In step (S4), the temperature during the deoxygenation reaction is 0~80℃ and the time is 3h~7h.

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