CN105968171A - Preparation method of sulforaphene derivative - Google Patents
Preparation method of sulforaphene derivative Download PDFInfo
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- CN105968171A CN105968171A CN201610320292.6A CN201610320292A CN105968171A CN 105968171 A CN105968171 A CN 105968171A CN 201610320292 A CN201610320292 A CN 201610320292A CN 105968171 A CN105968171 A CN 105968171A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0819—Tripeptides with the first amino acid being acidic
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Abstract
The invention relates to a preparation of a sulforaphene derivative and belongs to the field of anti-cancer compounds. Compared with the conventional method, the method is characterized in that a crystallization method is mainly used for replacing the conventional preparation chromatography method; the preparation cost can be obviously reduced; the large-scale preparation can be achieved. The method mainly comprises the steps: reacting reduced glutathione with sulforaphene crude products; mixing organic solvents such as ethanol with water for reacting, wherein the organic solvents are mutually soluble with water; and crystallizing the reaction product. The purity of the crystallized product is 96%; large-batch preparation of sulforaphene-glutathione complexes can be achieved.
Description
Technical field
The present invention relates to preparation and the purifying process of a kind of raphanin derivative compound, especially raphanin-paddy
The preparation method of Guang sweet peptide conjugate.
Background technology
Tumor be body under various carcinogenic factor effects, the cell of local organization loses on gene level
The neoplasm its normal regulation grown being caused paraplasm and differentiation and formed.Neoplasm is once formed,
The most no longer stopping growing, his growth is not by normal body physiological regulation, but destroys normal structure and device
Official, this point is especially apparent in malignant tumor.Compared with benign tumor, malignant growth speed is fast,
In infiltrative growth, easily there is hemorrhage, downright bad, ulcer etc., and often have metastasis, cause human body to disappear
Thin, unable, anemia, inappetence, heating and serious organ function are impaired etc., ultimately cause trouble
Person is dead.
The Therapeutic Method of tumor has operative treatment, radiotherapy and Drug therapy (i.e. chemotherapy) etc., wherein
Chemotherapy is treatment means currently mainly.Chemotherapy can be cured a part of tumor patient or extend trouble
The life of person, occupies increasingly critical role in oncotherapy.But, traditional antitumor drug pair
The killing rate of cancer cell is relatively low or needs higher concentration, therefore creates many serious toxicities.
Along with growth in the living standard, people significantly improve for the treatment expection of cancer, it is desirable to curing cancer
When healthy be not greatly affected, thus, it is found that new, high efficiency anti-tumor compound shows
Obtain the most urgent.
Raphanin is a kind of active skull cap components extracted in Chinese medicine Semen Raphani, close with Sulforaphane structure,
The most therefore close with the anticancer effect of Sulforaphane.But due to its structural instability, it is impossible to long term storage,
Seriously limit its application.Raphanin can be combined with the material of the thiol containing types such as reduced glutathion, makes
Easily the group of degraded is protected, and remains its active anticancer, defines brand-new anti-cancer compounds
Thing.The invention mainly includes and have studied a kind of raphanin derivant, raphanin-glutathion conjugate
Preparation method, the method instead of and original utilizes sterling raphanin participate in reaction and utilize preparative high
The approach of effect liquid phase chromatogram purification.
Concrete grammar and the step of the present invention are as follows:
It is an object of the invention to provide a kind of raphanin derivant, the preparation of raphanin-glutathion conjugate
Method, the chemical formula of this conjugate is:
A kind of preparation method of raphanin derivant, it is characterised in that:
(1) reduced glutathion is dissolved in and reduced glutathion is dissolved in going of 1 to 1000 volume times
Ionized water is dissolved.
(2) adding raphanin in reduced glutathion solution, (raphanin purity is
0.1%-100%), reduced glutathion is 100:1 to 1:100 with the molar ratio of raphanin.
(3) control temperature is at 0 DEG C to 80 DEG C, reacts 1 to 144 hour.
(4) if reaction system solid content is more than 5%, then deionized water is added, by dilute for reactant liquor solid content
Release between 1%-5%.If reaction system solid content is less than 1%, then by Rotary Evaporators concentration of reaction solution,
It is 1%-5% to reactant liquor solid content.
(5) after being filtered to remove insoluble impurity, add in reactant liquor 0.1 to 500 volume times with
The organic solvent that water dissolves each other, control temperature, at 0 DEG C to 100 DEG C, stirs 1min to 120min, at-20 DEG C
Crystallize at 20 DEG C.
(6) washing with alcohol after filtering, is drying to obtain.
Except as otherwise noted, the added composition proportion of each step described herein is mass ratio.
In described preparation method, the organic solvent miscible with water added can be ethanol, methanol, second
Nitrile, the pharmaceutically acceptable organic solvent such as isopropanol.
Accompanying drawing explanation
Fig. 1 is the mass-spectrogram of compound.
Fig. 2 is the infared spectrum of compound.
Fig. 3 is compound1H-NMR collection of illustrative plates.
Fig. 4 is compound13C-NMR collection of illustrative plates.
Fig. 5 is the preparative hplc collection of illustrative plates of compound.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention will be further described, but the purpose of these embodiments is also
Do not lie in and limit the scope of the invention.
Embodiment 1
Being dissolved in 100ml deionized water by 10g reduced glutathion, being added thereto to 10g purity is
Mass percent 96% raphanin, 25 DEG C are reacted 16 hours.
Adding 300ml deionized water, 50 DEG C of stirring 30min dissolve, and are filtered to remove insoluble impurities, add
Entering the dehydrated alcohol of 1600ml, controlling temperature is 75 DEG C of stirring 1h, is placed at 4 DEG C crystallization.
Crystallization solid is filtered, with 100ml absolute ethanol washing 3 times, drying 14.8g, pure
Degree is the product of 97.5%.
Embodiment 2
Being dissolved in 500ml deionized water by 5g reduced glutathion, being added thereto to 10g purity is
50% raphanin, 35 DEG C are reacted 40 hours.
Utilize Rotary Evaporators 50 DEG C, pressure 45mbar, reaction system is concentrated into 200ml.Cross and filter
Remove insoluble impurity, add the absolute methanol of 1800ml, control temperature at 65 DEG C, stir 40min,
It is placed at 0 DEG C crystallization.
Crystallization solid is filtered, by 50ml absolute ethanol washing 3 times, drying 7.0g, purity
It it is the product of 92.9%.
Embodiment 3
Being dissolved in 100ml deionized water by 1g reduced glutathion, being added thereto to 5g purity is 20%
Raphanin, 40 DEG C are reacted 48 hours.
Utilize Rotary Evaporators 55 DEG C, pressure 50mbar, reaction system is concentrated into 20ml.Cross and filter
Remove insoluble impurity, add the acetonitrile of 180ml, control temperature at 80 DEG C, stir 20min, be placed in
Crystallize at-10 DEG C.
Crystallization solid is filtered, by 10ml absolute ethanol washing 3 times, drying 1.5g, purity
It it is the product of 95.3%.
Embodiment 4
Being dissolved in 200ml deionized water by 10g reduced glutathion, being added thereto to 15g purity is
75% raphanin, room temperature reaction 26 hours.
Add 100ml deionized water, 60 DEG C of stirring and dissolving 20min, be filtered to remove insoluble impurities, add
2000ml isopropanol, controls temperature and stirs 20min at 85 DEG C, be placed in crystallizing at room temperature.
Crystallization solid is filtered, with 100ml absolute ethanol washing 3 times, drying 15.1g, pure
Degree is the product of 94.5%.
Embodiment 5
Being dissolved in 200ml deionized water by 10g reduced glutathion, being added thereto to 15g purity is
75% raphanin, room temperature reaction 25 hours.
Add 100ml deionized water, 60 DEG C of stirring and dissolving 20min, be filtered to remove insoluble impurities, add
2000ml isopropanol, controls temperature and stirs 20min at 85 DEG C, be placed in crystallizing at room temperature.
Crystallization solid is filtered, with 100ml absolute ethanol washing 3 times, drying 15.1g, pure
Degree is the product of 95.5%.
Embodiment 6
One, material and method
1. experimental cell system and related chemistry reagent: CH27 H460 buys from U.S. ATCC
Cell bank, cultivates in RPMI-1640 (HyClone) culture medium containing 10% hyclone, with 0.25%
Pass on after trypsin and 0.02%EDTA conventional digestion.This experiment related chemistry reagent is purchased from
Sigma company.
2. the compound body outer suppressioning experiment to H460 hemocyte: the H460 cell of trophophase of taking the logarithm disappears
After chemical conversion individual cells, being inoculated in 96 orifice plates, 3000, every hole cell, containing 5%CO237 DEG C
Incubator is cultivated.After being dissolved with sterile deionized water by compound, cross 0.22 μm filter membrane degerming, with containing
Serum free culture system liquid dilutes so that the ultimate density of conjugate II is 1 μM, 2 μMs, 3 μMs, 5 μMs, 10 μMs,
20 μMs and 50 μMs, after H460 cell inoculated and cultured 24 hours, change into containing compound respective concentration
Culture fluid cultivate cell, negative control group culture medium adds isopyknic sterile deionized water and cultivates thin
Born of the same parents.After cultivating 48 hours the most respectively, every hole adds 20 μ L MTT, containing 5%CO237 DEG C of trainings
After foster case continues to hatch 3h, clean the solution in every hole, be separately added into the DMSO of 150 μ L, shaking table
Concussion 10min, measures the absorbance under the 490nm wavelength of every hole.Survive with the negative control group of 0 hour
On the basis of cell number, calculate the half-inhibition concentration (IC of cell growth50), experimental result data is shown in Table 1.
Two, experimental result
Table 1 shows, the growth of H460 people's Lung Squamous Carcinoma Cells is rised in value and had significant inhibition by compound.
After processing 48 hours, the half-inhibition concentration (IC of the H460 cell growth of compound pair50) it is 9.116 μMs.
Embodiment 7
One, material and method
1. experimental cell system and related chemistry reagent: Human cervical cancer cell lines Hela buys from U.S. ATCC
Cell bank, cultivates in RPMI-1640 (HyClone) culture medium containing 10% hyclone, with 0.25%
Pass on after trypsin and 0.02%EDTA conventional digestion.This experiment related chemistry reagent is purchased from
Sigma company.
2. the compound body outer suppressioning experiment to Hela hemocyte: the Hela cell dissociation of trophophase of taking the logarithm
After becoming individual cells, being inoculated in 96 orifice plates, 3000, every hole cell, containing 5%CO237 DEG C
Incubator is cultivated.After being dissolved with sterile deionized water by compound, cross 0.22 μm filter membrane degerming, with containing
Serum free culture system liquid dilutes so that the ultimate density of conjugate II is 1 μM, 2 μMs, 3 μMs, 5 μMs, 10 μMs,
20 μMs and 50 μMs, after Hela cell inoculated and cultured 24 hours, change into containing compound respective concentration
Culture fluid cultivate cell, negative control group culture medium adds isopyknic sterile deionized water and cultivates thin
Born of the same parents.After cultivating 48 hours the most respectively, every hole adds 20 μ L MTT, containing 5%CO237 DEG C of trainings
After foster case continues to hatch 3h, clean the solution in every hole, be separately added into the DMSO of 150 μ L, shaking table
Concussion 10min, measures the absorbance under the 490nm wavelength of every hole.Survive with the negative control group of 0 hour
On the basis of cell number, calculate the half-inhibition concentration (IC of cell growth50), experimental result data is shown in Table 1.
Two, experimental result
Table 1 shows, the growth of Hela human cervical carcinoma cell is rised in value and had significant inhibition by compound.
After processing 48 hours, the half-inhibition concentration (IC of the Hela cell growth of compound pair50) it is 14.92 μMs.
Embodiment 8
One, material and method
1. experimental cell system and related chemistry reagent: human lung cancer cell line H446 buys thin from U.S. ATCC
Born of the same parents storehouse, cultivates in RPMI-1640 (HyClone) culture medium containing 10% hyclone, with 0.25%
Pass on after trypsin and 0.02%EDTA conventional digestion.This experiment related chemistry reagent is purchased from
Sigma company.
2. the compound body outer suppressioning experiment to H446 hemocyte: the H446 cell of trophophase of taking the logarithm disappears
After chemical conversion individual cells, being inoculated in 96 orifice plates, 3000, every hole cell, containing 5%CO237 DEG C
Incubator is cultivated.After being dissolved with sterile deionized water by compound, cross 0.22 μm filter membrane degerming, with containing
Serum free culture system liquid dilutes so that the ultimate density of conjugate II is 1 μM, 2 μMs, 3 μMs, 5 μMs, 10 μMs,
20 μMs and 50 μMs, after H446 cell inoculated and cultured 24 hours, change into containing compound respective concentration
Culture fluid cultivate cell, negative control group culture medium adds isopyknic sterile deionized water and cultivates thin
Born of the same parents.After cultivating 48 hours the most respectively, every hole adds 20 μ L MTT, containing 5%CO237 DEG C of trainings
After foster case continues to hatch 3h, clean the solution in every hole, be separately added into the DMSO of 150 μ L, shaking table
Concussion 10min, measures the absorbance under the 490nm wavelength of every hole.Survive with the negative control group of 0 hour
On the basis of cell number, calculate the half-inhibition concentration (IC of cell growth50), experimental result data is shown in Table 1.
Two, experimental result
Table 1 shows, the growth of H446 human lung carcinoma cell is rised in value and had significant inhibition by compound.
After processing 48 hours, the half-inhibition concentration (IC of the H446 cell growth of compound pair50) it is 6.098
μM。
Embodiment 9
One, material and method
1. experimental cell system and related chemistry reagent: Human skin melanoma cell line A375 is bought from the U.S.
ATCC cell bank, cultivates in RPMI-1640 (HyClone) culture medium containing 10% hyclone,
Pass on after 0.25% trypsin and 0.02%EDTA conventional digestion.This experiment related chemistry reagent
It is purchased from Sigma company.
2. the compound body outer suppressioning experiment to A375 hemocyte: the A375 cell of trophophase of taking the logarithm disappears
After chemical conversion individual cells, being inoculated in 96 orifice plates, 3000, every hole cell, containing 5%CO237 DEG C
Incubator is cultivated.After being dissolved with sterile deionized water by compound, cross 0.22 μm filter membrane degerming, with containing
Serum free culture system liquid dilutes so that the ultimate density of conjugate II is 1 μM, 2 μMs, 3 μMs, 5 μMs, 10 μMs,
20 μMs and 50 μMs, after A375 cell inoculated and cultured 24 hours, change into containing compound respective concentration
Culture fluid cultivate cell, negative control group culture medium adds isopyknic sterile deionized water and cultivates thin
Born of the same parents.After cultivating 48 hours the most respectively, every hole adds 20 μ L MTT, containing 5%CO237 DEG C of trainings
After foster case continues to hatch 3h, clean the solution in every hole, be separately added into the DMSO of 150 μ L, shaking table
Concussion 10min, measures the absorbance under the 490nm wavelength of every hole.Survive with the negative control group of 0 hour
On the basis of cell number, calculate the half-inhibition concentration (IC of cell growth50), experimental result data is shown in Table 1.
Two, experimental result
Table 1 shows, the growth of A375 Human skin melanoma cell is rised in value to have and significantly pressed down by compound
Effect processed.After processing 48 hours, the half-inhibition concentration (IC of the A375 cell growth of compound pair50)
It it is 17.515 μMs.
Embodiment 10
One, material and method
1. experimental cell system and related chemistry reagent: human breast carcinoma cell lines MCF-7 is bought from the U.S.
ATCC cell bank, cultivates in RPMI-1640 (HyClone) culture medium containing 10% hyclone,
Pass on after 0.25% trypsin and 0.02%EDTA conventional digestion.This experiment related chemistry reagent
It is purchased from Sigma company.
2. the compound body outer suppressioning experiment to MCF-7 hemocyte: the MCF-7 of trophophase of taking the logarithm is thin
After born of the same parents are digested to individual cells, being inoculated in 96 orifice plates, 3000, every hole cell, containing 5%CO2
37 DEG C of incubators in cultivate.After being dissolved with sterile deionized water by compound, cross 0.22 μm filter membrane degerming,
Dilute with containing serum free culture system liquid so that the ultimate density of conjugate II is 1 μM, 2 μMs, 3 μMs, 5 μMs,
10 μMs, 20 μMs and 50 μMs, after MCF-7 cell inoculated and cultured 24 hours, change into containing compound
The culture fluid of respective concentration cultivates cell, adds isopyknic aseptic deionization in negative control group culture medium
Aquaponic cell.After cultivating 48 hours the most respectively, every hole adds 20 μ L MTT, containing 5%CO2
37 DEG C of incubators continue to hatch 3h after, clean the solution in every hole, be separately added into the DMSO of 150 μ L,
Shaking table concussion 10min, measures the absorbance under the 490nm wavelength of every hole.With the negative control group of 0 hour
On the basis of survivaling cell number, calculate the half-inhibition concentration (IC of cell growth50), experimental result data is shown in
Table 1.
Two, experimental result
Table 1 shows, the growth of MCF-7 human breast cancer cell is rised in value to have and significantly suppressed effect by compound
Really.After processing 48 hours, the half-inhibition concentration (IC of the MCF-7 cell growth of compound pair50) it is
9.599μM。
Embodiment 11
One, material and method
1. experimental cell system and related chemistry reagent: Non-small cell lung carcinoma cell line A549 is bought from the U.S.
ATCC cell bank, cultivates in RPMI-1640 (HyClone) culture medium containing 10% hyclone,
Pass on after 0.25% trypsin and 0.02%EDTA conventional digestion.This experiment related chemistry reagent
It is purchased from Sigma company.
2. the compound body outer suppressioning experiment to A549 hemocyte: the A549 cell of trophophase of taking the logarithm disappears
After chemical conversion individual cells, being inoculated in 96 orifice plates, 3000, every hole cell, containing 5%CO237 DEG C
Incubator is cultivated.After being dissolved with sterile deionized water by compound, cross 0.22 μm filter membrane degerming, with containing
Serum free culture system liquid dilutes so that the ultimate density of conjugate II is 1 μM, 2 μMs, 3 μMs, 5 μMs, 10 μMs,
20 μMs and 50 μMs, after A549 cell inoculated and cultured 24 hours, change into containing compound respective concentration
Culture fluid cultivate cell, negative control group culture medium adds isopyknic sterile deionized water and cultivates thin
Born of the same parents.After cultivating 48 hours the most respectively, every hole adds 20 μ L MTT, containing 5%CO237 DEG C of trainings
After foster case continues to hatch 3h, clean the solution in every hole, be separately added into the DMSO of 150 μ L, shaking table
Concussion 10min, measures the absorbance under the 490nm wavelength of every hole.Survive with the negative control group of 0 hour
On the basis of cell number, calculate the half-inhibition concentration (IC of cell growth50), experimental result data is shown in Table 1.
Two, experimental result
Table 1 shows, the growth of A549 Non-small cell lung carcinoma cell is rised in value to have and significantly pressed down by compound
Effect processed.After processing 48 hours, the half-inhibition concentration (IC of the A549 cell growth of compound pair50)
It it is 9.154 μMs.
Embodiment 12
One, material and method
1. experimental cell system and related chemistry reagent: Non-small cell lung carcinoma cell line H1299 is bought from beautiful
State's ATCC cell bank, cultivates in RPMI-1640 (HyClone) culture medium containing 10% hyclone
In, pass on after 0.25% trypsin and 0.02%EDTA conventional digestion.This experiment related chemistry
Reagent is purchased from Sigma company.
2. the compound body outer suppressioning experiment to H1299 hemocyte: the H1299 cell of trophophase of taking the logarithm
After being digested to individual cells, being inoculated in 96 orifice plates, 3000, every hole cell, containing 5%CO2's
37 DEG C of incubators are cultivated.After being dissolved with sterile deionized water by compound, cross 0.22 μm filter membrane degerming,
Dilute with containing serum free culture system liquid so that the ultimate density of conjugate II is 1 μM, 2 μMs, 3 μMs, 5 μMs,
10 μMs, 20 μMs and 50 μMs, after H1299 cell inoculated and cultured 24 hours, change into containing compound
The culture fluid of respective concentration cultivates cell, adds isopyknic aseptic deionization in negative control group culture medium
Aquaponic cell.After cultivating 48 hours the most respectively, every hole adds 20 μ L MTT, containing 5%CO2
37 DEG C of incubators continue to hatch 3h after, clean the solution in every hole, be separately added into the DMSO of 150 μ L,
Shaking table concussion 10min, measures the absorbance under the 490nm wavelength of every hole.With the negative control group of 0 hour
On the basis of survivaling cell number, calculate the half-inhibition concentration (IC of cell growth50), experimental result data is shown in
Table 1.
Two, experimental result
Table 1 shows, the growth of H1299 Non-small cell lung carcinoma cell is rised in value and had significantly by compound
Inhibition.After processing 48 hours, the half-inhibition concentration (IC of the H1299 cell growth of compound pair50)
It it is 8.333 μMs.
Embodiment 13
One, material and method
1. experimental cell system and related chemistry reagent: human hepatoma cell line HepG2 buys from U.S. ATCC
Cell bank, cultivates in RPMI-1640 (HyClone) culture medium containing 10% hyclone, with 0.25%
Pass on after trypsin and 0.02%EDTA conventional digestion.This experiment related chemistry reagent is purchased from
Sigma company.
2. the compound body outer suppressioning experiment to HepG2 hemocyte: the HepG2 cell of trophophase of taking the logarithm
After being digested to individual cells, being inoculated in 96 orifice plates, 3000, every hole cell, containing 5%CO2's
37 DEG C of incubators are cultivated.After being dissolved with sterile deionized water by compound, cross 0.22 μm filter membrane degerming,
Dilute with containing serum free culture system liquid so that the ultimate density of conjugate II is 1 μM, 2 μMs, 3 μMs, 5 μMs,
10 μMs, 20 μMs and 50 μMs, after HepG2 cell inoculated and cultured 24 hours, change into containing compound
The culture fluid of respective concentration cultivates cell, adds isopyknic aseptic deionization in negative control group culture medium
Aquaponic cell.After cultivating 48 hours the most respectively, every hole adds 20 μ L MTT, containing 5%CO2
37 DEG C of incubators continue to hatch 3h after, clean the solution in every hole, be separately added into the DMSO of 150 μ L,
Shaking table concussion 10min, measures the absorbance under the 490nm wavelength of every hole.With the negative control group of 0 hour
On the basis of survivaling cell number, calculate the half-inhibition concentration (IC of cell growth50), experimental result data is shown in
Table 1.
Two, experimental result
Table 1 shows, the growth of HepG2 human liver cancer cell is rised in value and had significant inhibition by compound.
After processing 48 hours, the half-inhibition concentration (IC of the HepG2 cell growth of compound pair50) it is 8.776
μM。
Embodiment 14
One, material and method
1. experimental cell system and related chemistry reagent: the breast cancer cell line of people's TNBC hypotype
MDA-MB-453 buys from U.S.'s ATCC cell bank, cultivates containing 10% hyclone
In RPMI-1640 (HyClone) culture medium, conventional with 0.25% trypsin and 0.02%EDTA
Pass on after digestion.This experiment related chemistry reagent is purchased from Sigma company.
2. the compound body outer suppressioning experiment to MDA-MB-453 hemocyte: trophophase of taking the logarithm
After MDA-MB-453 cell dissociation becomes individual cells, it is inoculated in 96 orifice plates, 3000, every hole cell,
Containing 5%CO237 DEG C of incubators in cultivate.After compound is dissolved with sterile deionized water, mistake
0.22 μm filter membrane is degerming, dilutes with containing serum free culture system liquid so that the ultimate density of conjugate II is 1 μM,
2 μMs, 3 μMs, 5 μMs, 10 μMs, 20 μMs and 50 μMs, in MDA-MB-453 cell inoculated and cultured
After 24 hours, change the culture fluid containing compound respective concentration into and cultivate cell, negative control group culture medium
The isopyknic sterile deionized water of middle addition cultivates cell.After cultivating 72 hours the most respectively, every hole adds
20 μ L MTT, containing 5%CO237 DEG C of incubators continue to hatch 3h after, that cleans in every hole is molten
Liquid, is separately added into the DMSO of 150 μ L, shaking table concussion 10min, measures under the 490nm wavelength of every hole
Absorbance.On the basis of the negative control group survivaling cell number of 0 hour, the half calculating cell growth presses down
Concentration (IC processed50), experimental result data is shown in Table 1.
Two, experimental result
Table 1 shows, the growth of MDA-MB-453 human liver cancer cell is rised in value to have and significantly pressed down by compound
Effect processed.After processing 72 hours, the half suppression of the MDA-MB-453 cell growth of compound pair is dense
Degree (IC50) it is 3.08 μMs.
Embodiment 15
One, material and method
1. experimental cell system and related chemistry reagent: the breast cancer cell line of people's TNBC hypotype
MDA-MB-231 buys from U.S.'s ATCC cell bank, cultivates containing 10% hyclone
In RPMI-1640 (HyClone) culture medium, conventional with 0.25% trypsin and 0.02%EDTA
Pass on after digestion.This experiment related chemistry reagent is purchased from Sigma company.
2. the compound body outer suppressioning experiment to MDA-MB-231 hemocyte: trophophase of taking the logarithm
After MDA-MB-231 cell dissociation becomes individual cells, it is inoculated in 96 orifice plates, 3000, every hole cell,
Containing 5%CO237 DEG C of incubators in cultivate.After compound is dissolved with sterile deionized water, mistake
0.22 μm filter membrane is degerming, dilutes with containing serum free culture system liquid so that the ultimate density of conjugate II is 1 μM,
2 μMs, 3 μMs, 5 μMs, 10 μMs, 20 μMs and 50 μMs, in MDA-MB-231 cell inoculated and cultured
After 24 hours, change the culture fluid containing compound respective concentration into and cultivate cell, negative control group culture medium
The isopyknic sterile deionized water of middle addition cultivates cell.After cultivating 72 hours the most respectively, every hole adds
20 μ L MTT, containing 5%CO237 DEG C of incubators continue to hatch 3h after, that cleans in every hole is molten
Liquid, is separately added into the DMSO of 150 μ L, shaking table concussion 10min, measures under the 490nm wavelength of every hole
Absorbance.On the basis of the negative control group survivaling cell number of 0 hour, the half calculating cell growth presses down
Concentration (IC processed50), experimental result data is shown in Table 1.
Two, experimental result
Table 1 shows, the growth of MDA-MB-231 human liver cancer cell is rised in value to have and significantly pressed down by compound
Effect processed.After processing 72 hours, the half suppression of the MDA-MB-231 cell growth of compound pair is dense
Degree (IC50) it is 8.71 μMs.
Embodiment 16
One, material and method
1. experimental cell system and related chemistry reagent: the breast cancer cell line of people's TNBC hypotype
MDA-MB-436 buys from U.S.'s ATCC cell bank, cultivates containing 10% hyclone
In RPMI-1640 (HyClone) culture medium, conventional with 0.25% trypsin and 0.02%EDTA
Pass on after digestion.This experiment related chemistry reagent is purchased from Sigma company.
2. the compound body outer suppressioning experiment to MDA-MB-436 hemocyte: trophophase of taking the logarithm
After MDA-MB-436 cell dissociation becomes individual cells, it is inoculated in 96 orifice plates, 3000, every hole cell,
Containing 5%CO237 DEG C of incubators in cultivate.After compound is dissolved with sterile deionized water, mistake
0.22 μm filter membrane is degerming, dilutes with containing serum free culture system liquid so that the ultimate density of conjugate II is 1 μM,
2 μMs, 3 μMs, 5 μMs, 10 μMs, 20 μMs and 50 μMs, in MDA-MB-436 cell inoculated and cultured
After 24 hours, change the culture fluid containing compound respective concentration into and cultivate cell, negative control group culture medium
The isopyknic sterile deionized water of middle addition cultivates cell.After cultivating 72 hours the most respectively, every hole adds
20 μ L MTT, containing 5%CO237 DEG C of incubators continue to hatch 3h after, that cleans in every hole is molten
Liquid, is separately added into the DMSO of 150 μ L, shaking table concussion 10min, measures under the 490nm wavelength of every hole
Absorbance.On the basis of the negative control group survivaling cell number of 0 hour, the half calculating cell growth presses down
Concentration (IC processed50), experimental result data is shown in Table 1.
Two, experimental result
Table 1 shows, the growth of MDA-MB-436 human liver cancer cell is rised in value to have and significantly pressed down by compound
Effect processed.After processing 72 hours, the half suppression of the MDA-MB-436 cell growth of compound pair is dense
Degree (IC50) it is 3.45 μMs.
Embodiment 17
One, material and method
1. experimental cell system and related chemistry reagent: the breast cancer cell line of people's TNBC hypotype
MDA-MB-468 buys from U.S.'s ATCC cell bank, cultivates containing 10% hyclone
In RPMI-1640 (HyClone) culture medium, conventional with 0.25% trypsin and 0.02%EDTA
Pass on after digestion.This experiment related chemistry reagent is purchased from Sigma company.
2. the compound body outer suppressioning experiment to MDA-MB-468 hemocyte: trophophase of taking the logarithm
After MDA-MB-468 cell dissociation becomes individual cells, it is inoculated in 96 orifice plates, 3000, every hole cell,
Containing 5%CO237 DEG C of incubators in cultivate.After compound is dissolved with sterile deionized water, mistake
0.22 μm filter membrane is degerming, dilutes with containing serum free culture system liquid so that the ultimate density of conjugate II is 1 μM,
2 μMs, 3 μMs, 5 μMs, 10 μMs, 20 μMs and 50 μMs, in MDA-MB-468 cell inoculated and cultured
After 24 hours, change the culture fluid containing compound respective concentration into and cultivate cell, negative control group culture medium
The isopyknic sterile deionized water of middle addition cultivates cell.After cultivating 72 hours the most respectively, every hole adds
20 μ L MTT, containing 5%CO237 DEG C of incubators continue to hatch 3h after, that cleans in every hole is molten
Liquid, is separately added into the DMSO of 150 μ L, shaking table concussion 10min, measures under the 490nm wavelength of every hole
Absorbance.On the basis of the negative control group survivaling cell number of 0 hour, the half calculating cell growth presses down
Concentration (IC processed50), experimental result data is shown in Table 1.
Two, experimental result
Table 1 shows, the growth of MDA-MB-468 human liver cancer cell is rised in value to have and significantly pressed down by compound
Effect processed.After processing 72 hours, the half suppression of the MDA-MB-468 cell growth of compound pair is dense
Degree (IC50) it is 3.59 μMs.
Embodiment 18
One, material and method
1. experimental cell system and related chemistry reagent: the breast cancer cell line MCF-7 of people's ER+ hypotype buys
From U.S.'s ATCC cell bank, cultivate and train at the RPMI-1640 (HyClone) containing 10% hyclone
Support in base, pass on after 0.25% trypsin and 0.02%EDTA conventional digestion.This experiment is correlated with
Chemical reagent is purchased from Sigma company.
2. the compound body outer suppressioning experiment to MCF-7 hemocyte: the MCF-7 of trophophase of taking the logarithm is thin
After born of the same parents are digested to individual cells, being inoculated in 96 orifice plates, 3000, every hole cell, containing 5%CO2
37 DEG C of incubators in cultivate.After being dissolved with sterile deionized water by compound, cross 0.22 μm filter membrane degerming,
Dilute with containing serum free culture system liquid so that the ultimate density of conjugate II is 1 μM, 2 μMs, 3 μMs, 5 μMs,
10 μMs, 20 μMs and 50 μMs, after MCF-7 cell inoculated and cultured 24 hours, change into containing compound
The culture fluid of respective concentration cultivates cell, adds isopyknic aseptic deionization in negative control group culture medium
Aquaponic cell.After cultivating 72 hours the most respectively, every hole adds 20 μ L MTT, containing 5%CO2
37 DEG C of incubators continue to hatch 3h after, clean the solution in every hole, be separately added into the DMSO of 150 μ L,
Shaking table concussion 10min, measures the absorbance under the 490nm wavelength of every hole.With the negative control group of 0 hour
On the basis of survivaling cell number, calculate the half-inhibition concentration (IC of cell growth50), experimental result data is shown in
Table 1.
Two, experimental result
Table 1 shows, the growth of MCF-7 human liver cancer cell is rised in value and had significant inhibition by compound.
After processing 72 hours, the half-inhibition concentration (IC of the MCF-7 cell growth of compound pair50) it is 3.48
μM。
Embodiment 19
One, material and method
1. experimental cell system and related chemistry reagent: the breast cancer cell line ZR-75-1 of people's ER+ hypotype purchases
Buy from U.S.'s ATCC cell bank, cultivate at the RPMI-1640 (HyClone) containing 10% hyclone
In culture medium, pass on after 0.25% trypsin and 0.02%EDTA conventional digestion.This experiment phase
Close chemical reagent and be purchased from Sigma company.
2. the compound body outer suppressioning experiment to ZR-75-1 hemocyte: the ZR-75-1 of trophophase of taking the logarithm
After cell dissociation becomes individual cells, being inoculated in 96 orifice plates, 3000, every hole cell, containing 5%CO2
37 DEG C of incubators in cultivate.After being dissolved with sterile deionized water by compound, cross 0.22 μm filter membrane degerming,
Dilute with containing serum free culture system liquid so that the ultimate density of conjugate II is 1 μM, 2 μMs, 3 μMs, 5 μMs,
10 μMs, 20 μMs and 50 μMs, after ZR-75-1 cell inoculated and cultured 24 hours, change into containing chemical combination
The culture fluid of thing respective concentration cultivates cell, negative control group culture medium adds isopyknic aseptic go from
Sub-Aquaponic cell.After cultivating 72 hours the most respectively, every hole adds 20 μ L MTT, containing 5%CO2
37 DEG C of incubators continue to hatch 3h after, clean the solution in every hole, be separately added into the DMSO of 150 μ L,
Shaking table concussion 10min, measures the absorbance under the 490nm wavelength of every hole.With the negative control group of 0 hour
On the basis of survivaling cell number, calculate the half-inhibition concentration (IC of cell growth50), experimental result data is shown in
Table 1.
Two, experimental result
Table 1 shows, the growth of ZR-75-1 human liver cancer cell is rised in value to have and significantly suppressed effect by compound
Really.After processing 72 hours, the half-inhibition concentration (IC of the ZR-75-1 cell growth of compound pair50)
It it is 2.82 μMs.
Embodiment 20
One, material and method
1. experimental cell system and related chemistry reagent: the breast cancer cell line ZR-75-1 of people's ER+ hypotype purchases
Buy from U.S.'s ATCC cell bank, cultivate at the RPMI-1640 (HyClone) containing 10% hyclone
In culture medium, pass on after 0.25% trypsin and 0.02%EDTA conventional digestion.This experiment phase
Close chemical reagent and be purchased from Sigma company.
2. the compound body outer suppressioning experiment to ZR-75-1 hemocyte: the ZR-75-1 of trophophase of taking the logarithm
After cell dissociation becomes individual cells, being inoculated in 96 orifice plates, 3000, every hole cell, containing 5%CO2
37 DEG C of incubators in cultivate.After being dissolved with sterile deionized water by compound, cross 0.22 μm filter membrane degerming,
Dilute with containing serum free culture system liquid so that the ultimate density of conjugate II is 1 μM, 2 μMs, 3 μMs, 5 μMs,
10 μMs, 20 μMs and 50 μMs, after ZR-75-1 cell inoculated and cultured 24 hours, change into containing chemical combination
The culture fluid of thing respective concentration cultivates cell, negative control group culture medium adds isopyknic aseptic go from
Sub-Aquaponic cell.After cultivating 72 hours the most respectively, every hole adds 20 μ L MTT, containing 5%CO2
37 DEG C of incubators continue to hatch 3h after, clean the solution in every hole, be separately added into the DMSO of 150 μ L,
Shaking table concussion 10min, measures the absorbance under the 490nm wavelength of every hole.With the negative control group of 0 hour
On the basis of survivaling cell number, calculate the half-inhibition concentration (IC of cell growth50), experimental result data is shown in
Table 1.
Two, experimental result
Table 1 shows, the growth of ZR-75-1 human liver cancer cell is rised in value to have and significantly suppressed effect by compound
Really.After processing 72 hours, the half-inhibition concentration (IC of the ZR-75-1 cell growth of compound pair50)
It it is 2.82 μMs.
Embodiment 21
One, material and method
1. experimental cell system and related chemistry reagent: the breast cancer cell line Sk-br-3 of people's HER2+ hypotype
Buy from U.S.'s ATCC cell bank, cultivate at the RPMI-1640 (HyClone) containing 10% hyclone
In culture medium, pass on after 0.25% trypsin and 0.02%EDTA conventional digestion.This experiment phase
Close chemical reagent and be purchased from Sigma company.
2. the compound body outer suppressioning experiment to Sk-br-3 hemocyte: the Sk-br-3 of trophophase of taking the logarithm is thin
After born of the same parents are digested to individual cells, being inoculated in 96 orifice plates, 3000, every hole cell, containing 5%CO2
37 DEG C of incubators in cultivate.After being dissolved with sterile deionized water by compound, cross 0.22 μm filter membrane degerming,
Dilute with containing serum free culture system liquid so that the ultimate density of conjugate II is 1 μM, 2 μMs, 3 μMs, 5 μMs,
10 μMs, 20 μMs and 50 μMs, after Sk-br-3 cell inoculated and cultured 24 hours, change into containing compound
The culture fluid of respective concentration cultivates cell, adds isopyknic aseptic deionization in negative control group culture medium
Aquaponic cell.After cultivating 72 hours the most respectively, every hole adds 20 μ L MTT, containing 5%CO2
37 DEG C of incubators continue to hatch 3h after, clean the solution in every hole, be separately added into the DMSO of 150 μ L,
Shaking table concussion 10min, measures the absorbance under the 490nm wavelength of every hole.With the negative control group of 0 hour
On the basis of survivaling cell number, calculate the half-inhibition concentration (IC of cell growth50), experimental result data is shown in
Table 1.
Two, experimental result
Table 1 shows, the growth of Sk-br-3 human liver cancer cell is rised in value and had significant inhibition by compound.
After processing 72 hours, the half-inhibition concentration (IC of the Sk-br-3 cell growth of compound pair50) it is 2.82
μM。
Table 1 is the IC of 48 hours in kinds of tumor cells of formula II50Value
Table 2 is formula II in the different subtype breast carcinoma IC of 72 hours50Value
Table 1
| Cell line | Hela | H446 | A375 | MCF-7 | H460 | A549 | H1299 | HepG2 |
| IC50(uM) | 14.92 | 6.098 | 17.515 | 9.599 | 9.116 | 9.154 | 8.333 | 8.776 |
Table 2
Claims (7)
1. a raphanin derivant, its chemical formula is as follows:
2. the derivant described in claim 1 is in the application prepared in the medicine of the tumor treating people.
3. the use in the medicine that preparation is used for treatment or prophylaxis of cancer of the derivant described in claim 1
On the way.
4. pharmaceutical composition, comprises the derivant described in claim 1 and pharmaceutical excipients.
5., according to the pharmaceutical composition described in claim 4, it is tablet, capsule, injectable powder, liquid
Agent or suspended form.
6. the method preparing a kind of raphanin derivant as claimed in claim 1, it is characterised in that:
(1) reduced glutathion is dissolved in and reduced glutathion is dissolved in going of 1 to 1000 volume times
Ionized water is dissolved；
(2) in reduced glutathion solution, raphanin is added, reduced glutathion and raphanin
Molar ratio is 100:1 to 1:100；
(3) control temperature is at 0 DEG C to 80 DEG C, reacts 1 to 144 hour；
(4) if reaction system solid content is more than 5%, then deionized water is added, by dilute for reactant liquor solid content
Release between 1%-5%；If reaction system solid content is less than 1%, then pass through Rotary Evaporators
Concentration of reaction solution, is 1%-5% to reactant liquor solid content；
(5) after being filtered to remove insoluble impurity, add in reactant liquor 0.1 to 500 volume times with
The organic solvent that water dissolves each other, control temperature, at 0 DEG C to 100 DEG C, stirs 1min to 120min,
Crystallize at-20 DEG C to 20 DEG C；
(6) washing with alcohol after filtering, is drying to obtain.
Method the most according to claim 6, it is characterised in that: organic solvent is ethanol, methanol,
Acetonitrile or isopropanol.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102816203A (en) * | 2011-06-10 | 2012-12-12 | 上海医药工业研究院 | Substituted quinoline compound, and preparation method, medicine combination and application thereof |
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2016
- 2016-05-15 CN CN201610320292.6A patent/CN105968171A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102816203A (en) * | 2011-06-10 | 2012-12-12 | 上海医药工业研究院 | Substituted quinoline compound, and preparation method, medicine combination and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| HU WANG ET AL: "Development and Validation of an LC–MS–MS Method for the Simultaneous Determination of Sulforaphane and its Metabolites in Rat Plasma and its Application in Pharmacokinetic Studies", 《JOURNAL OF CHROMATOGRAPHIC SCIENCE》 * |
| YUESHENG ZHANG: "Role of glutathione in the accumulation of anticarcinogenic isothiocyanates and their glutathione conjugates by murine hepatoma cells", 《CARCINOGENESIS》 * |
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