CN113861114A - Morphinane and dibenzyl isoquinoline alkaloid compound and preparation method and application thereof - Google Patents

Morphinane and dibenzyl isoquinoline alkaloid compound and preparation method and application thereof Download PDF

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CN113861114A
CN113861114A CN202111307194.6A CN202111307194A CN113861114A CN 113861114 A CN113861114 A CN 113861114A CN 202111307194 A CN202111307194 A CN 202111307194A CN 113861114 A CN113861114 A CN 113861114A
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李宁
陈刚
侯悦
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Abstract

The invention belongs to the technical field of medicines, relates to alkaloid compounds, a preparation method and medical application thereof, and particularly relates to morphinan and dibenzylisoquinoline alkaloid compounds, a preparation method thereof and application thereof in preparing medicines for preventing or treating neurodegenerative diseases and antitumor medicines. Morphinane and bisbenzylisoquinoline alkaloid compounds and pharmaceutically acceptable salts thereof, which are one or more of the following structures.

Description

Morphinane and dibenzyl isoquinoline alkaloid compound and preparation method and application thereof
The invention name of the original application: alkaloid compound and preparation method and application thereof
Original application No.: 2019105741807
Application date of the original case: 2019.06.28
Technical Field
The invention belongs to the technical field of medicines, and relates to morphinan and dibenzyl isoquinoline alkaloid compounds, a preparation method and application thereof, in particular to 4 morphinan and 2 dibenzyl isoquinoline alkaloids, a preparation method and application thereof.
Background
Stephania cepharantha Hayata is dry root tuber of Stephania cepharantha of Stephania of Menispermaceae, also called Stephania cepharantha and Stephania cepharantha. Mainly distributed in places such as south of Yangtze river and the like, is a common folk herbal medicine, and has the effects of clearing away heat and toxic materials, relieving swelling and pain, resisting bacteria and diminishing inflammation.
As a traditional Chinese medicine, the name of ' Baiyao ' is recorded in ' Xinxie Bencao ' at the earliest time, and is called ' pungent, warm and nontoxic in taste; mainly treating incised wound and promoting tissue regeneration. The following are recorded in Ben Cao (materia Medica of the book Jing) and Ben Cao gang mu (compendium of the materia Medica).
In recent years, researches show that the dibenzyl isoquinoline alkaloid stephanine extracted and separated from stephanotis has good whitening effect in clinical application. However, there are few reports on the structure and biological activity of other alkaloids in stephanotis. The invention provides structures of 6 new alkaloids separated and identified from stephanotis, a preparation method and application thereof in developing medicaments for resisting tumors and treating neurodegenerative diseases.
Disclosure of Invention
The invention aims to provide a series of alkaloid compounds, a preparation method and a novel medical application thereof.
The alkaloid and pharmaceutically acceptable salts, isomers or solvates thereof provided by the invention have the following structures:
Figure BDA0003340719570000011
R1、R2is hydroxy, C1-C4 acyl;
Figure BDA0003340719570000021
R3is C1-C4 aldehyde group, R4、R9Is C1-C4 alkoxy, R5Is hydrogen, R6And R7Formation of methylenedioxy, R8Is C1-C4 alkyl;
Figure BDA0003340719570000022
R1' is C1-C4 alkyl, R2'、R3'、R5'、R7' is C1-C4 alkoxy, R4' is hydrogen, R6' is a C1-C4 aldehyde group;
the following 6 specific compounds are preferred in the present invention
Figure BDA0003340719570000023
The invention also provides a preparation method of the alkaloid 1,2,3,4,5 and 6, which comprises the following steps:
(1) extracting root tuber of Stephania cepharantha Hayata with 50-100% methanol or ethanol under reflux or ultrasonic heating, and recovering extractive solution to obtain crude extract;
(2) dissolving the crude extract obtained in the step (1) by using methanol, mixing the dissolved crude extract with a macroporous resin, sequentially eluting by using ethanol-water-formic acid and ethanol-water-triethylamine systems with different pH values and different concentrations, and recovering to obtain an ethanol-water-triethylamine eluate;
(3) separating the ethanol-water-triethylamine eluate obtained in the step (2) by silica gel column chromatography, and separating by using a mixed solvent of petroleum ether and ethyl acetate, namely 100: 10-1: 1. petroleum ether and acetone mixed solvent 100: 10-1: 1. chloroform-acetone mixed solvent 100: 1-100: 15. dichloromethane and acetone mixed solvent 100: 1-100: 15. chloroform-methanol mixed solvent 100: 1-100: 15. dichloromethane and methanol mixed solvent 100: 1-100: 15 gradient elution;
(4) separating the 100: 1-5: 1 flow obtained in the step (3) by ODS chromatography, and carrying out gradient elution by using a mixed solvent of methanol and water or a mixed solvent of acetonitrile and water as a mobile phase;
(5) and (3) further separating the methanol and water fractions 1: 9-9: 1 and the acetonitrile and water fractions 1: 9-8: 2 obtained in the step (4) by preparative HPLC-UV chromatography, and eluting by using a mixed solvent of methanol and water 3: 7-8: 2 or a mixed solvent of acetonitrile and water 1: 9-5: 5 as a mobile phase to obtain alkaloids 1,2,3 and 4.
(6) Eluting the fractions obtained in the step (4) by using a mixed solvent of methanol and water with a ratio of 3: 7-6: 4 or a mixed solvent of acetonitrile and water with a ratio of 1: 9-4: 6 as a mobile phase to obtain alkaloids 5 and 6.
The invention provides a preparation method of alkaloids 1,2,3,4,5 and 6, wherein the extraction method in the step (1) is heating reflux extraction or heating ultrasonic extraction for 1-4 times, and the used solvents are as follows: 50 to 100 percent of methanol or ethanol aqueous solution. The medicinal materials are as follows: the weight-volume ratio of the solvent is 1: 5-1: 15g/mL, preferably 1: 8-1: 12 g/mL.
The preparation method of the alkaloids 1,2,3,4,5 and 6 comprises the following steps of (1) dissolving a crude extract by using methanol/ethanol, mixing the crude extract according to the mass ratio of the crude extract to the mass ratio of 3: 1-6: 1, drying at room temperature, eluting by using ethanol-water-formic acid (20:70: 10-40: 55:5) and ethanol-water-triethylamine (60:35: 5-80: 10:10) for 8-12 column volumes, preferably 10 column volumes, and recovering ethanol-water-triethylamine partial eluates under reduced pressure.
According to the preparation method of the alkaloids 1,2,3,4,5 and 6, the volume ratio of the mixed solvent of the eluting solvent petroleum ether and ethyl acetate and the mixed solvent of the petroleum ether and acetone in the step (3) is 100: 10-1: 1, preferably 10: 1-10: 4; the volume ratio of the mixed solvent of dichloromethane and acetone, the mixed solvent of chloroform and acetone, the mixed solvent of dichloromethane and methanol, or the mixed solvent of chloroform and methanol is 100: 1-100: 15, preferably 100:1 to 100: 8.
According to the preparation method of the alkaloids 1,2,3,4,5 and 6, in the step (4), the mixed solvent of methanol and water is 1: 9-9: 1, preferably 3: 7-9: 1; the volume ratio of the acetonitrile-water mixed solvent is 1: 9-8: 2, preferably 1:9 to 5: 5.
According to the preparation method of the alkaloids 1,2,3,4,5 and 6, the ratio of the mobile phase methanol to the water mixed solvent in the step (5) is 3: 7-8: 2, preferably 4: 6-8: 2; the ratio of the acetonitrile to the water mixed solvent is 1: 9-5: 5, preferably 3: 7-5: 5.
According to the preparation method of the alkaloids 1,2,3,4,5 and 6, the ratio of the mobile phase methanol to the water mixed solvent in the step (6) is 3: 7-6: 4, preferably 4: 6-6: 4; the ratio of the acetonitrile to the water mixed solvent is 1: 9-4: 6, preferably 2: 8-4: 6.
The invention uses LPS-induced microglia cell over-activation model to evaluate the anti-neuritis activity of the prepared new morphinan alkaloids 1,2,3 and 4 bisbenzylisoquinoline alkaloids 5 and 6. The results show that compounds 5 and 6 are able to significantly inhibit LPS-induced microglial release of NO. Therefore, the novel bisbenzylisoquinoline alkaloid prepared by the invention can be applied to the development of medicines for treating neurodegenerative diseases.
The invention evaluates the anti-lung cancer activity of the prepared novel alkaloids 1,2,3,4,5 and 6. The results show that the compounds 1-6 can obviously inhibit the growth of the A549 cells under the condition of not influencing the survival rate of the lung cancer cells A549, thereby playing the role of preventing and treating the lung cancer. Therefore, the novel alkaloid prepared by the invention can be applied to the development of anti-lung cancer drugs.
The invention evaluates the anti-liver cancer activity of the prepared novel alkaloids 1,2,3,4,5 and 6. The results show that 1-6 can obviously inhibit the growth of HepG2 cells under the condition of not influencing the survival rate of the liver cancer cell HepG2, thereby playing the role of preventing and treating the lung cancer. Therefore, the novel alkaloid prepared by the invention can be applied to the development of anti-lung cancer drugs.
The invention provides a method for preparing and identifying 2 bisbenzylisoquinoline alkaloids and 4 morphinan alkaloids by taking radix stephaniae tetrandrae as a raw material for the first time, and systematically evaluates the application of the bisbenzylisoquinoline alkaloids and the 4 morphinan alkaloids in medicines for preventing or treating neurodegenerative diseases and cancers.
Detailed Description
The following examples further illustrate the invention but are not intended to limit the invention thereto.
Example 1
(1) Heating and ultrasonically extracting 1kg of rhizoma Dioscoreae with 50% ethanol for 4 times (2 hr per time; 12L per time), and recovering extractive solution under reduced pressure to obtain crude extract;
(2) dissolving the crude extract obtained in the step (1) by using ethanol, mixing the samples according to the mass ratio of macroporous resin to the crude extract of 5:1, drying the samples at room temperature, sequentially eluting the samples by using ethanol-water-formic acid (20:70:10) and ethanol-water-triethylamine (70:25:5) for 12 column volumes, and recovering the ethanol-water-triethylamine part eluate under reduced pressure.
(3) Separating the ethanol-water-triethylamine eluate obtained in the step (2) by silica gel column chromatography, and eluting with a mixed solvent of petroleum ether and ethyl acetate at a ratio of 100:1,100:3,100:7,100:10 and 100: 15;
(4) performing ODS (ODS chromatography) separation on the 100: 5-100: 10 flow obtained in the step (3), and performing gradient elution by using methanol/water 1:9,3:7,5:5,7:3,9:1 as a mobile phase;
(5) separating the fraction of methanol and water (3: 7-9: 1) obtained in the step (4) by HPLC-UV chromatography, detecting at 210nm with the flow rate of 4mL/min, and eluting with methanol and water (68: 32) as a mobile phase to obtain a compound 1 (t)R＝23.0min),2(tR＝34.5min),3(tR47.3min) (yield 0.00005% each);
(6) separating the sub-fraction obtained in the step (4) by HPLC-UV chromatography at 210nm with a flow rate of 4mL/min, eluting with acetonitrile water (38:62) as the mobile phase to obtain compound 4 (t)R＝35.0min),5(tR42.4min) and 6 (t)R51.3min) (yield 0.00005% each).
The structure of the morphinan alkaloid is identified according to the physicochemical properties and spectral data of the morphinan alkaloid 1,2 and 3 and the bisbenzylisoquinoline alkaloid 4,5 and 6.
The structural identification data for morphinan-type alkaloid 1 are as follows:
yellow powder (CH)3OH)，
Figure BDA0003340719570000051
The improved bismuth potassium iodide reaction is positive, and the HR-ESI-MS gives an excimer peak (M + H)]+m/z：446.1815(calcd.446.1811for C23H27NO8H) In combination with it1H-NMR and13C-NMR data (see Table 1) suggest a molecular formula of C23H27NO8The unsaturation degree was 11. By passing1The coupling constants of the key hydrogens in the H-NMR spectrum and the NOESY spectrum determine the relative configuration of Compound 1,1the coupling constant of H-6 and H-7 on the H-NMR spectrum was 3.2Hz, and it was thus determined to be in the cis configuration. The absolute configuration of compound 1 was determined using CD exciton chirality. Compound 1 has a positive compton effect at 241nm and a negative compton effect at 215nm, suggesting that the absolute configuration of the compound is determined to be 6S, 7S, 9R, 13S.
The structural identification data for morphinan-type alkaloid 2 are as follows:
white Crystal (CH)3OH)，
Figure BDA0003340719570000052
Improving positive reaction of bismuth potassium iodide, and giving an excimer ion peak (M + H) by HR-ESI-MS]+m/z:362.1604,(calcd.362.1607for C19H23NO4H) In combination with it1H-NMR and13C-NMR data (see Table 1) suggest a molecular formula of C19H23NO4The unsaturation degree was 9. From the NMR data, it was found that Compound 2 had the same parent nucleus as Compound 1. The relative configuration and absolute configuration of the compound are determined by the same method as that of the compound 1.1The coupling constant of H-6 and H-7 on the H-NMR spectrum was 3.3Hz, and it was thus determined to be in the cis configuration. The ECD of compound 2 was a positive Comton effect at 238nm and a negative Comton effect at 214nm, suggesting that the absolute configuration of the compound was determined to be 6S, 7S, 9R, 13S.
The structural identification data for morphinan-type alkaloid 3 are as follows:
white powder (CH)3OH), improving the reaction of bismuth potassium iodide to be positive,
Figure BDA0003340719570000061
Figure BDA0003340719570000062
HR-ESI-MS gives the peak of the excimer ion [ M + Na ]]+m/z：426.1529(calcd.426.1523for C21H25NO7Na) in combination with it1H-NMR and13C-NMR data (see Table 2) suggest a molecular formula of C21H25NO7The unsaturation degree was 10.1The coupling constant of H-6 and H-7 on the H-NMR spectrum was 2.4Hz, and therefore the relative configuration was determined to be the cis configuration. The absolute configuration of the compound 3 is determined by a CD exciton chiral method, the ECD of the compound 3 has a positive Comton effect at 241nm and a negative Comton effect at 209nm, and the absolute configuration of the compound is determined to be 6S, 7S, 9R and 13S.
The structural identification data for morphinan-type alkaloid 4 are as follows:
white powder (CH)3OH), improving the reaction of bismuth potassium iodide to be positive,
Figure BDA0003340719570000063
Figure BDA0003340719570000064
HR-ESI-MS gives the peak of the excimer ion [ M + Na ]]+m/z：426.1529(calcd.426.1523for C21H25NO7Na), in combination1H-NMR and13C-NMR data (see Table 2) suggest a molecular formula of C21H25NO7The unsaturation degree was 10. The coupling constants for H-6 and H-7 for the compounds were 2.9Hz, suggesting a cis configuration. The absolute configuration of the compound 4 is also determined by a CD exciton chiral method, and is positive Comton effect at 239nm and negative Comton effect at 210nm, which indicates that the absolute configuration of the compound is determined to be 6S, 7S, 9R and 13S.
The structural identification data of the bisbenzylisoquinoline alkaloid 5 are as follows:
yellow powder (CH)3OH)，
Figure BDA0003340719570000065
The improved bismuth potassium iodide reaction is positive, and the HR-ESI-MS gives an excimer peak (M + H)]+m/z:621.2601(calcd.621.2595for C37H36N2O7H) In combination with it1H-NMR and13C-NMR data (see Table 3) suggest a molecular formula of C37H36N2O7The unsaturation degree was 20. The possibility that compound 5 is a bisbenzylisoquinoline alkaloid was presumed from the molecular weight and NMR spectra. The absolute configuration of the compound 5 is determined according to the nuclear magnetic spectrum law and ECD of the key positions of the bisbenzylisoquinoline. 6-OCH when the 1, 1' positions of the two asymmetric carbon atoms in a molecule of the repandine-oxocanthin type form RS or SR3The bit shift is about 3.60, when the double RR or SS is formed, 6-OCH3The displacement is around 3.35. 6-OCH3Chemical shift 3.71 was observed and compound 5 was determined to be either the RS or SR configuration. The absolute configuration is determined by using a TDDFT method to calculate the ECD, the measured spectrum is compared with the ECD spectrum obtained by calculation, the measured ECD of the compound 5 is well fitted with the ECD of the 1R, 1'S, and the absolute configuration of the compound B5 is determined to be 1R, 1' S.
The structural identification data of the bisbenzylisoquinoline alkaloid 6 are as follows:
yellow powder (CH)3OH)，
Figure BDA0003340719570000073
The improved bismuth potassium iodide reaction is positive, and the HR-ESI-MS gives an excimer peak (M + H)]+m/z：637.2914(calcd.637.2908for C38H40N2O7H) In combination with it1H-NMR and13C-NMR data presume that the molecular formula is C38H40N2O7The unsaturation degree was 19. It was speculated from the molecular weight and NMR data (see Table 3) that Compound 6 was probably a bisbenzylisoquinoline type alkaloid. The relative and absolute configuration determination of compound 6 was performed in the same manner as compound 5. 6' -OCH3Chemical shift 3.69, so compound 6 is in either the RS or SR configuration. The ECD of the compound 6 is found to be well fitted with the ECD of 1R, 1'S, and the absolute configuration of the compound B6 is determined to be 1R, 1' S.
TABLE 1NM of Compounds 1,2R data (CDCl)3,600MHz,1H-NMR；150MHz 13C-NMR)
Figure BDA0003340719570000071
TABLE 2 NMR data (CDCl) for compounds 3,43,600MHz,1H-NMR；150MHz 13C-NMR)
Figure BDA0003340719570000072
Figure BDA0003340719570000081
TABLE 3 NMR data (CDCl) for Compounds 5,63,600MHz,1H-NMR；150MHz 13C-NMR)
Figure BDA0003340719570000082
Figure BDA0003340719570000091
Example 2
(1) Extracting radix Stephaniae Cepharanthae 1kg with 100% ethanol for 1 time, 2 hr each time (15L each time), heating and ultrasonic extracting, and concentrating the extractive solution to obtain crude extract;
(2) dissolving the crude extract obtained in the step (1) by using ethanol, mixing the crude extract according to the mass ratio of macroporous resin to crude extract of 4:1, drying the mixture at room temperature, sequentially eluting the mixture for 10 column volumes by using ethanol-water-formic acid (30:65:5) and ethanol-water-triethylamine (60:35:5), and recovering the ethanol-water-triethylamine partial eluate under reduced pressure.
(3) Separating the ethanol-water-triethylamine eluate obtained in the step (2) by silica gel column chromatography, and eluting by using a mixed solvent of petroleum ether and acetone at a ratio of 100:1,100:3,100:7,100:10 and 100: 15;
(4) performing ODS (ODS chromatography) separation on the 100: 5-100: 10 flow obtained in the step (3), and performing gradient elution by using methanol/water 1:9,3:7,5:5,7:3,9:1 as a mobile phase;
(5) separating the fraction of methanol and water (3: 7-9: 1) obtained in the step (4) by HPLC-UV chromatography, detecting at 210nm with the flow rate of 4mL/min, and eluting with methanol and water (68: 32) as a mobile phase to obtain a compound 1 (t)R＝23.0min),2(tR＝34.5min),3(tR47.3min) (yield 0.00005% each);
(6) separating the sub-fraction obtained in the step (4) by HPLC-UV chromatography at 210nm with a flow rate of 4mL/min, eluting with acetonitrile water (38:62) as the mobile phase to obtain compound 4 (t)R＝35.0min),5(tR42.4min) and 6 (t)R51.3min) (yield 0.00005% each).
The structural identification of morphinane alkaloids 1,2,3,4 and bisbenzylisoquinoline alkaloids 5,6 is shown in example 1.
Example 3
(1) Extracting radix Stephaniae Cepharanthae 1kg with 85% methanol for 2 times (each time for 2 hr, 12L), heating under reflux, and concentrating the extractive solution to obtain crude extract;
(2) dissolving the crude extract obtained in the step (1) by using methanol, mixing the sample according to the mass ratio of macroporous resin to the crude extract of 5:1, drying the mixture at room temperature, sequentially eluting 10 column volumes by using ethanol-water-formic acid (20:70:10) and ethanol-water-triethylamine (80:10:10), and recovering ethanol-water-triethylamine part eluate under reduced pressure.
(3) Separating the ethanol-water-triethylamine eluate obtained in the step (2) by silica gel column chromatography, and eluting with dichloromethane/methanol mixed solvent 100:1,100:2,100:4,100:6,100: 10;
(4) performing ODS (ODS chromatography) separation on the 100: 1-100: 6 flow obtained in the step (3), and performing gradient elution by using acetonitrile/water 1:9,3:7,6:4 and 8:2 as mobile phases;
(5) separating the fraction of methanol and water (3: 7-9: 1) obtained in the step (4) by HPLC-UV chromatography, detecting at 210nm with the flow rate of 4mL/min, and eluting with methanol and water (68: 32) as a mobile phase to obtain a compound 1 (t)R＝23.0min),2(tR＝34.5min),3(tR47.3min) (yield 0.00005% each);
(6) separating the sub-fraction obtained in the step (4) by HPLC-UV chromatography at 210nm with a flow rate of 4mL/min, eluting with acetonitrile water (38:62) as the mobile phase to obtain compound 4 (t)R＝35.0min),5(tR42.4min) and 6 (t)R51.3min) (yield 0.00005% each).
The structural identification of morphinane alkaloids 1,2,3,4 and bisbenzylisoquinoline alkaloids 5,6 is shown in example 1.
Example 4
(1) Extracting radix Stephaniae Cepharanthae 1kg with 75% ethanol for 3 times (each time for 2 hr, 10L), heating under reflux, and concentrating the extractive solution to obtain crude extract;
(2) dissolving the crude extract obtained in the step (1) by using ethanol, mixing the samples according to the mass ratio of macroporous resin to the crude extract of 6:1, drying the samples at room temperature, sequentially eluting 8 column volumes by using ethanol-water-formic acid (30:65:5) and ethanol-water-triethylamine (70:25:5), and recovering ethanol-water-triethylamine part eluate under reduced pressure.
(3) Separating the ethanol-water-triethylamine eluate obtained in the step (2) by silica gel column chromatography, and eluting with chloroform/methanol mixed solvent 100:1,100:2,100:4,100:6,100: 10;
(4) separating the 100: 1-100: 6 flow obtained in the step (3) by ODS chromatography, and carrying out gradient elution by taking methanol/water 1:9,3:7,5:5,7:3,9:1 as a mobile phase;
(5) separating the fraction of methanol and water (3: 7-9: 1) obtained in the step (4) by HPLC-UV chromatography, detecting at 210nm with the flow rate of 4mL/min, and eluting with methanol and water (68: 32) as a mobile phase to obtain a compound 1 (t)R＝23.0min),2(tR＝34.5min),3(tR47.3min) (yield 0.00005% each);
(6) separating the sub-fraction obtained in the step (4) by HPLC-UV chromatography at 210nm with a flow rate of 4mL/min, eluting with acetonitrile water (38:62) as the mobile phase to obtain compound 4 (t)R＝35.0min),5(tR42.4min) and 6 (t)R51.3min) (yield 0.00005% each).
The structural identification of morphinane alkaloids 1,2,3,4 and bisbenzylisoquinoline alkaloids 5,6 is shown in example 1.
Example 5
(1) Extracting radix Stephaniae Cepharanthae 1kg with 65% methanol for 3 times (2 hr each time (8L each time), heating, ultrasonic extracting, and concentrating the extractive solution to obtain crude extract;
(2) dissolving the crude extract obtained in the step (1) by using methanol, mixing the sample according to the mass ratio of macroporous resin to the crude extract of 4:1, drying the mixture at room temperature, sequentially eluting 8 column volumes by using ethanol-water-formic acid (40:55:5) and ethanol-water-triethylamine (70:25:5), and recovering the ethanol-water-triethylamine part eluate under reduced pressure.
(3) Separating the ethanol-water-triethylamine eluate obtained in the step (2) by silica gel column chromatography, and eluting with dichloromethane/methanol mixed solvent 100:1,100:2,100:4,100:6,100: 10;
(4) performing ODS (ODS chromatography) separation on the 100: 1-100: 6 flow obtained in the step (3), and performing gradient elution by using acetonitrile/water 1:9,3:7,6:4 and 8:2 as mobile phases;
(5) separating the fraction of methanol and water (3: 7-9: 1) obtained in the step (4) by HPLC-UV chromatography, detecting at 210nm with the flow rate of 4mL/min, and eluting with methanol and water (68: 32) as a mobile phase to obtain a compound 1 (t)R＝23.0min),2(tR＝34.5min),3(tR47.3min) (yield 0.00005% each);
(6) separating the sub-fraction obtained in the step (4) by HPLC-UV chromatography at 210nm with a flow rate of 4mL/min, eluting with acetonitrile water (38:62) as the mobile phase to obtain compound 4 (t)R＝35.0min),5(tR42.4min) and 6 (t)R51.3min) (yield 0.00005% each).
The structural identification of morphinane alkaloids 1,2,3,4 and bisbenzylisoquinoline alkaloids 5,6 is shown in example 1.
EXAMPLE 6 Activity of alkaloids 1,2,3,4,5 and 6 prepared in examples 1-5 to inhibit excessive activation of microglia cells
(1) The experimental principle is as follows: the chronic inflammatory reaction mediated by the microglia activation is an important link in the generation and development process of neurodegenerative diseases, and the inhibition of the microglia activation can become a new target point for drug discovery. LPS activates microglia to release NO, proinflammatory cytokines, active oxygen and the like. The experiment evaluates the anti-inflammatory activity of the neomorphinan alkaloid 1,2,3,4 and the bisbenzylisoquinoline alkaloid 5,6 obtained from the stephanotis by establishing a screening model for activating BV2 microglia abnormally and taking the activated microglia to release NO as an index.
(2) The experimental method comprises the following steps:
culture of mouse microglia line BV2
All glassware and metal instruments (culture bottles, pipettes, solution bottles, etc.) used in cell culture and model building were autoclaved at 121 ℃ for 30min to completely remove the contaminated LPS. A cell culture solution containing 10% fetal calf serum was prepared on the basis of DMDM medium. Microglia at about 4 × 105cells/ml at 5% CO2And subculturing in a culture bottle at 37 ℃, wherein the adherent cells account for about 70-80% of the bottom area of the culture bottle by the third day, digesting the adherent cells by pancreatin, and subculturing to another culture bottle. BV-2 after frozen and thawed in an ultra-low temperature refrigerator at minus 80 ℃ is taken as the first generation, and BV-2 cells of 3 rd to 8 th generations are selected for experiments.
② process for preparing medicine
All 6 compounds were in powder form and dissolved in DMSO. The stock solution was prepared at a concentration of 100mM and stored at-20 ℃. It was diluted with DMEM medium at the time of use to 100. mu.M, 30. mu.M, 10. mu.M and 1. mu.M in this order. The final concentration of DMSO is less than 1 ‰.
③ Griess method for detecting inhibition of compound to LPS activated microglia
Taking BV2 microglia in logarithmic growth phase, and adjusting the cell density to 0.2X 10 by using fresh DMEM medium containing 10% fetal calf serum5cells/ml, seeded in 96-well plates, 100. mu.l/well, 5% CO at 37 ℃2Culturing in the incubator. And replacing the cells with serum-free fresh culture solution after 24 hours of adherent culture, and simultaneously adding drugs. The 6 compounds were co-administered with LPS at 1,10, 30, 100. mu.M. Blank control was also set. The final concentration of LPS in each administration group was 100 ng/ml. Continuously culturing for 24h after adding medicine into cells, collecting supernatant, and detecting NO in the supernatant by Griess colorimetric method2-And (4) content.
MTT method for detecting influence of compound on survival rate of microglia cell
Taking BV2 microglia cultured in logarithmic growth phase, and adjusting the cell density to 0.2X 10 by using fresh DMEM medium containing 10% fetal calf serum5cells/ml, seeded in 96-well plates, 100. mu.l/well, 5% CO at 37 ℃2Culturing in the incubator. After the cells are cultured for 24 hours adherent, the cells are changed into fresh culture solution, and meanwhile, the cells are treated by adding medicine. The 6 compounds were co-administered with LPS at 1,10, 30, 100. mu.M. Blank control was also set. The final concentration of LPS in each administration group was 100 ng/ml. After adding the drug, the cells were cultured for 24 hours, MTT solution (10. mu.l/well) was added to the cell fluid, the cells were incubated with 0.25mg/ml MTT at 37 ℃ for 3 hours, the culture fluid was aspirated, and 150. mu.l of DMSO solution was added to determine the OD value of the optical density. And (3) processing data, namely processing the data by using corresponding software of a microplate reader, calculating an average value of OD values of 3 holes of each sample, and calculating the cell survival rate (CV%) by using the average value according to the following formula.
Percent cell survival%
Fifthly, statistical method
All data were examined using the SPSS (13.0) statistical software package. Results are expressed as mean ± standard error, and the global differences were evaluated, and the means between groups was analyzed by One-Way ANOVA analysis for homogeneity of variance and by Dunnett's test analysis for comparison between groups. The multiple sample homogeneity of variance test was conducted using a Leven test, where the variances were uniform when p >0.05, the differences in mean among the groups were tested using Dunnett's two-sided T, and the differences in mean among the groups were tested using Dunnett T3 when p <0.05 and the variances were not uniform.
⑥IC50Is calculated by
Calculating IC by nonlinear regression fitting of parameters such as each dosage and inhibition rate50The experimental results are as follows: see Table 4
TABLE 4 microglial activation test results for neoalkaloids 1,2,3,4,5 and 6
Figure BDA0003340719570000131
According to experimental results, the survival rate of the microglia BV2 is not affected by the novel alkaloids 1-6 prepared in examples 1-5, and the compounds 1-6 can inhibit the excessive activated BV2 cells from releasing NO to different degrees, so that the neuroinflammation can be prevented and treated. The new bisbenzylisoquinoline alkaloids 5 and 6 can obviously inhibit the excessively activated BV2 cells from releasing NO, thereby playing roles in preventing and treating neuroinflammation and having more obvious effect.
Example 7 inhibition of A549 cells by Alkaloids 1,2,3,4,5 and 6 prepared in examples 1-5
(1) Principle of experiment
The MTT method is a reaction based on reduction of tetramethyl azozolium (3- (4, 5-dimethylthiozol-2-yl) -2,5-diphenyltetrazolium bromide, MTT) in a matrix. The principle is that succinic dehydrogenase reduces exogenous MTT to water-insoluble blue-violet crystalline Formazan (Formazan) and deposits the Formazan in cells. The number of living cells was indirectly reflected by measuring the absorption of crystal violet at a wavelength of 570nm using a microplate reader. In recent years, the method has been widely applied to screening of a large number of antitumor drugs, cytotoxicity tests and the like.
(2) The experimental method comprises the following steps:
culture of lung cancer cell line A549
And (3) recovering the A549 cell line frozen in the liquid nitrogen, and continuously carrying out passage for 3 times until the cell growth state is good and stable. Three kinds of cells with good growth state are taken and inoculated on different 96-well plates, 2.0 x 10 of each well4A cell. Slowly shaking the inoculated 96-well plate to make the cells in the well uniformly distributed, and placing in 5% CO2Culturing at 37 ℃ for 24h in a constant-temperature incubator until the cells adhere to the wall.
② process for preparing medicine
All 6 compounds were in powder form and dissolved in DMSO. The stock solution was prepared at a concentration of 100mM and stored at-20 ℃.
③ MTT detection compound has inhibition effect on lung cancer cell line A549
Culture medium for stock solutionDilution to gradient concentration: 0. 10, 25, 50 and 100 mu M, sucking out the culture solution in a 96-well plate, adding a sample diluted by the culture solution, adding medicine to A549 cells, and placing in 5% CO2Incubate at 37 ℃ for 48h in a constant temperature incubator.
The incubated cells were incubated with 2mg/ml MTT for 4h at 37 deg.C, the supernatant removed and crystals were dissolved by adding 100. mu.L DMSO per well. The crystals were gently shaken until they were completely dissolved, and the Optical Density (OD) value was measured at 570nm in a microplate reader, and the cell inhibition ratio of each group was calculated with the blank control group (OD) value being 100%. The cell inhibition rate [% 1- (average value of OD value of drug addition/average value of OD value of control group) ] × 100%.
Fourthly, statistical method
All data were examined using the SPSS (13.0) statistical software package. Results are expressed as mean ± standard error, and the global differences were evaluated, and the means between groups was analyzed by One-Way ANOVA analysis for homogeneity of variance and by Dunnett's test analysis for comparison between groups. The multiple sample homogeneity of variance test was conducted using a Leven test, where the variances were uniform when p >0.05, the differences in mean among the groups were tested using Dunnett's two-sided T, and the differences in mean among the groups were tested using Dunnett T3 when p <0.05 and the variances were not uniform.
⑤IC50Is calculated by
Calculating IC by nonlinear regression fitting of parameters such as each dosage and inhibition rate50The experimental results are as follows: see Table 5
TABLE 5A 549 inhibition assay results for novel alkaloids 1,2,3,4,5 and 6
Figure BDA0003340719570000141
Figure BDA0003340719570000151
According to experimental results, the novel alkaloids 1-6 prepared in examples 1-5 do not affect the survival rate of the lung cancer cell line A549, and the compounds 1-6 can inhibit the growth of the A549 cells to different degrees, so that the effects of preventing and treating lung cancer are achieved, wherein the effect of the compound 5 is more obvious.
EXAMPLE 8 inhibition of HepG2 cells by the alkaloids 1,2,3,4,5 and 6 prepared in examples 1-5
(1) Principle of experiment
The MTT method is a reaction based on reduction of tetramethyl azozolium (3- (4, 5-dimethylthiozol-2-yl) -2,5-diphenyltetrazolium bromide, MTT) in a matrix. The principle is that succinic dehydrogenase reduces exogenous MTT to water-insoluble blue-violet crystalline Formazan (Formazan) and deposits the Formazan in cells. The number of living cells was indirectly reflected by measuring the absorption of crystal violet at a wavelength of 570nm using a microplate reader. In recent years, the method has been widely applied to screening of a large number of antitumor drugs, cytotoxicity tests and the like.
(2) The experimental method comprises the following steps:
culture of human liver cancer cell HepG2
And (3) recovering the HepG2 cell line frozen in liquid nitrogen, and continuously passaging for 3 times until the cell growth state is good and stable. Three kinds of cells with good growth state are taken and inoculated on different 96-well plates, 2.0 x 10 of each well4A cell. Slowly shaking the inoculated 96-well plate to make the cells in the well uniformly distributed, and placing in 5% CO2Culturing at 37 ℃ for 24h in a constant-temperature incubator until the cells adhere to the wall.
② process for preparing medicine
The six compounds were all in powder form and dissolved in DMSO. The stock solution was prepared at a concentration of 100mM and stored at-20 ℃.
③ MTT detection compound has the inhibiting effect on human liver cancer cell HepG2
Stock was diluted with medium to gradient concentration: 0. 10, 25, 50 and 100 mu M, sucking out the culture solution in a 96-well plate, adding a sample diluted by the culture solution, adding medicine to HepG2 cells, and placing in 5% CO2Incubate at 37 ℃ for 48h in a constant temperature incubator.
The incubated cells were incubated with 2mg/ml MTT for 4h at 37 deg.C, the supernatant removed and crystals were dissolved by adding 100. mu.L DMSO per well. The crystals were gently shaken until they were completely dissolved, and the Optical Density (OD) value was measured at 570nm in a microplate reader, and the cell inhibition ratio of each group was calculated with the blank control group (OD) value being 100%. The cell inhibition rate [% 1- (average value of OD value of drug addition/average value of OD value of control group) ] × 100%.
Fourthly, statistical method
All data were examined using the SPSS (13.0) statistical software package. Results are expressed as mean ± standard error, and the global differences were evaluated, and the means between groups was analyzed by One-Way ANOVA analysis for homogeneity of variance and by Dunnett's test analysis for comparison between groups. The multiple sample homogeneity of variance test was conducted using a Leven test, where the variances were uniform when p >0.05, the differences in mean among the groups were tested using Dunnett's two-sided T, and the differences in mean among the groups were tested using Dunnett T3 when p <0.05 and the variances were not uniform.
⑤IC50Is calculated by
Calculating IC by nonlinear regression fitting of parameters such as each dosage and inhibition rate50The experimental results are as follows: see Table 6
TABLE 6 results of HepG2 inhibition experiments with the novel alkaloids 1,2,3,4,5 and 6
Figure BDA0003340719570000161
The experimental results show that the novel alkaloids 1-6 prepared in the examples 1-6 have obvious inhibition effect on the growth of HepG2 cells within the range of 1-100 μ M, and have the potential of developing and preventing and treating liver cancer.

Claims (8)

1. Morphinane and bisbenzylisoquinoline alkaloid compounds and pharmaceutically acceptable salts thereof, characterized in that they are one or more of the following structures,
Figure FDA0003340719560000011
2. a process for the preparation of morphinane and bisbenzylisoquinoline alkaloid compounds and pharmaceutically acceptable salts thereof according to claim 1, comprising the steps of:
(1) extracting Stephania chinensis (Stephania cepharantha Hayata) with ethanol or methanol under reflux or ultrasonic extraction, and recovering extractive solution to obtain crude extract;
(2) dissolving the ethanol crude extract obtained in the step (1) by using ethanol, purifying by using macroporous resin column chromatography, carrying out gradient elution by using ethanol-water mixed solvents with different pH values, and recovering to obtain ethanol-water-triethylamine part eluate;
(3) separating the ethanol-water-triethylamine part eluate obtained in the step (2) by silica gel column chromatography, and performing gradient elution by using a mixed solvent of petroleum ether and ethyl acetate, a mixed solvent of petroleum ether and acetone, a mixed solvent of chloroform and acetone, a mixed solvent of dichloromethane and acetone, a mixed solvent of chloroform and methanol or a mixed solvent of dichloromethane and methanol;
(4) separating the flow obtained in the step (3) by ODS chromatography, and carrying out gradient elution by using a mixed solvent of methanol and water or a mixed solvent of acetonitrile and water as a mobile phase;
(5) and (3) further separating the fractions of methanol and water in a ratio of 3: 7-9: 1 and acetonitrile and water in a ratio of 1: 9-5: 5 obtained in the step (4) by preparative HPLC-UV chromatography, and eluting by using a mixed solvent of methanol and water or a mixed solvent of acetonitrile and water as a mobile phase to obtain alkaloids 1,2,3 and 4.
(6) Eluting the fractions obtained in the step (4) by using a mixed solvent of methanol and water with a ratio of 3: 7-6: 4, or a mixed solvent of acetonitrile and water with a ratio of 1: 9-4: 6 as a mobile phase to obtain alkaloids 5 and 6.
3. The method of claim 2, wherein: the extraction method in the step (1) is heating reflux extraction or heating ultrasonic extraction for 1-4 times, the volume concentration of methanol or ethanol is 50-100%, and the volume concentration of stephanotis is as follows: the weight-volume ratio of the methanol to the ethanol is 1: 5-1: 15 g/mL; the macroporous resin adsorption elution method in the step (2) adopts ethanol to dissolve the crude extract, samples are mixed according to the mass ratio of the macroporous resin to the crude extract of 3: 1-6: 1, after the samples are dried at room temperature, ethanol-water-formic acid and ethanol-water-triethylamine in the volume ratio of 20:70: 10-40: 55:5 and 60:35: 5-80: 10:10 are respectively used for eluting for 8-12 column volumes in sequence, and partial eluates of the ethanol-water-triethylamine are recovered under reduced pressure.
4. The method of claim 2, wherein: the volume ratio of the mixed solvent of the eluting solvent petroleum ether and ethyl acetate to the mixed solvent of the petroleum ether and acetone in the step (3) is 100: 10-1: 1, and the volume ratio of the mixed solvent of dichloromethane and acetone, the mixed solvent of chloroform and acetone, the mixed solvent of dichloromethane and methanol or the mixed solvent of chloroform and methanol is 100: 1-100: 15.
5. the method of claim 2, wherein: the mixed solvent of methanol and water in the step (4) is 1: 9-9: 1, the volume ratio of the acetonitrile-water mixed solvent is 1: 9-8: 2.
6. the method of claim 2, wherein: the mixed solvent of methanol and water, and the mixed solvent of acetonitrile and water in the step (5), wherein the volume ratio of the mixed solvent of methanol and water is 3: 7-8: 2, the volume ratio of the acetonitrile-water mixed solvent is 1: 9-5: 5.
7. a pharmaceutical composition comprising morphinane and bisbenzylisoquinoline alkaloid compounds of claim 1, and pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier.
8. Use of morphinane and bisbenzylisoquinoline alkaloid compounds of claim 1 and pharmaceutically acceptable salts thereof or the pharmaceutical composition of claim 7 for the preparation of a medicament for the prevention or treatment of neurodegenerative diseases or cancer.
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