CN101284792A - Bisbenzylisoquinoline compounds, preparation method and applications - Google Patents
Bisbenzylisoquinoline compounds, preparation method and applications Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- BJWWOUUGCAPHOV-UHFFFAOYSA-N 1,3-dibenzylisoquinoline Chemical class C=1C2=CC=CC=C2C(CC=2C=CC=CC=2)=NC=1CC1=CC=CC=C1 BJWWOUUGCAPHOV-UHFFFAOYSA-N 0.000 title 1
- 150000001875 compounds Chemical class 0.000 claims abstract description 50
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 28
- 229930013930 alkaloid Natural products 0.000 claims abstract description 25
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 20
- 239000002904 solvent Substances 0.000 claims abstract description 20
- 238000000605 extraction Methods 0.000 claims abstract description 13
- 239000012043 crude product Substances 0.000 claims abstract description 12
- 239000003960 organic solvent Substances 0.000 claims abstract description 8
- 239000002246 antineoplastic agent Substances 0.000 claims abstract description 6
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 claims abstract description 6
- 238000010828 elution Methods 0.000 claims abstract description 6
- 150000003797 alkaloid derivatives Chemical class 0.000 claims abstract description 5
- 229940041181 antineoplastic drug Drugs 0.000 claims abstract description 4
- 239000002253 acid Substances 0.000 claims abstract description 3
- 230000007935 neutral effect Effects 0.000 claims abstract description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 48
- 235000008671 Calycanthus floridus Nutrition 0.000 claims description 25
- 235000008670 Calycanthus occidentalis Nutrition 0.000 claims description 25
- 244000148992 Lindera benzoin Species 0.000 claims description 25
- 235000004520 Lindera benzoin Nutrition 0.000 claims description 25
- 235000008262 Lindera benzoin var. benzoin Nutrition 0.000 claims description 25
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 20
- 239000000284 extract Substances 0.000 claims description 14
- 239000012141 concentrate Substances 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 10
- 239000003814 drug Substances 0.000 claims description 9
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 8
- 230000002829 reductive effect Effects 0.000 claims description 8
- 239000000741 silica gel Substances 0.000 claims description 8
- 229910002027 silica gel Inorganic materials 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 7
- 238000010992 reflux Methods 0.000 claims description 6
- 230000001105 regulatory effect Effects 0.000 claims description 6
- 238000001556 precipitation Methods 0.000 claims description 5
- 238000007670 refining Methods 0.000 claims description 5
- 238000010898 silica gel chromatography Methods 0.000 claims description 5
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 4
- 238000000638 solvent extraction Methods 0.000 claims description 4
- 238000002347 injection Methods 0.000 claims description 3
- 239000007924 injection Substances 0.000 claims description 3
- 239000000463 material Substances 0.000 claims description 3
- 229910052500 inorganic mineral Inorganic materials 0.000 claims description 2
- 239000011707 mineral Substances 0.000 claims description 2
- 239000002552 dosage form Substances 0.000 claims 1
- 239000000546 pharmaceutical excipient Substances 0.000 claims 1
- 229940124531 pharmaceutical excipient Drugs 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 9
- 210000004881 tumor cell Anatomy 0.000 abstract description 7
- 238000002474 experimental method Methods 0.000 abstract description 6
- 238000000034 method Methods 0.000 abstract description 3
- 241000146029 Lindera aggregata Species 0.000 abstract 2
- 230000001476 alcoholic effect Effects 0.000 abstract 1
- -1 bisbenzylisoquinoline compound Chemical class 0.000 abstract 1
- 238000009833 condensation Methods 0.000 abstract 1
- 230000005494 condensation Effects 0.000 abstract 1
- 230000005764 inhibitory process Effects 0.000 abstract 1
- 239000002994 raw material Substances 0.000 abstract 1
- 238000011084 recovery Methods 0.000 abstract 1
- 238000004062 sedimentation Methods 0.000 abstract 1
- 239000003440 toxic substance Substances 0.000 abstract 1
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 12
- 210000004027 cell Anatomy 0.000 description 10
- HJKAMHCROYZVKL-UHFFFAOYSA-N linderegatine Natural products COc1ccc(CC2NCCc3cc(OC)c(O)cc23)cc1Oc4ccc(cc4)C(=O)C5=NCCc6cc(OC)c(O)cc56 HJKAMHCROYZVKL-UHFFFAOYSA-N 0.000 description 8
- LZJRNLRASBVRRX-ZDUSSCGKSA-N Boldine Chemical compound CN1CCC2=CC(O)=C(OC)C3=C2[C@@H]1CC1=C3C=C(OC)C(O)=C1 LZJRNLRASBVRRX-ZDUSSCGKSA-N 0.000 description 6
- 238000003810 ethyl acetate extraction Methods 0.000 description 6
- KYVJVURXKAZJRK-UHFFFAOYSA-N (+)-laurolitsine Natural products N1CCC2=CC(O)=C(OC)C3=C2C1CC1=C3C=C(OC)C(O)=C1 KYVJVURXKAZJRK-UHFFFAOYSA-N 0.000 description 5
- BHLYRWXGMIUIHG-HNNXBMFYSA-N (S)-reticuline Chemical compound C1=C(O)C(OC)=CC=C1C[C@H]1C2=CC(O)=C(OC)C=C2CCN1C BHLYRWXGMIUIHG-HNNXBMFYSA-N 0.000 description 5
- 230000001472 cytotoxic effect Effects 0.000 description 5
- TWNQGVIAIRXVLR-UHFFFAOYSA-N oxo(oxoalumanyloxy)alumane Chemical compound O=[Al]O[Al]=O TWNQGVIAIRXVLR-UHFFFAOYSA-N 0.000 description 5
- 238000001035 drying Methods 0.000 description 4
- 238000002329 infrared spectrum Methods 0.000 description 4
- 230000002107 myocardial effect Effects 0.000 description 4
- 239000012071 phase Substances 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- 230000001988 toxicity Effects 0.000 description 4
- 231100000419 toxicity Toxicity 0.000 description 4
- 238000002211 ultraviolet spectrum Methods 0.000 description 4
- 229920002472 Starch Polymers 0.000 description 3
- 239000003513 alkali Substances 0.000 description 3
- 230000000259 anti-tumor effect Effects 0.000 description 3
- MHVCSDLBQKSFQV-HNNXBMFYSA-N boldine Natural products COc1cc2c(C[C@@H]3N(C)CCc4cc(C)c(OC)c2c34)cc1O MHVCSDLBQKSFQV-HNNXBMFYSA-N 0.000 description 3
- LZJRNLRASBVRRX-UHFFFAOYSA-N boldine trifluoroacetic acid salt Natural products CN1CCC2=CC(O)=C(OC)C3=C2C1CC1=C3C=C(OC)C(O)=C1 LZJRNLRASBVRRX-UHFFFAOYSA-N 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- 229930005325 imidazole alkaloid Natural products 0.000 description 3
- 150000002460 imidazoles Chemical class 0.000 description 3
- 239000004615 ingredient Substances 0.000 description 3
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- RUPUUZZJJXCDHS-UHFFFAOYSA-N (+)-orientaline Natural products C1=C(O)C(OC)=CC(CC2C3=CC(O)=C(OC)C=C3CCN2C)=C1 RUPUUZZJJXCDHS-UHFFFAOYSA-N 0.000 description 2
- 235000011114 ammonium hydroxide Nutrition 0.000 description 2
- 238000000119 electrospray ionisation mass spectrum Methods 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- KYVJVURXKAZJRK-LBPRGKRZSA-N laurolistine Chemical compound N1CCC2=CC(O)=C(OC)C3=C2[C@@H]1CC1=C3C=C(OC)C(O)=C1 KYVJVURXKAZJRK-LBPRGKRZSA-N 0.000 description 2
- 150000002611 lead compounds Chemical class 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 150000003810 morphinanes Chemical class 0.000 description 2
- 210000004165 myocardium Anatomy 0.000 description 2
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- BNUZUOWRDKPBQR-UHFFFAOYSA-N reticuline Natural products CN1CCC2=CC(OC)=CC=C2C1CC1=CC=C(OC)C(O)=C1 BNUZUOWRDKPBQR-UHFFFAOYSA-N 0.000 description 2
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- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- 238000005084 2D-nuclear magnetic resonance Methods 0.000 description 1
- JGSARLDLIJGVTE-UHFFFAOYSA-N 3,3-dimethyl-7-oxo-6-[(2-phenylacetyl)amino]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid Chemical compound O=C1N2C(C(O)=O)C(C)(C)SC2C1NC(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-UHFFFAOYSA-N 0.000 description 1
- 244000080208 Canella winterana Species 0.000 description 1
- 235000008499 Canella winterana Nutrition 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 208000007342 Diabetic Nephropathies Diseases 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- QAAIWYNFNWDEAF-UHFFFAOYSA-N Laurolitsine Natural products COc1c(O)cc2CCNC3Cc4cc(O)ccc4c1c23 QAAIWYNFNWDEAF-UHFFFAOYSA-N 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
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- 229910052799 carbon Inorganic materials 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
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- 150000002596 lactones Chemical class 0.000 description 1
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- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 description 1
- 239000012982 microporous membrane Substances 0.000 description 1
- INAXVFBXDYWQFN-XHSDSOJGSA-N morphinan Chemical group C1C2=CC=CC=C2[C@]23CCCC[C@H]3[C@@H]1NCC2 INAXVFBXDYWQFN-XHSDSOJGSA-N 0.000 description 1
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Abstract
The invention provides a bisbenzylisoquinoline compound in lindera aggregate and the preparation method thereof. The lindera aggregate dried root is used as the raw material and is extracted by using an alcoholic solvent, the extractant is produced into extracturm through decompressing concentration, after the extracturm is dissolved and suspended by water and acidified by dilute acid, the neutral component of the extracturm is removed by extraction with an organic solvent, the water phase is adjusted to the pH value of 10 by weak base, extraction is performed by the organic solvent after solvent recovery, the total alkaloid crude product is obtained, fine alkaloid crude is prepared after the crude product is resolved in alcohol and sedimentation process is performed by adding an amount of water, eluting parts are colleted after the chloroform-carbinol system elution and the water-carbinol gradient elution, and the compound can be prepared after removing the solvent by condensation. The compound is used for performing activity inhibition experiments to L1210 and K562 tumor cell strains, results show that both L1210 and K562 tumor cell strains have strong cell-toxicant activity and present dose-effect relationships. The compound can be applied to the preparation of anticancer drugs, and has the above structural formula.
Description
Technical field
The invention belongs to medical technical field, being specifically related to a kind of is the method and the application in the preparation antitumor drug thereof of a kind of new imidazole alkaloid of feedstock production with the Chinese medicine root of three-nerved spicebush.
Background technology
The root of three-nerved spicebush is traditional conventional Chinese medicine, is one of important warm stomach medicines for regulating qi and alleviating pain, has the effect of warming spleen and stomach for dispelling cold, regulating QI to relieve pain.Modern study is found the complex chemical composition of the root of three-nerved spicebush; contain abundant furans sesquiterpene and lactone thereof, flavones, volatile oil, morphinane alkaloid etc., have antiviral, antibacterial, antitumor, regulate digestive tube, excited cardiac muscle, improve central nervous system function, relieving inflammation and relaxing pain, control diabetic nephropathy, protection liver, regulate pharmacological actions such as coagulation function.Contained alkaloid mostly is morphinane alkaloid in the root of three-nerved spicebush, laurolitsine (laureolistine) wherein, boldine (boldine) and d-Reticuline (reticuline) are three kinds of main morphinane alkaloids, ubiquity in canella (chemical ingredients of the root of three-nerved spicebush and pharmacological action. Wang Zhengtao, Xu Luoshan. Chinese wild plant resource, 1999,18 (3), 5-10.).Modern pharmacological research shows: Radix Linderae extract is obvious to murine sarcoma S180 restraining effect.But the Radix Linderae extract inducing mouse produces cell growth inhibitory factor, suppress tumor growth, prolong the lifetime of suffering from lung cancer in mice, and normal cell is not shown any toxicity, curative effect and dosage positive correlation (plant of the root of three-nerved spicebush and pharmacological research overview. Wang Junwei, Ruan Bing. Zhejiang Journal of Traditional Chinese Medicine, 2006,41 (11), 675-677.).But do not find to contain in the root of three-nerved spicebush bisbenzylisoquinoline alkaloid at present as yet, more do not see such alkaloidal antitumor cell cytotoxic activity report in the root of three-nerved spicebush.
Summary of the invention
The purpose of this invention is to provide the Bisbenzylisoquinolincompounds compounds that extracts in a kind of root of three-nerved spicebush (Linderegatine I), has following structural formula:
Second purpose of the present invention provides the preparation method of Bisbenzylisoquinolincompounds compounds (I), realize by following steps: root of three-nerved spicebush dried root is thinly sliced, pulverize the back and press medicinal material/weight of solvent/10 times of amount alcohol of volume ratio adding, room temperature lixiviate or refluxing extraction 2-3 time, more shallow to the extract color.United extraction liquid, concentrating under reduced pressure gets medicinal extract, with suitable quantity of water this medicinal extract suspendible is dissolved, to remove the non-alkaloid composition with the Semi-polarity organic solvent extraction after the mineral acid acidifying, water is regulated pH to 10 with weak base again, with Semi-polarity organic solvent extraction three times, organic solvent layer water backwash again obtains the crude product of total alkaloids to neutral after solvent recuperation; This crude product is dissolved with an amount of alcohol, adding water again keeps pure content≤60% to make precipitation fully, centrifugal or filter after promptly get and make with extra care total alkaloids, get refining root of three-nerved spicebush total alkaloids, with silica gel mixed sample, add the chromatographic column top that silica gel is housed, with the chloroform-methanol solvent systems from 50: 1 → 25: 1 wash-outs, collect chloroform: 25: 1 polarity sections of methyl alcohol wash-out part, concentrate to remove desolvate after through anti-phase C-18 silica gel chromatography, from 80: 20 → 40: 60 gradient elutions, collect water with the water-methanol solvent systems: 40: 60 wash-out parts of methyl alcohol concentrate except that getting the purpose Compound I after desolvating.
Another object of the present invention provides the application of Bisbenzylisoquinolincompounds compounds in the preparation antitumor drug stronger cytotoxic activity.
Be that anoxic reoxygenation model discrimination is found when having the medicine of myocardium protecting action with H9C2 myocardial cell; total alkaloids can increase the survival rate of cell and reduce the activity of cell LDH under the low concentration of 10,30 μ g/ml, but under the higher concentration of 90 μ g/ml the myocardial cell is had certain toxicity.Therefore infer that the trace ingredients in the total alkaloids may have stronger cytotoxic activity.Subsequently research emphasis is placed on the acquisition of trace active composition.Under activity experiment instructs, obtained Compound I through multistage chromatographic separation (comprising silica gel, aluminum oxide, reverse phase silica gel and gel chromatography), by multiple spectroscopic technique test, comprise UV spectrum (UV), infrared spectra (IR), mass spectrum (MS), nucleus magnetic resonance (NMR), the conclusive evidence structure is the imidazole alkaloid that contains highly unsaturated conjugated system after spectral information is carried out detailed parsing.This Compound I is mixed with 90 μ M/L, and 30 μ M/L and three concentration of 10 μ M/L are to L1210, and the K562 tumor cell line suppresses activity experiment, and the result shows that three concentration all have stronger cytotoxic activity, and presents dose-effect relationship.
Usefulness of the present invention is:
(1) from the root of three-nerved spicebush, finds imidazole alkaloid first, the preparation method and the spectral data of Compound I is provided, lay a good foundation for continuing from the root of three-nerved spicebush, to find this compounds of novel structure from now on highly unsaturated conjugated system;
(2) find that first Compound I has strong restraining effect to tumour cell, Compound I is carried out structure activity study, after the area of computer aided appropriate design, might obtain active stronger antitumor lead compound or medicine by chemosynthesis;
(3) Compound I does not almost have toxicity tumour cell being produced under the concentration of strong inhibitory or killing effect to normal cell, points out this compound to have and is developed to the anti-cancer agent prospect preferably.
Description of drawings
Fig. 1 is the high-efficient liquid phase chromatogram of root of three-nerved spicebush total alkaloids.
Embodiment
The present invention is further described with accompanying drawing in conjunction with specific embodiments.
The physics and chemistry of embodiment 1 Compound I and UV, IR, MS spectral data are as follows:
Compound I is a light yellow solid, [α]
D 20-16 ° (c 1.0, MeOH); Electrospray ionization mass spectrum (ESIMS) (positive ion type positive) m/z 595[M+H]
+(100); (negative ion type negative) m/z 593 (100); UV (MeOH) λ
MaxNm (log ε): 207 (5.30), 288 (4.87); IR (KBr) v
MaxCm
-13405,2937,2842,1664,1588,1510,1450,1371,1276,1228,1135,1028,756.
Compound I
1H NMR,
13C NMR data and ownership are referring to table 1, and the ownership of each hydrogen, carbon signal obtains by test two dimensional NMR spectrum (the relevant spectrum of carbon-hydrogen, the long-range relevant spectrum of carbon-hydrogen, nuclear Overhauser effect two-dimensional correlation spectrum).
Table 1. Compound I
1H (400MHz) and
13C (100MHz) NMR data and signal ownership (CD
3The OD solvent)
| No | δ H(multi，J in Hz) | δ C | No | δ H(multi，J in Hz) | δ C |
| 1 | 167.8 | 1′ | 4.45(dd，6.1，8.0) | 57.3 | |
| 3 | 3.79(2H，m) | 47.8 | 3′ | a 3.37(1H，m) b 3.15(1H，m) | 40.6 |
| 4 | 2.83(2H，m) | 26.0 | 4′ | 2.89(2H，m) | 26.9 |
| 4a | 131.4 | 4′a | 124.6 | ||
| 5 | 6.90(1H，s) | 111.9 | 5′ | 6.68(1H，s) | 112.6 |
| 6 | 152.6 | 6′ | 148.8 | ||
| 7 | 146.4 | 7′ | 146.2 | ||
| 8 | 6.67(1H，s) | 114.4 | 8′ | 6.48(1H，s) | 114.4 |
| 8a | 120.3 | 8a′ | 126.4 | ||
| α | 194.4 | α′ | a 3.25(1H，dd，14.0，6.1) b 3.03(1H，dd，14.0，8.0) | 40.7 | |
| 9 | 130.3 | 9′ | 130.8 | ||
| 10，14 | 7.91(2H，d，8.8) | 133.6 | 10′ | 6.93(1H，s) | 124.8 |
| 11，13 | 6.91(2H，d，overlap) | 117.0 | 11′ | 144.0 | |
| 12 | 165.0 | 12′ | 152.3 | ||
| 6-OCH 3 | 3.90(3H，s) | 56.5 | 13′ | 7.13(1H，d，8.4) | 114.6 |
| 14′ | 7.20(1H，d，8.3) | 128.7 | |||
| 6′-OCH 3 | 3.78(3H，s) | 56.3 | |||
| 12′-OCH 3 | 3.74(3H，s) | 56.3 |
Root of three-nerved spicebush 5kg dried root is thinly sliced, and pulverizes the back and adds 50L ethanol, and diacolation extracts secondary, 7 days for the first time, adds 50L ethanol behind the filtrate collection and continues diacolation 5 days.Concentrating under reduced pressure gets medicinal extract after merging extracted twice liquid, with 10L water this medicinal extract suspendible is dissolved, with 2N HNO
3Use equal-volume ethyl acetate extraction three times after the acidifying, ethyl acetate layer discards after reclaiming solvent.Water is used 5%Na again
2CO
3Regulate pH to 10, with equal-volume ethyl acetate extraction secondary, the combined ethyl acetate extraction liquid is washed till neutrality with distilled water, obtains the crude product of 32.3g total alkaloids after the reclaim under reduced pressure ethyl acetate.With the 3L dissolve with ethanol, adding water again, to make ethanol content be 60% with this crude product, makes precipitation fully after leaving standstill, and filters, promptly get 12.8g after the drying and make with extra care total alkaloids.
Get above-mentioned refining root of three-nerved spicebush total alkaloids, with the 15g silica gel mixed sample, add the chromatographic column top that 600g silica gel is housed, with the chloroform-methanol solvent systems from 50: 1 → 25: 1 wash-outs, collect chloroform: 25: 1 polarity sections of methyl alcohol wash-out part, concentrate remove desolvate after through anti-phase C-18 silica gel chromatography, with the water-methanol solvent systems from 80: 20 → 40: 60 gradient elutions, collect water: 40: 60 wash-out parts of methyl alcohol concentrate except that getting the 316mg Compound I after desolvating.
Root of three-nerved spicebush 3kg dried root is thinly sliced, and pulverizes the back and adds 30L methyl alcohol, and the refluxing extraction secondary 2 hours for the first time, is added 24L methyl alcohol and continued refluxing extraction 1 hour behind the extracting liquid filtering.Concentrating under reduced pressure gets medicinal extract after merging extracted twice liquid, and with this medicinal extract suspendible dissolving, to use equal-volume chloroform extraction three times after the 3N HCl acidifying, chloroform layer discards after reclaiming solvent with 6L water.Water is regulated pH to 10 with ammoniacal liquor again, and with equal-volume chloroform extraction secondary, the combined chloroform extraction liquid is washed till neutrality with distilled water, obtains the crude product of 21.7g total alkaloids behind the reclaim under reduced pressure chloroform.With the 3L dissolve with methanol, adding water again, to make methanol content be 50% with this crude product, makes precipitation fully after leaving standstill, and filters, promptly get 8.6g after the drying and make with extra care total alkaloids.
Get above-mentioned refining root of three-nerved spicebush total alkaloids, mix sample with the 10g aluminum oxide, add the chromatographic column top that the 260g aluminum oxide is housed, with the chloroform-methanol solvent systems from 50: 1 → 30: 1 wash-outs, collect chloroform: 30: 1 polarity sections of methyl alcohol wash-out part, concentrate remove desolvate after through gel Sephadex LH-20 column chromatography, be eluent with methyl alcohol, TLC detects the stream part that obtains to contain Compound I, concentrates except that after desolvating and obtains the 198mg Compound I.
Higher through the root of three-nerved spicebush total alkaloids purity that embodiment 1-3 method obtains, mainly contain three kinds of compositions, the high-efficient liquid phase chromatogram HPLC normalization method records three compound (compound as, b, c) content sum surpasses 85%, contain a trace ingredients Compound I in addition, referring to accompanying drawing 1, wherein 1 is compound a (norboldine); 2: compound b (boldine); 3: compound c (reticuline); 4: Compound I (linderegatine), structural formula is as follows:
Compound a compound b compound c
The pilot scale preparation of embodiment 4 Compound I
Root of three-nerved spicebush 100kg dried root is thinly sliced, and pulverizes the back and adds 1000L ethanol, and the refluxing extraction secondary 2 hours for the first time, is added 800L ethanol and continued refluxing extraction 1 hour behind the extracting liquid filtering.Concentrating under reduced pressure gets medicinal extract after merging extracted twice liquid, and with this medicinal extract suspendible dissolving, to use equal-volume ethyl acetate extraction three times after the 2N HCl acidifying, ethyl acetate layer discards after reclaiming solvent with 200L water.Water is regulated pH to 10 with ammoniacal liquor again, and with equal-volume ethyl acetate extraction secondary, the combined ethyl acetate extraction liquid is washed till neutrality with distilled water, obtains the crude product of 766g total alkaloids after the reclaim under reduced pressure ethyl acetate.With 80L alcohol dissolving, adding water again, to make ethanol content be 40%, makes precipitation fully after leaving standstill with this crude product, filters, promptly get 362g after the drying and make with extra care total alkaloids.Get above-mentioned refining root of three-nerved spicebush total alkaloids, mix sample with the 400g aluminum oxide, add the chromatographic column top that the 8kg aluminum oxide is housed, with the chloroform-methanol solvent systems from 50: 1 → 30: 1 wash-outs, collect chloroform: 30: 1 polarity sections of methyl alcohol wash-out part, concentrate to remove desolvate after through anti-phase C-18 silica gel chromatography, with the water-methanol solvent systems from 80: 20 → 40: 60 gradient elutions, collect water: 40: 60 wash-out parts of methyl alcohol, TLC detects the stream part that obtains to contain Compound I, merges to concentrate except that after desolvating to obtain the 7.9g Compound I.
The pharmacology activity research of embodiment 5 root of three-nerved spicebush total alkaloidss and Compound I
5.1. experiment material
L1210, K562 tumor cell line, PAA serum, H-DMEM
5.2. experimental technique
5.2.1. medicine preparation: root of three-nerved spicebush total alkaloids (LAA) is mixed with 90 μ g/ml, 30 μ g/ml and three concentration of 10 μ g/ml, Compound I (Linderegatine) is mixed with 90 μ M/L, 30 μ M/L and three concentration of 10 μ M/L.
5.2.2. with H9C2, L1210, the K562 cell strain is inoculated in 96 orifice plates by 10000 cells/well, adds the medicine of various concentration, cultivates after 48 hours, hatches 4 hours with MTT, detects cell survival rate.
5.2.3. statistical method: all data are carried out statistical study with t-text.
5.3. the result is referring to table 2.
Table 2. root of three-nerved spicebush total alkali and Linderegatine cytotoxic activity test data
Pass through cellulotoxic experiment, root of three-nerved spicebush total alkali produces certain toxicity to the myocardial cell under the higher concentration of 90 μ g/ml as can be known, and Compound I all has very strong cytotoxic activity to two strain tumour cell L1210 and K562 under the low concentration of 10,30 μ M, and under the concentration of 10 μ M, normal myocardial cell is not almost had toxicity, point out this compound to have the antitumor value of potential, be expected to be developed to anti-cancer agent or research and develop as lead compound.
The preparation of embodiment 6. Compound I (Linderegatine) preparation
6.1. make tablet
Get Compound I (Linderegatine) 3.0g and starch 10g mixing, add 10% starch slurry 3g and make softwood, add Magnesium Stearate 0.3g, be pressed into 100 behind the dry starch 2g mixing, promptly.Every contains root of three-nerved spicebush total alkali 30mg.
6.2. make injection
Get Compound I (Linderegatine) 1.0g, add 10g N.F,USP MANNITOL, add the injection water to the 1000ml heating for dissolving, every bottle of 2ml is sub-packed in the cillin bottle of 5ml behind 0.22 μ m filtering with microporous membrane, every bottle contains Linderegatine 2.0mg, put into the cold moving drying machine freeze-drying of vacuum, roll lid pack after the taking-up promptly.
Claims (5)
1. the preparation method of a Bisbenzylisoquinolincompounds compounds is characterized in that having following structural formula:
2. the preparation method of a Bisbenzylisoquinolincompounds compounds, it is characterized in that realizing by following steps: root of three-nerved spicebush dried root is thinly sliced, pulverize the back and press medicinal material/weight of solvent/10 times of amount alcohol of volume ratio adding, room temperature lixiviate or refluxing extraction 2-3 time, more shallow to the extract color.United extraction liquid, concentrating under reduced pressure gets medicinal extract, with suitable quantity of water this medicinal extract suspendible is dissolved, to remove the non-alkaloid composition with the Semi-polarity organic solvent extraction after the mineral acid acidifying, water is regulated pH to 10 with weak base again, with Semi-polarity organic solvent extraction three times, organic solvent layer water backwash again obtains the crude product of total alkaloids to neutral after solvent recuperation; This crude product is dissolved with an amount of alcohol, adding water again keeps pure content≤60% to make precipitation fully, centrifugal or filter after promptly get and make with extra care total alkaloids, get refining root of three-nerved spicebush total alkaloids, with silica gel mixed sample, add the chromatographic column top that silica gel is housed, with the chloroform-methanol solvent systems from 50: 1 → 25: 1 wash-outs, collect chloroform: 25: 1 polarity sections of methyl alcohol wash-out part, concentrate to remove desolvate after through anti-phase C-18 silica gel chromatography, from 80: 20 → 40: 60 gradient elutions, collect water with the water-methanol solvent systems: 40: 60 wash-out parts of methyl alcohol concentrate except that getting the purpose Compound I after desolvating.
3. the application of the described Bisbenzylisoquinolincompounds compounds of claim 1 in the preparation antitumor drug.
4. the application of Bisbenzylisoquinolincompounds compounds according to claim 2 is characterized in that: the pharmaceutical excipient that described Bisbenzylisoquinolincompounds compounds and preparation allow is prepared into medicine.
5. the application of Bisbenzylisoquinolincompounds compounds according to claim 3 is characterized in that: the dosage form of described medicine is injection or tablet.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101463074B (en) * | 2009-01-22 | 2011-06-29 | 厦门大学 | Method for synthesizing cell toxic alkaloid and epimer thereof |
| CN102399186A (en) * | 2011-12-15 | 2012-04-04 | 成都普思生物科技有限公司 | Method for separating and purifying norisoboldine monomer |
| CN111606917A (en) * | 2020-06-02 | 2020-09-01 | 杭州济蒿医药科技有限公司 | Abietane compound with C-ring-fused lactone ring novel skeleton and preparation method and application thereof |
| CN113214154A (en) * | 2020-02-04 | 2021-08-06 | 中国医学科学院药物研究所 | Trimethylbenzyl isoquinoline alkaloid, preparation method thereof, pharmaceutical composition and application |
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2008
- 2008-05-23 CN CN2008100616889A patent/CN101284792B/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101463074B (en) * | 2009-01-22 | 2011-06-29 | 厦门大学 | Method for synthesizing cell toxic alkaloid and epimer thereof |
| CN102399186A (en) * | 2011-12-15 | 2012-04-04 | 成都普思生物科技有限公司 | Method for separating and purifying norisoboldine monomer |
| CN102399186B (en) * | 2011-12-15 | 2013-09-25 | 成都普思生物科技有限公司 | Method for separating and purifying norisoboldine monomer |
| CN113214154A (en) * | 2020-02-04 | 2021-08-06 | 中国医学科学院药物研究所 | Trimethylbenzyl isoquinoline alkaloid, preparation method thereof, pharmaceutical composition and application |
| CN113214154B (en) * | 2020-02-04 | 2023-10-20 | 中国医学科学院药物研究所 | Tribenzyl isoquinoline alkaloid, preparation method, pharmaceutical composition and application thereof |
| CN111606917A (en) * | 2020-06-02 | 2020-09-01 | 杭州济蒿医药科技有限公司 | Abietane compound with C-ring-fused lactone ring novel skeleton and preparation method and application thereof |
| CN111606917B (en) * | 2020-06-02 | 2021-10-01 | 杭州济蒿医药科技有限公司 | Abietane compound with C-ring-fused lactone ring novel skeleton and preparation method and application thereof |
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