CN108379254A - Prevent the flavone compound of colorectal cancer - Google Patents
Prevent the flavone compound of colorectal cancer Download PDFInfo
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- CN108379254A CN108379254A CN201810380559.XA CN201810380559A CN108379254A CN 108379254 A CN108379254 A CN 108379254A CN 201810380559 A CN201810380559 A CN 201810380559A CN 108379254 A CN108379254 A CN 108379254A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
Abstract
The present invention relates to the flavone compounds of prevention colorectal cancer, belong to field of medicaments.To be solved by this invention is that existing treatment of colorectal cancer effect of drugs is not good enough, the problem of lacking effective medicine in particular for Advanced colorectal cancer, colorectal cancer recurrence and transfer, purposes of the technical solution there is provided I compound represented of formula, its pharmaceutically acceptable salt or its crystal in preparing treatment and/or preventing the drug of colorectal cancer.In vitro and in vivo experiments the result shows that, the compounds of this invention can significantly reduce colorectal cancer stem cells frequency, induce colorectal cancer stem cells differentiation, it is apparent to inhibit colorectal carcinoma growth, and without apparent toxic side effect, a kind of completely new, safe, effectively selection is provided for clinical treatment colorectal cancer.
Description
Technical field
The present invention relates to the flavone compounds of prevention colorectal cancer, belong to field of medicaments.
Background technology
In recent years, with the change of aging of population, life style and dietary structure, colorectal cancer (colorectal
Cancer, CRC) it has become and seriously threatens one of malignant tumour of health, and patient's rejuvenation trend is apparent, majority is suffered from
Person has been middle and advanced stage when medical, and therapeutic effect is not good enough, and postoperative recurrence and the rate of transform are high.With surgery operating technology and auxiliary treatment
Progress, 5 years overall survivals of CRC patient obtain a degree of improvement, but still have most middle and advanced stage patients postoperative
Die of the recurrence and transfer of colorectal cancer.Therefore, how to prevent the recurrence of CRC and transfer becomes to treat the difficult point of colorectal cancer
One of.
Invention content
The purpose of the present invention is to provide the flavone compounds of prevention colorectal cancer, to solve existing treatment of colorectal cancer
Effect of drugs is not good enough, is recurred in particular for Advanced colorectal cancer, colorectal cancer and transfer lacks asking for effective medicine
Topic.
The present invention provides I compound represented of formula, its pharmaceutically acceptable salt or its crystal prepare treatment and/or
Prevent the purposes in the drug of colorectal cancer：
Further, the drug is treatment and/or prevents Advanced colorectal cancer, postoperative recurrence of colorectal cancer, knot directly
The drug of intestinal cancer postoperative metastasis.
Further, the drug reduces colorectal cancer stem cells frequency.
Further, the drug-induced colorectal cancer stem cells differentiation.
Further, the Drug inhibition colorectal cancer cell proliferation.
Further, the drug promotes colorectal cancer cell apoptosis.
Further, the drug promotes colorectal cancer cell late apoptic.
Further, it is activity that the drug, which is with I compound represented of formula, its pharmaceutically acceptable salt or its crystal,
The preparation that pharmaceutically acceptable auxiliary material or complementary ingredient are prepared is added in ingredient.
Further, the preparation is oral preparation, injection or enema.
The present invention provides the pharmaceutical composition of prevention colorectal cancer, be with I compound represented of formula, it can pharmaceutically connect
The salt received or its crystal are active constituent, and the preparation that pharmaceutically acceptable auxiliary material or complementary ingredient are prepared is added.
The present invention provides the combination medicines of prevention colorectal cancer, shown in the formula I for being useful for simultaneously or separately being administered
Compound, its pharmaceutically acceptable salt or its crystal and 5 FU 5 fluorouracil.
Further, the weight proportion of active constituent is in the combination medicine：I compound represented of formula：5- fluorine
Uracil=(5~10)：(1~10).
Preferably, the weight proportion of active constituent is in the combination medicine：I compound represented of formula：5- fluorine is urinated
Pyrimidine=(5~10)：1.
Term " pharmaceutically acceptable " refers to certain carrier, load, diluent, auxiliary material, and/or to be formed by salt usual
In chemistry or physically with constitute the other compatible at split-phase of certain pharmaceutical dosage form, and physiologically mutually compatible with receptor.
Term " pharmaceutically acceptable salt " refers to the acid that the compounds of this invention is formed with inorganic and/or organic bronsted lowry acids and bases bronsted lowry
And/or basic salt, also include amphoteric ion salt (inner salt), further includes quaternary ammonium salt, such as alkylammonium salt.These salt can changed
Close being finally separating and being directly obtained in purifying for object.Can also be by the way that above compound and a certain number of acid or alkali is appropriate
(such as equivalent) is obtained by mixing.These salt may be formed precipitation and be collected with filter method in the solution, or molten
It recycles and obtains after agent evaporation, or be freeze-dried and be made after being reacted in aqueous medium.Heretofore described salt can be compound
Hydrochloride, sulfate, citrate, benzene sulfonate, hydrobromate, hydrofluoride, phosphate, acetate, propionate, fourth two
Hydrochlorate, oxalates, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate.
The method of application of the compounds of this invention or pharmaceutical composition is not particularly limited, and representative method of application includes
(but being not limited to)：Oral, parenteral (intravenous, intramuscular or subcutaneous) and local administration.
Solid dosage forms for oral medication includes capsule, tablet, pill, powder and granule.In these solid formulations
In type, reactive compound is mixed at least one conventional inert excipients (or carrier), such as sodium citrate or Dicalcium Phosphate, or with
Following compositions mix：(a) filler or bulking agent, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid；(b) it bonds
Agent, for example, hydroxymethyl cellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and Arabic gum；(c) moisturizer, example
Such as, glycerine；(d) disintegrant, for example, agar, calcium carbonate, potato starch or tapioca, alginic acid, certain composition silicates,
And sodium carbonate；(e) retarding solvent, such as paraffin；(f) absorbsion accelerator, for example, quaternary ammonium compound；(g) wetting agent, such as spermaceti
Alcohol and glycerin monostearate；(h) adsorbent, for example, kaolin；(i) lubricant, for example, talcum, calcium stearate, tristearin
Or mixtures thereof sour magnesium, solid polyethylene glycol, lauryl sodium sulfate,.In capsule, tablet and pill, dosage form also may include
Buffer.
Solid dosage forms such as tablet, sugar-pill, capsule, pill and granule can be used coating and shell material and prepare, such as casing and
Other materials well known in the art.They may include opacifying agent, also, reactive compound or compound in this composition
Release can discharge in certain part in the digestive tract in a delayed fashion.The example of adoptable embedding component is polymeric material
And wax material.When necessary, reactive compound also can be with one or more formation microencapsulation forms in above-mentioned excipient.
Liquid formulation for oral administration includes pharmaceutically acceptable lotion, solution, suspension, syrup or tincture.
In addition to active compounds, liquid dosage form may include the inert diluent routinely used in this field, such as water or other solvents, increase
Solvent and emulsifier, example know, ethyl alcohol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-BDO, dimethyl formyl
The mixture of amine and oil, especially cottonseed oil, peanut oil, maize germ, olive oil, castor oil and sesame oil or these substances
Deng.
Other than these inert diluents, composition also may include auxiliary agent, such as wetting agent, emulsifier and suspending agent, sweet taste
Agent, corrigent and fragrance.
In addition to active compounds, suspension may include suspending agent, for example, ethoxylation isooctadecane alcohol, polyoxyethylene
The mixture etc. of sorbierite and Isosorbide Dinitrate, microcrystalline cellulose, aluminium methoxide and agar or these substances.
Composition for parenteral injection may include physiologically acceptable sterile, aqueous or anhydrous solution, dispersion liquid,
Suspension or lotion, and the aseptic powdery for re-dissolving into sterile Injectable solution or dispersion liquid.It is suitable aqueous and
Nonaqueous carrier, diluent, solvent or excipient include water, ethyl alcohol, polyalcohol and its suitable mixture.
The dosage form of the compounds of this invention for local administration includes ointment, powder, patch, propellant and inhalant.
Active constituent aseptically with physiologically acceptable carrier and any preservative, buffer, or when necessary may need
Propellant be mixed together.
Pharmaceutically acceptable auxiliary material of the present invention refers to the substance being included in addition to the active ingredient (s in dosage form.
Pharmaceutically acceptable complementary ingredient of the present invention, it has certain physiological activity, but the addition of the ingredient
Leading position of the aforementioned pharmaceutical compositions in treatment of diseases will not be changed, and only play auxiliary effect, these auxiliary
Effect is only the utilization to the ingredient known activity, is the usual adjuvant treatment modality of field of medicaments.If by above-mentioned complementary
Ingredient is used cooperatively with pharmaceutical composition of the present invention, still should belong to the scope of protection of the invention.
The present invention provides the flavone compounds of prevention colorectal cancer.In vitro and in vivo experiments the result shows that, the present invention
Compound can significantly reduce colorectal cancer stem cells frequency, induction colorectal cancer stem cells differentiation, hence it is evident that inhibit Colon and rectum swollen
Tumor is grown, and without apparent toxic side effect, is provided for clinical treatment colorectal cancer a kind of completely new, safe, effectively
Selection.
Description of the drawings
Fig. 1 is colorectal cancer stem cells aspect graph in embodiment 3；
Fig. 2 is that PCR methods detect colon differentiation marker result figure in embodiment 3；
Fig. 3 is Immunofluorescence test colon differentiation marker result figure in embodiment 3；
Fig. 4 is influences of the compound YS434 to the cytotoxicity of colorectal cancer stem cells, proliferation and apoptosis in embodiment 4
Figure；
Fig. 5 is zebra fish vivo detection compound YS434 toxicity data figures in embodiment 4；
Fig. 6 is the drug combination result figure of compound YS434 and colorectal cancer classic chemotherapy drug in embodiment 4；
Fig. 7 is compound YS434 in embodiment 5 to the influence diagram derived from colorectal cancer stem cells source property transplantable tumor；
Fig. 8 is that compound YS434 induces colorectal cancer stem cells differentiation figure in vivo in embodiment 5.
Specific implementation mode
The raw material that is used in the specific embodiment of the invention, equipment are known product, pass through and buy commercial product and obtain.
The present invention provides I compound represented of formula, its pharmaceutically acceptable salt or its crystal prepare treatment and/or
Prevent the purposes in the drug of colorectal cancer：
Research finds that there are colorectal cancer stem cells (CoCSCs) in colorectal cancer.CoCSCs shows chemicotherapy
High resistance, the recurrence and transfer of CRC be postoperative colorectal cancer stem cells (CoCSCs) residual or because stadium relatively late
CoCSCs comes into caused by cycle, and CoCSCs plays critical function in the recurrence and transfer of CRC.Induce CoCSCs differentiation
Afterwards, the oncogenicity of tumour obviously weakens.
For the above situation, inventor has synthesized structure small molecule flavone compound YS434 as shown in formula I.Cell
Experiment shows that compound YS434 can induce the differentiation of CoCSCs, and after two weeks with compound processing CoCSCs, discovery can be bright
Aobvious to reduce stem cell frequency, just there are one colorectal cancer stem cells from 8.7 initial cells, are reduced to 22140 cells
There are one colorectal cancer stem cells for middle；Experiment in vivo shows to inject compound YS434 to transplantable tumor mouse peritoneal, can incite somebody to action
CoCSCs frequencies are reduced to 1/240.4 by 1/35.2.Above experiments have shown that the compounds of this invention YS434 can be largely
It exhausts CoCSCs, induces CoCSCs terminal differentiations, the postoperative recurrences of CRC can be prevented and turn by prompting the compound to a certain extent
It moves, a kind of new strategy and means is provided for complex treatment Advanced colorectal cancer.
In addition, the compounds of this invention YS434 is not shown in cell experiment and experiment in vivo, apparent poison is secondary to be made
With showing its good security.
Further, it can reach association when YS434 and existing colorectal cancer front-line chemotherapeutic agents 5 FU 5 fluorouracil use in conjunction
With the effect of synergy, 5 FU 5 fluorouracil is made just to reach good antitumous effect in low dosage, is clinical treatment colorectal cancer
Provide a kind of completely new, safe, effectively selection.
The preparation of 1 the compounds of this invention YS434 of embodiment
Preparation manipulation step includes：
A, 1 gram of intermediate 1 is added in 6 milliliters of triethyl orthoformates, 600 is slowly added dropwise under room temperature (15~25 DEG C)
Microlitre perchloric acid, is added dropwise, and after the stirring 2 hours of (15~25 DEG C) of room temperature, 60 milliliters of ether are added in reaction solution to analysing
Go out precipitation, filters, obtain red filter cake；The red filter cake is dissolved in 30 milliliters of water, 80 degree of heating stirrings 4 hours stop reaction,
Reaction solution is extracted three times with ethyl acetate, by combined acetic acid ethyl acetate extract concentration, mixes sample, column chromatography purifying.Obtain 600
Milligram intermediate 2.
B, 20 milliliters of tetrahydrofurans are added in 1 gram of meta nitro aniline, 3 grams of potassium carbonate, slowly added under room temperature (15~25 DEG C)
Entering 2 milliliters of chloracetyl chlorides, be added dropwise, moves to 60 degree and react 8 hours, the reaction was complete for the monitoring of TLC thin layers, and reaction solution is filtered,
Filtrate concentrates, then is extracted to concentrate ethyl acetate and water, by acetic acid ethyl acetate extract concentration, mixes sample, column chromatography purifying.
To intermediate 3.
C, 100 milligrams of 2,146 milligrams of intermediate, 3,100 milligrams of intermediate DBU are added to 3 milliliters of n-butanols, it first will be anti-
It answers (15~25 DEG C) of liquid room temperature to stir 0.5 hour, then is warming up to 90 degree and reacts 8 hours, it is cooling, reaction solution is filtered, filter cake is used
A small amount of ethyl alcohol washing, obtains inclined yellow-white filter cake, as intermediate 4.
D, 100 milligrams of intermediates, 4,30 milligrams of ammonium chlorides are added in the mixed liquor of 16 milliliters of ethyl alcohol and 4 milliliters of water, it will
Reaction solution is warming up to 80 degree of stirrings after ten minutes, is slowly added to 60 milligrams of iron powders, then 80 degree are reacted 3 hours, and the monitoring of TLC thin layers is anti-
It should finish, reaction solution is filtered, filtrate is concentrated to give inclined yellow solid, as intermediate 5.
E, 100 milligrams of intermediates 5 are added in 10 milliliters of pyridines, stirring at normal temperature after ten minutes, is added in reaction solution
80 milligrams of acyl chlorides, 65 degree of heating are reacted 8 hours, and the reaction was complete for the monitoring of TLC thin layers, reaction solution is concentrated, to concentrate acetic acid
Ethyl ester and water extract, then acetic acid ethyl acetate extract is concentrated, mixes sample, column chromatography purifying.Obtain faint yellow compound YS434.
The Structural Identification of the compounds of this invention YS434：
1H NMR (400MHz, DMSO) δ 10.20 (s, 1H), 9.89 (s, 1H), 8.22 (d, J=6.0Hz, 1H), 8.14
(s, 1H), 7.98 (d, J=9.5Hz, 1H), 7.87 (d, J=9.0Hz, 2H), 7.45 (s, 1H), 7.33 (s, 1H), 7.26 (d,
J=8.1 Hz, 1H), 7.21-7.14 (m, 2H), 6.75 (d, J=9.0Hz, 2H), 6.28 (d, J=6.0Hz, 1H), 4.90 (s,
2H), 3.00(s,6H).
13C NMR(101MHz,DMSO)δ176.12(s),166.07(s),165.67(s),162.85(s),158.02
(s), 157.03(s),152.87(s),140.54(s),138.85(s),129.65(s),129.10(s),126.96(s),
121.48(s),118.96 (s),116.40(s),115.43(s),115.05(s),112.68(s),112.37(s),111.21
(s),102.30(s),67.85(s).
MS (ESI, cation) m/z 458.05 [M+H]+.
Influences of the 2 the compounds of this invention YS434 of embodiment to colorectal cancer stem cells frequency
1) experiment material
Main agents：DMEM/F2 culture mediums, EGF, FGF, B27, Glutamax, pancreatin etc. are purchased from Gibco BRL companies
(Invitrogen Corporation, USA), DMSO (dimethyl sulfoxide (DMSO)) are Sigma companies (USA) product.It is used when experiment
100%DMSO is configured to 10mM storing liquids, sets -20 DEG C of refrigerators and is kept in dark place spare, face the used time is diluted to institute with complete culture solution
Need concentration.The model of internal test is using the Balb/c purchased from Beijing Vital River Experimental Animals Technology Co., Ltd. SPF grades
Nude mice builds.
Colorectal cancer stem cells culture and passage
Culture：This experiment Human colorectal carcinoma stem cell used is tumor tissues row of the inventor from colorectal cancer patients
Obtained by original cuiture.Rectum cancer cell primary cell is detached with enzyme method with machinery, is added using added with serum-frees such as EGF, FGF
Add the serum-free DMEM/F12 culture mediums of object and 100U/mL penicillin, 100 μ g/mL streptomysins in 5%CO2, under the conditions of 37 DEG C into
Row colorectal cancer stem cells original cuiture.
Passage：When cell balling-up grows to a diameter of 60-100 μM, 600rpm, 5min collect cell ball, use
Accutase digests 5-10min, and culture medium terminates digestion, and 800rpm, 5min abandon supernatant, cell precipitation are resuspended with culture medium
Cell is planted in new culture dish.
2) experimental method
Colorectal cancer tumor stem cell frequency analysis：Tumour balling-up capability analysis (Tumor sphere formation
Assay), it is the most popular method for detecting tumor stem cell self-renewal capacity.Using flow cytometer, by single tumor cell
In sterile sorting to 96 orifice plates, tumour ball then is formed to tumour cell in each hole, is counted and is counted.Basic step
It is as follows：
(1) influences of the vitro detection compound YS434 to CoCSCs stem cell frequencies
(1) CoCSCs 72 is handled respectively with the compound YS434 and isometric DMSO of various concentration (5 μM, 10 μM) in advance
Hour, 1 week, 2 weeks, with pancreatin by different groups of CoCSCs be digested to it is unicellular after, collection the good tumour cell of growth conditions
Ball is digested to individual cells respectively.Cell is resuspended to debita spissitudo with sterile PBS in cell count.
(2) the sterile preparation of flow cytometer：The article on the surface and surrounding of flow cytometer is cleaned with thimerosal, then will
The pipe-line system of flow cytometer hypochlorite solution's soaked overnight；Second day, first rinse streaming pipe repeatedly with hypochlorite solution
Then road system 30 minutes rinses streaming pipe-line system 30 minutes with 75% ethyl alcohol, finally contain 0.5% tire ox with sterile
The PBS of serum is rinsed 30 minutes；Liquid stream is adjusted, sample loading is prepared.
(3) flow cytometer is used, CoCSCs individual cells to the phase that different number group is sorted with single cell mode should hole
In plate.In 1 cell, 10 cells and 100 cell sortings to 96 orifice plates；1000 cells and 10000 cell sortings arrive
In 48 orifice plates.
(4) 37 DEG C, 5%CO2Cell incubator in culture.Period uses Olympus IX71 inverted microscopes to swollen daily
The balling-up growth of oncocyte is observed continuously, after culture 12 to 14 days, to the tumour cell ball quantity that is newly formed in orifice plate and
The hole count that the tumour ball of the CoCSCs of different number group is formed is counted, and limiting dilution assay software is used in combination to be calculated.
(2) influences of the detection compound YS434 to internal stem cell frequency
(1) structure of Transplanted tumor model：Colorectal cancer cell ball is digested to single cell suspension with 0.05% pancreatin, is counted
Cell is resuspended at 1 × 10 number6/ ml is distributed into 100 μ l/ pipes and matrigel 1：1 mixing；After routine disinfection, in inoculation portion
Position (side abdominal subcutaneous) is subcutaneously moved under water with 1ml insulin injector for medical purpose injects the cell suspension of preparation, co-injection 9 after one section
BALB/C nude mices；Next day starts the case where continuously monitoring mice-transplanted tumor tumor formation, and is measured to transplantable tumor volume, works as tumour
Volume reaches about 200mm3When 9 BALB/C nude mices are randomly divided into 3 groups (two experimental groups and control groups), be given once daily
YS434 (two experimental groups, dosage are respectively 25mg/kg and 50mg/kg) and isometric solvent (control group, 5%DMSO+
10%Tween80+10%PEG400+75% water), intraperitoneal injection continues 10 days；
(2) each group transplantable tumor is removed, is digested to by primary culture method unicellular, sorted with flow cytometry, by body
Outer stem cell frequency detecting method is detected.
3) experimental result
It is dry to colorectal cancer thin respectively using the compound YS434 of various concentration and isometric solvent using above method
After born of the same parents handle 72 hours, 1 week and 2 weeks respectively, with the limiting dilution assay of the sterile sortings of Liu Shi, stem cell frequency is detected,
Specific stem cell frequency calculation method is calculated (http using online software://bioinf.wehi.edu.au/
Software/elda/index.htm), it the results are shown in Table 1~3：
Colorectal cancer stem cells frequency after 1 compound YS434 of table is handled 72 hours
Colorectal cancer stem cells frequency after 2 compound YS434 of table is handled 1 week
Colorectal cancer stem cells frequency after 3 compound YS434 of table is handled 2 weeks
From the results shown in Table 1, with compound YS434 processing 72 it is small after, colorectal cancer stem cells frequency is from control
The 1/15.7 of group, is reduced to the 1/19.4 of 5 μM, and when drug concentration is 10 μM, stem cell frequency is further reduced to 1/260.
From table 2 it can be seen that after being handled 1 week with compound YS434, colorectal cancer stem cells frequency declines more bright
It is aobvious, it is reduced to the 1/2737.3 of 1/51.5 and 10 μM of 5 μM.
From table 3 it can be seen that after being handled 2 weeks with compound YS434, colorectal cancer stem cells frequency is reduced to the 1/ of 5 μM
The 1/22140.4 of 84 and 10 μM.
The effect of colorectal cancer stem cells frequency is reduced in order to further verify compound YS434, and is had detected in vivo
Colorectal cancer stem cells frequency is illustrated to be operated as in method, with isometric solvent (5%DMSO+10%Tween80+
10%PEG400+75% water) as a control group, using compound YS434 as experimental group, wherein experimental group is divided into two subgroups,
Respectively 25mg/kg and 50mg/kg groups.Through drug-treated 10 days, tumour is taken out and carries out stem cell frequency inspection by preceding method
It surveys, the results are shown in Table 4：
Internal colorectal cancer stem cells frequency after 4 compound YS434 processing of table
From table 4, it can be seen that by the compounds of this invention by 25mg/kg, 50mg/kg be administered when, internal stem cell frequency by
1/35.2 is reduced to 1/95.4,1/240.4 respectively.
The experimental results showed that, the compounds of this invention YS434 can be substantially reduced colorectal cancer stem cells frequency, greatly above
Exhaust colorectal cancer stem cells to degree.
The effect of 3 the compounds of this invention YS434 induction colorectal cancer tumor stem cell differentiation of embodiment
1) experiment material
PrimeScriptTMRT reagent Kit are purchased from precious biological (Dalian), Mucin2, ANPEP, CK20 antibody
(Abcam), SYBR PCR kit for fluorescence quantitative (Qiagen).
2) experimental method
(1) cellular morphology is identified：Colorectal cancer stem cells ball is digested to unicellular, counting with 0.25% pancreatin；Per hole
1×105~3 × 106Cell inoculation is cultivated 4 hours in 6 orifice plates；5uM is separately added into, at the compound YS434 of 10uM
Reason 48 and 72 hours；Cellular morphology is observed using inverted microscope and is taken a picture.
(2) Real time RT-PCR methods detect colon differentiation marker：Collection colorectal cancer stem cells ball, 800rpm,
5min is centrifuged, supernatant is abandoned；It is primary that preheating PBS washing cells are added, 0.25% pancreatin are added, 37 DEG C are digested to individual cells；It takes
Cell is resuspended in 1ml FBS culture mediums, and postdigestive cell is planted in 6 orifice plates by a certain percentage, sets CO2In incubator, 5%
CO2Then culture 24 hours uses the compound YS434 of various concentration (1 μM, 5 μM, 10 μM) to handle 72 hours；When reaching processing
Between after, sop up culture medium, the PBS washing cells that preheating is added are primary, and it is thin that 0.5ml TRzol piping and druming fully cracking is added to every hole
Born of the same parents, by RNA extraction protocol extraction total serum IgEs and measured concentration；1 μ g RNA are taken to be tried by the cDNA reverse transcriptions of Takara companies
Agent box PrimeScriptTMRT reagent Kit operating instructions carry out reverse transcription to the RNA of extraction, are carried out after 5 times of dilution
PCR。
Real time PCR：Specific primer, goblet cell marker TFF3, Pan are designed for colon differentiation marker
Schwann Cells marker Lysozyme, endocrine cell marker ChgA, cell marker ANPEP, general differentiation marker are absorbed
CK20, row PCR system are as follows：
60 DEG C of progress PCR of annealing temperature are used on BioRad CFX96 real-time PCRs.
(3) Immunofluorescence test colon differentiation marker：Colorectal cancer stem cells ball is collected, 800rpm centrifuges 5min,
Abandon supernatant；It is primary that preheating PBS washing cells are added, 0.25% pancreatin are added, 37 DEG C are digested to individual cells；1ml FBS are taken to train
It supports base weight and hangs cell, postdigestive cell is planted by a certain percentage on the cell climbing sheet prepared in advance, CO is set2In incubator,
5%CO2Then culture 24 hours is handled 72 hours with final concentration of 5 μM of compound YS434；After reaching processing time, inhale
Falling culture medium, the PBS washing cells that preheating is added are primary, and suitable 4% paraformaldehyde is added, is advisable with covering slide,
Cell is fixed in static 30min at room temperature；PBS wash 2 times, 0.5% TritonX-100, be stored at room temperature 20min into
Row punching；TritonX-100 is sopped up, PBS is washed 3 times, 3% BSA is added, is stored at room temperature 30min and is closed；Sop up 3%
BSA, groupization pen draw a circle to approve tissue, be added dropwise primary antibody (colon cell differentiation specific marker), be placed in wet box, 4 DEG C overnight；It sops up
Primary antibody is washed twice with PBS, each 5min, is protected from light and fluorescence secondary antibody is added dropwise, be stored at room temperature 60min, is then protected from light and DAPI is added dropwise,
1min is stood at room temperature；DAPI is sopped up, is washed three times with PBS, each 5min；Finally with 100% glycerine mounting, be placed in just set it is glimmering
Viewed under light microscopy is simultaneously taken a picture.
3) experimental result
It is special from cytomorphology and enterocyte respectively in order to understand whether compound YS434 can induce CoCSCs to break up
The differentiation of two kinds of approach detection CoCSCs of anisotropic differentiation marker.
Using above method, with 10%FBS, compound YS434 (10 μ g/ml) and equivalent solvent DMSO respectively to Colon and rectum
Cancer stem cell is handled 72 hours, and Fig. 1 is shown in the variation of microscopically observation cellular morphology.
It will be seen from figure 1 that control group colorectal cancer stem cells are still in the spherical growth to suspend, and 10% FBS processing
Afterwards it can be seen that its adherent growth is at typical polygon colorectal cancer cell form, cellular morphology after compound YS434 processing
More pleomorphism.
In order to which whether further detection compound YS434 can induce CoCSCs to break up, respectively from mRNA level in-site and albumen
Level has detected a variety of intestines terminally differentiated cells specific biomarkers, as a result sees Fig. 2,3.
From figures 2 and 3, it will be seen that with after compound YS434 processing, the general differentiation marker CK20 of intestines, intestines goblet cell
Marker TFF3/Mucin2, absorptive cell,intestinal marker ANPEP, the Lysozyme expression of intestines paneth's cell marker is apparent, prompts
Compound YS434 can induce colorectal cancer stem cells to break up to intestines thesocyte.
4 the compounds of this invention YS434 of embodiment is to colorectal cancer stem cells toxicity, proliferation, apoptosis and the shadow of cell cycle
Ring 1) experiment material
CCK8 purchased from Medchem Express companies, EDU,Staining reaction liquid, AnnexinV-FITC/PI purchases
From BD pharmingen companies, PBS (pH 7.2~7.6), 0.5%Triton X-100, glycine solution (2mg/mL), %
Paraformaldehyde is this laboratory Sterile culture purchased from Sigma companies, zebra fish.
2) experimental method
CCK8 methods detect cytotoxicity：Colorectal cancer stem cells ball is digested to unicellular, counting with 0.25% pancreatin；It will
Cell is inoculated by the holes 2*104/ in 96 orifice plates, is cultivated 4 hours in 37 DEG C of incubators；It is separately added into the compound of various concentration
YS434 or 10%FBS；10 μ lCCK8 are added after continuing culture 72 hours per hole, tapping culture culture plate makes its mixing；Continue
Light absorption value is sequenced in 450nm after cultivating 2-4 hours.
EDU detects cell Proliferation：Colorectal cancer stem cells ball is digested to unicellular, counting with 0.25% pancreatin；Per hole
1×105~3 × 106Cell inoculation is cultivated 4 hours in 6 orifice plates；It is separately added at the compound YS434 of various concentration
Reason 48 and 72 hours；With cell culture medium EdU solution, about 20 μM of final concentration in 6 orifice plates is added and is incubated 2 hours；Centrifugation is received
Collect cell, with 0.25% pancreatin will treated that colorectal cancer stem cells are digested to is unicellular；Cell, 1500rpm is resuspended in PBS
5min is centrifuged, supernatant is abandoned；Often pipe is added 100~500 μ l'sStaining reaction liquid is resuspended cell, is protected from light, room temperature is incubated
After educating 10min, 1500rpm centrifuges 5min, abandons staining reaction liquid；PBS is detected after cleaning 1 time with flow cytometry.
AnnexinV-FITC/PI detects Apoptosis：Colorectal cancer stem cells ball is digested to 0.25% pancreatin slender
It is secondary (2000rpm centrifuge 5min) to wash cell with PBS, counts, collects 1~5 × 10 by born of the same parents5Cell/pipe；It is added 500 μ l's
Cell is resuspended in Binding Buffer；After 5 μ lAnnexinV-FITC mixings are added, 5 μ L Propidium Iodide are added, mix
It is even；Room temperature is protected from light, reacts 5~15min；Flow cytomery.
Zebra fish internal test compound YS434 cytotoxicities：About 600 pieces of the zebra fish-egg of contemporaneity is collected, respectively
Into 6 culture dishes, the compound YS434 of various concentration is used to handle fish-egg respectively, developmental stage is in stereoscopic micro- border at the same time
Lower observation zebra fish developmental state, has seen whether developmental deformity, dyskinesia and has taken a picture.
3) experimental result
Using above method, vitro cytotoxicities of the compound YS434 to colorectal cancer stem cells is had detected respectively, as a result
Apparent cytotoxicity (Fig. 4 A) is not found；Further Edu intakes detection cell Proliferation finds YS434 pairs of the compounds of this invention
Cell Proliferation significantly inhibits, and is especially especially apparent (Fig. 4) to the inhibiting effect of cell in higher concentration；Together
Sample, apoptosis detection finds that the compounds of this invention YS434 can be obviously promoted the late apoptic of cell, but is influenced on early apoptosis of cells
Unobvious (Fig. 4)；Zebra fish body vivo detection does not find equally to impact (Fig. 5) development of zebra fish.
Colorectal cancer front-line chemotherapeutic agents traditional as one 5-Fu still have the status that do not replace at present, but it has
There are bone marrow suppression, symptom of digestive tract and neurological symptom, is especially especially apparent in high dose.Traditional with colorectal cancer
It is found in the use in conjunction of chemotherapeutics 5 FU 5 fluorouracil, either still 72 hours at 48 hours, 1ug/ml 5-Fu and this change
It when closing object YS434 use in conjunction, can achieve the effect that 5-Fu 10ug/ml are applied alone (see Fig. 6).This illustrates the compounds of this invention
YS434 and 5-Fu, which exists, to act synergistically, and can substantially reduce the dosage of 5 FU 5 fluorouracil.
Above the experimental results showed that, the compounds of this invention YS434 without apparent cytotoxicity but it is capable of inhibiting cell proliferation and
Promote cell death.
The In vivo model of 5 the compounds of this invention YS434 of embodiment is tested
1) experiment material
6~8 week old nude mices purchased from Beijing China Fukang, Matrigel purchased from BD pharmingen companies, DMSO,
Tween80, PEG400 be purchased from the Shanghai Sigma company, 1ml syringes purchased from Sichuan new century medical high polymer product it is limited
Company.
2) experimental method
Colorectal cancer stem cells are digested to unicellular rear counting with 0.25% pancreatin, with 1 × 105A/0.1ml with
Matrigel presses 1:Flank after being inoculated in mouse subcutaneously after 1 mixing, waits for that tumour grows to 200mm3(about 10-14 days) afterwards, mouse with
Machine is grouped (n=8) and starts intraperitoneal injection.
Experiment packet：Drug solvent control group (5%DMSO+10%Tween80+10%PEG400+75% water)；
Compound YS434：20mg/kg q.d.；
Compound YS434：40mg/kg q.d.；
Each group drug is dissolved in 5%DMSO+10%Tween80+10%PEG400+75% water.
Observation index：It measures within every 3 days a mouse weights and tumour major diameter, minor axis and calculates gross tumor volume (major diameter × short
Diameter2× π/6), the reactions such as whether there is or not diarrhea for observation, twitch, fash, and weight is substantially reduced.
3) experimental result
In order to verify therapeutic effects of the compounds of this invention YS434 to the colorectal cancer in the sources CoCSCs in vivo, use with
Two drug concentration processing lotuses are respectively adopted with compound YS434 processing derived from the transplantable tumor of colorectal cancer stem cells in upper method
Tumor mouse, as a result, it has been found that tumour growth is obviously suppressed, removing tumour, weigh further verification compound YS434 certain
It can inhibit the growth of the colorectal cancer in the sources CoCSCs (see Fig. 7).In order to understand the side effect of the compound, in monitoring tumour
While growth, discovery is monitored to tumor-bearing mice weight, is made using the secondary of apparent weight loss is had no after this compound
With (Fig. 7).
In order to tentatively understand the mechanism that the compounds of this invention inhibits colorectal cancer to grow in vivo, further immunofluorescence inspection
It surveys terminal differentiation the marker CK20, goblet cell marker Mucin2 for finishing rectal cell and absorbs cell marker ANPEP,
As a result see Fig. 8.From figure 8, it is seen that can obviously induce these terminal differentiation marks in vivo using the compounds of this invention YS434
Remember the expression of object, this suggests that the cylinder therapeutic effect of compound YS434 is caused by inducing cell terminal differentiation.
Claims (10)
1. I compound represented of formula, its pharmaceutically acceptable salt or its crystal are preparing treatment and/or are preventing colorectal cancer
Purposes in drug：
2. purposes as described in claim 1, it is characterized in that：The drug is treatment and/or prevents Advanced colorectal cancer, ties
The drug that rectal cancer recurrence, colorectal cancer shift.
3. purposes as claimed in claim 1 or 2, it is characterized in that：The drug reduces colorectal cancer stem cells frequency.
4. purposes as claimed in claim 1 or 2, it is characterized in that：The drug-induced colorectal cancer stem cells differentiation.
5. purposes as claimed in claim 1 or 2, it is characterized in that：The Drug inhibition colorectal cancer cell proliferation.
6. purposes as claimed in claim 1 or 2, it is characterized in that：The drug promotes colorectal cancer cell apoptosis；Further
Ground, the drug promote colorectal cancer cell late apoptic.
7. the purposes as described in claim 1~6 any one, it is characterized in that：The drug be with I compound represented of formula,
Its pharmaceutically acceptable salt or its crystal are active constituent, and pharmaceutically acceptable auxiliary material is added or is prepared by complementary ingredient
Made of preparation.
8. purposes as claimed in claim 7, it is characterized in that：The preparation is oral preparation, injection or enema.
9. the pharmaceutical composition of colorectal cancer is prevented, it is characterized in that：Be with I compound represented of formula, its is pharmaceutically acceptable
Salt or its crystal are active constituent, and the preparation that pharmaceutically acceptable auxiliary material or complementary ingredient are prepared is added.
10. the combination medicine of colorectal cancer is prevented, it is characterized in that：Containing changing shown in the formula I for being useful for simultaneously or separately being administered
Close object, its pharmaceutically acceptable salt or its crystal and 5 FU 5 fluorouracil；Preferably, the weight proportion of active constituent is：I institute of formula
The compound shown：5 FU 5 fluorouracil=(5~10)：(1~10)；It is further preferred that the weight proportion of active constituent is：Formula I
Compound represented：5 FU 5 fluorouracil=(5~10)：1.
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| CN115487177A (en) * | 2022-08-15 | 2022-12-20 | 四川大学华西医院 | New use of flavonoid compound for treating ulcerative colitis |
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2018
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| Title |
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| I-LI CHEN等: "Synthesis and antiproliferative evaluation of amide-containing flavone and isoflavone derivatives", 《BIOORGANIC & MEDICINAL CHEMISTRY》 * |
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|---|---|---|---|---|
| CN115487177A (en) * | 2022-08-15 | 2022-12-20 | 四川大学华西医院 | New use of flavonoid compound for treating ulcerative colitis |
| CN115487177B (en) * | 2022-08-15 | 2023-11-24 | 四川大学华西医院 | New use of flavonoid compounds for treating ulcerative colitis |
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