CN108309975A - Micromolecular compound WB460 is preparing the application in treating pancreatic cancer drug - Google Patents
Micromolecular compound WB460 is preparing the application in treating pancreatic cancer drug Download PDFInfo
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- 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
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
- A61K31/4365—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system having sulfur as a ring hetero atom, e.g. ticlopidine
Abstract
The invention discloses micromolecular compound WB460 shown in one kind such as formula (I) to prepare the application in treating cancer of pancreas disease medicament.In vitro, which can inhibit proliferation, migration, invasion and the Clone formation of pancreatic cancer cell, inducing pancreatic cancer cell that apoptosis and cell-cycle arrest occurs；In vivo, WB460 inhibits the growth and transfer of cancer of pancreas.The present invention has a good application prospect.
Description
Technical field
The invention belongs to medicine and its preparation and applied technical fields, and in particular to prepared by micromolecular compound WB460
Treat the application in pancreas tumor medicine.
Background technology
Cancer of pancreas is a kind of tumor in digestive tract of high grade malignancy, early stage patient non-evident sympton and without sensitive and special
Label protein, prevent or early diagnosis relative difficulty, patient's poor prognosis, survival rate is referred to as " it in cancer less than 7% within 5 years
King ".In recent years, cancer of pancreas morbidity and mortality obviously rise, and ranked fourth position in the lethality of developed country's cancer of pancreas, prediction
It will become the second largest lethal cancer within 10 years in future.
Currently, according to different stages of development, the therapeutic modality of cancer of pancreas includes operation, radiotherapy and chemotherapy, and operation is clinical
Upper unique recoverable method.However, since Pancreas cancer patients usually make a definite diagnosis later, forfeiture radical cure chance, only 10-15%'s
Patient meets surgical condition, and 80% undergos surgery and the patient of auxiliary treatment can recur and dead.Patients with terminal utilizes Ji
His shore of west, gemcitabine and Erlotinib combination, gemcitabine and the combination of albumin taxol or chemicotherapy treat cancer of pancreas,
But final most of patient's cancer of pancreas is transferred to liver, lung, peritonaeum and death.
STAT3 (signal transducers and activators of transcription 3), i.e. signal turn
Lead with activating transcription factor 3, be transcriptional activation in the JAK-STAT3 signal paths that cell factor and growth factor are mediated because
One of son participates in many important biological processes such as proliferation, differentiation, apoptosis and immunological regulation of cell.Wherein STAT3 exists
Equal overexpression in many malignant tumours, and promote the occurrence and development of many blood tumors and solid tumor, including leukaemia, lymph
Tumor, cancer of pancreas, breast cancer, oophoroma, prostate cancer, lung cancer, head and neck cancer etc..
Currently, the direct inhibitor of STAT3 includes polypeptide, micromolecular compound class and oligonucleotides.Since STAT3 is more
The unstable and permeability of inhibitor peptides metabolism is poor, limits their applications in vivo；Oligonucleotides inhibitor is due to nucleic acid
The fast degradation of enzyme, bioavailability is poor, pharmacokinetics is poor and half-life short, therefore Clinical practice is limited；Small molecule
In compounds inhibitor STA-21, Cryptotanshinone, Stattic, BBI608 and JMC-9 due to all contain quinones or
Class quinones structure, clinically shows larger toxicity and active anticancer is relatively low.In spite of some promising researchs, but still
There is not the direct inhibitor of STAT3 to be approved for Clinical practice.Therefore it develops a kind of novel with good antitumor activity
STAT3 inhibitor is very necessary.
This application involves prepare the novel small molecule time for treating cancer of pancreas of one kind with China's independent intellectual property right
Drug is selected, and illustrates its mechanism of action, is laid the foundation for follow-up new drug development, there is important theory significance and wide application
Foreground.
Invention content
The present invention proposes one kind micromolecular compound of WB460 or pharmaceutically acceptable salt as shown in formula (I) and exists
The application in the drug for the treatment of cancer of pancreas is prepared, formula (I) WB460 is used to inhibit the proliferation of cancer of pancreas, growth, shifts, invades
It attacks, infiltrate and Clone formation etc..
Wherein, WB460 shown in formula (I) is a kind of micromolecular compound, molecular formula C18H14BrN5O3S, molecular weight are
460.31。
In present invention application, formula (I) the WB460 micromolecular compounds can selectively inhibit high expression STAT3 to live
The proliferation of the pancreatic cancer cell of property albumen, that is, p-STAT3 (Y705).
In present invention application, the cell cycle of formula (I) the WB460 micromolecular compounds retardance pancreas cancer cell strain；Its
In, formula (I) WB460 blocks pancreatic cancer cell to the M phases；Wherein, the pancreatic cancer cell includes PANC-1, BxPC-3.
In present invention application, the Apoptosis of formula (I) the WB460 micromolecular compound inducing pancreatic cancer cell line；Its
In, the pancreatic cancer cell includes PANC-1, BxPC-3.
In present invention application, formula (I) the WB460 micromolecular compounds inhibit the migration of pancreas cancer cell strain, invasion with
And infiltration；Wherein, the pancreatic cancer cell includes PANC-1, BxPC-3.
In present invention application, formula (I) the WB460 micromolecular compounds inhibit the Clone formation of pancreas cancer cell strain；Its
In, the pancreatic cancer cell includes PANC-1, BxPC-3.
In present invention application, formula (I) the WB460 micromolecular compounds inhibit the growth of pancreas cancer cell strain；Wherein, institute
It includes PANC-1 to state pancreatic cancer cell.
In present invention application, formula (I) the WB460 micromolecular compounds can inhibit pancreas in the subcutaneous lotus knurl model of mouse
Gland cancer is grown.
In present invention application, formula (I) the WB460 micromolecular compounds can be in mouse tail vein injection Lung metastases model
In, inhibit pancreas metastasis of cancer.
In present invention application, the hydrate or pharmaceutically acceptable salt or medicine of formula (I) the WB460 micromolecular compounds
Acceptable carrier etc. has effect identical with formula (I) WB460 micromolecular compounds on.
The invention also provides formula (I) WB460 micromolecular compounds to inhibit high expression STAT3 activity eggs in vitro and in vivo
The application of the proliferation of white pancreatic cancer cell and method；Wherein, the pancreatic cancer cell relative to low expression STAT3 albumen, it is described
Formula (I) WB460 micromolecular compounds become apparent the inhibiting effect of the pancreatic cancer cell of high expression STAT3 albumen.
The invention also provides WB460 micromolecular compounds answering in inhibiting pancreatic cancer cell growth shown in formula (I)
With and method.
The invention also provides a kind of pharmaceutical composition, including the formula (I) WB460 micromolecular compounds or pharmaceutically
Acceptable salt and pharmaceutically acceptable carrier.
The invention also provides formula (I) WB460 micromolecular compounds or pharmaceutically acceptable salts or pharmaceutically acceptable
Carrier and containing formula (I) WB460 pharmaceutical composition prepare treatment malignant tumour drug in application.Wherein, institute
State the malignant tumour that malignant tumour is high expression STAT3 activated proteins, including blood tumor and solid tumor, wherein the blood tumor
There are leukaemia, lymthoma etc., the solid tumor to have cancer of pancreas, breast cancer, oophoroma, prostate cancer, lung cancer, head and neck cancer etc..
The present invention screens to obtain micromolecular compound WB460, on a cellular level, WB460 and STAT3 protein bindings, and
It is apparent to lower STAT3 (Y705) phosphorylation, selectively inhibit the proliferation of high expression p-STAT3 (Y705) cell, inhibits simultaneously
Migration, invasion and the Clone formation of pancreatic cancer cell；Zoopery also indicates that WB460 can also inhibit the growth of cancer of pancreas and turn
It moves, and lower toxicity is shown under the drug concentration for playing effect.The drug for the treatment of cancer of pancreas of FDA approvals at present is
Gemcitabine, gemcitabine and Erlotinib combination, gemcitabine and the combination of albumin taxol.Although said medicine being capable of phase
To improving the life cycle of patient, but mean survival time (MST), less than 1 year, patient finally still dies of the transfer and recurrence of cancer of pancreas.This hair
The blank in the domestic field has successfully been filled up in the bright new breakthrough obtained in terms of targeting STAT3 protein for treatment cancers of pancreas, is had very
Big development prospect.The present invention provides important references to target the new drug development of STAT3 albumen anti-pancreatic cancers, has good
Application prospect.
Description of the drawings
Fig. 1 show Proliferation Ability figures of formula (I) WB460 to the different pancreatic cancer cell of STAT3 Expression of phosphorylated amounts.
Scheme the protein level that (1A) indicates STAT3, p-STAT3 (Y705), p-STAT3 (S727) in seven kinds of pancreatic cancer cells
Figure；
Scheming (1B) indicates formula (I) WB460 as the increase of drug concentration is on the active influence of different pancreatic cancer cells；
Scheme the half-inhibition concentration (IC50) that (1C) indicates formula (I) WB460 and BP-1-102 to different pancreatic cancer cells.
Fig. 2 show influences of formula (I) WB460 to the STAT3 albumen of pancreatic cancer cell STAT3 albumen and phosphorylation.
Scheming (2A) indicates formula (I) WB460 to STAT3, p-STAT3 (Y705), p- in pancreatic cancer cell PANC-1, BxPC-3
The influence of STAT3 (S727) albumen；
Scheme (2B) and indicates influences of the formula (I) WB460 to other 4 kinds of pancreatic cancer cell p-STAT3 (Y705) albumen.
Fig. 3 show formula (I) WB460 under various concentration and the combination figure of STAT3 full-length proteins.
Fig. 4 show the retardance of formula (I) WB460 inducing pancreatic Cancer Cell cycles.
Scheming (4A) indicates formula (I) WB460 with the increase of drug concentration, cell cycle in pancreatic carcinoma cells figure；
Scheming (4B) indicates formula (I) WB460 with the increase of drug concentration, cell cycle in pancreatic carcinoma cells statistical chart；
Scheme (4C) and indicates influences of the formula (I) WB460 to pancreatic cancer cell M phase marker proteins.
Fig. 5 show formula (I) WB460 inducing pancreatic cancer cell-apoptosis.
Scheming (5A) indicates formula (I) WB460 with the increase of drug concentration, the apoptosis figure of pancreatic cancer cell；
Scheming (5B) indicates formula (I) WB460 with the increase of drug concentration, the apoptosis statistical chart of pancreatic cancer cell.
Fig. 6 show formula (I) WB460 and inhibits pancreatic cancer cell migration, invasion and Clone formation.
Scheme (6A) and indicates increases of the formula (I) WB460 with drug concentration, the migration of pancreatic cancer cell and invasion figure；
Scheme (6B) and indicates increases of the formula (I) WB460 with drug concentration, the migration of pancreatic cancer cell and invasion statistical chart；
Scheming (6C) indicates formula (I) WB460 with the increase of drug concentration, the Clone formation figure of pancreatic cancer cell；
Scheming (6D) indicates formula (I) WB460 with the increase of drug concentration, the Clone formation statistical chart of pancreatic cancer cell.
Fig. 7 show formula (I) WB460 and inhibits internal pancreatic tumour growth.
Figure (7A) indicates that mouse, the subcutaneous pancreatic tumour figure of stripping are put to death in the administration of each group mouse after three weeks；
Scheme the gross tumor volume statistical chart of mouse during (7B) expression each group mouse is administered three weeks
Figure (7C) indicates that mouse, the subcutaneous pancreatic tumour weight statistical chart of stripping are put to death in the administration of each group mouse after three weeks；
Scheme the weight statistical chart of mouse during (7D) expression each group mouse is administered three weeks；
Figure (7E) indicates that mouse, the heart, liver, spleen, lung, the kidney HE figures of stripping are put to death in the administration of each group mouse after three weeks；
Figure (7F) indicates that mouse is put to death in the administration of each group mouse after three weeks, and the subcutaneous pancreatic tumour of stripping contaminates Ki67 immune groups
Change figure.
Fig. 8 show formula (I) WB460 and inhibits internal pancreatic tumour transfer.
Scheme the metastases figure that the living imaging system during (8A) expression each group mouse is administered six weeks is shown；
Scheme the mouse weight statistical chart during (8B) expression each group mouse is administered six weeks.
Specific implementation mode
In conjunction with following specific examples and attached drawing, the present invention is described in further detail, protection content of the invention
It is not limited to following embodiment.Without departing from the spirit and scope of the invention, those skilled in the art it is conceivable that change
Change and advantage is all included in the present invention, and using appended claims as protection domain.The process of the implementation present invention,
Condition, reagent, experimental method etc. are among the general principles and common general knowledge in the art in addition to the following content specially referred to,
Content is not particularly limited in the present invention.
Embodiment 1：The preparation of WB460.
It weighs N-BOC-4- piperidones, malononitrile, sulphur and L-PROLINE to be dissolved in DMF, be stirred to react at 60 DEG C
10h；Reaction solution is slowly dropped in the water of stirring after cooling, by solid filtering, the drying of precipitation, after purification by column chromatography
Obtain -3 cyano piperidine thiophthene derivative of intermediate 2-amino.By -3 cyano piperidine thiophthene derivative of intermediate 2-amino,
1- methylpyrazole -4- carboxylic acids, the chloro- 1- methyl pyridinium iodides of 2- and DMAP are dissolved in 10mL dichloromethane, are delayed in stirring
It is slow that triethylamine is added dropwise and is heated to reflux 4h, intermediate 2- (1- methyl -4- pyrazoles)-formamido -3- cyano-is obtained after column chromatography
6- tertbutyloxycarbonyl -4,5,6,7- tetrahydrochysenes [2,3-c] piperidines bithiophene.By intermediate 2- (1- methyl -4- pyrazoles)-formamide
Base -3- cyano -6- tertbutyloxycarbonyls -4,5,6,7- tetrahydrochysenes [2,3-c] piperidines bithiophene is dissolved in 10mL dichloromethane, in ice
Isosorbide-5-Nitrae-dioxane solution 1mL of 4N HCl gases is slowly added dropwise under bath, is stirred overnight under room temperature, solvent evaporated obtains 2- (1-
Methyl -4- pyrazoles)-formamido -3- cyano -6- hydrogen -4,5,6,7- tetrahydrochysenes [2,3-c] piperidines and thiophene 287mg, yield 100%；
5- bromine furans -2- carboxylic acids, EDC.HCl and HOBt are dissolved in the DMF of 5mL, 2- (1- methyl -4- pyrroles are added after reacting 30min
Azoles)-formamido -3- cyano -6- (2- glycolyls) -4,5,6,7- tetrahydrochysenes [2,3-c] piperidines bithiophene, the reaction was continued 3h,
Extraction, column chromatography obtain WB-460, and yield 51%, reaction process is shown below.1H NMR(500MHz,DMSO)δ11.53
(s, 1H), 8.47 (s, 1H), 8.07 (s, 1H), 7.13 (d, J=2.9Hz, 1H), 6.80 (d, J=3.2Hz, 1H), 4.76 (s,
2H),3.92-3.91(m,5H),2.76-2.75(m,2H)。
Embodiment 2：Formula (I) WB460 has by force the Cell Proliferation of Pancreatic Cancer Cell of high expression STAT3 (Y-705) phosphorylated protein
The protein level of STAT3 in strong inhibition and pancreatic cancer cell.
Technical method
(1) culture of cell
Pancreatic cancer cell PANC-1, BxPC-3 used in the present invention, MIAPaCa-2, SW1990, AsPC-1, Capan-2,
CFPAC-1 cells are purchased from Chinese Academy of Sciences's Shanghai cell bank, and cell is placed in 37 DEG C of constant incubators.
Cancer of pancreas PANC-1 cell culture is in the DMEM containing 10% fetal calf serum and 1% Sodium Pyruvate, MIAPaCa-2
Cell culture containing 10% fetal calf serum, 1% Sodium Pyruvate and 2.5% horse serum in, AsPC-1, BxPC-3 and Capan-
2 cell culture are in 1640 containing 10% fetal calf serum and 1% Sodium Pyruvate, tire ox of the CFPAC-1 cell culture 10%
In the IMEM of serum, SW1990 cell culture is in the L-15 of 10% fetal calf serum.
(2) immunoblotting (Western Blot)
Cell cleans cell with PBS, RIPA lysates and phosphatase is added after various concentration WB-460 processing 12-24h
Inhibitor, protease inhibitors lytic cell, then by cell pyrolysis liquid centrifugation, it is quantitative, denatured by boiling, use polyacrylamide gel
Protein example is separated by electrophoresis in SDS-PAGE, is then transferred on cellulose nitrate film, after BSA confining liquids close 1h, point
Not Yong STAT3, p-STAT3 (Y705), p-STAT3 (S727), β-actin primary antibody be incubated overnight at 4 DEG C, then with fluorescence
The secondary antibody of label is incubated 1h, finally sweeps the expression that film instrument detects albumen with Odyssey.
(3) MTS methods measure cell proliferation experiment
MTS experiments are a kind of methods with colorimetric method indirect determination living cells quantity.MTS is a kind of newly synthesized tetrazolium
Class compound is measured by being reduced into orange-yellow first a ceremonial jade-ladle, used in libation by the succinate dehydrogenase in living cells mitochondrial at 490nm
The light absorption value of reduzate and the quantity of living cells are proportional, to reflect cell viability situation.This experiment can be used for evaluating
The influence of compounds on pancreatic cancer cell multiplication, to judge inhibiting effect and the calculating of compounds on pancreatic cancer cell multiplication
IC50。
Each cell is with (1-10) × 103The density in a/hole is uniformly inoculated into 96 orifice plates per 100 μ L of hole, is placed on constant temperature incubation
In case after cell is adherent, corresponding culture medium is added in control group, and experimental group gives the compound of various concentration, drug-treated
It is taken out after 24-96h and observes cell state under the microscope, under the conditions of being protected from light, MTS is added, avoid light place exists after mixing
Constant incubator reads absorbance value with being measured at microplate reader 490nm, tests in triplicate, IC50 GraphPad5 Prism
Software is calculated.
Experimental result is as shown in Figure 1, figure (A) is STAT3, p-STAT3 (Y705), p-STAT3 in seven kinds of pancreatic cancer cells
(S727) protein level, it can be seen that the phosphorylated protein content of STAT3 is relatively low in SW1990 and AsPC-1 cells, Qi Tawu
The phosphorylated protein level of the STAT3 of kind pancreatic cancer cell is higher；It is thin to cancer of pancreas to scheme formula (I) WB460 that (B) is various concentration
The suppression curve of born of the same parents' proliferation, it can be seen that the cell Proliferation that (I) WB460 can obviously inhibit p-STAT3 (Y705) content high is right
Inhibit unobvious in p-STAT3 (Y705) content low pancreatic cancer cell AsPC-1, SW1990；Scheme (C) be formula (I) WB460 and
BP-1-102 is to the IC50 of Cell Proliferation of Pancreatic Cancer Cell, and the IC50 of display WB460 ratios BP-1-102 is low and it is for STAT3 activity eggs
The selective inhibiting effect of pancreatic cancer cells different Bai Hanliang.
Embodiment 3：Formula (I) WB460 lowers the phosphorylation of STAT3 (Y705).
Technical method
(1) immunoblotting (Western Blot)
Method is as described above
Experimental result is as shown in Fig. 2, from figure (A) it can be seen that formula (I) WB460 concentration gradients rely on ground significantly downward
The phosphorylation of the intracellular STAT3 of PANC-1, BxPC-3 (Y705), and for the phosphorylation of STAT3 albumen and STAT3 (S-727)
Level does not influence；Scheme (B) and shows that formula (I) WB460 concentration gradients rely on the STAT3 (Y705) that other pancreatic cancer cells are lowered on ground
Phosphorylation level.
Embodiment 4：Formula (I) WB460 can be combined with STAT3 full-length proteins.
Technical method
(1) surface plasma resonance technology (Surface Plasmon Resonance technology, SPR)
The technology is based on before ligand on SPR detection bio-sensing chips (Biosensor chip) and analyte effect
Along technology, compared with traditional means, SPR has without being marked, can be monitored in real time to sample, the protrusion such as sensitivity height is excellent
Point.
The STAT3 full-length proteins of purifying are carried out SPR detections by us.Operation is summarized as follows：Induction chip is inserted into
Bicore T200 detecting systems, with PBST solution equilibria systems.Testing protein is dissolved in sodium acetate solution, then passes through ammonia
Above-mentioned proteopexy on the individual vertical access of induction chip, is groped relevant parameter by base, after radix stabilization, is dissolved in
The untested compound level of the various concentration of PBST solution flows into chip, and experimental result data software into Mobile state by dividing
Analysis.Untested compound can be detected under which kind of concentration by the experiment can be combined with each other with STAT3 albumen, in conjunction with detach
The parameters such as time.
Experimental result is as shown in figure 3, formula (I) WB460 can be combined with STAT3 full-length proteins, and its dissociation constant KD values
It is 2.96 μM.
Embodiment 5：Cell cycle in pancreatic carcinoma cells can be arrested in the M phases by formula (I) WB460.
Technical method
(1) streaming dye PI detection cell cycle experiments
Cell cycle refers to cell since the last time, terminating division to the active procedure for dividing end next time, usually by
G0/G1 phases, S phases, G2 phases and M phases form.The G0/G1 phases：Cell starts the synthesis of RNA and protein, but the content of DNA is still two
Times body；The S phases：DNA starts to synthesize, and at this moment the content of cell nuclear dna is between G1 phases and G2 phases；When DNA replication dna terminates into
For tetraploid when, cell enters the G2 phases；The M phases：Cell proceeds by mitosis.Propidium iodide (PI) is the property be inserted into nucleic acid fluorescent
Dyestuff can be optionally embedded into nucleic acid DNA and RNA double-stranded helicals, in conjunction with amount and DNA content it is proportional, accordingly may be used
To reflect the cell quantity in different cycles by detecting the fluorescence intensity in conjunction with the fluorescent dye PI of DNA.Utilize streaming
Cell instrument fluorescence intensity, to reacting cells cycle stage.
By cancer of pancreas PANC-1, BxPC-3 cell, about (1-10) × 106A/hole is inoculated in 6cm wares, is positioned over incubator
In it is adherent to cell, the compound of various concentration is added.Digestion is collected cell and is added after cleaning a cell with PBS after 12-96h
Cell is resuspended in 75% ethyl alcohol for entering 1mL, and 4 DEG C overnight.Cell was centrifuged in second day, discard 75% ethyl alcohol, cell is cleaned with PBS
200-800 μ L PBS are added afterwards twice, and the PI of the RNA enzyme and 1-10 μ L of 1-10 μ L, mixing rear chamber is added under the conditions of being protected from light
Temperature, which is protected from light, is incubated 30min, is transferred to the streaming Guan Zhongyong flow cytomery cell cycles of 5mL, ModFit softwares is used in combination to unite
Count the cell proportion in each period.
(2) immunoblotting (Western Blot)
Method is as described above
Experimental result is as shown such as figure (4A) and (4B), formula (I) WB460 handled under 0.5 μM of concentration cancer of pancreas PANC-1,
After BxPC-3 cells, the cell proportion of G2/M phases rises to 93.22%, 92.08% by 11.45%, 21.98% respectively.By scheming
Shown in (4C), the histone H 3 (Ser10) of phosphorylation and the Ser/Thr-MPM2 of phosphorylation are M phase markers, we can be with
See increasing with WB460 concentration, (protein level of (Ser10) and p-Ser/Thr-MPM2 obviously rise p-H3.Therefore,
Cell cycle in pancreatic carcinoma cells can be arrested in the M phases by formula (I) WB460.
Embodiment 6：Formula (I) WB460 can will cause apoptosis of pancreatic cancer cell.
Technical method
(1) flow cytometer detection cell apoptosis assay
In normal cell, phosphatidyl serine (PS) is only distributed in the inside of cell membrane lipid bilayer, occurs in cell
Apoptosis early stage, PS is from the interior rollover of cell membrane lipid bilayer on the outside of film.Annexin-V as a kind of cardiolipin binding protein, with
PS has high affinity, therefore can be combined with the cell membrane of apoptosis early stage by the PS of exposure on the outside of cell.Therefore, Annexin-V
By one of the sensitive indexes as detection early apoptosis of cells.Propidium iodide (PI) is a kind of nucleic acid dye, it cannot have been penetrated
Whole cell membrane, but the cell of apoptosis middle and advanced stage and dead cell increase due to permeability of cell membrane, PI can through cell membrane and
Nucleus is set to incarnadine.When by Annexin-V and PI collective effects, it can be used for differentiating living cells, in apoptosis early stage and middle evening
The cell of phase.Cell can be divided into four classifications by the double dye methods of cell using flow cytomery, be showed by four quadrants
Out, it is respectively dead cell, normal cell, late apoptic and viable apoptotic cell.
By cancer of pancreas PANC-1, BxPC-3 cell, (1-10) × 105A/hole is inoculated in six orifice plates, is added after cell is adherent
Enter the compound of various concentration.After compound handles 12-96h, cell is collected.Since the dead cell under drug effect is swum in
In culture medium, when collecting cell culture medium need to be collected to centrifugation together.It is detected with apoptosis kit after washing 2 times with the PBS of precooling.
Experiment, which is divided into, does not contaminate group, single dye Annexin V groups, single dye PI groups, the bis- dye groups of PI and Annexin V (1 × binding of 100 μ L
Cell is resuspended in buffer), adding consistency group by being dyed from low to high.Group is not contaminated is not added with PI and Annexin V, it is single to contaminate PI
PI 1-10 μ L are added in group, and single Annexin V groups addition bis- dye groups of Annexin V 1-10 μ L, Annexin-V-PI that contaminate are added
Each 1-10 μ L of PI and Annexin V, mixing.It is protected from light at room temperature after being incubated 15min, 1 × binding of 200-500 μ L is added
Cell suspension is transferred in the streaming pipe of 5mL, with machine testing on flow cytometer by buffer under the conditions of being protected from light after mixing.It withers
The statistics for the cell died includes viable apoptotic cell and middle and advanced stage apoptotic cell, with FlowJo software data processings and is divided
Analysis.
Experimental result is as shown such as figure (5A) and (5B), formula (I) WB460 handled under 0.5 μM of concentration cancer of pancreas PANC-1,
After BxPC-3 cells, the apoptosis rate of pancreatic cancer cell has respectively reached 12%, 29.76%, shows that formula (I) WB460 can induce carefully
Born of the same parents' apoptosis.
Embodiment 7：Formula (I) WB460 can inhibit pancreatic cancer cell invasion, migration and Clone formation.
Technical method
(1) cells Transwell migration experiment
Metastases are the important links of tumor development.By cancer of pancreas PANC-1, BxPC-3 cell dissociation and count, cell
It is resuspended in serum-free and basal medium containing various concentration compound, cell is with (1-10) × 104A/hole (300 μ L)
It is seeded in the upper chamber of the cells Transwell, the DMSO of equivalent is added in control group.Then be added in lower room 700 μ L contain correspondence it is dense
Spend the culture medium containing 20% fetal calf serum of compound.Cell, which is placed in cell incubator, takes out cell use after culture 12-48h
4% paraformaldehyde fixes small ventricular cell, after 30min, with 2 ‰ crystal violet dye liquor by cell dyeing 15min, cleans cell,
Unbonded crystal violet is washed off, the upside of the cells Transwell is gently wiped with cotton swab, will not migrated to the cell wiping of downside
Fall.It takes pictures under the microscope after natural drying, counts the cell number under multiple visuals field.
(2) cells Transwell Matrigel
First matrigel is placed on and is melted on ice, matrigel is then configured to the PBS solution containing 10% matrigel with PBS,
The 100 μ L solution are added in each cells Transwell to blot it only with sucking pump after placing 0.5-1h in the incubator, after
Continuous experimental implementation is identical with migration experimental implementation.
(3) colony formation
Colony formation is the effective ways for detecting cancer cells in vitro proliferative capacity.By cancer of pancreas PANC-1, BxPC-3
Cell dissociation simultaneously counts, with every hole (1-10) × 103A cell is uniformly inoculated in six orifice plates, is added and is contained after cell is adherent
In the complete medium of various concentration compound.After culture 7-14 days, original culture medium is sopped up with sucking pump, is cleaned 1 time with PBS
Cell 30min is fixed with 4% paraformaldehyde again afterwards, 2 ‰ crystal violet dye liquors wash off cell dyeing 15min more with tap water
Remaining crystal violet dye liquor is taken pictures under the microscope after natural drying at room temperature, and cell clonal formation quantity is calculated.
Experimental result inhibits the cell migration of cancer of pancreas PANC-1, BxPC-3, invasion as shown in fig. 6, figure (6A) is compound
Figure, formula (I) WB460 can be dose-dependently to inhibit the invasion and migration of PANC-1, BxPC-3 pancreatic cancer cell, (6B) to be
Corresponding statistical chart.It is the Clone formation figure that compound inhibits cancer of pancreas PANC-1, BxPC-3 cell to scheme (6C), under 0.5 μM
Formula (I) WB460 can significantly inhibit the Clone formation of PANC-1, BxPC-3 pancreatic cancer cell, illustrate that compound has in vitro
The effect of good anti-pancreatic cancer cell Proliferation；It is statistical chart to scheme (6D).
Embodiment 8：Formula (I) WB460 can inhibit pancreatic tumour to grow
Technical method
(1) nude mice by subcutaneous lotus knurl is tested
Subcutaneous lotus knurl experiment is in mouse bare subcutaneous injection tumour cell, and tumour cell absorbs the nutrients in Mice Body
Matter, rapid proliferation form tumour, then test mice group, experimental group is administered processing to mouse, can observe
Function and effect of the drug to tumour.
By (1-10) × 106A pancreatic cancer cell PANC-1 be subcutaneously injected into immunodeficient mouse (BALB/c-nude, it is naked
Mouse, 5-6 weeks) right side dorsal sc, waits for that subcutaneous tumor grows to 100mm3When left and right, mouse is uniformly divided into four groups, i.e. blank pair
The DMSO of 50 μ L is injected intraperitoneally every other day according to group, the WB460 that 10mg/kg is dissolved in DMSO, high dose is injected intraperitoneally in low dose group every other day
The WB460 that 20mg/kg is dissolved in DMSO is injected intraperitoneally in group every other day, and the BP-1-102 of 20mg/kg is injected intraperitoneally in positive controls every other day,
The every two days volumes and mouse weight for measuring tumour, successive administration put to death mouse and remove subcutaneous tumor, weigh and clap after three weeks
According to.
(2) nude mice toxicity in vivo test experience
The heart of each group mouse, liver, spleen, lung, kidney are stripped out, after PBS washes one's face and rinses one's mouth, fixed using 4% paraformaldehyde,
After dehydration, paraffin embedding, slice, hematoxylin eosin stain, dehydration mounting, observes and take pictures under inverted microscope, detectionization
Close influences of the object WB460 to viscera tissue.
(3) immunohistochemical experiment
The cancer of pancreas subcutaneous tumor of each group mouse is stripped out, fixed, be dehydrated with 4% paraformaldehyde, paraffin embedding,
After slice, then through dewaxing, antigen retrieval, removal catalase, blocking antigen site, incubate Ki67 primary antibodies, secondary antibody, colour developing, Soviet Union
After another name for dyeing, dehydration mounting, observes and take pictures under the microscope, the expression for detecting Ki67 in each group mice pancreatic tumor contains
Amount.
Experimental result is as shown in fig. 7, figure (7A) shows that the pancreatic tumour of 20mg/kg is administered most in formula (I) WB460 every other day
It is small, illustrate that formula (I) WB460 can dose-dependently significantly inhibit the growth of pancreatic tumour；(7B) is the administration phase of statistics
Between gross tumor volume；(7C) is the weight for removing pancreatic tumour, and formula (I) WB460 can dose-dependently inhibit cancer of pancreas
The weight of tumour；The variation of mouse weight during figure (7D) is administration, the growth of BP-1-102 group mouse weights slow down；(7E) is each
Group mouse stripping the heart, liver, spleen, lung, kidney H＆E figure, it can clearly be seen that hepatic tissue has occurred in the liver of BP-1-102 group mouse
Focal necrosis illustrates the dosage that BP-1-102 is administered every other day in 20mg/kg with the infiltration (part of arrow meaning) of inflammatory cell
Under it is toxic to mouse；(7F) is the immunohistochemistry figure of the pancreatic tumour of each group mouse stripping, and Ki67 is the mark of cell Proliferation
Will object, expression quantity rise with the increase of cancer of pancreas grade malignancy, and the part of arrow meaning can be seen that WB460 groups from figure
The brown of mouse is shallower and few, and DMSO groups and BP-1-102 group brown are more and deep, illustrate that formula (I) WB460 can inhibit pancreas
The pernicious growth of gland cancer.
Embodiment 9：Formula (I) WB460 can inhibit pancreatic tumour to shift
Technical method
(1) nude mice tail vein shift experiment
Can mouse tail vein metastasis model can be used for detection compound inhibit the far-end transfer of tumour cell in vivo.
Due to the limitation of mouse capillary size, tumour cell is less able to by way of pulmonary circulation reach vein from arterial system
System, therefore tumour cell can be arrested in lung, it is easy to it grows and shifts in lung.
By (1-10) × 106A pancreatic cancer cell PANC-1-luc be injected into immunodeficient mouse (BALB/c-nude, it is naked
Mouse, 5-6 weeks) tail vein in, with fluorescence intensity in living imaging system detection Mice Body and mouse is uniformly divided within second day
Four groups, i.e. the DMSO of 50 μ L is injected intraperitoneally in blank control group every other day, and low dose group is injected intraperitoneally 10mg/kg and is dissolved in DMSO's every other day
The WB460 that 20mg/kg is dissolved in DMSO is injected intraperitoneally in WB460, high dose group every other day, and 20mg/ is injected intraperitoneally in positive controls every other day
The BP-1-102 of kg detects weekly fluorescence intensity in Mice Body and records data, measures mouse weight, successive administration six within every two days
Mouse is put to death after week.
Experimental results are shown in figure 8, and figure (8A) is that the bioluminescence of pancreatic cancer cell in Mice Body during being administered six weeks is strong
Degree figure, it can be seen that formula (I) WB460 can significantly inhibit the transfer of pancreatic cancer cell；The weight of mouse during figure (8B) is administration
Variation.
For above-described embodiment only for illustrating the technical concepts and features of the present invention, its object is to allow those skilled in the art
It cans understand the content of the present invention and implement it accordingly, it is not intended to limit the scope of the present invention.It is every according to the present invention
Equivalent change or modification made by the essence of content should all cover in the scope of the present invention.
Claims (12)
1. WB460 micromolecular compounds or pharmaceutically acceptable salt shown in formula (I) are preparing treatment malignancy disease
Application in drug；
2. application as described in claim 1, which is characterized in that the malignant tumour includes blood tumor and solid tumor；Wherein, institute
It includes leukaemia, lymthoma to state blood tumor, and the solid tumor includes cancer of pancreas, breast cancer, oophoroma, prostate cancer, lung cancer, head
Neck cancer.
3. application as claimed in claim 2, which is characterized in that formula (I) the WB460 micromolecular compounds are for inhibiting pancreas
Proliferation, growth, transfer, invasion, infiltration and the Clone formation of cancer.
4. application as claimed in claim 3, which is characterized in that formula (I) the WB460 micromolecular compounds inhibit STAT3 to live
Property albumen, that is, p-STAT3Y705 high expression pancreatic cancer cell proliferation.
5. application as claimed in claim 3, which is characterized in that formula (I) the WB460 micromolecular compounds inhibit cancer of pancreas thin
The growth of born of the same parents' strain.
6. application as claimed in claim 3, which is characterized in that formula (I) the WB460 micromolecular compounds inhibit cancer of pancreas thin
The Clone formation of born of the same parents.
7. application as claimed in claim 3, which is characterized in that formula (I) the WB460 micromolecular compounds retardance cancer of pancreas is thin
The cell cycle of born of the same parents' strain.
8. application as claimed in claim 3, which is characterized in that formula (I) the WB460 micromolecular compound inducing pancreatic cancers are thin
The Apoptosis of born of the same parents' strain.
9. application as described in claim 1, which is characterized in that formula (I) the WB460 micromolecular compounds can be with STAT3
Full-length proteins combine, and are preparing the application in inhibiting STAT3 signal paths to promote malignant progression drug.
10. a kind of pharmaceutical composition, which is characterized in that it includes formula as described in claim 1 (I) WB460 small molecules
Close object or pharmaceutically acceptable salt and pharmaceutically acceptable carrier.
11. formula (I) WB460 micromolecular compounds as described in claim 1 or pharmaceutically acceptable salt, or as right is wanted
Ask pharmaceutical composition the answering in preparing malignancy disease drug of the treatment with high expression STAT3 activated proteins described in 10
With.
12. application as claimed in claim 11, which is characterized in that the malignant tumour includes blood tumor and solid tumor；Wherein,
The blood tumor includes leukaemia, lymthoma, the solid tumor include cancer of pancreas, breast cancer, oophoroma, prostate cancer, lung cancer,
Head and neck cancer.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111909167A (en) * | 2019-05-07 | 2020-11-10 | 华东师范大学 | Novel piperidinothiophene derivative and application thereof in preparing medicine for treating psoriasis |
| CN112516145A (en) * | 2019-09-19 | 2021-03-19 | 华东师范大学 | Application of small molecular compound WK369 in preparation of medicines for treating ovarian cancer |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108558848A (en) * | 2018-04-19 | 2018-09-21 | 华东师范大学 | A kind of cycloalkane thiophthene derivative and preparation method thereof and medical usage |
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2018
- 2018-05-02 CN CN201810410933.6A patent/CN108309975A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108558848A (en) * | 2018-04-19 | 2018-09-21 | 华东师范大学 | A kind of cycloalkane thiophthene derivative and preparation method thereof and medical usage |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111909167A (en) * | 2019-05-07 | 2020-11-10 | 华东师范大学 | Novel piperidinothiophene derivative and application thereof in preparing medicine for treating psoriasis |
| CN111909167B (en) * | 2019-05-07 | 2021-11-19 | 华东师范大学 | Piperidinothiophene derivative and application thereof in preparation of medicine for treating psoriasis |
| CN112516145A (en) * | 2019-09-19 | 2021-03-19 | 华东师范大学 | Application of small molecular compound WK369 in preparation of medicines for treating ovarian cancer |
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Application publication date: 20180724 |