Matrine 14-position spiro derivative, its preparation method and application

Abstract

The application discloses matrine 14-position spiro derivative and a preparation method and application thereof, and belongs to the technical field of pharmacy. The application takes matrine as raw material, synthesizes matrine diazonium at the 14-position as a reaction intermediate, and synthesizes 26 matrine 14-position spiro derivatives through [3+2] cycloaddition reaction with acrylic ester compounds at normal temperature without a catalyst. The synthesized compounds are subjected to preliminary screening of in-vitro anti-tumor activity by selecting human liver cancer cells HepG2, human cervical cancer cells Hela and rat neuroglioma cells C6. The target compounds all show good anti-tumor activity, indicating that the compounds are anti-tumor drugs with development prospects, and can provide a reference for further development of matrine as an anti-tumor drug.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and particularly relates to matrine 14-spirocyclic derivatives, their preparation methods and applications. Background Technology

[0002] Matrine is a tetracyclic quinolone alkaloid extracted and isolated from the root of Sophora flavescens. It consists of two quinazonidine rings fused together and has the chemical formula C. 15 H 24 N₂O has a relative molecular mass of 248.36. Matrine's molecule has six chiral centers, including two chiral nitrogen atoms and four chiral carbon atoms. The absolute configurations of the chiral carbon atoms have been determined to be 5S, 6S, 7R, 11R. The structural formula is as follows:

[0003]

[0004] Matrine compounds have a wide range of pharmacological effects, including sedation, anti-inflammation, immunomodulation, antiviral, antitumor, antipyretic, antifibrotic, anti-allergic, analgesic, and antithrombotic effects. Matrine can inhibit tumor cell proliferation by regulating pathways such as miR-10b / PTEN and p53 / p21 / PCNA / eIF4E, and can also be used in combination with anticancer drugs such as cisplatin to promote apoptosis by inhibiting survivin and activating the caspase pathway. Although matrine has a wide range of pharmacological effects, its relatively weak pharmacological activity, short half-life, and toxic side effects on the central nervous system (e.g., matrine injection can cause central nervous system paralysis and spasms) limit its clinical use.

[0005] The structure-activity relationship of matrine's antitumor, antiviral, and insecticidal activities lacks systematic research. Furthermore, matrine's low bioavailability and toxic side effects on the central nervous system limit its clinical application. Therefore, researching the total synthesis of matrine and the synthesis of its derivatives and analogs can help discover and optimize synthetic routes, improve yields, develop new synthetic and improved methods, identify compounds with higher activity and better selectivity, and gain a deeper understanding of their structure-activity relationships, thus laying the foundation for the development of novel drugs. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention proposes a matrine 14-spirocyclic derivative, its preparation method, and its application. The matrine 14-spirocyclic derivative synthesized by this invention exhibits excellent antitumor activity and can be used to prepare antitumor drugs.

[0007] To achieve the above objectives, the present invention provides a matrine 14-spirocyclic derivative, which is a diastereomer, with the following structural formula:

[0008]

[0009] Where R is

[0010] Any one of them.

[0011] Preferably, the matrine 14-spirocyclic derivative is selected from the following structures:

[0012]

[0013] More preferably

[0014] Spirocyclic rings are ring systems composed of two rings fused together by a single atom, and are found in many natural products and synthetic drug molecules. Spirocyclic rings offer a good balance between conformational rigidity and flexibility, allowing for greater conformational adaptability to many proteins as biological targets. Introducing conformational restriction through spirocyclic rings can not only modulate binding potency and specificity but also potentially improve bioavailability and metabolic stability. This invention provides a class of matrine spirocyclic derivatives with a spirocyclic structure at the 14-position, exhibiting good activity.

[0015] This invention also provides a method for preparing the 14-position spirocyclic derivative of matrine, using matrine (1) as a raw material. Under the action of a strong base bis(trimethylsilyl)amino sodium, the H at the α-position of the lactam bond of matrine is removed to form a carbanion. Then, it undergoes nucleophilic addition with tert-butyl nitrite to obtain matrine oxime (2), which then reacts with hydrazine hydrate to generate matrine hydrazone (3). The oxazone is then oxidized by manganese dioxide to obtain the intermediate matrine diazo (4). The intermediate matrine diazo (4) undergoes a [3+2] cyclization reaction with an acrylate derivative to obtain the 14-position spirocyclic derivative of matrine with strong antitumor activity. The reaction route is as follows:

[0016]

[0017] This invention uses matrine as a raw material, synthesizing 14-position matrine diazo as a reaction intermediate. Under room temperature and catalyst-free conditions, it reacts with acrylate compounds via a [3+2] cycloaddition reaction to synthesize 26 matrine 14-position spirocyclic derivatives. The synthesized matrine 14-position spirocyclic derivatives were preliminarily screened for in vitro antitumor activity using human hepatocellular carcinoma cells (HepG2), human cervical carcinoma cells (HeLa), and rat glioma cells (C6). The target compounds (i.e., matrine 14-position spirocyclic derivatives) all exhibited good antitumor activity, indicating that these compounds are promising antitumor drugs and can provide a reference for further development of matrine as an antitumor drug.

[0018] Preferably, the preparation method of the matrine oxime (2) is as follows:

[0019] Sodium bis(trimethylsilyl)amino and matrine (1) dissolved in anhydrous tetrahydrofuran were mixed and stirred at room temperature for 40 min. Then tert-butyl nitrite was added and stirred at room temperature for 2 h. After the reaction was completed, the solvent was removed by vacuum distillation, and the mixture was extracted three times with ethyl acetate, concentrated, and column chromatography was performed to obtain matrine oxime (2).

[0020] Preferably, in the preparation method of matrine oxime (2), the ratio of sodium bis(trimethylsilyl)amino, matrine (1) and tert-butyl nitrite is 20 mL: 2.5 g: 12 mL.

[0021] The solvent used during column chromatography is a mixture of ethyl acetate and petroleum ether in a volume ratio of 2:1.

[0022] Preferably, the preparation method of the matrine hydrazone (3) is as follows:

[0023] Matrine oxime (2) was dissolved in ethanol, hydrazine hydrate was added, and the mixture was stirred and refluxed at 87°C for 48 hours. Ethanol was removed by vacuum distillation, and the mixture was extracted three times with dichloromethane and concentrated to obtain matrine hydrazone (3). It could be used directly in the next step of the reaction without purification.

[0024] The ratio of matrine oxime (2) to hydrazine hydrate was 1 g: 4 mL.

[0025] The concentration of the hydrazine hydrate is 80 wt%.

[0026] Preferably, the preparation method of the intermediate matrine diazonium (4) is as follows:

[0027] Take 2-3 eq of matrine hydrazone (3), add dichloromethane to dissolve, add 0.2 g of anhydrous magnesium sulfate, stir, then add 0.3 g of manganese dioxide, stir at room temperature for 4 h, monitor the reaction by thin layer chromatography (TLC), after the reaction is complete, filter, wash with dichloromethane, concentrate, and pass through column to obtain matrine diazo (4).

[0028] The solvent used for column chromatography was a mixture of ethyl acetate and petroleum ether in a volume ratio of 2:1.

[0029] Preferably, the specific method for the [3+2] cyclization reaction of the intermediate matrine diazo (4) with the acrylate derivative is as follows:

[0030] Matrine diazo (4) was dissolved in dichloromethane, and then an acrylate derivative was added. The mixture was stirred overnight at room temperature, and the reaction was monitored by TLC. After the reaction was complete, the solvent was removed by vacuum distillation, and the 14-spirocyclic derivative of matrine was obtained by column chromatography.

[0031] Preferably, in the specific method for the [3+2] cyclization reaction of the intermediate matrine diazo (4) with the acrylate derivative, the equivalent ratio of the matrine diazo (4) to the acrylate derivative is (0.8-1):(3-4).

[0032] The solvent used for column chromatography was a mixture of ethyl acetate and petroleum ether in a volume ratio of 1:1.

[0033] The present invention also provides the application of the matrine 14-spirocyclic derivative in the preparation of anticancer drugs, wherein the cancer in the anticancer drugs is liver cancer, cervical cancer or glioma.

[0034] Compared with the prior art, the present invention has the following advantages and technical effects:

[0035] The method for preparing the 14-spirocyclic derivative of matrine provided by this invention features readily available raw materials, simple operation, streamlined synthesis steps, mild reaction conditions, and high product yield. Experiments show that the 14-spirocyclic derivative of matrine of this invention has a strong inhibitory effect on the proliferation of various cancer cells, and this type of compound can be used for tumor treatment. Detailed Implementation

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0038] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0039] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0040] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0041] In this embodiment of the invention, room temperature refers to 25±2℃.

[0042] The technical solution of the present invention will be further illustrated by the following embodiments.

[0043] Example 1

[0044] Preparation of G1 and G2:

[0045]

[0046] (1) Take a 250mL round-bottom flask, add 20mL of sodium bis(trimethylsilyl)amino and 2.5g of matrine 1 dissolved in anhydrous tetrahydrofuran, stir at room temperature for 40min, then add 12mL of tert-butyl nitrite, stir at room temperature for 2h. After the reaction is complete, remove the solvent by vacuum distillation, extract three times with ethyl acetate, concentrate, and pass through a column using ethyl acetate:petroleum ether = 2:1 (volume ratio) to obtain matrine oxime 2.

[0047] (2) Take 2g of matrine oxime 2, dissolve it in ethanol, add 8mL of hydrazine hydrate (80wt%), stir and reflux at 87℃ for 48h, remove ethanol by vacuum distillation, extract three times with dichloromethane, and concentrate to obtain matrine hydrazone 3, which can be used directly in the next reaction without purification. Matrine hydrazone 3: 1H NMR (500MHz, CDCl3) δ6.13 (s, 2H), 4.34 (dd, J = 12.8, 4.6Hz, 1H), 3.98-3.83 (m, 1 H),3.15(t,J=12.7Hz,1H),2.84-2.70(m,3H),2.49-2.37(m,1H),2.33-2.21(m,1 H),2.19-2.07(m,2H),1.99-1.85(m,2H),1.83(dt,J=13.7,2.4Hz,1H),1.81-1. 67(m,2H),1.69-1.57(m,2H),1.56-1.49(m,1H),1.46-1.31(m,3H),1.21(s,1H).

[0048] (3) Take 2-3 eq of matrine hydrazone 3, dissolve it in dichloromethane, add 0.2 g of anhydrous magnesium sulfate, stir, then add 0.3 g of manganese dioxide, and stir at room temperature for 4 h. Monitor the reaction by thin-layer chromatography (TLC). After the reaction is complete, filter, wash with dichloromethane, concentrate, and pass through a column using ethyl acetate:petroleum ether = 2:1 (v / v) to obtain matrine diazonium 4. Matrine diazonium 4: 1 HNMR(500MHz, CDCl3)δ4.31(dd,J=12.8,4.6Hz,1H),3.92-3.84(m,1H),3.16(t,J=12.6Hz,1H),2.82(d d,J=23.7,11.5Hz,2H),2.77-2.67(m,1H),2.70-2.61(m,1H),2.14(d,J=4.4Hz,1H),2.10(td,J=8.8,4 .4Hz,1H),2.03-1.91(m,2H),1.87(t,J=2.7Hz,1H),1.82-1.71(m,2H),1.72-1.63(m,1H),1.58(td,J= 8.4,3.7Hz,1H),1.52-1.44(m,2H),1.42(d,J=2.9Hz,2H),1.30(d,J=2.5Hz,1H),1.27(d,J=2.7Hz,1H).

[0049] (4) Take 0.8-1 eq matrine diazonium 4 into a 100 mL round bottom flask, add dichloromethane to dissolve it, then add 3-4 eq benzyl acrylate, stir at room temperature overnight, monitor the reaction by TLC, remove the solvent by vacuum distillation after the reaction is complete, and pass the solution through a column using ethyl acetate: petroleum ether = 1:1 (volume ratio) to obtain a pair of diastereomers G1 and G2 of the 14-position spirocyclic derivative of matrine.

[0050] G1: 14S-(3'-Benzoxycarboxyl)dihydropyrazolespiro[4.5]matrine, pale yellow solid, yield 76%, melting point 82.1℃~85.9℃. 1 H NMR (600MHz, CDCl3) δ7.44-7.38(m,2H),7.40-7.33(m,2H),7.35-7.29(m,1H),6.58(s,1H),5.27(s,2H),4.29(dd,J= 12.8,4.4Hz,1H),3.94-3.84(m,1H),3.74(d,J=17.3Hz,1H),3.13(t,J=12.8Hz,1H),2.82(dd,J=30.6,11.3Hz,2H),2 .78(d,J=17.3Hz,1H),2.12(d,J=3.5Hz,1H),2.05(d,J=2.7Hz,1H),2.03-1.95(m,3H),1.92-1.84(m,2H),1.80-1.73 (m,2H),1.73-1.64(m,1H),1.67-1.58(m,1H),1.52(dd,J=13.5,4.6Hz,2H),1.48-1.42(m,3H),1.27(d,J=1.8Hz,1H). 13 C NMR (151MHz, CDCl3) δ169.16,162.16,141.55,135.68,128.53,128.46,128.28,68.23,66.69,63.44,57 .01,53.34,51.41,42.99,42.56,35.14,31.58,29.69,28.62,27.52,22.53,20.90.HRMS(ESI)calcdfor C 25 H 32 N4O3[M+H + ],437.2474,found,437.2344.

[0051] G2: 14R-(3'-Benzoxycarboxyl)dihydropyrazolespiro[4.5]matrine, pale yellow solid, yield 76%, melting point 83.7℃~86.1℃. 1H NMR (600MHz, CDCl3) δ7.43-7.38(m,2H),7.39-7.33(m,2H),7.35-7.29(m,1H),6.68(d,J=1.5Hz,1H),5.26(s,2H), 4.26(dd,J=12.8,4.4Hz,1H),3.85(td,J=10.2,6.0Hz,1H),3.28(d,J=17.2Hz,1H),3.11(t,J=12.8Hz,1H),2.90(dd ,J=17.2,1.5Hz,1H),2.87-2.76(m,2H),2.22-2.16(m,1H),2.09(t,J=2.7Hz,1H),2.02-1.94(m,3H),1.95-1.87(m ,2H),1.71(dd,J=9.1,4.2Hz,1H),1.68-1.60(m,2H),1.54(tt,J=12.9,4.3Hz,2H),1.47-1.41(m,4H),1.26(s,1H). 13 C NMR (151MHz, CDCl3) δ170.58,162.15,140.81,135.64,128.53,128.28,68.89,66.74,63.26,57.0 5,53.27,43.66,43.51,42.32,35.23,31.86,27.59,26.01,24.47,21.03,20.63.HRMS(ESI)calcd for C 25 H 32 N4O3[M+H + ],437.2474,found,437.2343.

[0052] Compounds G3-G26 were prepared using the same experimental methods as compounds G1 and G2. The structural identification data for compounds G3-G26 are as follows:

[0053] Compound G3: 14S-[3'-(2'-trifluoromethyl)benzooxyformyl]dihydropyrazolespiro[4.5]matrine, yellow solid, yield 68%, melting point 83.7℃~85.2℃. 1H NMR (600MHz, CDCl3) δ7.71-7.66(m,1H),7.63(d,J=7.9Hz,1H),7.60-7.53(m,1H),7.43(t,J=7.7Hz,1H),6.64(s,1H),5.47(s,2H),4. 29(dd,J=12.8,4.4Hz,1H),3.91(dd,J=10.4,6.4Hz,1H),3.75(d,J=17.2Hz,1H),3.14(t,J=12.8Hz,1H),2.89-2.77(m,2H),2.80(dd, J=17.4,1.0Hz,1H),2.13(s,1H),2.11-2.02(m,1H),2.02-1.94(m,2H),1.93-1.87(m,1H),1.82-1.76(m,1H),1.75(q,J=6.1Hz,1H),1 .71-1.64(m,2H),1.65-1.58(m,2H),1.53(d,J=13.2Hz,2H),1.46(t,J=3.8Hz,1H),1.44(dd,J=6.3,3.7Hz,2H),1.27(d,J=1.3Hz,1H). 13 C NMR (151MHz, CDCl3) δ169.08,161.82,141.22,134.09,132.15,129.74,128.66,128.15,126.02,125.98,68.34,63.39 ,62.82,57.08,57.05,53.41,52.11,42.93,42.64,35.23,31.60,29.70,27.58,26.32,22.53,20.58.HRMS(ESI)calcd for C 26 H 31 F3N4O3[M+H + ],505.2348,found,505.2384.

[0054] Compound G4: 14R-[3'-(2'-trifluoromethyl)benzooxyformyl]dihydropyrazolespiro[4.5]matrine, yellow solid, yield 68%, melting point 84.1℃~86.5℃. 1H NMR (600MHz, CDCl3) δ7.67(dd,J=8.0,1.2Hz,1H),7.63(d,J=7.8Hz,1H),7.59-7.53(m,1H),7.42(t,J=7.6Hz,1H),6.76(d,J=1.4Hz,1H ),5.45(s,2H),4.26(dd,J=12.8,4.4Hz,1H),3.86(td,J=10.0,6.0Hz,1H),3.29(d,J=17.2Hz,1H),3.12(t,J=12.8Hz,1H),2.92(dd,J=1 7.2,1.5Hz,1H),2.82(dd,J=30.9,11.4Hz,2H),2.24-2.17(m,1H),2.10(d,J=2.8Hz,1H),2.03-1.98(m,2H),1.99-1.91(m,2H),1.92-1. 86(m,1H),1.75-1.70(m,2H),1.69-1.62(m,1H),1.59-1.49(m,2H),1.45(d,J=3.7Hz,2H),1.43(d,J=3.6Hz,2H),1.26(d,J=1.5Hz,1H). 13 C NMR (151MHz, CDCl3) δ170.50,161.85,140.30,134.10,132.17,129.70,128.10,127.93,125.97,125.93,68.98,63.24 ,62.77,57.08,57.07,53.30,43.67,43.40,42.35,35.24,31.84,27.62,26.04,24.42,21.05,20.66.HRMS(ESI)calcd for C 26 H 31 F3N4O3[M+H + ],505.2348,found,505.2398.

[0055] Compound G5: 14S-[3'-(4'-fluoro)benzooxycarboxyl]dihydropyrazolespiro[4.5]matrine, yellow solid, yield 70%, melting point 82.5℃~84.7℃. 1H NMR (600MHz, CDCl3) δ7.45-7.36(m,2H),7.09-7.01(m,2H),6.52(s,1H),5.24(s,2H),4.29(dd,J=12.8,4.5H z,1H),3.91(d,J=11.4Hz,1H),3.73(d,J=17.3Hz,1H),3.14(t,J=12.7Hz,1H),2.83(dd,J=30.4,10.7Hz,2H), 2.78(dd,J=17.3,1.1Hz,1H),2.13(s,1H),2.11-2.00(m,2H),1.99(d,J=14.5Hz,2H),1.90(d,J=13.9Hz,1H), 1.81-1.72(m,1H),1.70-1.63(m,3H),1.53(dd,J=13.8,4.7Hz,3H),1.49-1.40(m,3H),1.27(d,J=2.0Hz,1H). 13 C NMR (151MHz, CDCl3) δ169.13,163.53,162.09,141.75,131.53,130.58,130.53,115.54,115.39,68.24,66.02,6 4.58,63.41,57.07,53.39,43.04,42.64,35.24,31.62,29.70,27.58,26.35,22.55,20.57.HRMS(ESI)calcdfor C 25 H 31 FN4O3[M+H + ],455.2380,found,455.2427.

[0056] Compound G6: 14R-[3'-(4"-fluoro)benzooxycarboxyl]dihydropyrazolespiro[4.5]matrine, yellow solid, yield 72%, melting point 83.1℃~84.7℃. 1H NMR (600MHz, CDCl3) δ7.43-7.36(m,2H),7.04(t,J=8.7Hz,2H),6.66(d,J=1.5Hz,1H),5.22(s,2H),4.26( dd,J=12.8,4.5Hz,1H),3.89-3.82(m,1H),3.28(d,J=17.2Hz,1H),3.12(t,J=12.7Hz,1H),2.90(dd,J=17 .3,1.5Hz,1H),2.87-2.77(m,2H),2.24-2.17(m,1H),2.12-2.08(m,2H),2.03-1.94(m,2H),1.96-1.88(m ,2H),1.75-1.63(m,2H),1.59-1.50(m,2H),1.48-1.45(m,2H),1.45-1.42(m,3H),1.27(d,J=1.9Hz,1H). 13 CNMR(151MHz, CDCl3)δ170.56,163.54,162.13,140.84,131.50,130.64,130.59,115.53,115.38,68.91,66.04 ,64.05,63.24,57.08,53.30,43.49,42.35,35.26,31.90,27.62,26.05,24.52,21.07,20.68.HRMS(ESI)calcd for C 25 H 31 FN4O3[M+H + ],455.2380,found,455.2447.

[0057] Compound G7: 14S-[3'-(4'-cyano)benzooxycarboxyl]dihydropyrazolespiro[4.5]matrine, yellow solid, yield 71%, melting point 84.2℃~86.8℃. 1H NMR (600MHz, CDCl3) δ7.67(d,J=1.8Hz,1H),7.66(d,J=1.9Hz,1H),7.54-7.50(m,2H),6.61(s,1H),5.32(s,2H),4.28(d d,J=12.8,4.4Hz,1H),3.93(td,J=8.6,5.9Hz,1H),3.74(d,J=17.3Hz,1H),3.15(t,J=12.7Hz,1H),2.83(dd,J=34.9,7.1 Hz,2H),2.79(dd,J=17.4,1.1Hz,1H),2.13(s,1H),2.09-2.03(m,1H),2.04-1.95(m,2H),1.93-1.87(m,1H),1.82-1.76( m,1H),1.74(d,J=9.9Hz,2H),1.69-1.64(m,2H),1.55(tt,J=13.2,4.5Hz,2H),1.49-1.41(m,4H),1.27(d,J=1.5Hz,1H). 13 C NMR (151MHz, CDCl3) δ169.02,161.90,141.04,132.40,132.28,128.51,118.61,112.03,68.37,65.42,64.13, 63.36,57.12,57.09,53.45,42.91,42.70,35.31,31.68,29.70,27.63,26.41,22.58,21.06.HRMS(ESI)calcd for C 26 H 31 N5O3[M+H + ],462.2427,found,462.2488.

[0058] Compound G8: 14R-[3'-(4"-cyano)benzooxyformyl]dihydropyrazolespiro[4.5]matrine, yellow solid, yield 73%, melting point 84.9℃~87.7℃. 1H NMR (600MHz, CDCl3) δ7.66(d,J=8.4Hz,2H),7.54-7.49(m,2H),6.76(d,J=1.4Hz,1H),5.30(d,J=1.6Hz,2H),4.25(dd,J=1 2.9,4.4Hz,1H),3.87(td,J=10.2,6.0Hz,1H),3.28(d,J=17.2Hz,1H),3.13(t,J=12.8Hz,1H),2.91(dd,J=17.2,1.5Hz,1H ),2.88-2.76(m,2H),2.25-2.18(m,1H),2.10(d,J=2.9Hz,1H),2.05-1.97(m,2H),1.99-1.92(m,2H),1.89(d,J=12.6Hz,1 H),1.77-1.67(m,2H),1.66-1.59(m,1H),1.61-1.50(m,2H),1.49-1.45(m,2H),1.46-1.41(m,2H),1.27(d,J=1.9Hz,1H). 13 CNMR(151MHz, CDCl3)δ170.41,161.95,141.02,140.11,132.39,128.54,118.61,112.03,69.05,65.44,63 .79,63.21,57.08,53.31,43.74,42.37,35.28,31.87,27.64,26.07,24.50,21.08,20.69.HRMS(ESI)calcd for C 26 H 31 N5O3[M+H + ],462.2427,found,462.2484.

[0059] Compound G9: 14S-[3'-(3'-methyl)benzooxyformyl]dihydropyrazolespiro[4.5]matrine, pale yellow solid, yield 66%, melting point 86.2℃~89.7℃. 1H NMR (600MHz, CDCl3) δ7.25(t,J=7.5Hz,1H),7.25-7.19(m,2H),7.14(d,J=7.5Hz,1H),6.64(d,J=1.5Hz,1H),5.23(s,2H), 4.26(dd,J=12.8,4.3Hz,1H), 3.86(dt,J=10.2,5.1Hz,1H), 3.29(d,J=17.2Hz,1H), 3.12(t,J=12.8Hz,1H), 2.90(dd,J=17. 3,1.5Hz,1H),2.87-2.75(m,2H),2.36(s,3H),2.23-2.17(m,1H),2.10(t,J=2.8Hz,1H),2.03-1.96(m,3H),1.95-1.91(m, 1H),1.89(dd,J=7.3,4.1Hz,1H),1.76-1.67(m,1H),1.69-1.61(m,1H),1.60-1.49(m,2H),1.48-1.42(m,4H),1.27(s,2H). 13 C NMR (151MHz, CDCl3) δ170.61,162.17,141.13,138.21,135.51,129.36,129.07,128.42,125.68,68.86,66.87,63 .80,63.25,57.10,53.31,43.59,42.38,35.28,31.95,29.71,27.66,26.07,24.52,21.11,20.72.HRMS(ESI)calcd for C 26 H 34 N4O3[M+H + ],451.2631,found,451.2672.

[0060] Compound G10: 14R-[3'-(3"-methyl)benzooxyformyl]dihydropyrazolespiro[4.5]matrine, pale yellow solid, yield 80%, melting point 87.1℃~89.3℃. 1H NMR (600MHz, CDCl3) δ7.25(t,J=7.5Hz,1H),7.24-7.20(m,2H),7.14(d,J=7.5Hz,1H),6.67(s,1H),5.23(s,2H),4 .27(dd,J=12.9,4.4Hz,1H),3.87(td,J=10.2,6.0Hz,1H),3.29(d,J=17.2Hz,1H),3.13(t,J=12.8Hz,1H),2.91(d ,J=17.2Hz,1H),2.88-2.77(m,2H),2.37(s,3H),2.25-2.17(m,1H),2.10(d,J=2.8Hz,1H),1.99(dd,J=8.4,3.9Hz ,2H),1.96-1.92(m,1H),1.76-1.67(m,1H),1.60-1.51(m,1H),1.48-1.47(m,3H),1.46-1.45(m,5H),1.27(s,2H). 13 C NMR (151MHz, CDCl3) δ170.65,162.19,141.04,138.21,135.52,129.36,129.07,128.42,125.68,68.88,66.86,63.50 ,63.27,57.12,57.10,53.30,43.58,42.36,35.26,31.94,29.71,27.65,26.08,24.52,21.10,20.71.HRMS(ESI)calcd for C 26 H 34 N4O3[M+H + ],451.2631,found,451.2679.

[0061] Compound G11: 14S-[3'-(3,5"-difluoro)benzooxyformyl]dihydropyrazolespiro[4.5]matrine, yellow solid, yield 60%, melting point 83.5℃~86.7℃. 1H NMR (600MHz, CDCl3) δ6.96-6.90(m,2H),6.75(tt,J=8.9,2.4Hz,1H),5.22(s,2H),4.32-4.26(m,1H ),3.92(s,1H),3.73(d,J=17.3Hz,1H),3.20-3.10(m,1H),2.81(d,J=35.7Hz,2H),2.78(d,J=17.3H z,1H),2.12(d,J=15.4Hz,1H),2.09-2.03(m,2H),2.02-1.96(m,1H),1.97-1.83(m,2H),1.78(dd,J =17.9,6.4Hz,2H),1.71-1.62(m,3H),1.59-1.51(m,2H),1.50-1.41(m,3H),1.27(d,J=18.3Hz,2H). 13 CNMR(151MHz, CDCl3)δ169.09,163.89,162.24,162.15,161.88,139.57,110.89,110.72,103.59,68 .32,65.08,63.54,56.82,53.20,42.75,34.88,31.51,29.69,23.77,22.60,20.55.HRMS(ESI)calcd for C 25 H 30 F2N4O3[M+H + ],473.2286,found,473.2323.

[0062] Compound G12: 14R-[3'-(3,5"-difluoro)benzooxyformyl]dihydropyrazolespiro[4.5]matrine, yellow solid, yield 82%, melting point 84.6℃~87.7℃. 1H NMR (600MHz, CDCl3) δ6.93 (dt, J=6.3, 2.1Hz, 2H), 6.76 (dt, J=8.9, 2.4Hz, 1H), 6.74 (s, 1H), 5.21 (s, 2H), 4. 26(dd,J=12.9,4.4Hz,1H),3.93-3.84(m,1H),3.29(d,J=17.2Hz,1H),3.13(t,J=12.8Hz,1H),2.91(dd,J=17 .2,1.5Hz,1H),2.83(dd,J=31.5,11.3Hz,2H),2.26-2.18(m,1H),2.11(s,1H),2.05-1.95(m,4H),1.91-1.87 (m,1H),1.76-1.69(m,2H),1.70-1.61(m,1H),1.59-1.49(m,2H),1.46(dt,J=11.7,3.7Hz,4H),1.26(s,1H). 13 C NMR (151MHz, CDCl3) δ170.43,163.89,162.15,161.90,140.05,139.47,111.00,110.83,103.61,69.03,65.18 ,63.29,57.03,53.24,43.61,43.33,42.28,35.18,31.79,29.69,27.50,24.47,20.92,20.53.HRMS(ESI)calcd for C 25 H 30 F2N4O3[M+H + ],473.2286,found,473.2315.

[0063] Compound G13: 14S-[3'-(2"-bromo-5"-methoxy)benzooxycarboxyl]dihydropyrazolespiro[4.5]matrine, yellow oil, yield 64%. 1H NMR (600MHz, CDCl3) δ7.43(d,J=8.8Hz,1H),7.02(d,J=3.1Hz,1H),6.74(dd,J=8.7,3.1Hz,1H),5.29(d,J =1.9Hz,2H),4.28(dd,J=12.8,4.4Hz,1H),3.95-3.88(m,1H),3.79(s,3H),3.73(s,1H),3.13(d,J=16.2Hz ,1H),2.82(d,J=34.4Hz,2H),2.79(dd,J=17.3,1.1Hz,1H),2.18-2.10(m,1H),2.09-2.00(m,1H),1.99(s ,2H),1.88(d,J=12.9Hz,1H),1.78-1.72(m,3H),1.70-1.61(m,2H),1.58-1.49(m,3H),1.49-1.40(m,4H). 13 C NMR (151MHz, CDCl3) δ169.08,161.90,159.04,140.83,135.98,133.33,115.58,115.29,

[0064] 113.40,68.32,66.02,63.44,56.94,55.55,53.30,44.78,42.84,34.98,31.56,29.68,27.29,22.56,20.74,19.75.HRMS(ESI)calcd for C 26 H 33 BrN4O3[M+H + ],545.1685,found,545.1692.

[0065] Compound G14: 14R-[3'-(2"-bromo-5"-methoxy)benzooxycarboxyl]dihydropyrazolespiro[4.5]matrine, a yellow oil, yield 80%. 1H NMR (600MHz, CDCl3) δ7.44(d,J=8.8Hz,1H),7.03(d,J=3.1Hz,1H),6.74(dd,J=8.8,3.1Hz,1H),6.72(d,J=1.4Hz,1H),5.2 9(s,2H),4.27(dd,J=12.9,4.4Hz,1H),3.93-3.82(m,1H),3.79(s,3H),3.30(d,J=17.2Hz,1H),3.13(t,J=12.8Hz,1H),2. 92(dd,J=17.2,1.5Hz,1H),2.88-2.75(m,2H),2.25-2.17(m,1H),2.11(s,1H),2.04-1.96(m,3H),1.94(dd,J=13.1,3.4Hz ,1H),1.92-1.85(m,1H),1.72(d,J=10.9Hz,2H),1.70-1.59(m,2H),1.54(tdd,J=13.4,6.5,3.5Hz,2H),1.49-1.38(m,4H). 13 CNMR(151MHz, CDCl3)δ170.50,161.89,159.06,140.35,135.94,133.31,115.52,115.41,113.41,68.98,66.10 ,63.31,57.00,55.56,53.22,43.43,42.25,35.16,31.79,27.47,25.91,24.42,20.89,20.49.HRMS(ESI)calcd for C 26 H 33 BrN4O3[M+H + ],545.1685,found,545.1684.

[0066] Compound G15: 14S-[3'-(3"-nitro-4"-fluoro)benzooxycarboxyl]dihydropyrazolespiro[4.5]matrine, yellow solid, yield 56%, melting point 88.8℃~90.7℃. 1H NMR (600MHz, CDCl3) δ8.12 (dd, J=7.0, 2.3Hz, 1H), 7.73-7.67 (m, 1H), 7.29 (dd, J=10.5, 8.6Hz, 1H), 6.6 9(s,1H),5.28(d,J=2.7Hz,2H),4.26(dd,J=12.7,4.4Hz,1H),3.95-3.86(m,1H),3.72(d,J=17.3Hz,1H) ,3.13(t,J=12.7Hz,1H),2.87-2.74(m,1H),2.77(dd,J=17.3,1.1Hz,1H),2.13-2.04(m,3H),2.02-1.89 (m,3H),1.93-1.85(m,1H),1.84-1.69(m,2H),1.71-1.61(m,2H),1.58-1.48(m,2H),1.48-1.40(m,4H). 13 CNMR(151MHz, CDCl3)δ168.95,161.89,156.12,154.36,140.57,135.55,132.99,126.14,118.76,68.40,64.56,64 .01,63.34,57.11,57.08,53.44,42.78,40.84,35.28,32.82,31.66,27.63,26.38,22.53,21.05.HRMS(ESI)calcd for C 25 H 30 FN5O5[M+H + ],500.2231,found,500.2210.

[0067] Compound G16: 14R-[3'-(3"-nitro-4"-fluoro)benzooxycarboxyl]dihydropyrazolespiro[4.5]matrine, yellow solid, yield 88%, melting point 87.6℃~90.1℃. 1H NMR (600MHz, CDCl3) δ8.12(dd,J=7.0,2.3Hz,1H),7.76-7.67(m,1H),7.29(dd,J=10.5,8.6Hz,1H),6.79(d,J=1.4Hz ,1H),5.26(s,2H),4.24(dd,J=12.8,4.4Hz,1H),3.85(td,J=10.0,6.0Hz,1H),3.27(d,J=17.1Hz,1H),3.11(t,J=12 .8Hz,1H),2.89(dd,J=17.2,1.4Hz,1H),2.86-2.75(m,2H),2.24-2.17(m,1H),2.11-2.07(m,1H),2.04-1.95(m,2H) ,1.98-1.91(m,2H),1.91-1.85(m,1H),1.76-1.65(m,3H),1.67-1.59(m,1H),1.58-1.50(m,1H),1.48-1.39(m,5H). 13 C NMR (151MHz, CDCl3) δ170.35,161.91,156.13,154.37,139.71,135.63,132.94,126.20,118.76,69.10,64.58,63 .77,63.19,57.20,57.07,53.30,43.24,42.34,35.25,31.79,27.63,26.05,24.45,21.06,20.67.HRMS(ESI)calcd for C 25 H 30 FN5O5[M+H + ],500.2231,found,500.2223.

[0068] Compound G17: 14S-[3'-(2"-bromo-6"-fluoro)benzooxycarboxyl]dihydropyrazolespiro[4.5]matrine, yellow solid, yield 62%, melting point 82.1℃~84.7℃. 1H NMR (600MHz, CDCl3) δ7.52(dd,J=8.8,5.2Hz,1H),7.22(dd,J=9.1,3.1Hz,1H),6.92(td,J=8.3,3.1Hz,1H),6.61(s,1 H),5.32(s,2H),4.30(dd,J=12.9,4.4Hz,1H),3.93(s,1H),3.78(d,J=17.3Hz,1H),3.15(t,J=12.8Hz,1H),2.90-2.76 (m,2H),2.83(dd,J=17.2,1.1Hz,1H),2.13(s,1H),2.13-2.04(m,2H),1.98(dt,J=27.6,14.0Hz,3H),1.91(d,J=14.8 Hz,1H),1.86-1.74(m,3H),1.68(dd,J=11.2,2.8Hz,1H),1.59-1.51(m,2H),1.50-1.44(m,3H),1.27(d,J=1.8Hz,1H). 13 C NMR (151MHz, CDCl3) δ169.09,162.81,161.70,161.18,137.31,133.98,133.95,133.50,116.68,68.34,65.43,63.54 ,57.01,56.95,56.89,42.85,40.42,34.94,31.47,29.70,27.42,26.10,23.78,22.63,20.74,20.23.HRMS(ESI)calcd for C 25 H 30 BrFN4O3[M+H + ],533.1485,found,533.1710.

[0069] Compound G18: 14R-[3'-(2"-bromo-6"-fluoro)benzooxycarboxyl]dihydropyrazolespiro[4.5]matrine, yield 82%, melting point 83.5℃~86.8℃. 1H NMR (600MHz, CDCl3) δ7.52(dd,J=8.8,5.2Hz,1H),7.23(dd,J=9.1,3.1Hz,1H),6.92(td,J=8.3,3.1Hz,1H),6.73(d,J=1.5Hz,1H ),5.30(d,J=3.6Hz,2H),4.28(dd,J=12.9,4.4Hz,1H),3.87(td,J=10.0,6.0Hz,1H),3.32(d,J=17.2Hz,1H),3.13(t,J=12.8Hz,1 H),2.94(dd,J=17.2,1.5Hz,1H),2.89-2.76(m,2H),2.26-2.19(m,1H),2.10(q,J=3.3Hz,2H),2.04-1.98(m,1H),1.96(dd,J=12 .8,3.3Hz,1H),1.93-1.87(m,1H),1.73(d,J=12.6Hz,2H),1.70-1.63(m,1H),1.61-1.50(m,2H),1.50-1.41(m,5H),1.27(s,1H). 13 C NMR (151MHz, CDCl3) δ170.44,162.85,161.76,140.17,137.30,133.91,116.66,116.60,116.51,69.03,65.47,63 .81,63.25,57.10,57.08,53.32,43.40,42.38,35.27,31.89,27.63,26.07,24.46,21.08,20.69.HRMS(ESI)calcd forC 25 H 30 BrFN4O3[M+H + ],533.1485,found,533.1702.

[0070] Compound G19: 14S-[3'-(2,6"-dichloro)benzooxyformyl]dihydropyrazolespiro[4.5]matrine, yellow solid, yield 58%, melting point 84.1℃~87.7℃. 1H NMR (600MHz, CDCl3) δ7.34(d,J=8.1Hz,2H),7.24(dd,J=8.6,7.5Hz,1H),6.58(s,1H),5.53(d,J=1.5Hz,2H), 4.29(dd,J=12.8,4.5Hz,1H),3.89(d,J=13.4Hz,1H),3.72(d,J=17.4Hz,1H),3.12(q,J=12.2Hz,1H),2.89-2. 80(m,2H),2.78(dd,J=17.4,1.1Hz,1H),2.13(s,1H),2.08-2.01(m,1H),2.01-1.94(m,1H),1.90(d,J=13.5Hz ,2H),1.76(t,J=12.6Hz,1H),1.69-1.62(m,4H),1.58-1.50(m,1H),1.50-1.42(m,4H),1.27(d,J=2.0Hz,2H). 13 C NMR (151MHz, CDCl3) δ161.99,140.08,137.16,131.12,130.63,128.44,68.16,63.04,61.73,56.62,5 3.51,42.91,42.02,35.15,32.10,31.32,29.70,28.07,25.77,22.56,20.65,19.66.HRMS(ESI)calcd forC 25 H 30 Cl2N4O3[M+H + ],505.1695,found,505.1892.

[0071] Compound G20: 14R-[3'-(2,6"-dichloro)benzooxyformyl]dihydropyrazolespiro[4.5]matrine, yellow solid, yield 88%, melting point 83.6℃~86.1℃. 1H NMR (600MHz, CDCl3) δ7.33(d,J=8.0Hz,2H),7.23(dd,J=8.6,7.5Hz,1H),6.66(d,J=1.4Hz,1H),5.51(s,2H),4. 26(dd,J=12.8,4.4Hz,1H),3.85(s,1H),3.28(d,J=17.2Hz,1H),3.12(t,J=12.8Hz,1H),2.90(dd,J=17.3,1.5H z,1H),2.82(dd,J=32.4,11.8Hz,2H),2.23-2.15(m,1H),2.09(s,1H),2.02-1.94(m,2H),1.96-1.86(m,2H),1. 71(d,J=12.8Hz,2H),1.69-1.58(m,1H),1.56-1.51(m,1H),1.47-1.44(m,3H),1.44-1.39(m,3H),1.26(s,1H). 13 C NMR (151MHz, CDCl3) δ170.53,162.01,140.29,137.23,131.04,130.59,128.42,68.85,63.91,63.30,61 .82,57.01,53.21,43.53,42.25,35.17,32.81,31.82,29.70,27.17,25.90,24.42.HRMS(ESI)calcdfor C 25 H 30 Cl2N4O3[M+H + ],505.1695,found,505.1876.

[0072] Compound G21: 14S-[3'-(4"-biphenyl)benzoxycarboxyl]dihydropyrazolespiro[4.5]matrine, yellow solid, yield 66%, melting point 88.4℃~90.3℃. 1H NMR (600MHz, CDCl3) δ7.65-7.55(m,4H),7.52-7.47(m,2H),7.48-7.42(m,2H),7.40-7.33(m,1H),6.62(s,1H),5. 32(s,2H),4.29(dd,J=12.8,4.4Hz,1H),3.89(d,J=12.3Hz,1H),3.76(d,J=17.3Hz,1H),3.14(t,J=12.7Hz,1H),2 .83(d,J=31.3Hz,2H),2.80(dd,J=17.3,1.1Hz,1H),2.12(s,1H),2.10-2.00(m,2H),1.97(q,J=13.5Hz,3H),1.92 -1.86(m,1H),1.79-1.70(m,2H),1.68-1.64(m,1H),1.63-1.48(m,2H),1.48-1.39(m,4H),1.27(d,J=1.1Hz,1H). 13 C NMR (151MHz, CDCl3) δ169.13,162.23,141.23,140.70,134.72,128.98,128.80,127.42,127.28,127.12,68.26,66 .43,63.40,60.34,57.05,53.38,42.97,42.58,35.19,31.61,29.71,27.58,26.29,22.52,20.98.HRMS(ESI)calcd for C 31 H 36 N4O3[M+H + ],513.2787,found,513.2969.

[0073] Compound G22: 14R-[3'-(4"-biphenyl)benzoxycarboxyl]dihydropyrazolespiro[4.5]matrine, yellow solid, yield 84%, melting point 88.9℃~91.0℃. 1H NMR (600MHz, CDCl3) δ7.61-7.56(m,4H),7.52-7.47(m,2H),7.45(dd,J=8.4,7.0Hz,2H),7.39-7.33(m,1H),6.68(d,J=1.5 Hz,1H),5.31(s,2H),4.27(dd,J=12.8,4.4Hz,1H),3.86(td,J=10.1,6.0Hz,1H),3.30(d,J=17.2Hz,1H),3.12(t,J=12.7Hz ,1H),2.92(dd,J=17.3,1.5Hz,1H),2.82(dd,J=30.4,11.4Hz,2H),2.24-2.16(m,1H),2.09(s,1H),2.04-1.95(m,3H),1.96 -1.85(m,2H),1.78-1.66(m,3H),1.66-1.60(m,1H),1.59-1.49(m,1H),1.47-1.46(m,1H),1.45-1.43(m,3H),1.27(s,1H). 13 CNMR(151MHz, CDCl3)δ170.57,162.19,141.26,140.89,140.74,134.66,129.06,128.79,127.41,127.30,127.15,68.9 2,66.52,63.81,63.26,57.08,53.30,43.54,42.35,35.25,31.90,27.62,26.04,24.50,21.07,20.67.HRMS(ESI)calcd for C 31 H 36 N4O3[M+H + ],513.2787,found,513.2956.

[0074] Compound G23: 14S-(3'-α-naphthoxycarboxyl)dihydropyrazolespiro[4.5]matrine, pale yellow solid, yield 68%, melting point 90.4℃~93.3℃. 1H NMR (600MHz, CDCl3) δ8.07 (dd, J=8.3, 1.2Hz, 1H), 7.91-7.85 (m, 1H), 7.85 (d ,J=8.3Hz,1H),7.60(dd,J=7.0,1.2Hz,1H),7.59-7.53(m,1H),7.54-7.48(m ,1H),7.45(dd,J=8.3,7.0Hz,1H),6.68(d,J=4.1Hz,1H),5.73(q,J=12.6Hz, 2H),4.26(dd,J=12.8,4.4Hz,1H),3.88-3.81(m,1H),3.72(d,J=17.3Hz,1H), 3.10(t,J=12.7Hz,1H),2.80(dd,J=30.2,11.9Hz,2H),2.74(d,J=17.0Hz,1H ),2.07(d,J=3.1Hz,1H),1.97(dd,J=7.1,4.7Hz,2H),1.97-1.83(m,3H),1.8 2(dt,J=16.0,3.0Hz,1H),1.76-1.67(m,2H),1.69-1.62(m,1H),1.64-1.59( m,1H),1.61-1.53(m,1H),1.55-1.43(m,2H),1.42-1.32(m,1H),1.27(s,2H). 13 CNMR(151MHz, CDCl3)δ169.08,162.26,141.31,133.70,131.66,131.19,129.27,128.64,127.58,126.65,125.91,125.27,123.62, 68.25,64.91,63.50,63.33,57.10,57.07,53.39,42.95,42.61,35.18,31.55,27.61,26.30,22.43,21.05,20.63.HRMS(ESI)calcd for C 29 H 34 N4O3[M+H + ],487.2631,found,487.2802.

[0075] Compound G24: 14R-(3'-α-naphthoxycarboxyl)dihydropyrazolespiro[4.5]matrine, pale yellow solid, yield 78%, melting point 89.6℃~92.1℃. 1H NMR (600MHz, CDCl3) δ8.04(dd,J=8.5,1.1Hz,1H),7.88-7.80(m,2H),7.58(dd,J=7.0,1.2Hz,1H),7.57-7.51(m,1H),7.53-7 .46(m,1H),7.43(dd,J=8.3,7.0Hz,1H),6.79(d,J=1.4Hz,1H),5.70(s,2H),4.23(dd,J=12.8,4.3Hz,1H),3.85-3.76(m,1H) ,3.25(d,J=17.2Hz,1H),3.09(t,J=12.7Hz,1H),2.85(dd,J=17.3,1.4Hz,1H),2.84-2.73(m,2H),2.15-2.07(m,1H),2.05(q ,J=2.7Hz,1H),2.01-1.86(m,4H),1.86-1.76(m,2H),1.74-1.58(m,2H),1.55-1.48(m,1H),1.46-1.33(m,5H),1.26(s,1H). 13 C NMR (151MHz, CDCl3) δ170.50,162.24,140.36,133.67,131.64,131.16,129.24,128.63,127.59,126.64,125.90,125.28,123.60, 68.89,64.90,63.76,63.21,57.06,57.04,53.24,43.45,42.30,35.21,31.76,27.61,26.00,24.29,21.04,20.63.HRMS(ESI)calcd for C 29 H 34 N4O3[M+H + ],487.2631,found,487.2777.

[0076] Compound G25: 14S-[3'-(4"-tert-butyl)benzooxyformyl]dihydropyrazolespiro[4.5]matrine, yellow solid, yield 64%, melting point 86.4℃~88.5℃. 1H NMR (600MHz, CDCl3) δ7.40-7.32(m,4H),6.61(s,1H),5.24(s,2H),4.28(dd,J=12.8,4.4Hz,1H),3.89(t d,J=9.2,5.3Hz,1H),3.72(d,J=17.3Hz,1H),3.12(t,J=12.7Hz,1H),2.87-2.74(m,2H),2.78(s,1H),2.1 1(d,J=3.0Hz,1H),2.03(tt,J=10.9,2.7Hz,2H),2.02-1.94(m,2H),1.91-1.83(m,1H),1.77-1.71(m,3H ),1.70-1.61(m,2H),1.63-1.55(m,1H),1.54-1.49(m,1H),1.46-1.40(m,3H),1.32(s,9H),1.26(s,1H). 13 C NMR (151MHz, CDCl3) δ169.15,162.21,151.31,141.73,132.67,128.46,125.45,68.21,66.59,63.38,63.12,57. 12,57.08,53.42,43.05,42.64,35.23,34.59,31.62,31.31,29.70,26.38,22.50,21.05,20.64.HRMS(ESI)calcd for C 29 H 34 N4O3[M+H + ],493.3100,found,493.3233.

[0077] Compound G26: 14R-[3'-(4"-tert-butyl)benzooxyformyl]dihydropyrazolespiro[4.5]matrine, yellow solid, yield 84%, melting point 87.1℃~89.2℃. 1H NMR (600MHz, CDCl3) δ7.38-7.30(m,4H),6.80-6.76(m,1H),5.20(d,J=1.7Hz,2H),4.23(dd,J=12.8,4.3Hz,1 H),3.83(td,J=10.0,6.0Hz,1H),3.25(dd,J=17.3,1.7Hz,1H),3.09(t,J=12.7Hz,1H),2.91-2.84(m,1H),2.8 5-2.73(m,2H),2.20-2.12(m,1H),2.07(t,J=2.7Hz,1H),2.00-1.91(m,3H),1.90(dd,J=13.0,3.3Hz,1H),1. 89-1.83(m,1H),1.75-1.62(m,3H),1.63-1.51(m,2H),1.45-1.38(m,4H),1.30(d,J=1.6Hz,9H),1.25(s,1H). 13 C NMR (151MHz, CDCl3) δ170.58,162.21,151.27,140.66,132.65,128.50,125.43,68.87,66.57,63.23,57.09, 57.07,53.28,43.65,42.32,35.24,34.57,31.83,31.32,27.65,26.06,24.41,21.07,20.68.HRMS(ESI)calcd forC 29 H 34 N4O3[M+H + ],493.3100,found,493.3230.

[0078] The structural formulas of the synthesized matrine 14-spirocyclic derivatives are shown in Table 1.

[0079] Table 1. Structural formulas of matrine 14-position spirocyclic derivatives

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086] Application Example 1: The following experiments further illustrate the excellent effects of the matrine 14-spirocyclic derivative of the present invention.

[0087] The in vitro antitumor activity of the above-mentioned matrine 14-spirocyclic derivative was studied, wherein the tumors were human hepatocellular carcinoma cells HepG2, human cervical cancer cells HeLa, and rat glioma cells C6.

[0088] Experimental method: Cancer cell lines were obtained, resuscitated, and passaged. After 24 hours, the cells reached the logarithmic growth phase. A plate of cells in good growth condition was taken, and trypsin was added to digest the cells to prepare a solution containing 2 × 10⁻⁶ cells per milliliter. 4 ~4×10 4 Cell suspensions were seeded at 150 μL per well in 96-well plates, with blank, control, and sample groups, each group having 3 parallel wells. All 96-well cell culture plates were placed in a cell culture incubator at 37℃ and 5% CO2 for 24 h until cell adhesion. The culture medium was aspirated, and culture medium containing different concentrations of compounds was added (the 14-spirocyclic derivative of matrine was dissolved in dimethyl sulfoxide (DMSO) and then diluted with culture medium to the desired solvent concentration), and cultured for 48 h. The MTT assay was performed, with 15 μL of 5 mg / mL MTT added to each well, incubated for 4 h, the supernatant was discarded, 150 μL of DMSO was added, the mixture was shaken and the absorbance of each well was measured at 490 nm using a microplate reader, and the cell proliferation inhibition rate was calculated.

[0089] Proliferation inhibition rate (%) = [1 - (OD value of experimental group - OD value of blank group) / (OD value of negative control group - OD value of blank group)] × 100%.

[0090] The above experiment was repeated 3 times, and the IC was calculated. 50 value.

[0091] The experimental results are shown in Table 2.

[0092] Table 2. Inhibition of HepG2, Hela, and C6 cell proliferation by matrine 14-spirocyclic derivative.

[0093]

[0094] Experimental data show that most of the matrine 14-spirocyclic derivatives synthesized in this invention have good antitumor activity. When matrine was used as a control drug and the same samples were administered as the synthesized matrine 14-spirocyclic derivatives, the inhibition rates were compared and found that most target compounds had strong inhibitory effects on HepG2, Hela, and C6 cells. Among the compounds, G21, G22, G25, and G26 had strong inhibitory effects on tumor cells and had highly efficient antitumor proliferation activity.

[0095] The above are merely preferred embodiments of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A matrine 14-spirocyclic derivative, characterized in that, It is a diastereomer, with the following structural formula: Where R is , , , , , , , , , , , and Any one of them.

2. A method for preparing the matrine 14-spirocyclic derivative according to claim 1, characterized in that, Using matrine 1 as a raw material, under the action of a strong base bis(trimethylsilyl)amino sodium, the H at the α-position of the matrine lactam bond is removed to form a carbanion. Then, a nucleophilic addition reaction occurs with tert-butyl nitrite to give matrine oxime 2, followed by reaction with hydrazine hydrate to generate matrine hydrazone 3. Finally, oxidation with manganese dioxide yields the intermediate matrine diazo 4. The intermediate matrine diazo 4 undergoes a [3+2] cyclization reaction with acrylate derivatives to obtain a spirocyclic derivative of matrine at the 14-position. The reaction route is as follows: 。 3. The method for preparing the matrine 14-spirocyclic derivative according to claim 2, characterized in that, The preparation method of the matrine oxime 2 is as follows: Sodium bis(trimethylsilyl)amino and matrine 1 dissolved in anhydrous tetrahydrofuran were mixed and stirred at room temperature for 40 min. Then tert-butyl nitrite was added and stirred at room temperature for 2 h. After the reaction was completed, the mixture was distilled under reduced pressure, extracted with ethyl acetate, concentrated, and column filtered to obtain matrine oxime 2.

4. The method for preparing the matrine 14-spirocyclic derivative according to claim 3, characterized in that, The ratio of sodium bis(trimethylsilyl)amino, matrine 1, and tert-butyl nitrite in the liquid solution is 20 mL: 2.5 g: 12 mL. The solvent used during column chromatography is a mixture of ethyl acetate and petroleum ether in a volume ratio of 2:

1.

5. The method for preparing the matrine 14-spirocyclic derivative according to claim 2, characterized in that, The preparation method of the matrine hydrazone 3 is as follows: Matrine oxime 2 was dissolved in ethanol, hydrazine hydrate was added, the mixture was stirred and refluxed at 87°C for 48 hours, then distilled under reduced pressure, extracted with dichloromethane, and concentrated to obtain matrine hydrazone 3. The ratio of matrine oxime 2 to hydrazine hydrate in the liquid solution is 1g:4mL.

6. The method for preparing the matrine 14-spirocyclic derivative according to claim 2, characterized in that, The preparation method of the intermediate matrine diazonium 4 is as follows: Dissolve 2-3 eq of matrine hydrazone 3 in dichloromethane, add 0.2 g of anhydrous magnesium sulfate, stir, then add 0.3 g of manganese dioxide, stir at room temperature for 4 h, monitor the reaction by thin-layer chromatography, after the reaction is complete, filter, wash with dichloromethane, concentrate, and pass through column to obtain matrine diazonium 4. The solvent used for column chromatography was a mixture of ethyl acetate and petroleum ether in a volume ratio of 2:

1.

7. The method for preparing the matrine 14-spirocyclic derivative according to claim 2, characterized in that, The specific method for the [3+2] cyclization reaction of the intermediate matrine diazonium 4 with acrylate derivatives is as follows: Matrine diazonium 4 was dissolved in dichloromethane, and then an acrylate derivative was added. The mixture was stirred overnight at room temperature. The reaction was monitored by thin-layer chromatography. After the reaction was complete, the mixture was distilled under reduced pressure and column chromatography was used to obtain the 14-spirocyclic derivative of matrine.

8. The method for preparing the matrine 14-spirocyclic derivative according to claim 7, characterized in that, The equivalent ratio of matrine diazonium 4 to acrylate derivatives is (0.8~1):(3~4). The solvent used for column chromatography was a mixture of ethyl acetate and petroleum ether in a volume ratio of 1:

1.

9. The application of the matrine 14-spirocyclic derivative according to claim 1 in the preparation of anticancer drugs, characterized in that, The cancer mentioned in the anticancer drug is liver cancer, cervical cancer, or glioma.

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