CN104163823B - camptothecin and artesunate conjugate as well as preparation method and application thereof - Google Patents
camptothecin and artesunate conjugate as well as preparation method and application thereof Download PDFInfo
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- CN104163823B CN104163823B CN201410181237.4A CN201410181237A CN104163823B CN 104163823 B CN104163823 B CN 104163823B CN 201410181237 A CN201410181237 A CN 201410181237A CN 104163823 B CN104163823 B CN 104163823B
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- VSJKWCGYPAHWDS-FQEVSTJZSA-N camptothecin Chemical compound C1=CC=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 VSJKWCGYPAHWDS-FQEVSTJZSA-N 0.000 title claims abstract description 91
- FIHJKUPKCHIPAT-AHIGJZGOSA-N artesunate Chemical compound C([C@](OO1)(C)O2)C[C@H]3[C@H](C)CC[C@@H]4[C@@]31[C@@H]2O[C@@H](OC(=O)CCC(O)=O)[C@@H]4C FIHJKUPKCHIPAT-AHIGJZGOSA-N 0.000 title claims abstract description 68
- 229960004991 artesunate Drugs 0.000 title claims abstract description 67
- 238000002360 preparation method Methods 0.000 title claims abstract description 24
- KLWPJMFMVPTNCC-UHFFFAOYSA-N Camptothecin Natural products CCC1(O)C(=O)OCC2=C1C=C3C4Nc5ccccc5C=C4CN3C2=O KLWPJMFMVPTNCC-UHFFFAOYSA-N 0.000 title abstract description 6
- 229940127093 camptothecin Drugs 0.000 title abstract description 6
- VSJKWCGYPAHWDS-UHFFFAOYSA-N dl-camptothecin Natural products C1=CC=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)C5(O)CC)C4=NC2=C1 VSJKWCGYPAHWDS-UHFFFAOYSA-N 0.000 title abstract description 6
- 239000003960 organic solvent Substances 0.000 claims abstract description 29
- 238000006243 chemical reaction Methods 0.000 claims abstract description 27
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 claims abstract description 26
- 238000003756 stirring Methods 0.000 claims abstract description 17
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims abstract description 13
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims abstract description 13
- 239000002246 antineoplastic agent Substances 0.000 claims abstract description 6
- 229940041181 antineoplastic drug Drugs 0.000 claims abstract description 6
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 30
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 22
- 239000000243 solution Substances 0.000 claims description 21
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
- 206010028980 Neoplasm Diseases 0.000 claims description 18
- 206010006187 Breast cancer Diseases 0.000 claims description 13
- 208000026310 Breast neoplasm Diseases 0.000 claims description 13
- 229960000549 4-dimethylaminophenol Drugs 0.000 claims description 12
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- 150000001875 compounds Chemical class 0.000 claims description 11
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- -1 suction filtration Substances 0.000 claims description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 239000000463 material Substances 0.000 claims description 8
- 201000007270 liver cancer Diseases 0.000 claims description 7
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 6
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical class [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 6
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- 201000001275 rectum cancer Diseases 0.000 claims description 2
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- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 12
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- BLUAFEHZUWYNDE-NNWCWBAJSA-N artemisinin Chemical compound C([C@](OO1)(C)O2)C[C@H]3[C@H](C)CC[C@@H]4[C@@]31[C@@H]2OC(=O)[C@@H]4C BLUAFEHZUWYNDE-NNWCWBAJSA-N 0.000 description 8
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- 229910052742 iron Inorganic materials 0.000 description 5
- 238000011580 nude mouse model Methods 0.000 description 5
- 231100000419 toxicity Toxicity 0.000 description 5
- 230000001988 toxicity Effects 0.000 description 5
- 210000004881 tumor cell Anatomy 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 241000699660 Mus musculus Species 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- UCFGDBYHRUNTLO-QHCPKHFHSA-N topotecan Chemical compound C1=C(O)C(CN(C)C)=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 UCFGDBYHRUNTLO-QHCPKHFHSA-N 0.000 description 4
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- 229960002190 topotecan hydrochloride Drugs 0.000 description 3
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 2
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- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- 240000000011 Artemisia annua Species 0.000 description 1
- 101100045694 Caenorhabditis elegans art-1 gene Proteins 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
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- 230000000078 anti-malarial effect Effects 0.000 description 1
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- 239000012930 cell culture fluid Substances 0.000 description 1
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- UWKQSNNFCGGAFS-XIFFEERXSA-N irinotecan Chemical compound C1=C2C(CC)=C3CN(C(C4=C([C@@](C(=O)OC4)(O)CC)C=4)=O)C=4C3=NC2=CC=C1OC(=O)N(CC1)CCC1N1CCCCC1 UWKQSNNFCGGAFS-XIFFEERXSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention discloses a camptothecin and artesunate conjugate shown in formula (I), and a preparation method thereof comprises the following steps: dissolving camptothecin shown in a formula (II) and artesunate shown in a formula (III) in an organic solvent, stirring and reacting at 15-50 ℃ under the action of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 4-dimethylaminopyridine, tracking and monitoring by TLC (thin layer chromatography) until the reaction is not carried out any more, and carrying out post-treatment on a reaction solution to obtain a camptothecin and artesunate conjugate shown in a formula (I); the camptothecin and artesunate conjugate can be used for preparing antitumor drugs;
Description
(1) technical field
The present invention relates to a kind of camptothecine and Artesunate conjugate and preparation method thereof, and prepare the application in antitumor drug.
(2) background technology
Camptothecine derives from Chinese Plants camplotheca acuminata, and due to the mechanism of action of its uniqueness, in cancer therapy drug research field, a large amount of camptothecin derivatives is synthesized and research.Camptothecine extremely derivative all serves good inhibition for various cancer cells, such as: lung cancer, prostate cancer, mammary cancer, colorectal carcinoma, cancer of the stomach, ovarian cancer, melanoma, lymphoma etc.But camptothecine is not applied to clinical, because himself high toxicity and poor solvability.The another kind of Artemisinin extracted from composite family artemisia sweet wormwood employs more than 2000 year as anti-malaria medicaments always, and Artemisinin extremely derivative is considered to safely and effectively always.Research finds, Artemisinin can be combined with Transferrins,iron complexes in vivo, and himself chemical structure contains a peroxide bridge, when with iron ion in conjunction with time can form a free radical, thus play restraining effect.Transferrins,iron complexes is the functional protein being responsible for transhipment iron ion in organism, and relative to normal cell, the demand of cancer cells to iron ion is higher, this to such an extent as to Artemisinin can play better action effect to cancer cells.The transferrin content on such as breast cancer cell surface is Normocellular 5-15 times, and Transferrins,iron complexes is expressed on breast cancer cell, and breast cancer cell absorbs a large amount of iron ion, and normal cell does not then have.Therefore the selective action of Artemisinin to cancer cells is because this compound can be combined with Transferrins,iron complexes in vivo, and Transferrins,iron complexes can transport to the position that iron ion content is high.
Therefore utilize the mechanism that Artemisinin can be combined with Transferrins,iron complexes, Artemisinin is used as carrier to use, and camptothecine has for the higher restraining effect of various cancer cells, but the feature that himself toxicity is large, we are by camptothecine and artemisinin derivative---Artesunate coupling, while making it have better inhibition to cancer cells, reduce self toxicity.
(3) summary of the invention
The object of the invention is to provide a kind of camptothecine and Artesunate conjugate and preparation method thereof, and preparing the application in antitumor drug, camptothecine of the present invention and Artesunate conjugate all have inhibited proliferation in various degree to liver cancer cell (SMMC-7721), human breast cancer cell (MCF-7) etc.
For achieving the above object, the technical solution used in the present invention is:
Camptothecine shown in a kind of formula (I) and Artesunate conjugate:
The preparation method of a kind of camptothecine of the present invention and Artesunate conjugate, described preparation method is: be dissolved in organic solvent a by Artesunate shown in camptothecine formula (II) Suo Shi and formula (III), under the effect of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and DMAP (DMAP), in 15 ~ 50 DEG C of stirring reactions, TLC tracking monitor no longer carries out to reaction, reaction solution aftertreatment, obtains the camptothecine shown in formula (I) and Artesunate conjugate.
The preparation method of camptothecine of the present invention and Artesunate conjugate, wherein said organic solvent a is the solvent that side reaction does not occur with it solubilized reaction substrate, as chloroform or methylene dichloride etc., is preferably chloroform; The volumetric usage of described organic solvent a for 1 ~ 10mL/mmol, can be preferably 3 ~ 5mL/mmol with the molar amount of camptothecine.
The preparation method of camptothecine of the present invention and Artesunate conjugate, wherein said camptothecine can be 1:1 ~ 10 with the ratio of the amount of substance that feeds intake of Artesunate, is preferably 1:3;
The ratio of the amount of substance that feeds intake of described camptothecine and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride can be 1:1 ~ 5, is preferably 1:3;
The quality consumption of described DMAP for 1 ~ 20mg/mmol, can be preferably 5 ~ 10mg/mmol with the molar amount of camptothecine.
The preparation method of camptothecine of the present invention and Artesunate conjugate, recommends feeding method as follows: under room temperature, to be first dissolved in chloroform by Artesunate, dissolve completely and add EDCI, stir 30 minutes, then add DMAP, continue stirring 30 minutes, finally add camptothecine, stirring reaction; Further, preferred stirring reaction 3 hours at 25 DEG C.
The preparation method of camptothecine of the present invention and Artesunate conjugate, described post-treating method is:
After reaction terminates, in reaction solution, add organic solvent b, suction filtration, filtrate is successively with saturated sodium-chloride water solution and saturated sodium bicarbonate aqueous solution washing, use anhydrous magnesium sulfate drying again, suction filtration, filtrate reduced in volume is to dry, and gained enriched material carries out silica gel column chromatography, eluent is methylene dichloride and the methyl alcohol mixed liquor of volume ratio 100:1, collect the elutriant containing target compound, concentrate drying, obtains the camptothecine shown in formula (I) and Artesunate conjugate; Described organic solvent b is chloroform or methylene dichloride, and its volumetric usage is 2 times of organic solvent a volume.
Concrete, the preparation method of recommendering folder invention camptothecine and Artesunate conjugate is: under room temperature, Artesunate is dissolved in organic solvent a, dissolve completely and add 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, stir 30 minutes, add DMAP again, continue stirring 30 minutes, finally add camptothecine, stirring reaction 3 hours at 25 DEG C, after reaction terminates, organic solvent b is added in reaction solution, suction filtration, filtrate is successively with saturated sodium-chloride water solution and saturated sodium bicarbonate aqueous solution washing, use anhydrous magnesium sulfate drying again, suction filtration, filtrate reduced in volume is to dry, gained enriched material carries out silica gel column chromatography, eluent is methylene dichloride and the methyl alcohol mixed liquor of volume ratio 100:1, collect the elutriant containing target compound, concentrate drying, obtain the camptothecine shown in formula (I) and Artesunate conjugate, wherein, described organic solvent a is chloroform, and the volumetric usage of described organic solvent a with the molar amount of camptothecine for 3 ~ 5mL/mmol, described camptothecine is 1:3 with the ratio of the amount of substance that feeds intake of Artesunate, the ratio of the amount of substance that feeds intake of described camptothecine and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is 1:3, the quality consumption of described DMAP with the molar amount of camptothecine for 5 ~ 10mg/mmol, in last handling process, described organic solvent b is chloroform or methylene dichloride, and its volumetric usage is 2 times of organic solvent a volume.
In the present invention, organic solvent a and organic solvent b all refers to organic solvent, and wherein, label a, b are just for distinguishing organic solvent used in different operating step.
Camptothecine of the present invention and Artesunate conjugate can be used for preparing antitumor drug, and described camptothecine and Artesunate conjugate are 10 ~ 60mg/kg body weight as the quality consumption of antitumor inhibitor to common mouse.
Described tumour as cerebral tumor, oral carcinoma, laryngocarcinoma, the esophageal carcinoma, lung cancer, cancer of the stomach, liver cancer, kidney, prostate cancer, colorectal carcinoma, mammary cancer, ovarian cancer, carcinoma of the pancreas, bladder cancer, skin carcinoma, leukemia, sarcoma, the rectum cancer, glioma and HIV (human immunodeficiency virus) (HIV), but is not only confined to this.
Camptothecine of the present invention and Artesunate conjugate can also combine with the acceptable pharmaceutical excipient of human body makes various formulation, comprises solution, injection, tablet, capsule, ointment, gelifying agent etc.Camptothecine of the present invention and Artesunate conjugate can together with conventional pharmaceutical adjuvants, and with the form of pharmaceutical composition by oral, snuffing enters, the mode of stomach or administered parenterally is applied to patient.For time oral, conventional solid preparation can be made into as tablet, pulvis, granula, capsule etc.; During for administered parenterally, the solution of injection, water or oleaginous suspension can be made into; The externally used solution agent of percutaneous drug delivery, lotion, liniment, ointment, paste, patch; The gargle of mucosa delivery, sublingual tablet, suppository, film; The enema, suppository, rectal capsule bolt etc. of rectal administration.
Compared with prior art, beneficial effect of the present invention is mainly reflected in: the camptothecine and the Artesunate conjugate that the invention provides a kind of novelty, and this conjugate has anti-tumor activity, can be used in preparing antitumor drug, camptothecine of the present invention and Artesunate conjugate have inhibited proliferation (detailed in Example 3) in various degree to liver cancer cell (SMMC-7721), human breast cancer cell (MCF-7), the IC of this conjugate
50basic and camptothecine maintains an equal level.And shown by experimentation on animals, this conjugate reduces (embodiment 4) relative to camptothecine to a great extent to the injury of body.
(4) accompanying drawing explanation
Fig. 1 is the situation that camptothecine and Artesunate conjugate suppress proliferative activity o f tumor;
Fig. 2 is that camptothecine and Artesunate conjugate are on the impact of Mouse Weight;
In figure, the implication of each label is: CPT---camptothecine group, ART---Artesunate group, C-Q---conjugate group, Control---control group.
(5) embodiment
Below by specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this.
Embodiment 1: the preparation (1) of camptothecine and Artesunate conjugate
Artesunate (15mmol) and methylene dichloride (10mL) is added in 25mL round-bottomed flask, 25 DEG C of stirring and dissolving, add EDCI (15mmol) again, 25 DEG C are stirred 0.5h, then DMAP (50mg) is added, 25 DEG C are stirred 0.5h, finally add camptothecine (5mmol), 25 DEG C of stirring reaction 3h, obtain faint yellow suspension liquid, after reaction terminates, methylene dichloride (20mL) is added in reaction solution, suction filtration, filtrate is successively with saturated sodium-chloride water solution and saturated sodium bicarbonate aqueous solution washing, use anhydrous magnesium sulfate drying again, suction filtration, filtrate reduced in volume is to dry, gained enriched material carries out silica gel column chromatography, eluent is methylene dichloride and the methyl alcohol mixed liquor of volume ratio 100:1, collect the elutriant containing target components, namely the camptothecine shown in formula (I) and Artesunate conjugate is obtained after concentrate drying, productive rate 80%.
MP:194.8-195.3℃
1H-NMR(500MHz,CDCl3):8.3742(s,1H),8.2587(d,J＝8.5HZ,2H),7.9331(d,J＝8.15HZ,1H),7.8157(t,J＝7.5HZ,1H),7.6525(t,J＝7.4HZ,1H),7.2865(d,J＝5.5HZ,1H),5.679,(m,J＝9.85HZ,2H),5.4117,(d,J＝17.15HZ,1H),5.2991(s,1H),5.2606(d,J＝3.75HZ,1H),5.1177(s,1H),2.8763(m,J＝6,85HZ,2H),2.8623(m,J＝9.69HZ,2H),2.4341(m,J＝3.55HZ,2H),2.3180(m,J＝7.2125HZ,2H),1.9937(m,J＝9.8152HZ,3H),1.8264(m,J＝3.258HZ,1H),1.6931(m,J＝4.2625HZ,1H),1.6159(d,J＝5.33HZ,1H),1.5121(m,J＝4.48HZ,1H),1.3301(m,J＝7.45HZ,4H),0.9940(m,J＝7.475HZ,3H),0.9353(m,J＝7.2375HZ,5H),0.7791(d,J＝7.1HZ,3H)；
13C-NMR171.1203,170.6127(ArtC＝O),167.3167(CPT-16a),157.3081(CPT-21),152.4506(CPT-6),148.8204(CPT-15),146.2229(CPT-8),145.7012(CPT-13),131.0144(CPT-12),130.4693(CPT-3),129.7100(CPT-2),128.6331(CPT-7),128.1745(CPT-11),128.1107(CPT-10),127.8704(CPT-16),120.2405(CPT-9),104.2652(ART-12),96.3067(ART-4),92.2332(ART-5),91.2771(ART-6),79.8964(CPT-20),76.1379(CPT-14),51.4200(ART-1),49.9400(CPT-5),45.1595(ART-7),37.0777(ART-10),36.0987(ART-3),33.9923(ART-9),31.8131(CPT-19),31.6769(ART-11),28.8540,28.5795(ART-O＝C-C-C＝O-),25.7284(ART-15),24.4896(ART-2),21.8608(ART-8),20.1093(ART-14),11.9571(ART-13),7.5473(CPT-18),IR＝(Ar-H:2929.03,2875.63C＝O:1750.98,1663.72)HRMSm/z[M+H]
+:715.2840(CalcdforC
39H
43N
2O
11:715.2867).
Embodiment 2: the preparation (2) of camptothecine and Artesunate conjugate
Artesunate (10mmol) and chloroform (20mL) is added in 25mL round-bottomed flask, 30 DEG C of stirring and dissolving, add EDCI (10mmol) again, 30 DEG C are stirred 0.5h, then DMAP (50mg) is added, 25 DEG C are stirred 0.5h, finally add camptothecine (3mmol), 30 DEG C of stirring reaction 3h, obtain faint yellow suspension liquid, after reaction terminates, methylene dichloride (40mL) is added in reaction solution, suction filtration, filtrate is successively with saturated sodium-chloride water solution and saturated sodium bicarbonate aqueous solution washing, use anhydrous magnesium sulfate drying again, suction filtration, filtrate reduced in volume is to dry, gained enriched material carries out silica gel column chromatography, eluent is methylene dichloride and the methyl alcohol mixed liquor of volume ratio 100:1, collect the elutriant containing target components, namely the camptothecine shown in formula (I) and Artesunate conjugate is obtained after concentrate drying, productive rate 85%.
Embodiment 3: shown in formula (I), compound on tumor cell inhibitory effect is active
1) test materials
Liver cancer cell (SMMC-7721), human breast cancer cell (MCF-7), all purchased from the American Type Culture Collection council of Chinese Academy of Sciences cell bank.
Camptothecine and Artesunate standard substance are purchased from lark prestige bio tech ltd.
Cell maintenance medium is 2% serum-concentration substratum, and SMMC-7721 uses RPMI1640 substratum, and MCF-7 uses DMEM substratum.
The camptothecine prepare embodiment 1 and Artesunate conjugate, camptothecine, Artesunate, dimethyl sulfoxide (DMSO) (DMSO) are diluted with cell maintenance medium respectively, often kind of sample does 6 concentration gradients: 0.032,0.16,0.8,4,20,100 μm of ol/L, and each concentration gradient does 4 multiple holes.
2) detection method
By the human liver cancer cell (SMMC-7721), the human breast cancer cell (MCF-7) that are in logarithmic phase, be configured to single cell suspension, with 10 with corresponding nutrient solution
5the density in individual/hole is inoculated in 96 orifice plates, CO
2in cell culture incubator after 37 DEG C of cultivation 24h, discard cell culture fluid, be divided into 5 groups: group 1 is experimental group (conjugate prepared by cell maintenance medium dilution embodiment 1, concentration is 0.032,0.16,0.8,4,20,100 μm of ol/L); Group 2 is blank group (only adding cell maintenance medium); Group 3 is negative control group (adding the isocyatic DMSO of cell maintenance medium dilution); Group 4 is positive controls (adding the isocyatic camptothecin standard product of cell maintenance medium dilution), and group 5 is also positive controls (adding the isocyatic Artesunate standard substance of cell maintenance medium dilution).Every hole adds reference substance or the medicine of 100 μ L, CO
2in incubator 37 DEG C hatch 48h after, every hole adds the MTT solution of the 5mg/mL of 20 μ L, after continuing to hatch 4h, discard liquid in hole, add 100 μ LDMSO, vibrate after fully dissolving to purple precipitation, the absorbance value (OD value) in each hole when being 492nm with microplate reader mensuration wavelength.Calculate the mean OD value in multiple hole, calculate the growth inhibition ratio of compound on intracellular according to mean OD value: inhibiting rate=1-[(experimental group OD value-blank group OD value)/(negative control group OD value-blank group OD value)] × 100%.The results are shown in Figure shown in 1.
Compound shown in formula (I) is determined to two kinds of tumour cells: the cell inhibitory effect of liver cancer cell (SMMC-7721), human breast cancer cell (MCF-7) is active, sees Fig. 1 to growth of tumour cell inhibit activities with mtt assay.As seen from the figure, compound shown in formula (I) has all showed Developing restraint activity in various degree to two kinds of tumour cells.By the analysis to detected result, camptothecine of the present invention is close with camptothecine to the inhibiting rate of tumour cell with Artesunate conjugate, higher than Artesunate inhibition.And experimental result display, along with increasing of concentration, the inhibition of conjugate is progressively higher than camptothecine.
Embodiment 4: shown in formula (I), compound activity in vivo is evaluated
1) test materials
Medicine: camptothecine prepared by embodiment 1 and Artesunate conjugate, camptothecine, Artesunate, topotecan hydrochloride.Described medicine first uses DMSO and dissolve with ethanol respectively, use normal saline dilution respectively again, in each drug solution of dilution gained, the volumetric concentration of DMSO is 5%, the volumetric concentration of ethanol is 5%, drug level is according to the weight regulation (volume injected is 200 μ L, and dosage is 0.023mmol/kg/2days) of mouse.
Test is bought from Shanghai Slac Experimental Animal Co., Ltd. with nude mice.
2) detection method
This experiment have chosen four to five week ages female BAl BIc/c nude mice, treat nude mouse weigh 18 ± 2g time, human breast cancer cell (MCF-7 cell) is inoculated in nude mice oxter, and (cell concn of inoculated tumour is 1 × 10
7individual every, cell mouse).When the volume of nude mouse tumor grows to 200mm
3during left and right, getting 35 successful mouse of tumor inoculation, be divided into 5 groups at random, is camptothecine and Artesunate conjugate group, camptothecine group, Artesunate group, topotecan hydrochloride group respectively, often organizes 7 mouse.After overnight fasting, intraperitoneal administration, administration group is according to 0.023mmol/kg/2days dosed administration, volume injected is 200 μ L (regulating drug for injection concentration according to Mouse Weight), control group injecting normal saline 200 μ L, be administration every other day, be administered four times altogether, within the 8th day, put to death and get tumour and weigh.Tumour inhibiting rate result is as follows:
Table 1 different pharmaceutical is to the tumour inhibiting rate of human breast cancer cell
| Topotecan hydrochloride | Camptothecine | Artesunate | Conjugate | |
| Tumour inhibiting rate (%) | 70.21±1.32 | 45.31±1.34 | 40.21±0.34 | 50.19±1.31 |
From table inhibiting tumor assay result we can show that the tumour inhibiting rate of conjugate is higher than Artesunate and camptothecine, not as good as clinical medicine topotecan hydrochloride, the solvability of this and compound has certain relation.
By monitoring (as shown in Figure 2) Mouse Weight, we can find out camptothecine having the greatest impact to Mouse Weight, average mice body weight by the 17.8g of camptothecine, drop to 11.9g after 8 days, toxicity is very large; And to the substantially identical 17g of Artesunate group Mouse Weight and control group, toxicity can be ignored substantially; Drop to 15.8g to the Mouse Weight of conjugate group after 8 days, illustrate that conjugate toxicity is between Artesunate and camptothecine, far below camptothecine.
Claims (9)
1. the camptothecine shown in a formula (I) and Artesunate conjugate:
2. the preparation method of a camptothecine as claimed in claim 1 and Artesunate conjugate, it is characterized in that described preparation method is: be dissolved in organic solvent a by Artesunate shown in camptothecine formula (II) Suo Shi and formula (III), under the effect of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and DMAP, in 15 ~ 50 DEG C of stirring reactions, TLC tracking monitor no longer carries out to reaction, reaction solution aftertreatment, obtains the camptothecine shown in formula (I) and Artesunate conjugate;
3. the preparation method of camptothecine as claimed in claim 2 and Artesunate conjugate, is characterized in that described organic solvent a is chloroform or methylene dichloride.
4. the preparation method of camptothecine as claimed in claim 2 and Artesunate conjugate, is characterized in that the volumetric usage of described organic solvent a with the molar amount of camptothecine for 1 ~ 10mL/mmol.
5. the preparation method of camptothecine as claimed in claim 2 and Artesunate conjugate, is characterized in that described camptothecine is 1:1 ~ 10 with the ratio of the amount of substance that feeds intake of Artesunate; The ratio of the amount of substance that feeds intake of described camptothecine and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is 1:1 ~ 5.
6. the preparation method of camptothecine as claimed in claim 2 and Artesunate conjugate, is characterized in that the quality consumption of described DMAP with the molar amount of camptothecine for 1 ~ 20mg/mmol.
7. the preparation method of camptothecine as claimed in claim 2 and Artesunate conjugate, is characterized in that described post-treating method is:
After reaction terminates, in reaction solution, add organic solvent b, suction filtration, filtrate is successively with saturated sodium-chloride water solution and saturated sodium bicarbonate aqueous solution washing, use anhydrous magnesium sulfate drying again, suction filtration, filtrate reduced in volume is to dry, and gained enriched material carries out silica gel column chromatography, eluent is methylene dichloride and the methyl alcohol mixed liquor of volume ratio 100:1, collect the elutriant containing target compound, concentrate drying, obtains the camptothecine shown in formula (I) and Artesunate conjugate; Described organic solvent b is chloroform or methylene dichloride, and its volumetric usage is 2 times of organic solvent a volume.
8. the preparation method of camptothecine as claimed in claim 2 and Artesunate conjugate, it is characterized in that described preparation method is: under room temperature, Artesunate is dissolved in organic solvent a, dissolve completely and add 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, stir 30 minutes, add DMAP again, continue stirring 30 minutes, finally add camptothecine, stirring reaction 3 hours at 25 DEG C, after reaction terminates, organic solvent b is added in reaction solution, suction filtration, filtrate is successively with saturated sodium-chloride water solution and saturated sodium bicarbonate aqueous solution washing, use anhydrous magnesium sulfate drying again, suction filtration, filtrate reduced in volume is to dry, gained enriched material carries out silica gel column chromatography, eluent is methylene dichloride and the methyl alcohol mixed liquor of volume ratio 100:1, collect the elutriant containing target compound, concentrate drying, obtain the camptothecine shown in formula (I) and Artesunate conjugate, wherein, described organic solvent a is chloroform, and the volumetric usage of described organic solvent a with the molar amount of camptothecine for 3 ~ 5mL/mmol, described camptothecine is 1:3 with the ratio of the amount of substance that feeds intake of Artesunate, the ratio of the amount of substance that feeds intake of described camptothecine and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is 1:3, the quality consumption of described DMAP with the molar amount of camptothecine for 5 ~ 10mg/mmol, in last handling process, described organic solvent b is chloroform or methylene dichloride, and its volumetric usage is 2 times of organic solvent a volume.
9. camptothecine as claimed in claim 1 and Artesunate conjugate are preparing the application in antitumor drug, it is characterized in that described tumour is cerebral tumor, oral carcinoma, laryngocarcinoma, the esophageal carcinoma, lung cancer, cancer of the stomach, liver cancer, kidney, prostate cancer, colorectal carcinoma, mammary cancer, ovarian cancer, carcinoma of the pancreas, bladder cancer, skin carcinoma, leukemia, sarcoma, the rectum cancer or glioma.
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