CN109369579A - A kind of preparation of new compound and its application in terms for the treatment of of cancer - Google Patents
A kind of preparation of new compound and its application in terms for the treatment of of cancer Download PDFInfo
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- CN109369579A CN109369579A CN201811299205.9A CN201811299205A CN109369579A CN 109369579 A CN109369579 A CN 109369579A CN 201811299205 A CN201811299205 A CN 201811299205A CN 109369579 A CN109369579 A CN 109369579A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
- C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
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Abstract
The invention belongs to biomedicine fields, provide a kind of application of the design and preparation method and the compound of new compound in terms of cell growth control mechanism and treatment of cancer.Shown by biological activity test, compound of the present invention has significant negative regulation to act on STAT3 cellular signal transduction, and there is significant inhibiting effect to the proliferation of a variety of cancer cells such as lung cancer, breast cancer, colon cancer and leukaemia, it is shown that the compound has potential significance to clinical treatment and cell the regulatory mechanism research of cancer.The compound great exploitation potential, has a good application prospect, preparation method simple process, needs easy to implement, being suitble to scale industrial production.
Description
Technical field
The present invention relates to biomedicine field, more particularly to a kind of preparation of novel acylhydrazone schiff base compounds and
Application in terms for the treatment of of cancer.
Background technique
The newest cancer data of China of National Cancer Center publication in 2017 is shown, in China, annual new cancer cases
Up to 4,290,000, the 20% of global new cases is accounted for, dead 2,810,000.Lung cancer is the cancer of disease incidence, the double rates first of the death rate, cream
Gland cancer is the highest cancer of women's tumor incidence.The morbidity and mortality of colon cancer and leukaemia also remain high.Cancer
Have become one of the factor for influencing Chinese national health and causing heavy economic losses.The method-of traditional treatment cancer is performed the operation, is put
It treats, chemotherapy and biological therapy have certain limitation (newest cancer data of China in 2017 to the control of the tumour patient state of an illness
[J] China's clinical tumor and rehabilitation, 2017 (5): 574-574).2001, the appearance of first generation targeted drug-Gleevec was given
The clinical treatment of cancer patient brings new favourable turn and hope.The anticancer targeting medicine of the newest listing in the world in 2017 is in China
Occupation rate of market only 1% or so.5 anti-cancer drugs of Chinese food Drug Administration (CFDA) reply listing in 2017 do not have
There is an innovation target spot, the original targeting new drug of China is very few, totally reflects the domestic research and development water for targeting original drug
It is American-European after falling to normal price.How to accomplish that " double wounds " (biological targets are relevant to disease to control for the first time for clinic in terms of medicament research and development
The novel targets and novel compounds for the treatment of) it is the touchstone for testing our Chinese Medicine research and development strength.Seek new target spot and has latent
The lead compound of power has breakthrough in terms of specific clinical therapy of tumor, is medicine research and development scientific research personnel when business
It is anxious.The excited growth and breeding for promoting kinds of tumor cells of JAKs-STAT3 signal transduction, tumour relevant to STAT3 includes head
Neck cancer, lung cancer, breast cancer, prostate cancer, colorectal cancer, chronic myelocytic leukemia, acute myeloblastic leukemia and multiple
Myeloma etc..U.S. Food and Drug Administration (FDA) given an written reply in the clinical trial stage based on STAT3 albumen target spot
Anticancer drug up to more than 30, recent clinical test results show that such drug is huge in terms of future tumors clinical treatment
Potentiality and the open market space (Johnson D E., et al., Nature Reviews Clinical Oncology,
2018,15 (4): 234).It is found by the applicant that schiff base compounds belong to STAT3 inhibitor, such compound inhibit STAT3 it is thin
The mechanism of born of the same parents' signal transduction activation is clear, induces the significant effect of associated tumor cells apoptosis.STAT3 inhibitor class anti cancer target
The features such as drug has target spot new and anticancer spectrum width.This compound is researched and developed by Henan Province is sharp up to Pharmaceutical Technology Co., Ltd.
Schiff base compounds are because its unique structure feature, i.e. core group contain the N atom with lone pair electrons, to have
There is good pairing ability, and the group both sides can react to obtain the different derivative of property with various types of groups
Object, this makes it very characteristic in practical application, is especially widely used in terms of chemistry, biology.Schiff bases containing O, S, N
And its metal complex, the attention of people is constantly subjected to its good antibacterial, anticancer and antiviral bioactivity.Especially
Research of the Ercefuryl in terms for the treatment of of cancer had certain progress (Nelson E A., et al., Blood, 2008,
112 (13): 5095-5102).
The present inventor intends to solve clinical cancer targeted therapy problem.Advanced drug evaluation system and preparation in the reference world
While technique, the targeting type for grinding management regulation and the clinical Chinese independent intellectual property rights of method for transformation exploitation with its efficient medicine is new
Medicine.Compound of the present invention belongs to Schiff bases, and synthesis cost is cheap, it is contemplated that and price is only the 1/3 of Conmana price,
The 1/20 of Gleevec, researches and develops the novel targets targeting type new drug for being successfully expected to achieve Chinese first independent research, and feature is to treat
Imitate significant, broad spectrum anticancer and lower-price characteristic.
Summary of the invention
The main purpose of the present invention is to provide a kind of preparations of New Schiff Base class compound, novel high living as STAT3
Property, application of the hypotoxicity inhibitor in terms for the treatment of of cancer.
For achieving the above object, the present invention provides the preparation of the compound of general formula and treatment of cancer sides
The application in face.
One aspect of the present invention is related to the preparation method of above compound, includes the following steps:
I) using nepal Gold methyl ester as raw material, by itself and N- piperidine ethanol, triphenylphosphine dissolved is cold in tetrahydrofuran
To 0 DEG C, then diisopropyl azodiformate is slowly added dropwise, reaction is finally stirred at room temperature overnight, obtains intermediate
(II):
Ii intermediate (II) and hydration hydrazine reaction 12h) are obtained into intermediate (III):
Iii intermediate (III)) is reacted into 12h with 4- nitryl furfural and obtains compound described in claim 1:
Wherein, the heating temperature refers to 50-100 DEG C.
The purpose of the present invention is find with high STAT3 inhibiting effect and with the noval chemical compound of low toxicity.
The present invention relates to above-mentioned compound 4- (2- (piperidin-1-yl) ethyoxyl) methyl benzoate derivatives, its pharmacy
The solvate of upper acceptable salt, the solvate of the derivative or the salt is controlled in preparation for treating or assisting
Purposes in treatment/or prevention mammal in the drugs such as lung cancer, breast cancer, colon cancer and leukaemia.Specifically, the lactation
Animal is the mankind.
Another aspect of the present invention is related to above-mentioned compound 4- (2- (piperidin-1-yl) ethyoxyl) methyl benzoate and spreads out
The solvate of biology, the solvate of its pharmaceutically acceptable salt, the derivative or the salt is in preparation for controlling
All tumor cell proliferations relevant to STAT3 cellular signal transduction and migration in treatment or adjuvant treatment/or prevention mammal
Purposes in drug.Specifically, the mammal is the mankind.
Another aspect of the present invention is related to above-mentioned compound 4- (2- (piperidin-1-yl) ethyoxyl) methyl benzoate and spreads out
The solvate of biology, the solvate of its pharmaceutically acceptable salt, the derivative or the salt is in preparation for controlling
Purposes in treatment or adjuvant treatment/or prevention mammal in all diseases relevant to STAT3 cellular signal transduction.Specifically
Ground, the mammal are the mankind.
The present invention will be further described below.
All documents recited in the present invention, their full content are incorporated by reference into the present invention, and if these
Meaning expressed by document and when the inconsistent present invention, is subject to statement of the invention.In addition, the various terms that the present invention uses
With phrase have well known to a person skilled in the art general senses, nonetheless, the present invention is remained desirable at this to these terms
It is described in more detail and explains with phrase, the term and phrase referred to is if any inconsistent with common art-recognized meanings, with institute of the present invention
Subject to the meaning of statement.
In the method for synthetic compound of formula i of the present invention, the various raw material for reacting used are those skilled in the art's roots
It can be prepared according to existing knowledge, or can be made from the method as well known to document, or quotient can be passed through
What industry was bought.Intermediate used in the above reaction scheme, raw material, reagent, reaction condition etc. can be according to this field skills
Art personnel, which have knowledge and can make, suitably to be changed.
Compound of formula I of the invention can be applied in combination with other active components, as long as it does not generate other detrimental effects,
Such as allergic reaction.
Reactive compound shown in formula I can be used as anticancer drug exclusive use, or can with it is one or more
Other anti-tumor drugs are used in combination.Combination therapy by by each therapeutic component simultaneously, sequence or separate administration and realize.
Detailed description of the invention
Fig. 1 is STAT3 (Signal Transducer and Activator of Transcription Type-3) letter
Number conducting system schematic diagram: cell factor (Cytokine) triggers Dimerized (dimerization) of receptor in conjunction with receptor
Or polymer (oligomerization), and make JAKs phosphorylation (phosphorylation) to activating STAT3 signal,
Make STAT3 is attached to combine on receptor by the phosphorylation of the specific tyrosine of the termination environment C in recipient cell after birth;After autophosphorylation,
STAT3 forms the binary parallel conformation of bidirectional crossed interaction;It wears in film transporte to cells core, and starts with specific DNA
Son combines, and induces the expression of related anti-apoptotic gene Bcl-2, Survivin, Bcl-XL and c-Jun etc..
Fig. 2 a is compound HN2SM-001 structural formula in embodiment 1;Fig. 2 b is compound HN2SM-001 in embodiment 1
Nuclear-magnetism figure.
Fig. 3 is the experiment knot of compound HN2SM-001 and the virtual molecular docking of STAT3 Protein S H2 functional domain in embodiment 1
Fruit, compound HN2SM-001 are presented on functional domain activated interface, and nonpolar hydrogen atom (H) is unmarked, in STAT3 albumen
Above the section SH2, have with the key amino acid of HN2SM-001 interaction of molecules: lysine 591 and arginine 609 and silk ammonia
Acid 611,613,636 and glutamic acid 612,638 respectively with LYS591, ARG609, SER611, GLU612, SER613, SER636 and
GLU638 label;The β-pleated sheet in the section STAT3 Protein S H2, α spiral and random coil are respectively with delaying straight band, hurricane band and tubule
It indicates.It is virtually docking the results show that compound HN2SM-001 and STAT3 Protein S H2 functional domain phosphorylation site
ARG609, specific amino acid GLU638 and the hydrophobic selection cavity of drug have strong interaction, infer the compound for effect
STAT3 signal transduction inhibitor in STAT3 phosphorylation site.
Fig. 4 is compound HN2SM-001 induced breast cancer cancer cell line apoptosis MTT experiment, the knot of MTT cell experiment in figure
Fruit is characterized with IC50 (μM) value.Should the experimental results showed that, compound HN2SM-001 can effectively induced breast cancer MDA-MB-
The apoptosis of 231 and MCF-7 cell strain.
Fig. 5 is compound HN2SM-001 induction colon cancer cancer cell line apoptosis MTT experiment, the knot of MTT cell experiment in figure
Fruit is characterized with IC50 (μM) value.Should the experimental results showed that, compound HN2SM-001 can effectively induce colon cancer HCT-116
With the apoptosis of HT-29 cell strain.
Fig. 6 is compound HN2SM-001 inducing leukemia cell strain apoptosis MTT experiment, the result of MTT cell experiment in figure
It is characterized with IC50 (μM) value.Should the experimental results showed that, compound HN2SM-001 can effectively inducing leukemia MOLT-13 and
The apoptosis of Jurkat cell strain.
Fig. 7 is compound HN2SM-001 induction lung cancer cell line apoptosis MTT experiment, in figure the result of MTT cell experiment with
IC50 (μM) value is characterized.Should the experimental results showed that, compound HN2SM-001 can effectively induce lung cancer PC9 and HCC827 thin
The apoptosis of born of the same parents' strain.
Table 1 is that the IC50 value that compound HN2SM-001 inhibits the increment of different type cell strain summarizes.
The IC50 value that 1 HN2-SM-001 of table inhibits the increment of different type cell strain
Fig. 8 is the result of compound HN2SM-001 protein immunoblot.The result of protein immunoblot experiment is with by electrophoresis
Total cellular protein after separation is transferred on solid support film from gel, according to the special principle of antigen-antibody, is passed through respectively
The corresponding expressing quantity of STAT3, p-STAT3, β-Actin antibody test.As a result as schemed, it can be seen that acted in different pharmaceutical
Under, increase with drug concentration, the expressing quantity of STAT3 and β-Actin is constant, and p-STAT3 expression quantity is on a declining curve, changes
Close the expression that object HN2SM-001 obviously inhibits p-STAT3.
Fig. 9 is compound HN2SM-001 inverse transcription polymerase chain reaction test (PCR) result.Utilize sxemiquantitative RT-
The related anti-tune of PCR measurement dies expression conditions, after verifying drug various concentration acts on MDA-MB-231, the expression of related gene
Situation.After the drug effect MDA-MB-231 cell of various concentration, inverted by RNA sample extracting, rna content measurement, sample
It records and measures gene expression amount for cDNA, PCR amplification β-Actin and Survivin gene, gel electrophoresis, the anti-tune of comparison dies gene and exists
Expression under the effect of different pharmaceutical concentration.It can be seen that compound HN2SM-001 has obvious inhibition to Survivin expression
Effect.
Specific embodiment
The present invention is further described below by specific preparation embodiment and simulation and biological test example, still,
It is used it should be appreciated that these embodiments and test example are only used for specifically describing in more detail, but should not be understood as being used for
Present invention is limited in any form.
The present invention carries out general and/or specific description to the material and test method arrived used in test.Though
So used many materials and operating method are it is known in the art that still the present invention still exists to achieve the purpose of the present invention
This is described in detail as far as possible.It will be apparent to those skilled in the art that hereinafter, if not specified, material used in the present invention
Material and operating method are well known in the art.
In the present invention, unless otherwise stated, in which: (i) temperature is indicated with degree Celsius (DEG C), is operated under room temperature environment
It carries out;More specifically, the room temperature refers to 20-30 DEG C;(ii) organic solvent is dry with conventional drying method, and the evaporation of solvent makes
It is evaporated under reduced pressure with Rotary Evaporators, bath temperature is not higher than 60 DEG C;(iii) reaction process is tracked with thin-layer chromatography (TLC);(iv) it produces eventually
Object has satisfied proton magnetic resonance (PMR) (1H-NMR) and mass spectrum (MS) data.
Embodiment 1: the synthesis of compound HN2SM-001
Compound HN2SM-001
1) synthesis of 4- (2- (piperidin-1-yl) ethyoxyl) methyl benzoate:
Nepal Gold methyl ester (1.52g, 10mmol) solid is added N- piperidine ethanol (1.93g, 15mmol), triphenylphosphine
(2.62g, 10mmol) is dissolved in 15mL anhydrous tetrahydro furan and is stirred in ice bath, and azoformic acid is added dropwise in 0.5h
Diisopropyl ester (2mL, 10mmol);TLC detects fully reacting, and column chromatogram chromatography obtains white solid powder, is 4- (2- (piperidines-
1- yl) ethyoxyl) methyl benzoate.
2) synthesis of 4- (2- (piperidin-1-yl) ethyoxyl) benzoyl hydrazine:
4- (2- (piperidin-1-yl) ethyoxyl) methyl benzoate (2.63g, 10mmol) solid, mixes with 8mL hydrazine hydrate,
80 DEG C of reflux 12h are heated to, acicular crystal is precipitated after being fully cooled, using ethyl alcohol, ether washs respectively, obtains target compound
That is 4- (2- (piperidin-1-yl) ethyoxyl) benzoyl hydrazine.
3) synthesis of 4- (2- (piperidin-1-yl) ethyoxyl) -2- (5- nitrofuran methylene) benzoyl hydrazine:
4- (2- (piperidin-1-yl) ethyoxyl) benzoyl hydrazine (2.63g, 10mmol) is dissolved in 15mL dehydrated alcohol, 5- nitre
Base furfural (2.1g, 15mmol) puts into the solution, 80 DEG C of back flow reaction 10h is heated to, after TLC detects fully reacting, by solution
It pours into 200mL distilled water, there is faint yellow solid precipitation, filter and use recrystallizing methanol solid, obtain faint yellow solid powder
Last 1.76g, i.e. compound HN2SM-001.1H NMR (300MHz, DMSO) δ 12.16 (s, 1H), 8.40 (s, 1H), 7.92 (d, J
=8.7Hz, 2H), 7.81 (d, J=3.8Hz, 1H), 7.26 (d, J=3.5Hz, 1H), 7.10 (d, J=8.6Hz, 2H), 4.22
(s, 2H), 2.88 (s, 2H), 2.64 (s, 4H), 1.56 (s, 4H), 1.43 (s, 2H)
Embodiment 2: molecular docking (docking) experiment
Method: the interaction mechanism in order to verify compound HN2SM-001 Yu STAT3 albumen, inventor use STAT3
Protein template of the phosphorylation tyrosine bond area (pY-705) in the area SH2 as computer virtual simulation (docking), it is empty
Quasi- docking region is concentrated mainly on phosphorylated tyrosine action site ARG609 and LYS591 near zone.The knot of STAT3 SH2
Structure coordinate is taken at Protein structure databases (PDB data bank, ID:1BG1).The method of molecular docking (docking): institute
The experiment of some computer coordination simulations (docking) is completed on the operating platform of sybyl X2.1.1, calculating used
The tool of machine coordination simulation (docking) is SUEFLEX DOCK.It (mainly include that phosphorylated tyrosine is made according to selected site
It is calculated with site ARG609 and LYS591), determine potential energy level (potential gradient) and makees computer coordination mould
The experiment (4-6) of quasi- (docking).Divided according to the score (Score) and conformation of simulation (docking) and interaction
Analysis.
Embodiment 3: induced breast cancer, colon cancer, the MTT experiment of lung cancer and leukaemia cancer cell apoptosis
Method: logarithmic growth phase breast carcinoma cell strain MDA-MB-231 and MCF-7, colon cancer cell line HCT-116 are collected
And HT-29, lung cancer cell line PC9 and HCC827, leukemia cell line Jurkat and MOLT-13 are counted, adjustment cell suspension is dense
Degree is 40000/mL, and 200 μ L cell suspensions, that is, 8000, every hole cell is added in every hole;Cell strain adds compound to handle, dense eventually
Degree is respectively 0.01,0.03,0.1,0.3,1,3,10,30,100 μM of nine gradient, and sets blank control, continues to cultivate 48h;Medicine
After object processing, 100 μ L (1mg/mL) of thiazolyl blue reagent, 37 DEG C of incubation 4h is added in every hole;Liquid in hole is discarded after incubation, is dripped
Then solid carbon dioxide part is added 100ul dimethyl sulfoxide, is reacted 7-8min on horizontal oscillator with filter paper exhaustion Liquid Residue, until blue
Purple crystal is completely dissolved;The OD value for being 570nm in absorbing wavelength with microplate reader measurement, records result.
Embodiment 4: protein immunoblot (Westernblot) experiment
1, cell culture and dosing
(1) logarithmic growth phase MDA-MB-231 cell, after being digested with pancreatin, with the RPMI- for containing 10% fetal calf serum
The single cell suspension that density is 300000/mL is made in 1640 culture mediums, and it is thin to 6 holes that 2mL cell suspension inoculation is added with every hole
In born of the same parents' culture plate.
(2) 37 DEG C, 5%CO2Incubator is incubated for, and after cell is adherent, it is respectively 10,30,60 and 100 μM that final concentration, which is added,
Drug, and set blank control group.After 1h in addition to blank control group, it is 1mg/mL interleukin 6 (IL-6) that concentration, which is added, in each group
30 μ L stimulate cell, interleukin 6 (IL-6) final concentration of 30ng/mL.
2, cell cracking, protein extraction and determining the protein quantity
(1) upper layer culture medium is removed, is washed twice in six porocyte culture plates with phosphate buffer (PBS).Pre-cooling is added
RIPA cell pyrolysis liquid (protease inhibitors, inhibitors of phosphatases and phenylmethylsulfonyl fluoride and lysate are with 1: 100 ratio
Example is added mixes in advance) 100 μ L.Cell pyrolysis liquid is scraped off with cell scraping clean in advance, be collected into one it is clean
In 1.5mL centrifuge tube.
(2) it places on ice, cracks 30min, be vortexed every 6min primary.
(3) 4 DEG C, 12000rpm, it is centrifuged 12min.
(4) after being centrifuged, cell conditioned medium is collected, is divided into two parts: 5 μ L being taken to be added in the centrifuge tube of 1.5mL, be used for BCA
Protein content is surveyed, 1 × phosphate buffer (PBS) mixing for adding 45 μ L is spare;Remaining 60 μ L of cell conditioned medium quantitative,
5 × SDS sample-loading buffer (Loading Buffer) 15 μ L is added, boils 8min after mixing in boiling water, -20 DEG C of ice after centrifugation
It is saved in case.
(5) determination of protein concentration, specific steps:
A, with 1 × phosphate buffer (PBS) diluted protein standard items, table handling is pressed:
B, BCA working solution is prepared: according to the number of standard items and sample to be tested, A needed for calculating in total and B hybrid working
The amount of liquid.In the ratio of BCA reagent A and B volume ratio 50: 1, working solution is prepared, vortex oscillation mixes spare.
C, it by protein standard liquid and the sample supernatant diluted with phosphate buffer (PBS) (10 times of dilutions), respectively takes
25 μ L are added in 96 new orifice plates.Then it is separately added into the BCA working solution that 200 μ L are prepared in advance again, mixes well.Make sure to keep in mind not
Generation bubble is blown and beaten, 96 orifice plate plate lids is covered tightly, reacts 30min in 37 DEG C of insulating boxs.
D, 96 orifice plates are taken out to restore to room temperature, 3-5min, the absorbance value under 562nm wavelength is measured in microplate reader, is made
Out standard curve and 1 μ L/Protein content of each sample is calculated in case albumen loading.
3, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
(1) fixed plastic plate, prepares 12% SDS-PAGE separation gel.
Separation gel: 10mL is prepared according to following table
(2) mixed separation gel is added separately in 2 blocks of offset plates, is added to the position from top 1.0cm, uses dehydrated alcohol
Offset plate is filled up, 30-45min is stood.
(3) after separation gelling is good, remaining dehydrated alcohol is poured out, and blotted remaining dehydrated alcohol only with filter paper.
(4) 5% concentration glue: 5mL is prepared according to following table
(5) prepared concentration glue is slowly added into offset plate, avoids generating bubble, be inserted into sample comb, stands 30-
45min。
(6) protein sample is taken out, 100 DEG C of heating water bath 5min, revolving speed 10000rpm are centrifuged 10min.
(7) offset plate is fixed in electrophoresis tank, SDS-PAGE electrophoretic buffer is added, extract sample comb, in sequence will
The protein sample handled well is added in sample cell.
(8) 80V electrophoresis 40min.
(9) voltage 120V electrophoresis about 1.5h is changed, until bromophenol blue runs out of colloid.
4, transferring film
(1) the SDS-PAGE glue that electrophoresis finishes is put into TBST buffer and is rinsed once, protein adhesive is put into transferring film buffering
It is impregnated in liquid.
(2) one layer of foam-rubber cushion is soaked in film transfer buffer, is clipped on transferring film instrument with tweezers, according to foam-rubber cushion, three
Layer filter paper, protein adhesive, Kynoar (PVDF) film, three layers of filter paper, foam-rubber cushion are sequentially put well, are aligned, are picked up and be put into transferring film instrument
On, when operation, filter paper, foam-rubber cushion are intended to soak in transferring film buffer.If having bubble application teat glass between every layer gently
Rolling is driven out of.
(3) transferring film instrument, 300mA transferring film 75min are opened.
(4) it takes the film out, is put into TBST buffer, the horizontal concussion instrument of 60rpm rinses 3 times, each 8min.
(5) 2h is closed with the horizontal concussion instrument room temperature of 5% bovine serum albumin(BSA) (BSA) confining liquid 20mL, 60rpm.
(6) with the 3mL antibody incubation liquid added with 3 μ L primary antibodies (STAT3 and p-STAT31: 1000), 4 DEG C of 60rpm level shakes
Instrument is swung to be incubated overnight.
(7) 10mL TBST is used, the horizontal concussion instrument of room temperature 60rpm washs pvdf film three times, each 10min.
(8) with the 20mL antibody incubation liquid added with 2 μ L secondary antibodies, the horizontal concussion instrument incubation pvdf membrane 2h of room temperature 60rpm.
(9) 10mL TBST is used, the horizontal concussion instrument of room temperature 60rpm washs pvdf membrane three times, each 10min.
(10) chemiluminescent substrate reagent solution A and solution B each 1mL, color development at room temperature 5min are taken.
(11) liquid on film is blotted with filter paper, is developed.
Embodiment 5: reverse transcriptase polymerase chain reaction tests (RT-PCR)
1, cell culture and dosing:
(1) logarithmic growth phase MDA-MB-231 cell, after being digested by pancreatin, with the RPMI- for containing 10% fetal calf serum
The single cell suspension that density is 100000/mL is made in 1640 culture mediums, and it is thin to 6 holes that 2mL cell suspension inoculation is added with every hole
In born of the same parents' flat-bottomed plates.
(2) 37 DEG C, 5%CO2Incubator is incubated for, and after cell is adherent, replaces fresh culture, experimental group is added containing difference
The drug 10,30,60,100 (μM) of concentration, it is that 1mg/mL interleukin 6 (IL-6) 30 μ L stimulates cell, Bai Jie that concentration is added after 1h
- 6 (IL-6) final concentration of 30ng/mL of element.
(3) after continuing culture for 24 hours, the total serum IgE for extracting group of cells is illustrated according to RNA extracts kit.
2, prepared by RNA sample:
(1) it after culture for 24 hours, discards supernatant, every hole adds TRIZOL 1mL, shakes up, and digests 5min on ice, uses liquid transfer gun head
Piping and druming, draws each boreliquid into 1.5mL centrifuge tube.
(2) plus 0.2mL chloroform, jog 15s are stored at room temperature 3min.
(3) 4 DEG C, 12000rpm centrifugation, 15min.Liquid is divided into three layers after centrifugation, and upper layer is colourless water sample layer RNA, intermediate
White is DNA, and bottom red is protein, and careful upper layer colourless liquid of drawing is transferred in a new centrifuge tube.
(4) it takes honest and upright and thrifty 0.4mL that isometric isopropanol is added, is stored at room temperature 10min.
(5) 4 DEG C, 12000rpm, it is centrifuged 10min.
(6) liquid in centrifuge tube is discarded, 75% ethyl alcohol 1mL is added.
(7) 4 DEG C, 7500rpm, it is centrifuged 5min, carefully discards supernatant, is placed in super-clean bench, forced air drying 5-10min.
(8) 20-30 μ L coke diethyl phthalate (DEPC) aqueous solution is added, it is spare that packing is stored in -70 DEG C of refrigerators.
(9) total serum IgE K5500 ultramicrospectrophotometer detectable concentration and the purity of 2 μ L are taken.According to OD260/OD280
Ratio identify total serum IgE purity.OD260/OD280 illustrates that purity is preferable between 1.8-2.0.
(10) total serum IgE of 5 μ L is taken to mix with 1 μ L of sample-loading buffer, through 1% Ago-Gel (containing GleRed), 120V, electricity
It swims 30min (electrophoretic buffer is 1 × TAE), the quality of RNA is observed under Labworks image acquisition and analysis software.If RNA is feasible without degrading
It tests in next step.
3, the synthesis of cDNA
Reverse transcriptase polymerase chain reaction (Reverse Transcription-Polymerase Chain Reaction,
RT-PCR), experimental principle: the total serum IgE first in extraction group of cells, using mRNA therein as template, in reverse transcriptase
Under catalytic action, synthesized under random primer or poly thymidine (Oligo (dT)) or the guidance of gene-specific primer mutual
The single-stranded cDNA of the DNA of benefit.Again using this cDNA as template, under the guidance of special primer, the PCR amplification for carrying out target gene is anti-
It answers.Since the cDNA is obtained by template reverse transcription of mRNA, so the objective gene sequence of amplification is not comprising introne
Codified gene order.
(1) synthesis of cDNA
A. the centrifuge tube of no RNA enzyme is taken to sequentially add following solution under ice bath:
4 μ L of deoxyribonucleoside triphosphate mixture (dNTP Mix) 2.5mM/each;Primer mixture (Primer
Mix)2μL;RNA template (RNATemplate) 1ug;No RNA enzyme water (RNase-Free Water) adds to 15 μ L.Reaction condition:
70 DEG C of incubations 10min, rapid ice bath 2min.
B. following component is sequentially added in ice bath:
5 times of first chain buffer (5 × First-Strand buffer) 4uL;1 μ L of reverse transcriptase (M-MLV).React item
Part: 42 DEG C of warm bath 50min, 85 DEG C of heating 5min terminate reaction.
C. it takes 2-3 μ L to be directly used in PCR and reacts or be stored in -20 DEG C.
(2) semiquantitive PCR amplifying target genes
A. following reaction system is added in PCR pipe in order on ice:
cDNA 2.5μL;1 μ L of forward primer (Forward primer) (10mM);Reverse primer (Reverse primer)
(10mM)1μL;2 times of PCR premix enzyme (2 × Taq PCR Mix) 12.5 μ L;Distilled water (ddH2O it) mends to 25 μ L.
B.PCR reaction cycle condition
It is recycled by following temperature:
1) 94 DEG C of 5min of initial denaturation;2) 94 DEG C of 30s are denaturalized;3) anneal 61 DEG C of 30s;4) extend 72 DEG C of 1min;5) it repeats
2-4 step 30 circulation;6) extend 72 DEG C of 5min;7) 4 DEG C of preservations.
C. result detects: taking 5 μ L reaction products after reaction, carries out agarose gel electrophoresis separation identification.
Claims (8)
1. a kind of Schiff base derivatives, it is characterised in that the structural formula of the Schiff base derivatives are as follows:
2. the method for preparing chemical combination described in claim 1, characterized by the following steps:
I) using nepal Gold methyl ester as raw material, by itself and N- piperidine ethanol, triphenylphosphine dissolved is cooled to zero in tetrahydrofuran
Degree, then diisopropyl azodiformate is slowly added dropwise, and reaction is finally stirred at room temperature overnight, obtains intermediate (II):
Ii intermediate (II) and hydration hydrazine reaction 12h) are obtained into intermediate (III):
Iii intermediate (III)) is reacted into 12h with 4- nitryl furfural and obtains compound described in claim 1:
3. a kind of Schiff bases compound and its pharmaceutically acceptable salt are preparing the application in anticancer drug.
4. application according to claim 3, wherein the cancer includes colon cancer.
5. application according to claim 3, wherein the cancer includes breast cancer.
6. application according to claim 3, wherein the cancer includes leukaemia.
7. application according to claim 3, wherein the cancer includes lung cancer.
8. application according to claim 3, wherein the cancer includes extremely relevant to STAT3 cellular signal transduction
Every other cancer.
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CN102961374A (en) * | 2012-12-12 | 2013-03-13 | 苏州大学 | Application of compound and STAT3 (Signal Transducer and Activator of Transcription) inhibitor |
WO2018183908A1 (en) * | 2017-03-31 | 2018-10-04 | Dana-Farber Cancer Institute, Inc. | Compositions and methods for treating ovarian tumors |
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CN102961374A (en) * | 2012-12-12 | 2013-03-13 | 苏州大学 | Application of compound and STAT3 (Signal Transducer and Activator of Transcription) inhibitor |
WO2018183908A1 (en) * | 2017-03-31 | 2018-10-04 | Dana-Farber Cancer Institute, Inc. | Compositions and methods for treating ovarian tumors |
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CN113336729A (en) * | 2021-05-31 | 2021-09-03 | 四川大学华西医院 | Nifuratel derivatives, and preparation method and application thereof |
CN113336729B (en) * | 2021-05-31 | 2022-05-27 | 四川大学华西医院 | Nifuratel derivatives, and preparation method and application thereof |
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