CN108395429A - A kind of compound and its preparation method and application - Google Patents
A kind of compound and its preparation method and application Download PDFInfo
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- CN108395429A CN108395429A CN201810067124.XA CN201810067124A CN108395429A CN 108395429 A CN108395429 A CN 108395429A CN 201810067124 A CN201810067124 A CN 201810067124A CN 108395429 A CN108395429 A CN 108395429A
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- C07D455/00—Heterocyclic compounds containing quinolizine ring systems, e.g. emetine alkaloids, protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine
- C07D455/03—Heterocyclic compounds containing quinolizine ring systems, e.g. emetine alkaloids, protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine containing quinolizine ring systems directly condensed with at least one six-membered carbocyclic ring, e.g. protoberberine; Alkylenedioxy derivatives of dibenzo [a, g] quinolizines, e.g. berberine
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Abstract
The invention discloses a kind of compound, the compound is prepared by alkyl substitution by berberrubine with honokiol under certain conditions.Meanwhile the purposes the invention also discloses the preparation method of the compound and the compound in being used to prepare tumor.Compound of the present invention is inventor under the guidance of tcm clinical medication experience for many years and on the basis of organic chemistry, purposefully, selectively honokiol and berberrubine are combined together by specific synthetic method, gained compound has the effect of Synergy and attenuation, has better effect in anti-tumor aspect.
Description
Technical field
The invention belongs to pharmaceutical chemistry and pharmaceutical technology field, and in particular to a kind of compound with antitumor action and
Preparation method.
Background technology
Tumour has become the principal disease for threatening contemporary mankind's health, and tumour is died of every year according to World Health Organization
Patient accounts for 60% or more of all Died Patients.Malignant tumour is called cancer, in the cases of cancer of numerous types, male cancer
It is lung cancer that the death rate is highest in patient, and the highest mainly breast cancer of the death rate in female cancer patients.It data show,
Cancer mortality is still rising, and tends to rejuvenation.There is operative treatment, chemotherapy, common for the treatment means of cancer
Radiotherapy, stereotaxis therapy, immunotherapy, endocrinotherapy, targeting therapy, cold therapy, heating therapy, gene are treated
Method.Wherein chemotherapy generally refers to drug to anticancer, and the different phase that these drugs can be bred in growth of cancer cells inhibits
Or cancer cell is killed, reach therapeutic purposes.Chemotherapy mainly applies to various types of leukaemia, and for not expert
Art and the patient insensitive to radiotherapy, account for critical role in treatment of cancer.
Cortex Magnoliae Officinalis is Magnoliacea plant point leaf Cortex Magnoliae Officinalis or the branch skin root skin that Magnolia bilola is dried, and main active is thickness
Plain phenol and honokiol, have anti-oxidant, anti-inflammatory, antitumor, antibacterial, antiviral pharmacological action, while also have liver protecting,
Protect the pharmacological actions such as nerve.
Jamaicin (Berberine) is a kind of common isoquinoline alkaloid, be primarily present in Berberidaceae, Ranunculaceae,
In the plants such as opium poppy section.The raw material jamaicin extracted now is the rhizome from medicinal plants such as the coptis, Cortex Phellodendri, radix berberidis, cloud companies
In obtain.The chemical composition of jamaicin and pharmacological action become the hot spot of research in recent years, in addition to being clinically used as wide spectrum
Outside antimicrobial and heat-clearing and detoxifying drug, important role is also played in the treatment of Other diseases, such as diabetes, tumour, painstaking effort
Pipe disease etc..It is cracked at high temperature by jamaicin and berberrubine can be obtained.
Using chemically synthesized method, the two molecule parent nucleus is purposefully stitched together, is in the prior art without appointing
What is open or report.In addition, can the compound being newly spliced into, increase the bioavilability of honokiol and berberrubine,
Can drug toxicity, certain pharmacological actions be enhanced, if can reach 1+1 >=2, can the effect of Synergy and attenuation protrude
The antitumor pharmacological action etc. of the two, these are also all in the prior art without any disclosure or report.
Invention content
Based on this, a kind of compound is provided it is an object of the invention to overcome above-mentioned the deficiencies in the prior art place, institute
Compound is stated than honokiol and berberrubine small toxicity, and is had the effects that preferably antitumor.
To achieve the above object, the technical solution adopted by the present invention is：A kind of compound, the structural formula of the compound
For：
Compound of the present invention by berberrubine with honokiol as obtained by specifically reacting and synthesize, change of the invention
The toxicity of honokiol and berberrubine can be significantly reduced by closing object, and can reach 1+1 >=2, play the work of Synergy and attenuation
With in anti-tumor aspect with better effect.
Meanwhile the invention also discloses the preparation methods of the compound comprising following steps：
(1) compound shown in formula (I) in solvent A is subjected to alkyl substitution with halogenated dose, obtained shown in formula (II)
Compound；
(2) by formula (II) compound obtained in step (1) under base catalyst and phase transfer catalyst effect, with formula
(III) substitution reaction occurs to be alkylated in solvent B for compound shown in get to the compound (formula (IV) and (V))
The preparation method of compound of the present invention is spelled the parent nucleus of honokiol and berberrubine by specific mode
It is combined, but this split is not blindness, but under the guidance of tcm clinical medication experience for many years and have
It on the basis of chemical machine, purposefully, selectively by the two combines, present inventor has found to rise after the two combines
To the effect of Synergy and attenuation.And the method for the present invention is easy to operate, can rapidly and efficiently obtain compound of the present invention.
The preferred embodiment of preparation method as compound of the present invention, halogenating agent selects in the step (1)
From at least one of 1,4- dibromobutanes, bis- iodobutanes of 1,4- and 1,4- dichloroetane；According to optimal yield, we select 1,
4- dibromobutanes.
The preferred embodiment of preparation method as compound of the present invention, halogenated dose and formula in the step (1)
(I) molar ratio of compound is 20:1~40:1；The molar ratio of the solvent A and formula (I) compound is 100:1~200:1.
The preferred embodiment of preparation method as compound of the present invention, step (2) Chinese style (III) chemical combination
The molar ratio of object and formula (II) compound is 1:1~4:1；The molar ratio of base catalyst and formula (II) compound is 5:1~20:1；
The molar ratio of phase transfer catalyst and formula (II) compound is 0.5:1~5:1；Solvent B and the molar ratio of formula (II) compound are
200:1~300:1.
The preferred embodiment of preparation method as compound of the present invention, reaction temperature is in the step (1)
30~100 DEG C, the reaction time is 3~12 hours；The more preferable embodiment of preparation method as compound of the present invention,
Reaction temperature is 70~80 DEG C in the step (1), and the reaction time is 8~10 hours.
The preferred embodiment of preparation method as compound of the present invention, the middle temperature reacted of the step (2)
It it is 30~100 DEG C, the reaction time is 4~15 hours；The more preferable embodiment party of preparation method as compound of the present invention
Formula, the middle temperature reacted of the step (2) is 70~80 DEG C, and the reaction time is 4~15 hours.
The preferred embodiment of preparation method as compound of the present invention, solvent A is selected from first in the step (1)
Alcohol, acetonitrile, DMF, acetone, ethyl alcohol, dimethyl sulfoxide (DMSO), tetrahydrofuran, dichloromethane, dimethyl sulfoxide (DMSO), dioxane and isopropyl
At least one of alcohol；The more preferable embodiment of preparation method as compound of the present invention, in the step (1)
Solvent A is DMF.
Present inventor has found, when the solvent A in the step (1) selects DMF, reaction that DMF reacts the step
Object dissolubility is preferable, and polarity is moderate, under the premise of not only ensure that product yield, but also is easy to post-process.
The preferred embodiment of preparation method as compound of the present invention, the base catalyst in the step (2)
Selected from least one of natrium carbonicum calcinatum, Anhydrous potassium carbonate and triethylamine；Phase transfer catalyst is selected from benzyl triethyl ammonium chlorination
Ammonium (TEBA), tetrabutylammonium bromide (TBAB), tetrabutylammonium chloride, 4-butyl ammonium hydrogen sulfate, tri-n-octyl methyl ammonium chloride, 12
At least one of alkyl trimethyl ammonium chloride and tetradecyl trimethyl ammonium chloride；Preparation as compound of the present invention
The more preferable embodiment of method, the base catalyst in the step (2) is Anhydrous potassium carbonate, and phase transfer catalyst is the tetrabutyl
Ammonium bromide (TBAB).
Present inventor has found that, when the base catalyst selects Anhydrous potassium carbonate, phase transfer is urged by numerous studies
The yield of agent selection tetrabutylammonium bromide (TBAB) product is apparently higher than other catalyst.
The preferred embodiment of preparation method as compound of the present invention, solvent B is selected from first in the step (2)
Alcohol, DMF, ethyl acetate, acetonitrile, acetone, pyridine, quinoline, tetrahydrofuran, dichloromethane, dimethyl sulfoxide (DMSO), dioxane and different
At least one of propyl alcohol；The more preferable embodiment of preparation method as compound of the present invention, in the step (2)
Solvent B be acetonitrile.
Present inventor is had found by numerous studies, when the solvent B in the step (2) selects acetonitrile, acetonitrile pair
The reactant solubility of step reaction is preferable, and moderate boiling point, polarity is moderate, under the premise of not only ensure that product yield, but also easily
In post-processing.
The most preferred embodiment of preparation method as compound of the present invention, the solvent A in the step (1) are
DMF；Solvent B in the step (2) is acetonitrile, and base catalyst is Anhydrous potassium carbonate, and phase transfer catalyst is tetrabutyl phosphonium bromide
Ammonium (TBAB).
Present inventor is by the study found that when the halogenating reaction temperature in the step (1) is 80 DEG C, reaction time
For 10h, the temperature of the alkyl substitution in step (2) is 80 DEG C, the reaction time is 12 hours, not only can guarantee yield, but also tool
There is preferable economy.
The specific method of the preferred embodiment of preparation method as compound of the present invention, the step (1) is：
The compound of formula (I) is added in clean three-necked flask, solvent A is added, is slowly added to halogenated dose, when the temperature to 80 DEG C,
HPLC monitoring reactions, stop reaction when raw material fundamental reaction is completely or product is not further added by, are cooled to room temperature, suction filtration is drying to obtain
Formula (II) compound.
The specific method of the preferred embodiment of preparation method as compound of the present invention, the step (2) is：
Formula (III) compound is added in clean three-necked flask, the molar ratio of formula (III) compound and formula (II) compound is 1:
1~4:1, base catalyst, phase transfer catalyst and solvent B, be warming up to be 70-80 DEG C in temperature at react 4~15 hours, reaction
After cooled down, filter, washed with dichloromethane, filtrate revolving recycling, obtain orange/yellow solid with re-crystallizing in ethyl acetate
Crude product.With CH2Cl2:CH3OH systems are eluant, eluent, and silica gel column chromatography carries out purifying to obtain compound of the present invention (IV and V).
The present invention also provides purposes of the compound in being used to prepare tumor.
Compound of the present invention can be used for preparing the drug for the treatment of tumour.
The compounds of this invention is inventor under the guidance of tcm clinical medication experience and organic chemistry for many years, there is mesh
, selectively berberrubine and honokiol be combined together by specific synthetic method, gained compound, which has, to be increased
The effect of attenuation is imitated, anti-tumor aspect has better effect.
Compared with the existing technology, beneficial effects of the present invention are：Compound provided by the invention is by berberrubine and and thickness
Plain phenol can significantly reduce the toxicity of honokiol, and can reach 1+1 >=2, rise as specifically reacting obtained by synthesis
To the effect of Synergy and attenuation, there is better effect in anti-tumor aspect；Compound of the present invention is to be used to prepare treatment swollen
There is important use in tumor medicine, new selection is provided to prepare tumor；The preparation of compound of the present invention
Method is simple, can be rapidly and efficiently obtain the compound of the present invention.
Description of the drawings
Fig. 1 is the high resolution mass spectrum figure of compound of the present invention (IV)；
Fig. 2 is the infrared spectrogram of compound of the present invention (IV)；
Fig. 3 is the ultraviolet spectrogram of compound of the present invention (IV)；
Fig. 4 is the hydrogen spectrogram of the nuclear magnetic resonance of compound of the present invention (IV)；
Fig. 5 is the carbon spectrogram of the nuclear magnetic resonance of compound of the present invention (IV)；
Fig. 6 is the nuclear magnetic resonance HMQC correlation spectrograms of compound of the present invention (IV)；
Fig. 7 is the nuclear magnetic resonance HMBC correlation spectrograms of compound of the present invention (IV)；
Fig. 8 is the nuclear magnetic resonance H-H Correlated Spectroscopies of compound of the present invention (IV)；
Fig. 9 is the high resolution mass spectrum figure of compound of the present invention (V)；
Figure 10 is the infrared spectrogram of compound of the present invention (V)；
Figure 11 is the ultraviolet spectrogram of compound of the present invention (V)；
Figure 12 is the hydrogen spectrogram of the nuclear magnetic resonance of compound of the present invention (V)；
Figure 13 is the carbon spectrogram of the nuclear magnetic resonance of compound of the present invention (V)；
Figure 14 is the nuclear magnetic resonance HMQC correlation spectrograms of compound of the present invention (V)；
Figure 15 is the nuclear magnetic resonance HMBC correlation spectrograms of compound of the present invention (V)；
Figure 16 is the nuclear magnetic resonance H-H Correlated Spectroscopies of compound of the present invention (V).
Specific implementation mode
To better illustrate the object, technical solutions and advantages of the present invention, below in conjunction with specific embodiment to the present invention
It is described further.
The structural formula difference of the compound of formula used in following embodiment (I)~(V) is as follows：
Embodiment 1
A kind of embodiment of the compounds of this invention, the compound are prepared using following preparation method：
(1) formula (I) compound is added in the three-necked flask of dried and clean, DMF, the DMF and formula (I) compound is added
Molar ratio is 100:1, it is slowly added to Isosorbide-5-Nitrae-dibromobutane, the molar ratio of the Isosorbide-5-Nitrae-dibromobutane and formula (I) compound is 20:
1；Temperature rises to 80 DEG C or so, and back flow reaction 10 hours, HPLC monitoring reactions, raw material fundamental reaction is completely or product is not further added by
When stop reaction, cool down, filter, it is dry to get formula (II) compound；
(2) formula (III) compound is added in clean three-necked flask, formula (III) compound is rubbed with formula (II) compound
You are than being 1:1, acetonitrile solution, Anhydrous potassium carbonate and tetrabutylammonium bromide (TBAB) is added, the acetonitrile being added is changed with formula (II)
The molar ratio for closing object is 200:1, the molar ratio of the Anhydrous potassium carbonate and formula (II) compound that are added is 5:1, four be added
The molar ratio of butylammonium bromide (TBAB) and formula (II) compound is 0.5:1；It is warming up at 80 DEG C and reacts 12 hours, reaction terminates
Postcooling, suction filtration, drying are further purified to get formula (IV) and (V) compound represented.
Formula (IV) compound yield that the present embodiment method is prepared is 45.5%, purity 93%；Formula (V) compound
Yield is 35.5%, purity 91%.
Embodiment 2
A kind of embodiment of the compounds of this invention, the compound are prepared using following preparation method：
(1) formula (I) compound is added in the three-necked flask of dried and clean, DMF, the DMF and formula (I) compound is added
Molar ratio is 150:1, it is slowly added to Isosorbide-5-Nitrae-dichloroetane, the molar ratio of the Isosorbide-5-Nitrae-dichloroetane and formula (I) compound is 25:
1；Temperature rises to 30 DEG C or so, and back flow reaction 10 hours, HPLC monitoring reactions, raw material fundamental reaction is completely or product is not further added by
When stop reaction, cool down, filter, it is dry.Up to formula (II) compound；
(2) formula (III) compound is added in clean three-necked flask, formula (III) compound is rubbed with formula (II) compound
You are than being 2:1, DMF, Anhydrous potassium carbonate and tetrabutylammonium chloride, the molar ratio of the DMF being added and formula (II) compound is added
It is 250:1, the molar ratio of the Anhydrous potassium carbonate and formula (II) compound that are added is 20:1, the tetrabutylammonium chloride that is added with
The molar ratio of formula (II) compound is 1:1；It is reacted 15 hours at being 70 DEG C in temperature, cooling after reaction, suction filtration, drying,
It is further purified to get formula (IV) and (V) compound represented.
Formula (IV) compound yield that the present embodiment method is prepared is 31.2%, purity 90%；Formula (V) compound
Yield is 25.1%, purity 87%.
Embodiment 3
A kind of embodiment of the compounds of this invention, the compound are prepared using following preparation method：
(1) formula (I) compound is added in the three-necked flask of dried and clean, methanol, the methanol and formula (I) chemical combination is added
Object molar ratio is 200:1, it is slowly added to-two iodobutane of Isosorbide-5-Nitrae ,-two iodobutane of the Isosorbide-5-Nitrae and the molar ratio of formula (I) compound are
25:1；Temperature rises to 100 DEG C, reacts 3 hours, and HPLC monitoring reactions stop when raw material fundamental reaction is completely or product is not further added by
It only reacts, cools down, filter, it is dry to get formula (II) compound；
(2) formula (III) compound is added in clean three-necked flask, formula (III) compound is rubbed with formula (II) compound
You are than being 4:1, be added dichloromethane, Anhydrous potassium carbonate and benzyltriethylammoinium chloride (TEBA), the dichloromethane being added with
The molar ratio of formula (II) compound is 200:1, the molar ratio of the Anhydrous potassium carbonate and formula (II) compound that are added is 8:1, institute
The benzyltriethylammoinium chloride (TEBA) of addition is 2 with the molar ratio of formula (II) compound:1；15 are reacted at being 30 DEG C in temperature
Hour, cooling after reaction, suction filtration, drying are further purified to get formula (IV) and (V) compound represented.
Formula (IV) compound yield that the present embodiment method is prepared is 34.1%, purity 87%；Formula (V) compound
Yield is 32.1%, purity 89%.
Embodiment 4
A kind of embodiment of the compounds of this invention, the compound are prepared using following preparation method：
(1) will formula (I) compound be added dried and clean three-necked flask in, be added tetrahydrofuran, the tetrahydrofuran with
Formula (I) compound mole ratio is 150:1, it is slowly added to Isosorbide-5-Nitrae-dibromobutane, the Isosorbide-5-Nitrae-dibromobutane and formula (I) compound
Molar ratio is 30:1；Temperature rises to 60 DEG C, reacts 10 hours, HPLC monitoring reactions, raw material fundamental reaction completely or product no longer
Stop reaction when increase, cool down, filters, it is dry to get formula (II) compound；
(2) formula (III) compound is added in clean three-necked flask, formula (III) compound is rubbed with formula (II) compound
You are than being 4:1, acetone, triethylamine and dodecyl trimethyl ammonium chloride, the acetone being added and formula (II) compound is added
Molar ratio is 200:1, the molar ratio of the triethylamine and formula (II) compound that are added is 15:1, the dodecyl front three being added
The molar ratio of ammonium chloride and formula (II) compound is 1：1；It is reacted 12 hours at being 60 DEG C in temperature, cooling after reaction,
It filters, is dry, being further purified, obtain formula (IV), the compound of (V) is to get chemical combination shown in formula (IV) and (V)
Object.
Formula (IV) compound yield that the present embodiment method is prepared is 21.1%, purity 90%；Formula (V) compound
Yield is 17.5%, purity 89%.
Embodiment 5
A kind of embodiment of the compounds of this invention, the compound are prepared using following preparation method：
(1) formula (I) compound is added in the three-necked flask of dried and clean, pyridine, the pyridine and formula (I) chemical combination is added
Object molar ratio is 120:1, it is slowly added to Isosorbide-5-Nitrae-dibromobutane, the Isosorbide-5-Nitrae-dibromobutane and the molar ratio of formula (I) compound are
40:1；Temperature rises to 80 DEG C, reacts 12 hours, and HPLC monitoring reactions stop when raw material fundamental reaction is completely or product is not further added by
It only reacts, cools down, filter, it is dry to get formula (II) compound.
(2) formula (III) compound is added in clean three-necked flask, formula (III) compound is rubbed with formula (II) compound
You are than being 2.5:1, be added dioxane, Anhydrous potassium carbonate and tetradecyl trimethyl ammonium chloride, the dioxane being added with
The molar ratio of formula (II) compound is 300:1, the molar ratio of the Anhydrous potassium carbonate and formula (II) compound that are added is 10:1, institute
The tetradecyl trimethyl ammonium chloride of addition is 2 with the molar ratio of formula (II) compound：1；Reaction 8 is small at being 70 DEG C in temperature
When, cooling after reaction, suction filtration, drying are further purified to get formula (IV) and (V) compound represented.
Formula (IV) compound yield that the present embodiment method is prepared is 33.1%, purity 91%；Formula (V) compound
Yield is 24.9%, purity 92%.
Embodiment 6
A kind of embodiment of the compounds of this invention, the compound are prepared using following preparation method：
(1) formula (I) compound, purity 90% are directly commercially available from market；
(2) formula (I) compound is added in the three-necked flask of dried and clean, the mixed solution of methanol and acetonitrile, institute is added
The mixed solution and formula (I) compound mole ratio for stating methanol and acetonitrile are 200:1, it is slowly added to Isosorbide-5-Nitrae-dibromobutane, described 1,
The molar ratio of 4- dibromobutanes and formula (I) compound is 28:1；Temperature rises to 60 DEG C, reacts 12 hours, HPLC monitoring reactions, former
Stop reaction when material fundamental reaction is completely or product is not further added by, cool down, filters, it is dry to get formula (II) compound.
(3) formula (III) compound is added in clean three-necked flask, formula (III) compound is rubbed with formula (II) compound
You are than being 3:1,95% ethyl alcohol, Anhydrous potassium carbonate and tetrabutylammonium bromide, 95% ethyl alcohol being added and formula (II) chemical combination is added
The molar ratio of object is 250:1, the molar ratio of the Anhydrous potassium carbonate and formula (II) compound that are added is 18:1, four fourths being added
The molar ratio of base ammonium bromide and formula (II) compound is 5：1；It is reacted 4 hours at being 100 DEG C in temperature, cooling after reaction,
It filters, is dry, being further purified to get formula (IV) and (V) compound represented.
Formula (IV) compound yield that the present embodiment method is prepared is 36.6%, purity 93%；Formula (V) compound
Yield is 29.9%, purity 93%.
Embodiment 7
The present embodiment is total to high resolution mass spectrum figure, infrared spectrogram, ultraviolet spectrogram, the nuclear-magnetism of compound of the present invention
The hydrogen that shakes spectrum, carbon spectrogram, nuclear magnetic resonance H-H Correlated Spectroscopies, C-H correlation spectrogram deciles are not researched and analysed, such as 1~16 institute of attached drawing
Show.
From attached drawing 1~8 it is found that the compounds of this invention (IV), m.p.230.6 DEG C, ESI-MS m/z:642.2788[M]+；
The compounds of this invention (IV), IR (KBr) νmax：3396.99,1636.3,1269.9,1397.17,1228.43,
1600.63,1506.13,1480.1cm-1；
The compounds of this invention (IV)1H-NMR and13C-NMR(MeOD-d2) as shown in table 1：
1 compound of table (IV)1H-NMR and13C-NMR(MeOD-d2)
From attached drawing 9~16 it is found that the compounds of this invention (V), m.p.233.6 DEG C, ESI-MS m/z:642.2787[M]+；
The compounds of this invention (V), IR (KBr) νmax：3396.99,1636.3,1269.9,1397.17,1228.43,
1600.63,1506.13,1480.1cm-1；
The compounds of this invention (V)1H-NMR and13C-NMR(MeOD-d2) as shown in table 2：2 compound of table (V)1H-NMR
With13C-NMR(MeOD-d2)
The anti-liver cancer efficacy of 8 the compounds of this invention of embodiment is tested
One, inhibiting effect of the compounds of this invention to Proliferation of Human Hepatoma Cell SMMC-7721 is inquired into
This experiment is using mtt assay detection the compounds of this invention to the inhibiting effect of human liver cancer cells Hep G2.
(1) material, instrument and reagent
Material and reagent：(DMSO prepares stoste to the compounds of this invention, dilutes before use, ensures DMSO concentration in reactant
1 × 10 is at least diluted in system-3), human liver cancer cells Hep G2 is quoted from Lanzhou University's Life Science College；Culture medium RPMI
1640 (U.S. Gibcobr l), calf serum (Hangzhou Chinese holly), other chemical reagent are that analysis is pure.
Laboratory apparatus：U.S. Precision Scientific CO2Incubator, superclean bench, inverted microscope, light
Learn microscope, microplate reader (BIO-RADModel 550 and BIO-RAD Model 680), fluorescence microscope, Horizontal electrophoresis tank, height
Fast refrigerated centrifuge, Costar tissue culture plates.
(2) experimental method
Cell culture：Human hepatocarcinoma BEL-7402 is inoculated in respectively containing 10% calf serum RPMI, 1640 culture mediums
In, 37 DEG C are set, 5%CO2It is cultivated in incubator, logarithmic growth phase cell is for testing.
By a concentration of 1 × 105The SMMC-7721 cells of the exponential phase of/mL, are inoculated in 96 orifice plates, per 100 μ L of hole,
In 5%CO2, cultivate for 24 hours under 37 DEG C of incubation conditions；The compounds of this invention that different diluted concentrations are separately added into corresponding aperture is new
100 μ l of fresh culture solution, 6 multiple holes of each concentration, setting zeroing hole and control wells, the PBS polishings of extra hole same volume,
37 DEG C are set, 5%CO248h is cultivated in incubator, suction is abandoned culture solution, washed with sterile PBS, cleaning 2 times per hole, every time 200 μ L.
Then culture medium (5mg/mL MTTs of the 100 μ L containing MTT is added per hole：Culture medium=1:4), continue in insulating box after cultivating 4h,
Culture solution is carefully sucked out, 150 μ L DMSO are added per hole, after oscillator oscillation, under being 490nm with microplate reader Detection wavelength
Absorbance value, record using 16.0 softwares of SPSS as a result, calculate IC50Value.
(3) experimental result
The compounds of this invention is as shown in table 3 to the inhibiting effect testing result of Proliferation of Human Hepatoma Cell SMMC-7721：
Half-inhibition concentration (μm ol/L) of the 3 invention compound of table to SMMC-7721 cells
By above-mentioned experimental result it is found that the IC of the compounds of this invention50Value is less than monomeric compound before synthesis, i.e., to human liver cancer
The inhibiting effect enhancing of cancer cell SMMC-7721 proliferation, but still it is weaker than positive control drug.Illustrate that the compounds of this invention can be effective
Inhibit the proliferation of human liver cancer cells Hep G2.
Two, research of the compounds of this invention to human liver cancer cell HepG2 apoptosis is inquired into
(1) Sulforhodamine B (SRB) decoration method detection the compounds of this invention is used to increase human liver cancer cell HepG2
The inhibiting effect grown.
Material and reagent：(DMSO prepares stoste to the compounds of this invention, dilutes before use, ensures DMSO concentration in reactant
1 × 10 is at least diluted in system-3), human liver cancer cell HepG2 is quoted from Guangdong pharmaceutical university Life Science College；DMEM culture mediums
(Ji Nuo biotech firms), calf serum (Hangzhou Chinese holly), other chemical reagent are that analysis is pure.
Laboratory apparatus：U.S. Precision Scientific CO2Incubator, superclean bench, inverted microscope, light
Learn microscope, microplate reader (BIO-RADModel 550 and BIO-RAD Model 680), fluorescence microscope, Horizontal electrophoresis tank, height
Fast refrigerated centrifuge, Costar tissue culture plates.
Cell culture：HepG 2 is inoculated in containing in 10% calf serum DMEM culture mediums, sets 37 DEG C, 5%CO2Incubator
Middle culture, logarithmic growth phase cell is for testing.
1, inhibiting effect of the srb assay detection the compounds of this invention to HepG2 cells
Principle：SulforhodamineB (SRB) is a kind of protein combination dye, pink, water-soluble.SRB can be with
It is combined with the basic amino acid in large biological molecule, is in good linear relationship in OD readings and the cell number of 511nm.Therefore it can
Quantifying as cell number.
Method：Respectively by the HepG2 cells of exponential phase with 10 × 104ml-1Concentration be inoculated in 96 well culture plates,
After culture for 24 hours, it is separately added into the 100 μ l of the compounds of this invention fresh medium of different diluted concentrations into corresponding aperture, continues to train
After supporting 48h, culture solution is discarded, 10% 100 μ l of trichloroacetic acid (TCA) are added, after standing 5min, by the 4 DEG C of fixations of orifice plate dislocation
1h outwells fixer, is washed with deionized water 5 times, dries.The SRB that 100 μ l0.4% are added per hole later dyes 10min, with 1%
Acetic acid wash 5 times, naturally dry.Then 150 μ l 10mmolL are added per hole-1Non-buffered Tris lye (unbuffered
Tris-base solution, pH10.5) SRB combined with protein is dissolved, so that it is fully dissolved with oscillator concussion 5min
Afterwards, the OD values per hole are measured at 515nm with microplate reader.Seek its cell proliferation rate (%)=(T-T0)/(C-T0)×100.Wherein
C indicates the cell OD values of control group；T indicates the cell OD values of dosing group；T0Indicate cell OD when control tablet measures dosing
Value.The cell being inoculated in tablet is fixed with TCA at once before dosing.If the final OD values of dosing group are more than T0, illustrate thin
Born of the same parents still grow after dosing.If the final OD values of dosing group are less than T0, cell is killed after illustrating dosing.Utilize recurrence side
Journey finds out drug concentration (the 50%inhibition concentration, IC of 50% proliferation inhibition rate50).From experimental result
It sees, the final cell number of dosing group is less than T0, then illustrate that drug has inhibiting effect to cell growth, cell is killed after dosing
Extremely.IC after handling 2 cells of HepG for 24 hours with the compounds of this invention group and control group50It is thin to this to carry out each compound of comparison for value
The toxic effect of born of the same parents, IC50Value is as shown in table 4：
Half-inhibition concentration (μm ol/L) of the 4 invention compound of table to HepG2 cells
By above-mentioned experimental result it is found that the IC of the compounds of this invention50Value is less than monomeric compound before synthesis, i.e., to human liver cancer
The inhibiting effect enhancing of cancer cell HepG2 proliferation, and it is suitable with positive drug.Illustrate that the compounds of this invention can effectively inhibit people liver
The proliferation of cancer cancer cell HepG2.
(2) cell clonal formation is tested
Exponential phase experimental cell strain is prepared into single cell suspension, is inoculated in six well culture plates, per 500, hole,
Every group of 3 multiple holes, 1. the compounds of this invention IV, 2. the compounds of this invention V, 3. each group corresponding concentration (0.1 × IC is added in cis-platinum50)
Drug, 4. blank control group (culture solution), 37 DEG C, cultivate 7 days in 5%CO2 environment, until there are naked eyes in six well culture plates
Visible white colonies spot terminates culture immediately.Culture solution is discarded, PBS buffer solution embathes cell 2 times, and again with methanol is fixed
5min, then Giemsa dyeing 0.5h, flowing water wash away dyeing liquor, dry at room temperature.Finally in inverted phase contrast fluorescence microscope (4
The number of cell clones of >=50 cells, cloning efficiency (%)=(clone's number/inoculation number) × 100% are counted under again).
(3) flow cyctometry detects Apoptosis
1. the cell of exponential phase, after pancreatin digestion, with 5 × 104The density of/ml is inoculated with, and DMEM trainings are added after adherent
Supporting base culture for 24 hours, 10 μ l of the compounds of this invention (200 μ g/ml) are added in treatment group, and 10 μ l of cis-platinum (20 μ g/ml) are added in control group,
Isometric culture solution is added in blank group.2. continuing to cultivate 48h, AnnexinV is marked as fluorescence in fluorescein (FITC)
Probe, with flow cytomery Apoptosis.
(4) cell cycle is detected
Exponential phase experimental cell strain is prepared into single cell suspension, is inoculated in six well culture plates, per hole 5 × 104
A/ml, every group of 3 multiple holes, 1. the compounds of this invention IV, 2. the compounds of this invention V, 3. cis-platinum group
Each group corresponding concentration (0.1 × IC is added50) drug, 4. blank control group (culture solution), 37 DEG C, 5%CO2 environment
After middle culture 48h, digestion 1., 2., 3. organize attached cell, EP pipes are collected, and precooling PBS purgings centrifuge (1500rpm, 15min),
Liquid is discarded supernatant, adds 75% ethyl alcohol 3mL of precooling to fix 0.5h, centrifuges (1500rpm, 15min) again, discard fixer, PBS is thin
5min is resuspended in born of the same parents, and sieve (400 mesh) filters 1 time, centrifuges (1000rpm, 10min), discards PBS, PI under the conditions of 4 DEG C of low-temperature darks
Dye liquor 0.5h, every group of acquisition >=10000 cell, flow cytomery one-color fluorescence cell cytometry, Multiplus
Software softwares analyze the cell cycle.
It is as shown in table 5 to each group cloning efficiency, apoptosis rate and the testing result of cell cycle：
Cloning efficiency, apoptosis rate and the cell cycle of 5 each group of table compare (N=6)
By above-mentioned experimental result it is found that the compounds of this invention there is certain inhibition to make human hepatoma cell HepG 2 proliferation
With；According to cell apoptosis assay as can be seen that the compounds of this invention can promote the apoptosis of hepatocellular carcinoma H22.
(5) cell migration scratch experiment
Exponential phase experimental cell strain is prepared into single cell suspension, is inoculated in six well culture plates, per hole 5 × 104
It is a, 37 DEG C, cultivate for 24 hours in 5%CO2 environment after, cell is carried out with 20 μ l pipettor gun heads to draw " one " word trace, per hole cut 1
Secondary, PBS is washed 2 times, elutes suspension cell 1. the compounds of this invention IV, 2. the compounds of this invention V, 3. cis-platinum group, 4. sky as possible
White control group (culture solution), then be placed in incubator and continue to cultivate, shoot respectively using phase contrast microscope cultivate 12h, for 24 hours, 36h traces
Close call, cut area healing rate (%)=(initial area-shooting time point area)/initial area × 100%.
Cell migration scratch experiment testing result is as shown in table 6：
The different time points cut healing rate of 6 each group of table compare (N=6)
Experimental result shows, the compounds of this invention has certain inhibiting effect to human hepatoma cell HepG 2 proliferation, can be with
Cancer cell clone and migration are reduced, blocks cellular promotes cancer cell-apoptosis in the G0/G1 phases.
The anti-breast cancer effect test of 9 the compounds of this invention of embodiment
One, the inhibiting effect that the compounds of this invention is proliferated human breast cancer cell line Bcap-37 is inquired into
This experiment is using mtt assay detection the compounds of this invention to the inhibiting effect of human breast cancer cell line Bcap-37.
(1) material, instrument and reagent
Material and reagent：(DMSO prepares stoste to the compounds of this invention, dilutes before use, ensures DMSO concentration in reactant
1 × 10 is at least diluted in system-3), human breast cancer cell line Bcap-37 is quoted from Guangdong pharmaceutical university Life Science College；Culture medium RPM
I1640 (U.S. Gibcobr l), calf serum (Hangzhou Chinese holly), other chemical reagent are that analysis is pure.
Laboratory apparatus：U.S. Precision Scientific CO2Incubator, superclean bench, inverted microscope, light
Learn microscope, microplate reader (BIO-RADModel 550 and BIO-RAD Model 680), fluorescence microscope, Horizontal electrophoresis tank, height
Fast refrigerated centrifuge, Costar tissue culture plates.
(2) experimental method
Cell culture：Human breast cancer cell line Bcap-37 is inoculated in respectively containing in 10% calf serum medium RPM I1640,
37 DEG C are set, 5%CO2It is cultivated in incubator, logarithmic growth phase cell is for testing.By a concentration of 1 × 105The logarithmic growth of/mL
The MCF-7 cells of phase, are inoculated in 96 orifice plates, in 5%CO2, cultivate for 24 hours under 37 DEG C of incubation conditions；It is separately added into corresponding aperture
Zeroing hole and control wells are arranged in the 100 μ l of the compounds of this invention fresh medium of different diluted concentrations, 6 multiple holes of each concentration,
The PBS polishings of extra hole same volume, set 37 DEG C, 5%CO248h is cultivated in incubator, culture solution is abandoned in suction, with sterile PBS
It washs, cleaning 2 times per hole, every time 200 μ L.Then culture medium (5mg/mL MTTs of the 100 μ L containing MTT is added per hole：Culture medium=
1:4), continue in insulating box after cultivating 4h, culture solution is carefully sucked out, 150 μ LDMSO are added per hole and are used after oscillator oscillation
Microplate reader detects absorbance value, and record using 16.0 softwares of SPSS as a result, calculate IC50Value.
(3) experimental result
Experimental result is as shown in table 7：
Half-inhibition concentration (μm ol/L) of 7 the compounds of this invention of table to MCF-7 cells
Experimental result shows the IC of the compounds of this invention50Value is less than monomeric compound before synthesis, i.e., thin to everybody breast cancer
The inhibiting effect enhancing of born of the same parents' MCF-7 proliferation, but still it is weaker than positive control drug.
Two, the inhibiting effect that the compounds of this invention is proliferated human breast cancer cell line MDA-MB-453 is inquired into
This experiment is using mtt assay detection the compounds of this invention to the inhibiting effect of human breast cancer cell line MDA-MB-453.
(1) material, instrument and reagent
Material and reagent：(DMSO prepares stoste to the compounds of this invention, dilutes before use, ensures DMSO concentration in reactant
1 × 10 is at least diluted in system-3), human breast cancer cell line MDA-MB-453 is quoted from Chinese Academy of Sciences's Shanghai cell bank；Culture medium L-15
(U.S.), glutamine (the silent winged generation that of match), calf serum (Hangzhou Chinese holly), other chemical reagent are that analysis is pure.
Laboratory apparatus：U.S. Precision Scientific CO2Incubator, superclean bench, inverted microscope, light
Learn microscope, microplate reader (BIO-RADModel 550 and BIO-RAD Model 680), fluorescence microscope, Horizontal electrophoresis tank, height
Fast refrigerated centrifuge, Costar tissue culture plates.
(2) experimental method
Cell culture：Human breast cancer cell line MDA-MB-453 is inoculated in respectively containing 15% calf serum, 0.2922g/L paddy ammonia
In Amide Medium L-15,37 DEG C are set, is cultivated in 100%air incubators, logarithmic growth phase cell is for testing.By concentration
It is 1 × 105The MDA-MB-453 cells of the exponential phase of/mL, are inoculated in 96 orifice plates, in 100%air, 37 DEG C of incubation conditions
Lower culture is for 24 hours；The compounds of this invention fresh medium 100 the μ l, Mei Genong of different diluted concentrations are separately added into corresponding aperture
6 multiple holes are spent, zeroing hole and control wells, the PBS polishings of extra hole same volume are set, set 37 DEG C, 100%air cultures
48h is cultivated in case, suction is abandoned culture solution, washed with sterile PBS, cleaning 2 times per hole, every time 200 μ L.Then 100 μ L are added per hole
Culture medium (5mg/mL MTT containing MTT：Culture medium=1:4), continue in insulating box after cultivating 4h, culture solution be carefully sucked out,
150 μ LDMSO are added per hole, after oscillator oscillation, detects absorbance value with microplate reader, records as a result, soft using SPSS 16.0
Part calculates IC50 values.
(3) experimental result
Experimental result is as shown in table 8：
Half-inhibition concentration (μm ol/L) of 8 the compounds of this invention of table to MDA-MB-453 cells
Experimental result shows the IC of the compounds of this invention50Value is less than monomeric compound before synthesis, i.e., to human breast carcinoma MDA-
The inhibiting effect of MB-453 cell Proliferations enhances, but still is weaker than positive control drug.
Three, the inhibiting effect that the compounds of this invention is proliferated human breast cancer cell MDA-MB-231 is inquired into
This experiment is using mtt assay detection the compounds of this invention to the inhibiting effect of human breast cancer cell MDA-MB-231.
(1) material, instrument and reagent
Material and reagent：(DMSO prepares stoste to the compounds of this invention, dilutes before use, ensures DMSO concentration in reactant
1 × 10 is at least diluted in system-3), human breast cancer cell MDA-MB-231 is quoted from Chinese Academy of Sciences's Shanghai cell bank；Culture medium L-15
(U.S.), glutamine (the silent winged generation that of match), bovine insulin (the holy biology of assist), calf serum (Hangzhou Chinese holly), other chemistry
Reagent is that analysis is pure.
Laboratory apparatus：U.S. Precision Scientific CO2Incubator, superclean bench, inverted microscope, light
Learn microscope, microplate reader (BIO-RADModel 550 and BIO-RAD Model 680), fluorescence microscope, Horizontal electrophoresis tank, height
Fast refrigerated centrifuge, Costar tissue culture plates.
(2) experimental method
Cell culture：Human breast cancer cell MDA-MB-231 is inoculated in respectively containing 10% calf serum, 0.01g/L glutamy
Amine in the culture medium L-15 of 0.01g/L bovine insulins, is set 37 DEG C, is cultivated in 100%air incubators, logarithmic growth phase cell
For testing.By a concentration of 1 × 105The MDA-MB-453 cells of the exponential phase of/mL, are inoculated in 96 orifice plates, 100%
It is cultivated for 24 hours under air, 37 DEG C of incubation conditions；The fresh training of the compounds of this invention of different diluted concentrations is separately added into corresponding aperture
100 μ l of nutrient solution, 6 multiple holes of each concentration, are arranged zeroing hole and control wells, the PBS polishings of extra hole same volume set 37
DEG C, 48h is cultivated in 100%air incubators, suction is abandoned culture solution, washed with sterile PBS, cleaning 2 times per hole, every time 200 μ L.So
Culture medium (5mg/mL MTTs of the 100 μ L containing MTT is added per hole afterwards：Culture medium=1:4), continue in insulating box after cultivating 4h, it is small
Heart be sucked out culture solution, per hole be added 150 μ LDMSO, oscillator oscillation after, with microplate reader detect absorbance value, record as a result,
IC is calculated using 16.0 softwares of SPSS50Value.
(3) experimental result
Experimental result is as shown in table 9：
Half-inhibition concentration (μm ol/L) of 9 the compounds of this invention of table to MDA-MB-231 cells
Experimental result shows the IC of the compounds of this invention50Value is less than monomeric compound before synthesis, i.e., to human breast carcinoma MDA-
The inhibiting effect of MB-231 cell Proliferations enhances, but still is weaker than positive control drug.
The anti-leukemia effect test of 10 the compounds of this invention of embodiment
By taking K562 Leukaemia as an example, by inquiring into the compounds of this invention in the inhibiting effect of tumor cell proliferation,
To study the anti-leukemia effect of the compound pair.
This experiment, trypan blue row's dye method, comet electrophoresis technology and the bis- dye methods of AO/EB detect the compounds of this invention to leukaemia
The inhibiting effect of cell cycling inhibiting to the damages of K562 cell DNAs and induces the apoptotic effects of K562 cells.
(1) material, instrument and reagent
Material and reagent：(DMSO prepares stoste to the compounds of this invention, dilutes before use, ensures DMSO concentration in reactant
1 × 10 is at least diluted in system-2), Leukemia K562 cell is quoted from Lanzhou University's Life Science College；Culture medium RPM
I1640 (U.S. Gibcobr l), yellow acyl rhodamine B (Sulforhodam ineB, SRB, U.S. Sigma), trypan blue (Sigma
Company), calf serum (Hangzhou Chinese holly), other chemical reagent are that analysis is pure.
Laboratory apparatus：U.S. Precision Scientific CO2Incubator, superclean bench, inverted microscope, light
Learn microscope, microplate reader (BIO-RADModel 550 and BIO-RAD Model 680), fluorescence microscope, Horizontal electrophoresis tank, height
Fast refrigerated centrifuge, Costar tissue culture plates.
Principle：Living cells has the ability for repelling trypan blue, due to the destruction of film completeness, cell, that is, quilt after cell death
Coloring.Cell suspension containing trypan blue is instilled in white blood cell count(WBC) plate cell, observation under an optical microscope counts living thin
Born of the same parents' (cell for not being dyed to blue).
Experimental method：A concentration of 10 × 104·ml-1The K562 cell suspensions in exponential phase, with 0.5ml
Hole-1It is inoculated in 24 well culture plates.It is placed in 37 DEG C, 5%CO2Preculture rear dosing for 24 hours in saturated humidity incubator.Continue to cultivate
24, it after 48h, takes 0.4% trypan blue solution 0.15ml to be added in 0.6ml cell suspensions, mixes well, drawn less after dyeing 5min
Mixed liquor is measured, blood cell counting plate cell is injected, counts living cells under light microscope, be repeated 3 times, take its average.Ask it thin
Born of the same parents' appreciation rate (%)=(T-T0)/(C-T0)×100.Wherein C indicates the cell number of control group；T indicates the cell number of dosing group；
T0Indicate cell number when control tablet measures dosing.If the final cell number of dosing group is more than T0, illustrate cell after dosing
Still it grows.If the final cell number of dosing group is less than T0, cell is killed after illustrating dosing.It is found out using regression equation
IC50.From the experimental results, the final cell number of dosing group is less than T0, then illustrate that drug has inhibiting effect to cell growth, adds
Cell is killed after medicine.IC50Value is as shown in table 10：
Half-inhibition concentration (μm ol/L) of 10 the compounds of this invention of table to K562 cells
Experimental result shows the IC of the compounds of this invention50Value is less than monomeric compound before synthesis, i.e., to K562 cell Proliferations
Inhibiting effect enhancing, but still be weaker than positive control drug.
The anti-lung cancer effect test of 11 the compounds of this invention of embodiment
One, the inhibiting effect that the compounds of this invention is proliferated human large cell lung cancer cell H-460 is inquired into
This experiment is using mtt assay detection the compounds of this invention to the inhibiting effect of human large cell lung cancer cell H-460.
(1) material, instrument and reagent
Material and reagent：(DMSO prepares stoste to the compounds of this invention, dilutes before use, ensures DMSO concentration in reactant
1 × 10 is at least diluted in system-3), human large cell lung cancer cell H-460 is quoted from Chinese Academy of Sciences's Shanghai cell bank；Culture medium RPM
I1640 (U.S. Gibcobr l), calf serum (Hangzhou Chinese holly), other chemical reagent are that analysis is pure.
Laboratory apparatus：U.S. Precision Scientific CO2Incubator, superclean bench, inverted microscope, light
Learn microscope, microplate reader (BIO-RADModel 550 and BIO-RAD Model 680), fluorescence microscope, Horizontal electrophoresis tank, height
Fast refrigerated centrifuge, Costar tissue culture plates.
(2) experimental method
Cell culture：Human large cell lung cancer cell H-460 is inoculated in respectively containing 10% calf serum medium RPM I1640
In, 37 DEG C are set, 5%CO2It is cultivated in incubator, logarithmic growth phase cell is for testing.By a concentration of 1 × 105The logarithm of/mL
The human large cell lung cancer cell NCI-H460 in growth period, is inoculated in 96 orifice plates, in 5%CO2, cultivate for 24 hours under 37 DEG C of incubation conditions；
It is separately added into the 100 μ l of the compounds of this invention fresh medium of different diluted concentrations into corresponding aperture, 6 multiple holes of each concentration,
Setting zeroing hole and control wells, the PBS polishings of extra hole same volume set 37 DEG C, 5%CO248h is cultivated in incubator,
Culture solution is abandoned in suction, is washed with sterile PBS, cleaning 2 times per hole, every time 200 μ L.Then 100 culture mediums of the μ L containing MTT are added per hole
(5mg/mL MTT：Culture medium=1:4), continue in insulating box after cultivating 4h, culture solution is carefully sucked out, 150 μ are added per hole
LDMSO after oscillator oscillation, detects absorbance value, record using 16.0 softwares of SPSS as a result, calculate IC with microplate reader50Value.
(3) experimental result
Experimental result is as shown in table 11：
Half-inhibition concentration (μm ol/L) of 11 the compounds of this invention of table to H-460 cells
Experimental result shows the IC of the compounds of this invention50Value is less than monomeric compound before synthesis, i.e., to H-460 cell Proliferations
Inhibiting effect enhancing, but still be weaker than positive control drug.
Two, the inhibiting effect that the compounds of this invention is proliferated human lung cancer cell A549 is inquired into
The inhibiting effect that this experiment is proliferated human lung cancer cell A549 using mtt assay detection the compounds of this invention.
(1) material, instrument and reagent
Material and reagent：(DMSO prepares stoste to the compounds of this invention, dilutes before use, ensures DMSO concentration in reactant
1 × 10 is at least diluted in system-3), human lung cancer cell A549 is quoted from Wuhan University's China typical culture collection center；Culture medium
RPM I1640 (U.S. Gibcobr l), calf serum (Hangzhou Chinese holly), other chemical reagent are that analysis is pure.
Laboratory apparatus：U.S. Precision Scientific CO2Incubator, superclean bench, inverted microscope, light
Learn microscope, microplate reader (BIO-RADModel 550 and BIO-RAD Model 680), fluorescence microscope, Horizontal electrophoresis tank, height
Fast refrigerated centrifuge, Costar tissue culture plates.
(2) experimental method
Cell culture：Human lung cancer cell A549 is inoculated in respectively containing in 10% calf serum medium RPM I1640, sets 37
DEG C, 5%CO2It is cultivated in incubator, logarithmic growth phase cell is for testing.By a concentration of 1 × 105The exponential phase of a/mL
Human lung cancer cell A549,96 orifice plates are inoculated in, in 5%CO2, cultivate for 24 hours under 37 DEG C of incubation conditions；Add respectively into corresponding aperture
Enter the 100 μ l of the compounds of this invention fresh medium of different diluted concentrations, zeroing hole and control is arranged in 6 multiple holes of each concentration
Hole, the PBS polishings of extra hole same volume, sets 37 DEG C, 5%CO248h is cultivated in incubator, culture solution is abandoned in suction, and use is sterile
PBS is washed, cleaning 2 times per hole, every time 200 μ L.Then culture medium (5mg/mL MTTs of the 100 μ L containing MTT is added per hole：Culture
Base=1:4), continue in insulating box after cultivating 4h, culture solution is carefully sucked out, 150 μ LDMSO, oscillator oscillation are added per hole
Afterwards, absorbance value is detected with microplate reader, record using 16.0 softwares of SPSS as a result, calculate IC50Value.
(3) experimental result
Experimental result is as shown in table 12：
Half-inhibition concentration (μm ol/L) of 12 the compounds of this invention of table to A549 cells
Experimental result shows the IC of the compounds of this invention50Value is less than monomeric compound before synthesis, i.e., to A549 cell Proliferations
Inhibiting effect enhancing, but still be weaker than positive control drug.
The effect test of the anti-human cervical cancer cell Hela of 12 the compounds of this invention of embodiment
(1) inhibiting effect of the mtt assay detection the compounds of this invention to human cervical carcinoma cell Hela is used.
Material and reagent：Human cervical cancer cell lines HeLa (Chinese Academy of Medical Sciences's tumour cell library)；DMEM culture mediums, tire
Cow's serum (U.S. Gibco)；MTT (U.S. Sigma)；Other chemical reagent are that analysis is pure
Laboratory apparatus：U.S. Precision Scientific CO2Incubator, superclean bench, inverted microscope, light
Learn microscope, microplate reader (BIO-RADModel 550 and BIO-RAD Model 680), fluorescence microscope, Horizontal electrophoresis tank, height
Fast refrigerated centrifuge, Costar tissue culture plates.
The culture of cell：Human cervical cancer cell lines HeLa use containing 10% fetal calf serum DMEM culture mediums 37 DEG C, 5%
CO2Under the conditions of cultivate, with 0.25% pancreatin -0.53mmolL- 1EDTA digests, and 2~3d is passed on 1 time.
MTT is analyzed：By the HeLa cell inoculations of exponential phase in 96 orifice plates, per hole 104A cell, every group sets 6
The compounds of this invention fresh medium culture 48h of different diluted concentrations is added in parallel hole, abandons old culture solution, and 200 μ L are added
0.2mg·mL- 1The serum free medium of MTT continues to abandon supernatant after cultivating 4h, and 150 μ L DMSO, oscillation dissolving royal purple is added
Se Jia Za precipitates.Absorbance (A492) is measured with microplate reader.Growth inhibition ratio=(1-A experimental groups/A control groups) × 100%.
IC is calculated according to the result of growth inhibition ratio50, as a result as shown in table 13：
Half-inhibition concentration (μm ol/L) of the 13 invention compound of table to HeLa cells
Experimental result shows the IC of the compounds of this invention50Value is less than monomeric compound before synthesis, i.e., to Hela cell Proliferations
Inhibiting effect enhancing, but still be weaker than positive control drug.
The effect test of the 13 thin MGC-803 of the compounds of this invention resisting human gastric cancer of embodiment
(1) material, instrument and reagent
Material and reagent：Human gastric cancer MGC-803 cell strains, cell strain derive from Shanghai life science institute；DMEM is trained
Support base (Hyclone companies), trypsase (Amresco companies), high-quality fetal calf serum (Gibco companies), tetramethyl azo azoles
Blue (MTT, Sigma company), dimethyl alum (DMSO, Sigma company).
Laboratory apparatus：U.S. Precision Scientific CO2Incubator, superclean bench, inverted microscope, light
Learn microscope, microplate reader (BIO-RADModel 550 and BIO-RAD Model 680), fluorescence microscope, Horizontal electrophoresis tank, height
Fast refrigerated centrifuge, Costar tissue culture plates.
(2) experimental method
The culture of cell：Human gastric cancer MGC-803 cells use the DMEM culture mediums containing 10% fetal calf serum in 37 DEG C, 5%CO2
Under the conditions of cultivate.
MTT is analyzed：By the MGC-803 cell inoculations of exponential phase in 96 orifice plates, per hole 104A cell, every group sets 6
The compounds of this invention fresh medium culture 48h of different diluted concentrations is added in a parallel hole, abandons old culture solution, and 200 μ L are added
0.2mg·mL- 1The serum free medium of MTT continues to abandon supernatant after cultivating 4h, and 150 μ L DMSO, oscillation dissolving royal purple is added
Se Jia Za precipitates.Absorbance is measured with microplate reader.Growth inhibition ratio=(1-A experimental groups/A control groups) × 100%.According to growth
The result of inhibiting rate calculates IC50。
(3) experimental result
Experimental result is as shown in table 14：
Half-inhibition concentration (μm ol/L) of 14 the compounds of this invention of table to MGC-803 cells
Experimental result shows the IC of the compounds of this invention50Value is less than monomeric compound before synthesis, i.e., increases to MGC-803 cells
The inhibiting effect enhancing grown, but still it is weaker than positive control drug.
The effect test of 14 the compounds of this invention anti-human prostate cell PC-3 of embodiment
(1) material, instrument and reagent
Material and reagent：Human prostate cell PC-3 is quoted from Shanghai life science institute；RPMI-1640 cultivates the (U.S.
Gibco companies), trypsase (Amresco companies), high-quality fetal calf serum (Gibco companies), Methyl thiazoly tetrazolium assay (MTT,
Sigma companies), dimethyl alum (DMSO, Sigma company).
Laboratory apparatus：U.S. Precision Scientific CO2Incubator, superclean bench, inverted microscope, light
Learn microscope, microplate reader (BIO-RADModel 550 and BIO-RAD Model 680), fluorescence microscope, Horizontal electrophoresis tank, height
Fast refrigerated centrifuge, Costar tissue culture plates.
(2) experimental method
The culture of cell：Human prostate cell PC-3 use containing 10% fetal calf serum RPMI-1640 culture mediums 37 DEG C,
5%CO2Under the conditions of cultivate.
MTT is analyzed：By the PC-3 cell inoculations of exponential phase in 96 orifice plates, per hole 104A cell, every group sets 6
The compounds of this invention fresh medium culture 48h of different diluted concentrations is added in parallel hole, abandons old culture solution, and 200 μ L are added
0.2mg·mL- 1The serum free medium of MTT continues to abandon supernatant after cultivating 4h, and 150 μ L DMSO, oscillation dissolving royal purple is added
Se Jia Za precipitates.Absorbance is measured with microplate reader.Growth inhibition ratio=(1-A experimental groups/A control groups) × 100%.According to growth
The result of inhibiting rate calculates IC50。
(3) experimental result
Experimental result is as shown in Table 15：
Half-inhibition concentration (μm ol/L) of 15 the compounds of this invention of table to PC-3 cells
Experimental result shows the IC of the compounds of this invention50Value is less than monomeric compound before synthesis, i.e., to PC-3 cell Proliferations
Inhibiting effect enhancing, but still be weaker than positive control drug.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than is protected to the present invention
The limitation of range is protected, although being explained in detail to the present invention with reference to preferred embodiment, those skilled in the art should
Understand, technical scheme of the present invention can be modified or replaced equivalently, without departing from the essence of technical solution of the present invention
And range.
Claims (10)
1. a kind of compound, which is characterized in that the structural formula of the compound is：
2. the preparation method of compound as described in claim 1, which is characterized in that include the following steps：
(1) compound shown in formula (I) in solvent A is subjected to alkyl substitution with halogenated dose, obtains chemical combination shown in formula (II)
Object；
(2) by formula (II) compound obtained in step (1) under base catalyst and phase transfer catalyst effect, with formula (III) institute
Show that substitution reaction occurs to be alkylated in solvent B for compound to get to the compound (formula (IV) and (V))
3. preparation method as claimed in claim 2, which is characterized in that be selected from Isosorbide-5-Nitrae-dibromo fourth for halogenated dose in the step (1)
At least one of alkane, bis- iodobutanes of 1,4- and 1,4- dichloroetane.
4. preparation method as claimed in claim 2, which is characterized in that the molar ratio of described halogenated dose and formula (I) compound is
20:1~40:1；The molar ratio of the solvent A and formula (I) compound is 100:1~200:1.
5. preparation method as claimed in claim 2, which is characterized in that step (2) Chinese style (III) compound and formula (II)
The molar ratio of compound is 1:1~4:1.
6. preparation method as claimed in claim 2, which is characterized in that base catalyst and formula (II) chemical combination in the step (2)
The molar ratio of object is 5:1~20:1；The molar ratio of phase transfer catalyst and formula (II) compound is 0.5:1~5:1；Solvent B with
The molar ratio of formula (II) compound is 200:1~300:1.
7. preparation method as claimed in claim 2, which is characterized in that alkyl substitution temperature is 30 in the step (1)
~100 DEG C, the reaction time is 3~12 hours；Preferably, reaction temperature is 70~80 DEG C, and the reaction time is 8~10 hours.
8. preparation method as claimed in claim 2, which is characterized in that the temperature of alkylated reaction is 30 in the step (2)
~100 DEG C, the reaction time is 4~15 hours；Preferably, reaction temperature is 70~80 DEG C, and the reaction time is 10~12 hours.
9. preparation method as claimed in claim 2, which is characterized in that in the step (1) solvent A be selected from methanol, acetonitrile,
At least one of DMF, acetone, ethyl alcohol, tetrahydrofuran, dichloromethane, dimethyl sulfoxide (DMSO), dioxane and isopropanol；It is described
Base catalyst in step (2) is selected from natrium carbonicum calcinatum, at least one of Anhydrous potassium carbonate and triethylamine；Phase transfer catalyst
Selected from benzyltriethylammoinium chloride (TEBA), tetrabutylammonium bromide (TBAB), tetrabutylammonium chloride, 4-butyl ammonium hydrogen sulfate, three
At least one of octylmethylammonium chloride, dodecyl trimethyl ammonium chloride and tetradecyl trimethyl ammonium chloride；The step
Suddenly the solvent B in (2) be selected from methanol, DMF, ethyl acetate, acetonitrile, acetone, pyridine, quinoline, dimethyl sulfoxide (DMSO), tetrahydrofuran,
At least one of dichloromethane, dioxane and isopropanol.
10. a kind of purposes of compound as described in claim 1 in being used to prepare tumor.
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| CN111689870A (en) * | 2020-07-20 | 2020-09-22 | 广东食品药品职业学院 | Honokiol-chlorambucil co-prodrug with lymphocyte leukemia resisting effect and preparation method and application thereof |
| WO2023169567A1 (en) * | 2022-03-11 | 2023-09-14 | 南京施江医药科技有限公司 | Application of tetracyclic compound in treatment of tumors |
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