CN101012227A - Novel 13-n-octylberberine derivative with antineoplastic action - Google Patents
Novel 13-n-octylberberine derivative with antineoplastic action Download PDFInfo
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- CN101012227A CN101012227A CN 200710019854 CN200710019854A CN101012227A CN 101012227 A CN101012227 A CN 101012227A CN 200710019854 CN200710019854 CN 200710019854 CN 200710019854 A CN200710019854 A CN 200710019854A CN 101012227 A CN101012227 A CN 101012227A
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Abstract
The invention discloses a new synthesizing method of 13-n-octyl berberine (1) and 13-n-octyl palmatine (2) as berberine derivant and medical acceptable salt and liposome, which is characterized by the following: inhibiting the growth of cancer cell obviously through S180 transplanted tumour model test under lower dose condition; saving cost.
Description
Technical field
The present invention relates to is the new berberinc derivate of the synthetic acquisition of raw material with Berberine and palmatine respectively, be 13-just-octyl berberine (1) and 13-just-octyl group palmatine (2), and pharmacy acceptable salt class and liposome have more notable antitumor activity, and their structural formula is as shown in the formula expression:
Technical background
Berberine (berberine) claim berberine again, and the main component for extracting in the plant roots and stems such as the Ranunculaceae coptis plant coptis, golden cypress, Radix Berberidis belongs to isoquinoline alkaloid.Berberine hydrochloride (also claim: the Berberine muriate claims traditionally: Berberine) be quaternary ammonium salt compound, the nations of China and India as the history in existing more than 3000 year of treatment digestive tract diseases medication (orchid advances, Yang Shilin, Zheng Yuquan, etc.; The progress of the coptis, herbal medicine, 2001,32 (2): 1139～1141), record in two ones of Pharmacopoeias of People's Republic of China.It is not normal etc. also to be used for the cardiovascular diseases such as the rhythm of the heart clinically, and its purposes is wider.But report (Wang Yusheng, Deng Wenlong, Xue Chunsheng as far back as existing berberine hydrochloride anticancer 50～sixties; Herbal pharmacology and application, second edition, Beijing: People's Health Publisher, 1995,1011), so far, studies show that:
(1) berberine hydrochloride all has restraining effect (Li XK, Motwani M, Tong W, et al to the growth and the propagation of many tumour cells; Huanglia, a Chinese herbal extract, inhibits cell growth by suppressing the expression ofcyclin B1 and inhibiting CDC2 kinase activity in human cancer cells, Mol Pharmacol, 2000,58 (6): 1287～1293; Inoue K, Kulsum U, Chowdhury SA, et.al; .Tumor-specific cytotoxicity andapoptosis-inducing activity of berberines.Anticancer Res.2005; 25 (6B): 4053-9; Jiang Yan, Hu Qun, etc.; Berberine is induced the acute T lymphocytic leukemia cell of people Study of apoptosis, Chinese Journal of New Drugs, 2004,13 (9): 796～799), as liver cancer, cancer of the stomach, colorectal carcinoma, the esophageal carcinoma, lung cancer, leukemia and bladder cancer etc., and be the dependency of action time and dosage; Berberine hydrochloride also has opposing people or mouse hepatocellular carcinoma cells (Lin H, Liu TY, Lui WY, et al; Up-regulationof multidrug resistance transporter expression by berberine in human and murine hepatoma cells, Cancer, 1999,1937～1942), the transferance (Lou Jinli of stomach cancer cell 85 (9):, Qiu Quanying, imitative elegant beautiful, etc.; The influence that Berberine is expressed gastric carcinoma cells propagation, cell cycle and CD44V6, Chinese Journal of Immunology, 2004,20 (5): 315～317);
(2) berberine hydrochloride can produce restraining effect to the growth of tumour cell, propagation by number of mechanisms, and promotes apoptosis (Hwang JM, Kuo HC, Tseng TH, the et.al of tumour cell; Berberine induces apoptosis through amitochondria/caspases pathway in human hepatoma cells.Arch Toxicol, 2006,80:62～73), with the performance antitumor action.The example hydrochloric acid Berberine not only can suppress tumour cell cyclin B by selectivity to the influence in growth of tumour cell cycle
1, do not influence cyclin A or E, cause the inhibition of CDK2 kinase activity, make tumour cell rest on G
2Phase; By inducing scavenger cell to produce active oxygen, produce the macrophage activating factor (MAF) lipopolysaccharides simultaneously, the obstruction tumor cell proliferation (Huang Linqing, Xu Chuanfu, Zhou Jinwen, etc.; The Berberine experimental Study on Antitumor Effect, Chinese Pharmacological circular, 1997,13 (2): 189); Berberine has more significantly influence to the rat liver gene expression profile, that its antitumor action may change with the expression that it causes a series of metabolism related genes is relevant (hot Hua Wen, Wu laughs at the spring, Liu Youying, etc.; Berberine hydrochloride causes the rat liver metabolism related gene and expresses the gene chip research that changes, Chinese Pharmacological circular, 2004,20 (10): 1122～1126);
(3) some berberinc derivates also have antitumor action, as Berberine demethylation compound, be between berberrubine and antitumor drug target-DNA topoisomerase I, the II interaction to be arranged, the activity that suppresses these enzymes, anti-tumor activity is (Kobayashi Y very obviously, Yamashita Y, Fujii N, et al; Inhibitors of DNA topoisomerase I and II isolated fromthe coptisrhizomes, Planta Med, 1995,61:414～418);
Berberine hydrochloride berberrubine (demethyl Berberine)
(4) report to the antitumor research of berberine hydrochloride mainly is the result of experiment in vitro so far, this major cause is: few so far experimentation on animals shows, berberine hydrochloride anti-tumor in vivo effect is obvious not as its in vitro effects, as dosage when 25 μ mol/kg and the 75 μ mol/kg, tumour inhibiting rate to murine sarcoma S180 only is respectively 30.0% and 41.3%, experimental result also similar (seeing: table 4 and table 5 in " embodiment ") in our body with above result, and external S180 cell inhibiting rate is reached 93% when berberine hydrochloride concentration is 80 μ M.The mechanism that above phenomenon occurs may be: berberine hydrochloride is quaternary ammonium salt structure (sees and go up structure iron), and lipotropy is poor, is difficult to the lipid film by cell, and difficulty reaches effective blood drug concentration in vivo.
In sum, the berberine hydrochloride antitumor action is very clear and definite, has anticancer broad spectrum, and its source is abundant, cheap, safe, is a kind of medicine that is worth further investigation aspect treatment and prevention of tumour.But can see that it comes with some shortcomings simultaneously, poor as lipotropy, bioavailability is not high yet, and these become the present invention carries out structural modification to Berberine major reason.
The present invention is by carrying out structural modification to Berberine, obtained not seeing the new alkylation berberinc derivate of bibliographical information: 13-just-octyl berberine (1) and 13-just-octyl group palmatine (2), its key character is, fat-soluble remarkable enhancing makes their hydrochloride all very easy dissolving in the not diffluent organic solvent of berberine salt.
Biological activity test studies show that, the related new compound 13-of this patent just-octyl berberine (1) and 13-just-octyl group palmatine (2), with and salt compare with similar compound with liposome and have following characteristics:
1, in the cell experiment system, they and its raw material Berberine and palmatine, and 13-just-hexyl Berberine and 13-just-the hexyl palmatine compares, all has stronger anti-tumor biological and learn active.
2, suppress in vivo in the experiment of tumor growth, 13-just-octyl berberine (1) and salt thereof and liposome antitumour activity also obviously be better than Berberine, 13-just-the hexyl Berberine, and their salt and liposome.
Summary of the invention
The object of the invention is: seeking is raw material with Berberine and palmatine directly, after handling, alkylation makes the new berberinc derivate of lipotropy better (being fat-soluble good), be 13-just-octyl berberine (1) and 13-just-octyl group palmatine (2), with and salt and liposome.
The object of the invention also is: prepared new alkylating berberinc derivate has more notable antitumor activity, can be used for treatment and prevention malignant tumour.
Technical scheme of the present invention is: 13-just-octyl berberine (1) and 13-just-octyl group palmatine (2) preparation method, comprise following two-step reaction:
1, be raw material with Berberine and palmatine respectively, through the reduction respectively corresponding dihydroberberine and dihydro palmatine;
2, respectively or down in Lewis (lewis) acid effect at weak acid, in ethanol directly and n-octaldehyde react, reflux a few hours, make corresponding 13-just-octyl berberine (1) and 13-just-octyl group palmatine (2).
With the Berberine is example, above-mentioned preparation 13-just-process of octyl berberine (1) can represent with following reaction formula:
Berberine hydrochloride dihydroberberine 13-n-octyl berberine (1)
Berberine novel derivative involved in the present invention be the represented 13-of following formula just-octyl berberine (1) and 13-just-octyl group palmatine (2), with and pharmacy acceptable salt and liposome:
Should be pointed out that the salt of compound of the present invention (1) and (2), it is characterized in that by pharmaceutically acceptable mineral acid and the formed salt of organic acid, wherein more excellent mineral acid comprises: hydrochloric acid, Hydrogen bromide, phosphoric acid, nitric acid, sulfuric acid; More excellent organic acid comprises: formic acid, acetate, propionic acid, Succinic Acid, naphthalene disulfonic acid (1,5), Asiatic Acid, oxalic acid, tartrate, lactic acid, Whitfield's ointment, phenylformic acid, valeric acid, diethylacetic acid, propanedioic acid, succsinic acid, fumaric acid, pimelic acid, hexanodioic acid, toxilic acid, oxysuccinic acid, thionamic acid, phenylpropionic acid, glyconic acid, xitix, nicotinic acid, Yi Yansuan, methylsulfonic acid, tosic acid, citric acid, and amino acid.
Should also point out, compound of the present invention (1) and (2) liposome, it is characterized in that by pharmaceutically acceptable preparation liposome material and obtained through preparation method commonly used, wherein more excellent liposome material comprises: Yelkin TTS, neutral phosphatide, cholesterol and negative charge phosphatide.
The present invention also aims to: seek 13-just-octyl berberine (1) and 13-just-octyl group palmatine (2), with and salt and the application of liposome in treatment and preventing cancer.Introduce the long linear octyl group by the 13-position in Berberine and palmatine molecule respectively, balance the polarity effect of quaternary ammonium salt in the molecule, it is bigger fat-soluble to make that new berberinc derivate 1 and 2 has, and then has strengthened biologic activity and bioavailability and have medical proper value.
Above-described compound 1 and 2, with and salt and liposome, its key character is that also fat-soluble remarkable enhancing easily enters cell, by growth, the propagation generation restraining effect of number of mechanisms to tumour cell, promote the apoptosis of tumour cell, the performance antitumor action can be used for malignant tumour, and the treatment of viral dermatosis, compound 1 and 2 also can be used for the treatment of cardiovascular disorder.
The present invention is by external mtt assay and the test of murine sarcoma S180 body inner model, and the result shows:
1, in the cell experiment system, they and its raw material Berberine and palmatine, and 13-just-hexyl Berberine and 13-just-the hexyl palmatine compares, all has stronger anti-tumor biological and learn active.
2, suppress in vivo in the experiment of tumor growth, 13-just-octyl berberine (1) and salt thereof and liposome antitumour activity also obviously be better than Berberine, 13-just-the hexyl Berberine, and their salt or liposome.
The malignant tumour that it should be noted that indication comprises leukemia, skin carcinoma and solid tumor; Wherein, described leukemia is an acute lymphoblastic leukemia, acute myeloblastic leukemia, promyelocytic leukemia, erythrocyte leucocythemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, T chronic myeloid leukemia and B cell leukemia; Described solid tumor and skin carcinoma are liver cancer, lung cancer, mouth neoplasm, ear cancer, nasopharyngeal carcinoma, tongue cancer, esophagus cancer, cancer of the stomach, colorectal carcinoma, carcinoma of the pancreas, mammary cancer, cervical cancer, ovarian cancer, prostate cancer, penile cancer, brain tumor and melanoma.
Should point out, described compounds 1 and 2, with and salt when being used for the treatment of and prevent various tumour, can be made into all suitable formulations in the pharmaceutical industry, described formulation comprises: needle injection, middle needle injection, big needle injection, powder needle injection, injectable emulsion, tablet, pill, capsule, paste, creme, patch, liniment, pulvis, sprays, implant, drops, suppository, ointment, confection; Corresponding liposome is mainly made injection mentioned above.
Described compounds 1 and 2, with and salt when being used for the treatment of and prevent various tumour, can adopt various administrations: oral administration, drug administration by injection, drug delivery implant, intracavitary administration, sublingual administration, anum administration, transdermal administration, interior external application: wherein said drug administration by injection can be: intravenous injection, intramuscular injection, subcutaneous injection, intracavitary administration.
In therapeutic process, described compounds 1 and 2, with and salt and liposome, when being used for the treatment of and prevent various tumour, can be single therapy, can also be and 2 kinds, 3 kinds or more multiple medicines thing combination therapy administration simultaneously, or different precedence administrations.Also can with the other therapies combination therapy, comprise here: unite use with radiotherapy, unite use, unite use, unite use, unite use with gene therapy with biological regulator with surgical operation with herbal medicine.
Embodiment
1 compound
1.1 instrument and reagent
Instrument: Mel-TEMP fusing point instrument (temperature is not calibrated); HP1100 LC/MSD mass spectrograph; (solvent is CDCl to BruckerAV-300 type nmr determination
3); Ultimate analysis ElementarVario ELIII Instrument measuring.
Solvent and reagent are commercially available analytical pure commodity, do not specify then not treated direct use.
1.2 13-just-octyl berberine (1)
5g berberine hydrochloride (13.4mmol) is dissolved in the 100mL5% sodium hydroxide solution, under the room temperature, splash into the stable sodium borohydride solution 50mL of alkali (wherein containing sodium borohydride 0.5g) while stirring, after dripping off, stirring at room 3 hours, the solid filtering that forms, filter cake is washed with water to neutrality, gets the dihydroberberine crude product.
Dihydroberberine 150mL80% dissolve with ethanol solution, behind adding 35mL acetate and the 5mL n-octaldehyde, reflux 3 hours.Reaction solution concentrates, and gets the red-brown dope.With the chloroform dissolving, add equal-volume 2% hydrochloric acid soln, leave standstill, separate out khaki color solid (for the raw material berberine hydrochloride of oxidation generation in the reaction), remove by filter.Gained filtrate concentrates, through column chromatography (CHCl
3/ CH
3OH, 95/5, v/v) separate, collect the 3rd component.Collect liquid and be concentrated into incomplete doing, add ether, separate out a large amount of isabelline 13-octyl berberines (1) solid, behind the filtration drying, get isabelline powder 3.6g, yield 56%, mp:(dec.); Purity is (HPLC) more than 98%;
ESI-MS:448.1[M-Cl]
+, molecular formula: C
28H
34ClNO
4, molecular weight: 484.07;
1H-NMR(CDCl
3，δ)：0.81(s，3H，CH
3)，1.11～1.16(m，8H，CH
2×4)，1.43～1.46(m，2H，CH
2)，1.78～1.81(m，2H，CH
2)，3.10(s，2H，CH
2)，3.17～3.22(m，2H，CH
2)，4.02(s，3H，OCH
3)，4.28(s，3H，OCH
3)，5.22(bs，2H，CH
2)，6.04(s，2H，OCH
2)，6.83(s，1H，Ph-H)，7.04(s，1H，Ph-H)，7.79～7.86(m，2H，Ph-H)，10.59(bs，1H，Ph-H)；
Ultimate analysis: C
28H
34ClNO
4Found (%): C69.68, H7.11, N2.86;
Calcd(％)：C69.41，H7.08，N2.89.
1.3 13-just-octyl group palmatine (2)
The preparation method is consistent with compound 1,13-just-octyl group palmatine (2) is the yellow solid powder, yield 62%, mp:(dec.); Purity is (HPLC) more than 98%;
ESI-MS:464.2[M-Cl]
+, molecular formula: C
29H
38ClNO
4, molecular weight: 500.07;
1H-NMR(CDCl
3，δ)：0.87(s，3H，CH
3)，1.27～1.32(m，8H，CH
2×4)，1.51～1.54(m，2H，CH
2)，1.89～1.91(m，2H，CH
2)，3.18(s，2H，CH
2)，3.25～3.31(m，2H，CH
2)，3.92(s，3H，OCH
3)，3.98(s，3H，OCH
3)，4.07(s，3H，OCH
3)，4.33(s，3H，OCH
3)，5.26(bs，2H，CH
2)，6.91(s，1H，Ph-H)，7.19(s，1H，Ph-H)，7.85～7.89(m，2H，Ph-H)，10.83(bs，1H，Ph-H)；
Ultimate analysis: C
29H
38ClNO
4Found (%): C69.71, H7.75, N2.69;
Calcd(％)：C69.65，H7.66，N2.80.
1.4 13-just-octyl berberine-Asia hydrochlorate (13-B-8-AA)
With 40mg13-just-octyl berberine (1) is dissolved in 5mL3%NaHCO
3In the water dissolution, behind the stirring 5min, with ethyl acetate extraction (5mL * 3), merge organic phase, drying is filtered, after concentrating, after in residue, adding the 5mL acetone solution, add Asiatic Acid (molecular weight is 488) 45mg, be stirred under the room temperature and be clear solution, continue again to stir 2 hours, be concentrated into driedly then, be product, the deep green solid.
1.5 13-just-the octyl berberine liposome
Get 20mg13-just-octyl berberine (1), 400mg ovum phosphine ester and 40mg cholesterol be added in the flask, adds an amount of ethanol, be stirred to dissolving and be clear solution, then, rotate in 40 ℃ of following vacuum and to do film forming and invest a bottle wall, add that to contain a little amino acid 5% D/W an amount of, after the ultrasonic homogenate, after the filter membrane degerming, pack in the ampoule, lyophilize, faint yellow 13-just-the octyl berberine liposome, seal stand-by, cryopreservation.Be diluted to the required concentration that contains with 5% D/W during use.
The research of 2 anti-tumor activities
2.1 anti tumor activity in vitro test
2.1.1 13-just-octyl berberine (1) and 13-just-test of octyl group palmatine (2) anti-tumor activity
2.1.1.1 experiment purpose: the mtt assay measuring is to multiple animal and the effect of human body tumour cell strain growth-inhibiting;
2.1.1.2 plant and instrument
(1) CO
2Cell culture incubator, PHARMA SCIENTIFIC;
(2) enzyme-linked immunosorbent assay instrument, Labsystems Dragon, Wellscan, Mk2;
(3) the vertical double Bechtop of single face, Suzhou Decontamination Equipment Plant;
(4) inverted biologic microscope, 37XB/37 * BTV, Shanghai opticinstrument six factories;
2.1.1.3 experiment material and preparation
(1) human liver cancer cell 7701 QGY, SMMC 7721, HepG-2, human lung adenocarcinoma cell A549, the former leukemia cell K562 of the chronic marrow of people, human leukemia cell CEM, mouse melanin tumor cell KIII and mouse lewis lung carcinoma cell;
(2) RPMI (1640) cell culture medium (GIBCO): contain 10% deactivation newborn calf serum (Saida biological Pharmaceutical Co., Ltd., Shanghai city); The L-glutaminase (the import packing, SANGON); Sodium.alpha.-ketopropionate; 1 * 10
5U.L
-1Penicillin, 100mg.L
-1Streptomycin sulphate; Sterile filtration, 4 ℃ of preservations;
(3) 0.25% trypsin solutions (Trypsin): available from Invitrogen company ,-20 ℃ of preservations.
(4) phosphate buffered saline buffer (PBS): NaCl8g, KCl0.2g, Na
2HPO
41.15g, KH
2PO
40.2g, be dissolved in the 1L distilled water, 121 ℃ of autoclave sterilization 20min, 4 ℃ of preservations;
(5) MTT (AMRESCO) solution: be made into 5mg/mL solution with PBS;
(6) lysate: every 100mL deionization distilled water contains SDS10g, isopropylcarbinol 5mL, vitriol oil 0.12mL;
(7) be subjected to the reagent thing.
Title: Berberine, prothionamide, 13-hexyl Berberine, 13-hexethal sodium Martin, 13-octyl berberine, 13-octyl group prothionamide;
Content: more than 98%;
Compound method: DMSO dissolving, the PBS dilution is desired concn.
2.1.1.4 experimental technique
Get the tumour cell that is in logarithmic phase, digestion back diluting cells suspension to 20 * 10
4Individual/mL, join in the 96 porocyte culture plates, every hole 90 μ L, 2 multiple holes add soup 10 μ L again, and concentration is respectively 100,10, and 1,10
-1, 10
-2, 10
-3μ g/mL, control group add isopyknic PBS damping fluid.37 ℃ of 5%CO
2, behind the cultivation 48h, every hole adds freshly prepared MTT solution 20 μ L/ holes, behind 37 ℃ of incubation 4h, every hole adds lysate 100 μ L/ holes, spends the night, and the 570nm place measures the OD value, with the inhibiting rate of each concentration of OD value calculating,, calculate IC according to the inhibiting rate straight-line regression of different concns
50(μ mol/L).
2.1.1-5 experimental result the results are shown in table 1 and the table 2:
Table 1 Berberine, 13-n-hexyl Berberine and 13-n-octyl berberine (1)
Suppress the comparison of tumor cell growth activity
| Cell strain | Berberine | 13-n-hexyl Berberine | 13-n-octyl berberine (1) | |||
| IC 50(μmol/L) | I | IC 50 (μmol/L) | I | IC 50(μmol/L) | I | |
| 7701QGY | 23.90 | 34.39 | 3.46 | 85.98 | 2.16 | 99.00 |
| SMMC7721 | 2.32 | 83.82 | 0.37 | ＞100 | 0.05 | ＞100 |
| HepG-2 | 120.76 | 45.90 | 5.04 | 98.17 | 3.45 | 99.16 |
| CEM | 46.32 | 34.85 | 3.99 | ＞100 | 0.43 | ＞100 |
| CEM/VCR | 123.87 | 34.64 | 11.65 | 82.15 | 5.86 | 88.08 |
| KIII | 87.52 | 6.05 | 29.50 | 44.90 | 16.96 | 51.88 |
| Lewis | 24.10 | 52.57 | 2.79 | 85.08 | 0.93 | 92.21 |
IC
50: medium lethal dose, the inhibiting rate (%) of I:10 μ g/ml.
Table 2 palmatine, 13-n-hexyl palmatine and 13-n-octyl group palmatine (2)
Suppress the comparison of tumor cell growth activity
| Cell strain | Palmatine | 13-n-hexyl palmatine | 13-n-octyl group palmatine (2) | |||
| IC 50 (μmol/L) | I | IC 50 (μmol/L) | I | IC 50 (μmol/L) | I | |
| 7701QGY | ＞258 | 25.18 | 12.22 | 41.26 | 1.40 | 99.27 |
| SMMC7721 | 24.13 | 43.66 | 0.70 | ＞100 | 0.02 | ＞100 |
| HepG-2 | ＞258 | 12.73 | 5.81 | 91.16 | 2.54 | 99.31 |
| CEM | 5.98 | 59.31 | 2.16 | 98.45 | 0.24 | ＞100 |
| CEM/VCR | 236.76 | 28.03 | 19.03 | 52.06 | 5.60 | 98.00 |
| KIII | 78.12 | 9.72 | 33.69 | 44.68 | 12.60 | 56.30 |
| Lewis | 32.18 | 47.86 | 2.39 | 90.59 | 0.34 | 92.21 |
IC
50: medium lethal dose, the inhibiting rate (%) of I:10 μ g/ml.
In last two tables: human liver cancer cell 7701 QGY, SMMC 7721, HepG-2, human leukemia cell CEM, CEM/VCR[are CEM vincristine(VCR) (VCR) drug-resistant cell strain], mouse melanin tumor cell KIII and mouse lewis lung carcinoma cell.
The result of table 1 and table 2 shows, 13-just-octyl berberine (1) and 13-just-octyl group palmatine (2) suppresses tumor cell growth activity and all is higher than their corresponding homologues, the carbochain that also demonstrates the straight chained alkyl that replaces along with the 13-position simultaneously increases, corresponding anti-tumor activity also increases thereupon, this may ascribe the carbochain growth to and make the corresponding fat-soluble increase of derivative, also more balance the polarity of quaternary ammonium salt in the molecule, make compound 1 and 2 be easy to pass through after birth, enter in the tumour cell, thereby, their IC
50Lower, the inhibition growth of tumour cell rate under the finite concentration is also inevitable bigger.
CEM/VCR is the vincristine(VCR) drug-resistant cell strain of CEM, the IC of CEM/VCR
50The obvious IC of value greater than CEM
50Value, Berberine and prothionamide do not have the reversing drug resistance effect in two tables, and compound 1 and 2 is very approaching to the I value of CEM/VCR and CEM inhibition respectively, embodies the effect of their reversing drug resistances.
2.1.2 13-just-octyl berberine-Asia hydrochlorate (13-B-8-AA) and 13-just-octyl berberine liposome (13-B-8 liposome)
The anti-tumor activity test
Testing method is consistent with 2.1.1, here with the positive contrast medicine of cis-platinum, also records Berberine-Asia hydrochlorate (B-AA) anti-tumor activity simultaneously, so that relatively, the results are shown in table 3:
Table 3 B-AA, 13-B-8-AA and 13-B-8 liposome are to the growth in vitro restraining effect of various tumor cell strains
| Cell strain | Cis-platinum | 13-B-8-AA | B-AA | The 13-B-8 liposome | ||||
| IC 50 μmol/L | I 20gmol/L | IC 50 μmol/L | I 20gmol/L | IC 50 μmol/L | I 20gmol/L | IC 50 μmol/L | I 20μmol/L | |
| L1210 | 28.60 | 47.11 | 7.99 | 67.03 | 0.91 | 55.43 | 3.70 | 64.66 |
| K562 | 58.10 | 43.74 | 2.23 | 74.50 | 0.86 | 67.69 | 33.58 | 45.16 |
| A549 | 58.73 | 41.94 | 12.10 | 52.48 | 5.84 | 54.46 | ＞100 | 27.07 |
The inhibiting rate of I:20gmol/L (%);
The leukemia cell L1210 of mouse, the former leukemia cell K562 of the chronic marrow of people, human lung adenocarcinoma cell A549.
Table 3 result shows, with respect to cis-platinum and B-AA, 13-B-8-AA has and suppresses the tumor growth effect more by force, the 13-B-8 liposome except that insensitive to lung carcinoma cell, the leukemia cell is still had active preferably, from they IC
50See that cellular toxicity descends to some extent than B-AA.
2.2 anti-tumor in vivo active testing
2.2.1 13-just-octyl berberine (1) anti-tumor activity test
2.2.1.1 test objective: test Berberine, 13-hexyl Berberine, 13-octyl berberine are to mouse S
180The restraining effect of sarcoma (solid-type);
2.2.1.2 be subjected to the reagent thing
Title: Berberine, 13-hexyl Berberine, 13-octyl berberine;
Content: 98%;
Compound method: small amount of ethanol dissolving back suspends with tween, is diluted to desired concn with physiological saline at last.
2.2.1.3 control sample
Title: Cyclophosphamide for injection (CTX); Source: Hua Lian pharmaceutical Co. Ltd;
Lot number: 040606; Specification: 200mg;
Compound method: physiological saline is formulated as desired concn
2.2.1.4 animal
Strain: Kunming mouse; Body weight: 19～21g;
Sex: the male and female dual-purpose, same sex is adopted in a collection of test;
The source: Chinese Academy of Sciences's Shanghai Experimental Animal Center provides; Credit number: SCXK (Shanghai) 2003-0003.
2.2.1.5 other material
Transplanted tumor: sarcoma S
180System, pharmacological room of Shanghai Institute of Pharmaceutical Industry goes down to posterity and keeps.
2.2.1.6 experimental procedure and method
Get well-grown S180 ascites, with physiological saline dilution in 1: 10, adjusting cell concn is 1～2 * 10
7Individual/mL, every mouse armpit subcutaneous vaccination 0.2mL, random packet is established coordinative solvent control group and endoxan positive controls (30mg/kg/d, ip * 7d), the administration group plays administration next day, the tail intravenously administrable is 7 days continuously, takes off cervical vertebra execution, and dissects and get the knurl piece in the 10th day, relatively each dosage group knurl is great little, and the result judges according to following formula:
2.2.1.7 test-results:
The results are shown in table 4 Berberine, palmatine, 13-hexyl Berberine, 13-hexyl palmatine, 13-octyl berberine, 13-octyl group palmatine mouse S180 sarcoma is had certain inhibiting rate, knurl is heavy, and relatively there were significant differences (P＜0.01) with control group, and along with the prolongation of 13 carbochains, tumour inhibiting rate has tangible rising.
Table 4 Berberine, 13-n-hexyl Berberine and 13-n-octyl berberine are to the tumor-inhibiting action of mouse S180 sarcoma
| Group | Dosage mg/kg | Dosage regimen | Number of animals | Administration begins body weight (g) | Knurl body weight (g) is gone in off-test | Knurl heavy (g) | Tumour inhibiting rate (%) |
| control | ip×7 | 10 | 19.48±1.45 | 19.99±2.20 | 2.20±0.93 | ||
| Berberine | 30 | ip×7 | 10 | 19.98±1.33 | 17.86±2.25 | 1.32±0.63 ** | 40.00 |
| 13-n-is own | 2.5 | ip×7 | 10 | 19.62±1.52 | 18.04±2.54 | 1.01±0.48 ** | 54.09 |
| The base Berberine | 1.0 | ip×7 | 10 | 20.56±2.03 | 19.50±2.18 | 1.45±0.31 ** | 34.09 |
| The 13-n-suffering | 2.5 | ip×7 | 10 | 19.89±1.54 | 17.11±1.06 | 0.88±0.28 ** | 60.00 |
| Base Berberine (1) | 1.0 | ip×7 | 10 | 19.17±1.12 | 20.20±2.22 | 1.02±0.25 ** | 53.64 |
| CTX | 30 | ip×7 | 10 | 20.23±1.25 | 20.28±2.31 | 0.43±0.28 ** | 80.46 |
Compare with control group
*P＜0.05,
*＜0.01
Table 4 result as seen, 13-n-octyl berberine (1) tumour inhibiting rate is greater than Berberine and 13-n-hexyl Berberine; In addition, the Berberine analog derivative has big toxicity under higher administration concentration, and the mouse body weight descends to some extent, and compound 1 is after administration concentration reduces, and toxic effect obviously descends, and tumour inhibiting rate decline is less, and compound 1 has the potential using value.
2.2.2 13-just-octyl berberine-Asia hydrochlorate (13-B-8-AA) and 13-just-octyl berberine liposome (13-B-8 liposome)
The anti-tumor activity test
Testing method is consistent with 2.2.1, records Berberine-Asia hydrochlorate (B-AA) anti-tumor activity simultaneously, so that relatively, the results are shown in table 5.
Table 5 result shows, 13-just-octyl berberine liposome (13-B-8 liposome) anti-tumor activity is suitable with CTX, but toxicity is more obvious, illustrate that liposome easily enters in the cell, form higher concentration, thereby body weight descends to some extent, and its toxicity can utilize methods such as conversion liposome material, prescription to be improved.
13-just-octyl berberine-Asia hydrochlorate (13-B-8-AA) subtracts greatly than Berberine-Asia hydrochlorate toxicity, this is consistent with the initial design objective of medicine, its antitumour activity also can improve by increasing drug dose or salify mode.
Specification sheets
| Table 5B-AA, 13-B-AA, the 13-B-8 liposome is to the tumour inhibiting rate of mouse S180 sarcoma (solid-type) | |||||||
| Group | Dosage mg/kg | Dosage regimen | Number of animals 0 day/10 days | Administration begins body weight (g) | Knurl body weight (g) is gone in off-test | Knurl heavy (g) | Tumour inhibiting rate (%) |
| Blank physiological saline | 0.5mL | ip×7 | 10/10 | 19.41±2.14 | 21.35±1.26 | 1.53±0.41 | |
| B-AA | 2.5 | ip×7 | 10/10 | 19.44±1.32 | 17.41±2.13 ** | 0.93±0.35 ** | 39.19 |
| 13-B-8-AA | 2.5 | ip×7 | 10/10 | 18.22±1.95 | 18.68±2.43 ** | 0.89±0.28 ** | 41.61 |
| 13-B-8-fat | 2.5 | ip×7 | 10/7 | 18.70±2.28 | 14.58±1.03 ** | 0.49±0.23 ** | 67.72 |
| CTX | 30 | ip×7 | 10/7 | 19.49±2.00 | 18.06±1.94 ** | 0.34±0.19 ** | 77.70 |
Compare with control group
*P＜0.05,
*P＜0.01
Claims (10)
1, institute of the present invention synthetic Berberine novel derivative be the represented 13-of following formula just-octyl berberine (1) and 13-just-octyl group palmatine (2), with and pharmacy acceptable salt and liposome:
The 13-of brand new just-octyl berberine (1) and 13-just-octyl group palmatine (2) is a raw material with Berberine and palmatine respectively, at first, through the reduction respectively corresponding dihydroberberine and dihydro palmatine, then, respectively weak acid or Lewis (lewis) acid effect down directly and n-octaldehyde react and make.
2, the salt of the described compound of claim 1 (1) and (2) is characterized in that, by pharmaceutically acceptable mineral acid and the formed salt of organic acid, wherein more excellent mineral acid comprises: hydrochloric acid, Hydrogen bromide, phosphoric acid, nitric acid, sulfuric acid; More excellent organic acid comprises: formic acid, acetate, propionic acid, Succinic Acid, naphthalene disulfonic acid (1,5), Asiatic Acid, oxalic acid, tartrate, lactic acid, Whitfield's ointment, phenylformic acid, valeric acid, diethylacetic acid, propanedioic acid, succsinic acid, fumaric acid, pimelic acid, hexanodioic acid, toxilic acid, oxysuccinic acid, thionamic acid, phenylpropionic acid, glyconic acid, xitix, nicotinic acid, Yi Yansuan, methylsulfonic acid, tosic acid, citric acid, and amino acid.
3, the described compound of claim 1 (1) and (2) liposome, it is characterized in that, make by pharmaceutically acceptable preparation liposome material and through preparation method commonly used, wherein more excellent liposome material comprises: Yelkin TTS, neutral phosphatide, cholesterol and negative charge phosphatide etc.
4, the described compound of claim 1～3 (1) and (2) and salt and liposome, its key character also is, fat-soluble remarkable enhancing, easily enter cell,, promote the apoptosis of tumour cell by growth, the propagation generation restraining effect of number of mechanisms to tumour cell, the performance antitumor action, can be used for malignant tumour, and the treatment of viral dermatosis, compound (1) and (2) also can be used for the treatment of cardiovascular disorder.
5, the described malignant tumour of claim 4 comprises leukemia, skin carcinoma and solid tumor.
6, the described application of compound of claim 5, it is characterized in that, described leukemia is an acute lymphoblastic leukemia, acute myeloblastic leukemia, promyelocytic leukemia, erythrocyte leucocythemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, T chronic myeloid leukemia and B cell leukemia.
7, the described application of compound of claim 5 is characterized in that, described solid tumor is a liver cancer, lung cancer, mouth neoplasm, ear cancer, nasopharyngeal carcinoma, tongue cancer, esophagus cancer, cancer of the stomach, colorectal carcinoma, carcinoma of the pancreas, mammary cancer, cervical cancer, ovarian cancer, prostate cancer, penile cancer, melanoma and brain tumor.
8, the described compounds of claim 1～3 can be made into all suitable formulations in the pharmaceutical industry when being used for the treatment of and prevent various tumour, and described formulation comprises: needle injection, middle needle injection, big needle injection, powder needle injection, injectable emulsion, tablet, pill, capsule, paste, creme, patch, liniment, pulvis, sprays, implant, drops, suppository, ointment, confection; Corresponding liposome is mainly made injection mentioned above.
9, the described compounds of claim 1～3 can adopt various administrations when being used for the treatment of and prevent various tumour: oral administration, drug administration by injection, drug delivery implant, intracavitary administration, anum administration, transdermal administration, interior external application; Wherein said drug administration by injection can be: intravenous injection, intramuscular injection, subcutaneous injection, intracavitary administration.
10, the described compounds of claim 1～3 can be a single therapy when being used for the treatment of and prevent various tumour, can also be and 2 kinds, 3 kinds or more multiple medicines thing combination therapy administration simultaneously, or different precedence administrations; Also can with the other therapies combination therapy, comprise here; Unite use with radiotherapy, unite use, unite use, unite use, unite use with gene therapy with biological regulator with surgical operation with herbal medicine.
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| CN101935319A (en) * | 2010-09-09 | 2011-01-05 | 陕西科技大学 | Berberine organic acid salt, berberine organic acid salt inclusion compound and preparation methods thereof |
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| CN101935319A (en) * | 2010-09-09 | 2011-01-05 | 陕西科技大学 | Berberine organic acid salt, berberine organic acid salt inclusion compound and preparation methods thereof |
| CN102746291A (en) * | 2011-04-19 | 2012-10-24 | 中国医学科学院医药生物技术研究所 | 13-substituted berberine derivatives and preparation method thereof, and uses of 13-substituted berberine derivatives as anti-tuberculosis drugs |
| CN102746291B (en) * | 2011-04-19 | 2015-07-01 | 中国医学科学院医药生物技术研究所 | 13-substituted berberine derivatives and preparation method thereof, and uses of 13-substituted berberine derivatives as anti-tuberculosis drugs |
| CN102351870A (en) * | 2011-08-25 | 2012-02-15 | 中山大学 | Method for preparing benzacridine derivative and application of benzacridine derivative as anti-cancer medicine |
| CN102351870B (en) * | 2011-08-25 | 2014-03-12 | 中山大学 | Method for preparing benzacridine derivative and application of benzacridine derivative as anti-cancer medicine |
| CN104211697A (en) * | 2013-06-04 | 2014-12-17 | 中山大学 | Berberine derivative and use thereof |
| CN104211697B (en) * | 2013-06-04 | 2016-08-10 | 中山大学 | Berberinc derivate and application thereof |
| CN106008560A (en) * | 2016-05-18 | 2016-10-12 | 中国医学科学院生物医学工程研究所 | Preparation method and application of diberberine derivative |
| CN107501259A (en) * | 2017-09-08 | 2017-12-22 | 厦门大学 | A kind of berberine derivative XMU Ber122 |
| CN113880830A (en) * | 2021-10-25 | 2022-01-04 | 南京中医药大学 | 9-O-aminoalkyl-13-alkyl disubstituted berberine derivative and preparation method and application thereof |
| CN113880830B (en) * | 2021-10-25 | 2023-09-22 | 南京中医药大学 | 9-O-aminoalkyl-13-alkyl disubstituted berberine derivative and preparation method and application thereof |
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