CN109223776A - Laurel diamine derivative is preparing the application in the drug for treating breast cancer - Google Patents
Laurel diamine derivative is preparing the application in the drug for treating breast cancer Download PDFInfo
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- CN109223776A CN109223776A CN201811034130.1A CN201811034130A CN109223776A CN 109223776 A CN109223776 A CN 109223776A CN 201811034130 A CN201811034130 A CN 201811034130A CN 109223776 A CN109223776 A CN 109223776A
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Classifications
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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
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/4164—1,3-Diazoles
- A61K31/4178—1,3-Diazoles not condensed 1,3-diazoles and containing further heterocyclic rings, e.g. pilocarpine, nitrofurantoin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/14—Drugs for genital or sexual disorders; Contraceptives for lactation disorders, e.g. galactorrhoea
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
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- Animal Behavior & Ethology (AREA)
- Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Epidemiology (AREA)
- Gynecology & Obstetrics (AREA)
- Pregnancy & Childbirth (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Endocrinology (AREA)
- Reproductive Health (AREA)
- Oncology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present invention provides a kind of laurel diamine derivatives to prepare the application in the drug for treating breast cancer, belongs to cancer drug field.Inventor studies discovery, these three laurel diamine derivatives as shown in Formulas I, Formula II and formula III can strongly regulate and control the canceration signal for the CXCR4 excitation that SDF-1 can induce, induction, which adjusts SDF-1, becomes part or lower efficiency agonist from the agonist of strength in the case where physiology perhaps pathology.Thus illustrate, these three compounds are capable of the targeting CXCR4 of specificity, efficiently kill the breast cancer cell that canceration occurs, and the therapeutic agent that can be used as breast cancer uses, and have a extensive future.
Description
Technical field
The present invention relates to drug fields, in particular to a kind of laurel diamine derivative in preparation for treating mammary gland
Application in the drug of cancer.
Background technique
Estimate that Female Breast Cancer in China Standardized incidence rate was in 2008 according to World Health Organization's International Cancer Research Center
21.6/10 ten thousand, huge due to Chinese population radix, annual new breast cancer case load accounts for global total up to 16.9 ten thousand
The 12.25% of case load is only second to the U.S. (18.2 ten thousand), ranks the whole world second, it is contemplated that in the year two thousand thirty China's women with breast cancer
Number fall ill up to annual 23.4 ten thousand, and leads to breast cancer deaths about 70,000 every year.
Recent studies indicate that take tamoxifen or Exemestane can reduce breast cancer people at highest risk hair
Disease, but the drug is adjusted just for the specificity of breast hormone secretion, does not have any specificity for breast cancer cell
Killing ability, and when cancer cell has been spread or after unlimited hyperplasia, including chemotherapy, mastectomy and all kinds of targets
It has been unable to control the state of an illness to drug, cancer overall diffusion can only be waited to cause patient dead.
Currently, there has been no explicitly for breast cancer carcinogenesis mechanism, can effectively inhibit small point of growth of cancer cells special target
Sub- drug.It reduces the disease incidence of breast cancer, research and develop the anti-breast cancer medicines of a line, have become the most important thing of the world of medicine.
Summary of the invention
The purpose of the present invention is to provide a kind of new medicine uses of laurel diamine derivative, i.e. laurel diamine derivative
In preparation for treating the application in the relevant drug of breast cancer, improves the quality of life of patient with breast cancer, mitigates drug use
Burden.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
Laurel diamine derivative is preparing the application in the drug for treating breast cancer, above-mentioned laurel diamine derivative
Selected from such as at least one of Formulas I, Formula II and formula III compound represented:
Laurel diamine derivative is preparing the application in the antagonist for inhibiting breast cancer cell CXCR4 to express, above-mentioned
Laurel diamine derivative is selected from least one of Formulas I as claimed in claim 1, Formula II and formula III compound represented.
Further, in preferred embodiments of the present invention, above-mentioned CXCR4 expression includes CXCR4 agonist induction
CXCR4 expression.
Further, in preferred embodiments of the present invention, above-mentioned CXCR4 agonist is SDF-1.
Further, in preferred embodiments of the present invention, above-mentioned laurel diamine derivative passes through specifically competition suppression
The combination of CXCR4 agonist and CXCR4 processed inhibits CXCR4 to express.
Laurel diamine derivative is preparing the application in the inhibitor for inhibiting breast cancer cell to shift, above-mentioned laurel
Diamine derivative is selected from least one of Formulas I as claimed in claim 1, Formula II and formula III compound represented.
Further, in preferred embodiments of the present invention, above-mentioned breast cancer cell transfer includes the cancer of SDF-1 induction
Cell transfer.
Compared with prior art, the invention has the benefit that
Inventors discovered through research that three kinds of laurel diamine derivatives are to cancer target as shown in Formulas I, Formula II and formula III
The inhibiting effect of PROTEIN C XCR4, and pass through the Inhibition test of SDF-1 combination CXCR4 or CXCR7, further illustrate these three
Compound has the inhibiting effect of specificity to SDF-1 combination CXCR4, and to SDF-1 combination CXCR7 unrestraint activity.Meanwhile
CXCR4 Chemotaxis test and SDF-1 is induced to induce CXCR4 chemotactic Inhibition test by SDF-1, it was confirmed that these three compounds
Both there is no the inhibitions to CXCR4 chemotactic ability, and also there is no the inhibitions of the inducing chemotactic ability to SDF-1.Finally,
It is experimentally confirmed that these three compounds have the function of cell killing to breast cancer cell line 4T1B, and to normal cell acellular poison
Property.
Experiment discovery, these three compounds too late positive drug AMD3100, P2G in the antagonist ability to CXCR4,
But it is the Reverse transcriptase of specificity to the combination of SDF-1 and CXCR4, and is inhibited for incomplete combination.Thus illustrate,
These three compounds can forcefully regulate and control the canceration signal for the CXCR4 excitation that SDF-1 can induce, and induction adjusts SDF-
1 becomes part or lower efficiency agonist from the agonist of strength in the case where physiology perhaps pathology.This regulation
Be advantageous in that, certain CXCR4/SDF-1 signal can be remained with maintain normal physiological function and CXCR4 due to
SDF-1 intense stimulus and lead to canceration signal.It can be seen that these three compounds are capable of the targeting CXCR4 of specificity, efficiently
The breast cancer cell that canceration occurs is killed, the therapeutic agent that can be used as breast cancer uses, and has a extensive future.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, below will to embodiment or
Attached drawing needed to be used in the description of the prior art is briefly described.
Fig. 1 is the suppression curve that test-compound combines 12G5 antibody combination CXCR4 receptor in experimental example 1；
Fig. 2 is the suppression curve that test-compound combines 2D7 antibody combination CCR5 receptor in experimental example 2；
Fig. 3 is test-compound in experimental example 3 to the suppression curve of chemotactic factor (CF) SDF-1 combination CXCR4；
Fig. 4 is test-compound in experimental example 3 to the suppression curve of chemotactic factor (CF) SDF-1 combination CXCR7；
Fig. 5 is the cytotoxicity experiment result figure of test-compound in experimental example 4；
Fig. 6 is the cell SDF-1 Chemotaxis test illustraton of model in experimental example 5；
Fig. 7 is test-compound in experimental example 5 to the influence diagram of cancer cell migration quantity；
Fig. 8 is test-compound in experimental example 5 to the influence diagram of SDF-1 induced cellular chemotaxis metastasis suppressor；
Fig. 9 is test-compound in experimental example 6 to the growth inhibition curve graph of breast cancer cell line；
Figure 10 is 10 in experimental example 7-5Acute toxicity of the test-compound (administered volume 0.1ml/10g) of M to mouse
Experimental result；
Figure 11 is 10 in experimental example 7-5Acute toxicity of the test-compound (administered volume 0.3ml/10g) of M to mouse
Experimental result；
Figure 12 is 10 in experimental example 7-3Acute toxicity of the test-compound (administered volume 0.1ml/10g) of M to mouse
Experimental result；
Figure 13 is 10 in experimental example 7-3Acute toxicity of the test-compound (administered volume 0.3ml/10g) of M to mouse
Experimental result.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
It will be appreciated that the following example is merely to illustrate the present invention, and it is not construed as limiting the scope of the invention.It is not specified in embodiment
Actual conditions person carries out according to conventional conditions or manufacturer's recommended conditions.Production firm is not specified in agents useful for same or instrument
Person is the conventional products that can be obtained by commercially available purchase.
Test-compound in following experimental examples of present embodiment are as follows: m2018A, m2018B and m2018C, Yi Jijie
Structure formula similar congener m2018D, m2018E and m2018F.
Compound m2018A, chemical name are as follows: N, N'- bis- (4,5- dihydro -1H- imidazoles -2-yl) -1,8- octane binary
Amine (N, N'-Di (4,5-dihydro-1H-imidazol-2-yl) -1,8-octane diamine), structure is as follows:
Compound m2018B,Its chemical name are as follows: N, N'- bis- (4,5- dihydro -1H- imidazoles -2-yl) -1,10- decane binary
Amine (N, N'-Di (4,5-dihydro-1H-imidazol-2-yl) -1,10-decane diamine), structure is as follows:
Compound m2018C,Its chemical name are as follows: N, N'- bis- (4,5- dihydro -1H- imidazoles -2-yl) -1,12- dodecane two
First amine (N, N'-Di (4,5-dihydro-1H-imidazol-2-yl) -1,12-dodecane diamine), structure is as follows:
Compound m2018D, structural formula are as follows:
Compound m2018E, structural formula are as follows:
Compound m2018F, structural formula are as follows::
Control drug in following experimental examples of present embodiment are as follows:
SDF-1 is the unique native agonist of source of people CXCR4, is called CXCL12, amino acid sequence are as follows:
GMKPVSLSYR CPCRFFESHV ARANVKHLKI LNTPNCALQI VARLKNNNRQ VCIDPKLKWI QEYLEKALNK。
P2G is SDF-1 mutain, amino acid sequence are as follows: GMKGVSLSYR CPCRFFESHV ARANVKHLKI
LNTPNCALQI VARLKNNNRQ VCIDPKLKWI QEYLEKALNK.P2G has strong antagonist ability (rather than similar SDF-
1 agonist as CXCR4, the reason is that the mutational site of P2G).
AMD3100 (Plerixafor, No. CAS is 110078-46-1), for the small of commercialization activation stem cell activation
Molecular drug, while being also that its structural formula of the antagonist of CXCR4 is as follows:
Maraviroc (abbreviation MVC) (Selzentry, No. CAS is 376348-65-1), for commercialization CCR5 antagonism
The small-molecule drug of agent, while being also the antagonist of CCR5.
Human MIP1 β eta Chemokine (abbreviation CCL4), amino acid sequence are as follows: MKLCVTVLSL
LMLVAAFCSP ALSAPMGSDP PTACCFSYTA RKLPRNFVVD YYETSSLCSQ PAVVFQTKRS KQVCADPSES
WVQEYVYDLE LN.CCL4 is 1 β of source of people macrophage inflammatory protein, is CCR5 receptor native agonist and chemotactic factor (CF).
Feature and performance of the invention are described in further detail with reference to embodiments:
The Inhibition test that 1 test-compound of experimental example combines 12G5 antibody CXCR4 receptor
CXCR4 is the biomarker on a cell membrane of cancer cell hyperplasia and pernicious division, and SDF-1 is
The natural chemokine and agonist of CXCR4.Therefore, by effectively inhibiting CXCR4 combination SDF-1, so that it may inhibit CXCR4
Signal, maintain normal cell physiological function, avoid hematopoietic differentiation cell (immunocyte and leucocyte) enter pernicious increasing
Raw formula.When CXCR4 is stimulated overflow, either positive adjusting or negative regulation all can cause CXCR4 intracellular
Signal Regulation is not normal, grows so as to cause cell unbalance.
12G5 is the commercial antibodies of a general source of mouse monoclonal as identification CXCR4.12G5 antibody pair
CXCR4 has stronger binding affinity, can be used to identify CXCR4.
In this experiment, in order to confirm test-compound whether can in conjunction with CXCR4 receptor and with CXCR4 receptor
Binding site, it is mono- that inventor's selection carries out the 12G5 anti-CXCR4 with fluorescent marker on A3.01T lymphocytic series
Clonal antibody membrane protein labelling.
One, experimental method:
A. it by way of conventional cell amplification, that is, uses and contains 1X RPMI1640 (THERMOFISCHER), 10%FBS
(calf serum, Biowest), 1x penicillin and 1x streptomysin (THERMOFISCHER) are with 1000000 cell/2 milliliter
Concentration, in 75cm2Culture dish is cultivated 48 hours, then carries out the amplification (expanding in proportion) no more than 5 generations again.By living thin
Born of the same parents' calculating instrument (Biorad TC-20) quantitatively uses 50000 cells as unified recipient cell total amount.
B. selecting Kd (dissociation constant) is 3.3nM, i.e., the concentration of 12G5 antibody is 3.3nM.Reaction system are as follows: with 25 μ l
Buffer mixes 50000+25 μ l of cell, 3.3 μM of 12G5 antibody constitute the antibody receptor cell combination model of 50 μ l volumes,
It is separately added into 0~10 again-5The test-compound (m2018A, m2018B, m2018C, m2018F) and control drug of M concentration
It is incubated for 1 hour by (SDF-1, P2G, AMD3100) at 4 DEG C.
C. after being incubated for 1 hour, with eluent (by 0.1 μM+0.01 μM of sodium azide+1% of EDTA+1X PBS buffer solution
BSA mix) de- liquid twice, carry out 1200rpm respectively every time, centrifugation in 5 minutes discards supernatant.Then, then
The F (ab ') that the Jackson ImmunoResearch of 1/1000 dilution (0.1ug/1ml) is provided is added in above-mentioned buffer
Goat-anti-mouse IgG (FITC coupling) is mixed, shading, is incubated for 30 minutes.Then it repeats with the eluent/buffer
Cleaning twice, respectively at being centrifuged 5min under 1200rpm, discards supernatant liquid.Then, cell is mixed using conventional method, obtained
The cell liquid of 100 μ l total volumes.
D. the fluorescent value degree of FITC in every hole, Mei Genong are measured using flow cytometer (Canto-II SORP, BD)
The repetition of 3 holes is spent, being set to B0 with CXCR4 receptor+12G5 antibody (being free of other test molecules), (Bmax, receptor maximum are identified
Amount), the concentration setting of test molecule are as follows: 10-12M~10-5M.It is analyzed using Flowjo (Treestar) software, then
Data analysis is carried out using PRISM software (Graphpad).
Two, experimental result
As a result as shown in Figure 1, with positive control SDF-1 (i.e. CXCL12, as most basic combination bioactivity point
Son, the ability that Competitive assays 12G5 is combined are most strong) it compares, test-compound m2018B, m2018C are combined in 12G5 antibody
The rejection ability of A3.01T cell (in normal state, expressing a large amount of CXCR4 membrane receptor) is lower, but with P2G and
AMD3100 is little compared to difference.Thus illustrate, combination of the two test-compounds m2018B, m2018C in identification CXCR4
The ability of point is substantially suitable with the ability of P2G and AMD3100.And m2018A, influence energy of the CXCR4 in conjunction with 12G5 antibody
Power is poorer than other tested molecules, and the rejection ability of m2018F is worst, and (molecule connects the carbon branch of two chief active groups
Chain is odd number, i.e. n=9).
M2018B, m2018C compare CXCR4 agonist SDF-1, CXCR4 antagonist P2G and AMD3100, only 1 to 2
The difference of the dissociation constant Kd of log, it is contemplated that too strong inhibition signal, the minimal maintenance and cell to CXCR4 signal are raw
Reason normality unfavorable therefore too strong agonist and antagonist can all have an impact to CXCR4 physiological function signal.The Kd of m2018A
It is more relatively poor than m2018B, m2018C and other controls.Thus illustrate, m2018A, m2018B, m2018C these three
Laurel diamine derivative can identification division or new CXCR4 binding site, have maintain SDF-1 lowest signal induce energy
Power, the antagonist that can be used as CXCR4 come using chemokine inhibiting induces canceration signal in conjunction with SDF-1 receptor leads to cancer
Become.
2 test-compound of experimental example tests the specific recognition of CXCR4
CCR5 receptor is the special chemokine receptors that T lymphocyte is expressed in active state.T is drenched
Bar cell, in the case where being activated or being activated by multi-signal, CXCR4 and CCR5 can be induced to express.CXCR4 and
CCR5 can sometimes be aggregated into heteromers (heterodimer), and irregular combination will lead to the signal intensity of membrane receptor and thin
The complete change of born of the same parents' physiological function leads to the membrane receptor signal mutagenesis of its generation so as to generate unpredictable side effect
Pernicious canceration signal.Therefore, in order to confirm three laurel diamine derivatives in the application to the specific recognition of CXCR4
Ability, spy carry out this experiment.
In order to confirm test-compound to the binding specificity of CXCR4 rather than for CCR5, inventor selects to use
A3.01-R5T lymphocyte, the cell express two receptors of CXCR4 and CCR5 simultaneously.In T lymphocyte system on cell membrane
The 2D7 anti-CCR5 monoclonal antibody membrane protein labelling with fluorescent marker is carried out on A3.01-R5.
One, experimental method
A. quantitatively using 50000 cells as unified recipient cell total amount, (amplification mode is such as
Embodiment 1), selecting Kd (dissociation constant) is 2.2nM, i.e., the concentration of CCR5 2D7 antibody is 2.2nM.Reactant
System are as follows: thin with the antibody receptor that the 2D7 antibody of 25 μ l buffers mixing, 50000+25 μ l of cell, 2.2nM are constituted 50 μ l volumes
Born of the same parents' binding model is added from 10-6The test-compound (m2018A, m2018B, m2018C, m2018D) of M concentration and right
According to drug (CCL4), it is incubated for 1 hour at 4 DEG C.
B. be incubated for 1 hour after, with eluent (by 0.1 μM+0.01 μM of sodium azide of EDTA+1 X PBS buffer solution+
1% BSA is mixed) take off liquid twice, carry out 1200rpm respectively every time, centrifugation in 5 minutes discards supernatant.Then, then
The F (ab ') that the Jackson ImmunoResearch of 1/1000 dilution (0.1ug/1ml) is provided is added in above-mentioned buffer
Goat-anti-mouse IgG (FITC coupling) is mixed, shading, is incubated for 30 minutes.Then it repeats with the eluent/buffer
Cleaning mixes cell twice, using conventional method, obtains the cell liquid of 100 μ l total volumes.
C. the fluorescent value degree of FITC in every hole, each concentration are measured using flow cytometer (CantoII SORP, BD)
3 holes repeat, and are not included with receptor+2D7 antibody and are set to B0 (Bmax, receptor maximum identified amount) (without other test molecules), surveyed
The concentration of examination molecule is set as 10-6M.It is analyzed using Flowjo (Treestar) software, then uses PRISM software
(Graphpad) data analysis is carried out.
Two, experimental result
As a result as shown in Fig. 2, since the T cell normality of expression CCR5 secretes natural chemokine CCL4, and based on homologous
Inhibit, Chemokines CC CL4 can the specific combination for inhibiting CCR5 and 2D7 antibody.Therefore, in the presence of CCL4,2D7 is anti-
The binding ability of body and CCR5 reduce by 70%, and test-compound m2018A, m2018B, m2018C and m2018D are anti-to 2D7
(0% rejection ability) is cut little ice in terms of body combination A3.01-R5 cell ability.
Thus illustrate, when T cell or megacaryocyte express CXCR4 and CCR5 simultaneously, m2018A, m2018B,
These three laurel diamine derivative nonrecognition of m2018C CCR5.In conjunction with the experimental result of experimental example 1, it may be said that bright:
These three laurel diamine derivatives of m2018A, m2018B, m2018C, T cell or megacaryocyte express simultaneously CXCR4 and
In the case of CCR5, can specific recognition CXCR4, without identifying CCR5.Therefore, m2018A, m2018B, m2018C this three
A laurel diamine derivative can become the CXCR4 antagonist of specificity.
3 test-compound of experimental example tests the specific recognition of SDF-1 combination CXCR4
The expression of CXCR4 and CXCR7 receptor be lymphocyte occur migration, activation, one of boundling and effect exist point
Sub- foundation.But under normal circumstances, the expression quantity of this receptor, agonist to the signal function of receptor make chemotactic factor (CF) by
Body becomes an advantageous signal path of cancer or poisoning intrusion.Using CXCR4 of the expression on cell membrane and
CXCR7 receptor inhibits the combination of SDF-1 and CXCR4 or CXCR7 as target, specificity, will be important targeting CXCR4
The important indicator of antagonist.
SDF-1 can be with specific recognition CXCR4 and CXCR7, in order to confirm that laurel diamine derivative in the application can be with
Specific inhibition SDF-1 combination CXCR4, rather than CXCR7, inventor prove through the following experiment.
One, experimental principle
SDF-1 will be coupled Tebium reagent (signal ligand), and Tebium reagent turns applied to the homologous Ford energy of HTRF
Move experiment；And CXCR4 and CXCR7 will be coupled the frizzled receptor reagent of upper HTRF.When the two generates the distance less than 100nm
In conjunction with rear, it will generate energy transfer (from Tebium ligand to receptor).By detecting the size of energy transfer, can determine
Measure the binding ability (that is, IC50 of antagonist) that test-compound inhibits SDF-1 and CXCR4 or CXCR7.
Two, experimental method
Experimental cell: selection human embryonic kidney cell 293T (Human Embryonic Kidney Cells 293), which is stablized, to be turned
Contaminate CXCR4 the CXCR7 receptor with Tag-lite molecular label, referred to as HEK-293T CXCR4-CLIP and HEK-293T
CXCR7-CLIP。
Quantifiable signal ligand, i.e. the SDF-1 chemotactic factor (CF) (abbreviation Tb-SDF-1) with the coupling of Tebium fluorescer, with
3.3 μM and 4.8 μM of Kd value it is thin respectively in connection with 5000 HEK-293T CXCR4-CLIP and HEK-293T CXCR7-CLIP
Born of the same parents, the specific steps are as follows:
Experimental cell system is that HEK-293T CXCR4-CLIP and the HEK-293T CXCR7-CLIP from CISBIO stablize
Cell line, commercialized cell.By -150 DEG C of cryo-conservations until after experiment starts, cell uses both cell lines
10%DMSO and 90% calf serum (filtering) recovery.After cell thaws, cell survival rate identification is carried out, cell survival rate is
95% or more.
Reaction system are as follows: the Tb-SDF-1+3 μ l various concentration (10 of 2 μ l cell (5000)+5 μ l, 3.3nM-5M to 10-12M test-compound (m2018B, m2018C etc.) and control drug (AMD3100)), are incubated for 1 at 20 DEG C after being mixed
Hour.
1X elution 2 times provided using Cisbio, the automatic upper original mold then carried using TECAN mechanical arm
Block carries out the data analysis of spectrophotometric.
Three, experimental result
Positive control AMD3100 is point for reducing SDF-1 and CXCR4 or CXCR7 Percentage bound (IC50) optimal at present
Son.
Test-compound with the combination of CXCR4 as shown in figure 3, compared with AMD3100, test-compound m2018A,
M2018B, m2018C reduce SDF-1 and the ability of CXCR4 Percentage bound is only second to AMD3100, and difference very little is (less than 1
log).And m2018E and m2018F can not effectively inhibit the combination of SDF-1 and CXCR4.
The combination of test-compound and CXCR7 are as shown in figure 4, AMD3100 can be effectively reduced SDF-1 in conjunction with CXCR7
Rate (reduces 80~100%), and at higher concentrations, m2018E also can reduce SDF-1 and CXCR7 Percentage bound.And testedization
Object m2018A, m2018B, m2018C and m2018F is closed almost not work to the combination of SDF-1 and CXCR7.
By Fig. 3 and Fig. 4 as it can be seen that the ability that m2018C reduces SDF-1 and CXCR4 Percentage bound be slightly weaker than m2018A,
M2018B illustrates that different carbon chain lengths will affect the combination of SDF-1.M2018A, m2018B, m2018C to SDF-1 with
The combination of CXCR7 does not almost work, while being able to suppress the combination of SDF-1 and CXCR4 again, and its binding affinity and suppression
The level of IC50 processed is only second to AMD3100.Thus illustrate, these three laurel diamine derivatives of m2018A, m2018B, m2018C
Can specificity steric hindrance SDF-1 inhibit CXCR4, and the lowest activity signal of CXCR4 can be kept；Due to the letter of SDF-1
It number plays a very important role to the regulation of CXCR4 function tool, the data of this experimental example illustrate again: m2018A, m2018B,
M2018C plays the importance that strength imitates CXCR4.
Meanwhile Fig. 3 and Fig. 4 are shown, AMD3100 is similar to the rejection ability of SDF-1 and CXCR4 and CXCR7, explanation
AMD3100 can identify the binding site of SDF-1 and CXCR7 and the binding site of SDF-1 and CXCR7 simultaneously, and take
Steric effect inhibits SDF-1 to fight for the binding site.Due to AMD3100 meeting Competitive assays CXCR7 signal, this can be generated not
The side effect and cytotoxicity that can be estimated.And these three laurel diamine derivatives of m2018A, m2018B, m2018C just for
CXCR4 rather than CXCR7 carry out steric hindrance effect, and Competitive assays SDF-1 bind receptor, Small side effects.In addition, comparing
The signal of SDF-1 and CXCR4 is all inhibited in AMD3100, the letter of m2018A, m2018B, m2018C to SDF-1 and CXCR4
Number this part inhibit, have certain advantage in terms of cytotoxicity and tolerance.
4 cytotoxicity of experimental example and biological tolerance experiment
One, experimental principle
PI (Propidium Iode) is a kind of specific stain agent of cell membrane.When cell is marked by positive PI,
Illustrate the Apoptosis and membranolysis, to reflect physiological-toxicity of the test-compound to cell membrane and cell of addition.
Two, experimental method
It is separated by PBMC human peripheral blood mononuclear cell by Ficoll, uses the CD4+T of Mitenyli Biotec
Cell kit filters out CD4+T cells (yield 30%), and the full periphery blood of 50ml can obtain 100 at (containing blood platelet),
000,000 or so CD4+T lymphocyte.
Using 100000CD4+T lymphocyte as experimental model, by being centrifuged in the environment of 1200rpm, 5min, 4 DEG C, go
Except supernatant, the mixing of 50 μ l buffers is then added, then be separately added into concentration be 1 μM, 10 μM of test-compound (m2018C,
M2018B, m2018A, m2018E) and positive control (antagonist AMD3100, CCR5 of CXCR4 antagonist MVC (CAS:
376348-65-1).After being mixed 24 hours, be put into 37 DEG C cell incubator cultivate, after 24 hours, elution twice, from
The heart removes supernatant.PI of the diluted concentration 1/10000 from THERMOFISCHER is added in cell, 10 points are cultivated at 20 DEG C
Then clock elutes twice, supernatant is removed in centrifugation, and 100 μ l buffers are added, utilize the SSC and PI of Canto II flow cytometer
Channel screen (determines the detected position of cell PI is not added as negative control), filters out the PI in the cell always detected
Positive quantity determines total survival rate of cell.
Three, experimental result
As shown in figure 5, under two 1 μM and 10 μM of experimental standard concentration, in addition to negative control m2018E micro- rubs 10
50% toxicity is generated under concentration to cell, remaining tested molecule mixes common be incubated for by 24 hours and T lymphocytes primary
Have no effect on the physiological status of cell.
M2018A, m2018B and m2018C usually only need 2 hours in the mechanism for generating antagonist to CXCR4
In conjunction with the composite construction for dissociating, generating stable cell-membrane receptor and antagonist, therefore, m2018A, m2018B and m2018C this
Three laurel diamine derivatives do not cause toxic effect to cell.
Three bioactive molecules m2018A, m2018B and m2018C to CXCR4 generate antagonist effect during simultaneously
Toxic effect is not caused to cell.The biological tolerance ability of these three bioactive molecules is strong, and it is very strong further to prove that its structure has
Targeting, targeting for CXCR4 by the access of SDF-1 signal stimulus induction canceration signal, while SDF-1 is lured
The cells survival and normal chemotactic activity signal (can be provided by CXCR7) led are without influence.Thus illustrate, these three work
Property molecule have very strong pharmacological activity to the normal immunological of cell and human body balance.
Influence of 5 test-compound of experimental example to cancer cell chemotaxis
One, experimental principle
Chemotaxis is a vital signs of cancer cell canceration, and the usual most basic active mechanism of cancer metastasis is
On cell adhesion molecule integrin (integrins), the interaction of desintegrins and chemotactic factor (CF) and
The complexing actions such as chemokine receptors be combined with each other, signals-modulating.SDF-1 is as the unique agonist of CXCR4, for occurring
The cell of canceration generates irreversible transferance.Cancer cell, damage and infected tissue and host cell can all raise
CXCR4 and SDF-1 expression, so that this transfer and the effect of chemotactic are more aobvious strong.
By the inducing action of chemotactic factor (CF), cell can generate the variation of polar cell film, and formed and tend to chemotactic factor (CF) height
The place polarization migration of concentration.This chemotactic process is by chemotactic factor (CF) concentration, the expression quantity and cell of chemokine receptors
Normality influence.
Two, experimental method
A. the ability of tested molecule chemotactic cancer metastasis is tested:
Cell chemotaxis experimental model is as shown in fig. 6, experiment uses the transwell of the offer of Corning company
Bicarbonate filter chamber (48wells), first respectively the upper and lower be separately added into 50ul and
Mixed liquor (the streptomysin (Gibco)+10% of the penicillin+100ug/ml of RPMI1640 culture medium+100ug/ml of 500ul
100% calf serum purchased from Biowest), it is put into 37 DEG C of incubators and cultivates 4 hours, 50ul then is added on upper layer and contains
There is the T lymphocyte of 5000 A3.01 expression CXCR4, the test-compound of 50ul difference gradient concentration is added in lower layer
(m2018B, m2018C and m2018A) and control molecule (SDF-1, P2G and AMD3100).
37 DEG C of incubators are put into, 5h to be migrated is waited.After 5 hours, lower confluent monolayer cells are all collected, the stream of 100ul is concentrated to
In the buffer of formula Cytometric Analysis, the channel of FSC and SSC then through flow cytometer CantoII carry out 180 seconds thin
Born of the same parents' reading Analysis (every 3 Kong Weiyi concentration), statistical analysis are past from upper layer in the tested molecule inducing cell of each concentration
The mobile cell quantity of lower layer, as a result as shown in Figure 7.
B. tested molecule is tested to the rejection ability of SDF-1 chemotactic cancer metastasis:
Cell chemotaxis experimental model is as shown in fig. 6, experiment uses the transwell of the offer of Corning company
Bicarbonate filter chamber (48X) is being separately added into 50ul's and 500ul in the upper and lower respectively first
(streptomysin (Gibco)+10% of the penicillin+100ug/ml of RPMI1640 culture medium+100ug/ml is purchased from mixed liquor
100% calf serum of Biowest), it is put into 37 DEG C of incubators and cultivates 4 hours, 50ul then is added on upper layer respectively and contains
Have 5000 A3.01 expression CXCR4 T lymphocyte and 50ul difference gradient concentration test-compound (m2018A,
M2018B and m2018C) and control molecule (P2G and AMD3100), certain density SDF-1 is added in lower layer.
37 DEG C of incubators are put into, 5h to be migrated is waited.After 5 hours, lower confluent monolayer cells are all collected, in the same way
It is detected, as a result as shown in Figure 8.
Three, experimental result:
As seen from Figure 7, the CXCR4 chemotactic process of SDF-1 induction causes a large amount of A3.01 T lymphocyte to move from upper layer
Lower layer is moved to, when SDF-1 concentration is 10-9.5M~10-8When the section M is sequentially increased, the migration quantity of A3.01T lymphocyte by
It is cumulative more；When SDF-1 concentration is greater than 10-8When M, the migration quantity of A3.01T lymphocyte is gradually decreased.Illustrate SDF-1 pairs
A3.01T lymphocyte has the concentration chemotactic effect of normal distribution.And test-compound (m2018A, m2018B and m2018C) is equal
Special chemotactic effect is not generated to CXCR4.
As seen from Figure 8, under chemotaxis, the combination of CXCR4 and chemotactic factor (CF) SDF-1 are the phases of a dosage effect
Interaction process；A large amount of SDF-1 pours in the chemotaxis that will affect CXCR4, generate internal actin (actin) and
The polymerization of filament albumen (filament) forms cell by the stronger chemotactic of activity mechanism (pseudopodia) of creeping
Local polarisation state migrates into lower layer (or moving in parallel) from upper layer.And its tested molecule is not then any to this generation
Chemotaxis.
Thus illustrate, SDF-1 is uniquely can be with the chemotactic factor (CF) of inducer T lymphocyte (expression CXCR4) in current experiment
And agonist.And all positive or negative controls in this experiment and m2018A, m2018B, m2018C these three laurels
Diamine derivative does not have the effect of inducer T lymphocyte Chemotaxis.And when T lymphocyte combines upper bioactive molecule
After m2018A, m2018B, m2018C, the inducing chemotactic ability of SDF-1 is substantially completely suppressed.
--- --- --- --- --- --- --- --- --- ----specificity experiments segmentation
Line --- --- --- --- --- --- --- --- ---
Cell suppression test of 6 test-compound of experimental example to breast cancer cell line
One, experimental principle:
It (is obtained from ATCC) using breast cancer cell line 4T1B, the cell experiment for breast cancer medicines screening.4T1B is thin
Born of the same parents are the breast cancer not dead cell system from mouse, very high with the eucaryotic cell structure and genetic homology of people.Due to current people
The physiological function of source breast cancer cell line is not possible to be received by the standard that breast cancer medicines are tested, therefore 4T1B cell line is
At present colon cancer drug test conventional isolated experiment it is required.
By 4T1B cell and tested molecule incubation in logarithmic growth state, using purchased from Roche ROCHE's
Cell Proliferation Reagent WST-1 cell growth detection reagent is detected.Contain a kind of spy in the reagent
Other salt ion WST-1, this molecule can be catalyzed discoloration (from shallow because of the mitochondrial dehydrogenase oxidizing ferment that is discharged of living cells
Red becomes peony), therefore, Apoptosis and killed can be determined by the variation degree of color in cell culture fluid
Degree and efficiency, so as to probe into the size of the tested molecular targeted pharmacological activity for killing breast cancer cell.
Two, experimentation:
In vitro culture meets the breast cancer cell line 4T1B of requirement of experiment, enters (culture plate before logarithmic growth phase to cell
Under intensive 80% degree), be added quantitative test-compound (m2018A, m2018B) and control molecule (AMD3100,
P2G), 3 testing sites are repeated.After cultivating for 24 hours, remove supernatant, after culture medium (be free of calf serum) elution, observation and
Control the adhesion situation of cell.Then WST-1 reagent is added with the concentration in the every hole 100ul/.After 10 minutes cracking reactions, use
Pipettor, which mixes well cell extract, to be made, and then under the measurement of spectrophotometer, calculates tested molecule to breast cancer cell
It is the 503nhibiting concentration IC of 4T1B50。
Three, experimental result
As shown in figure 9, control molecule AMD3100 and P2G, do not generate the growth of breast cancer cell line 4T1B any
Inhibiting effect, AMD3100 and P2G at higher concentrations (i.e. 10 instead-6M) induction can be generated to breast cancer cell line 4T1B
Growth.Especially AMD3100 (potent inhibitor of CXCR4) can generate induced growth to CT26 colon cancer cancer cell and make
With indirect proof potent inhibitor not has inhibiting effect to the CXCR4 cancer cell signals induced.And test-compound m2018A
And m2018B, under same concentrations (such as 10-6M), that is, realize and inhibit to the 100% of breast cancer cell, it is also not any dense
Recurrence status caused by degree changes.Thus illustrate, m2018A and m2018B can be powerful to breast cancer cell line 4T1B generation
Cell is apoptosis-induced and cell development inhibiting effect.On the basis of previous experiments, inventor it is reasonable to conclude that go out with
The similar compound m2018C of m2018A and m2018B structure also can generate inhibiting effect to breast cancer cell line 4T1B.
Thus illustrate, m2018A and m2018B and m2018C these three bioactive molecules can be used as potential anti cancer target
Small-molecule drug can establish chemical structure and functional target base for optimal candidate active anti-breast cancer cancerous tumor cell
Plinth.M2018A will become the lead compound of this kind of compound.
7 test-compound of experimental example is to the intracorporal Acute Toxicity of mouse
Above 6 external experimental verifications mechanism of action of the series test-compound, physiological function and safety, it is real
Test example 7 be intended to verify the series test-compound internal stability and potential a possibility that causing acute toxicity.
One, experimentation:
Use weight be about 10-15g birth Balb/c wild-type mice as test Acute Toxicity model,
200 mouse are randomly divided into 20 groups, and every group 10, hero is female fifty-fifty.It is respectively 10e with concentration-5M and 10e-3The m2018A of M,
M2018B, m2018C and AMD3100 are test medicine.The dosage of intraperitoneal injection, test medicine is respectively
The PBS buffer solution of same volume is injected intraperitoneally in 0.1ml/10g mouse and 0.3ml/10g mouse, blank control group.It gives daily
Medicine is primary, after successive administration 14 days, observes the physiological status of each group mouse and calculates its survival rate.
Three, experimental result:
As shown in Figure 10, the administration concentration of test medicine is 10-5M, when administered volume is 0.1ml/10g mouse, abdominal cavity note
After penetrating the 14th day observed after being administered, compared to other test-compounds, compound AMD3100 generates mouse stronger
Toxicity affects total survival rate (80%) of mouse.And the toxicity that compound m2018A, m2018B, m2018C generate mouse
Less than compound AMD3100.Meanwhile compound m2018A and m2018C opposite will be weaker than m2018B to the toxicity that mouse generates
(in Figure 10, the curve co-insides of the curve and PBS of m2018A and m2018C), in compound to be tested, only m2018B is to small
Mouse, which produces toxicity, leads to its death.And it ought at this concentration (i.e. 10-5M), administered volume is three times volume (that is, 0.3ml/
10g mouse) when, as shown in figure 11, since the possibility of volume relationship and potential experimental implementation, each test-compound perfusion is perfused
The death rate caused by 0.3ml is higher than the death rate of each group when perfusion volume is 0.1ml.M2018C and m2018B performance is close.
The safe sex expression of m2018A it is more preferable than other compounds, the survival rate of AMD3100 is minimum, but its lethal time than
M2018C wants late.
As shown in figure 12, the administration concentration of test medicine is 10-3In the case where M, administered volume is 0.1ml/10g mouse
When.Survival rate chart is consistent with Figure 11: the possibility since volume relationship and potential experimental implementation is perfused, each test-compound fill
It infuses the death rate caused by 0.1ml and is equal to 10-5M administration concentration, perfusion volume be 0.3ml when each group the death rate, m2018C and
M2018B performance is close.The safe sex expression of m2018A it is more preferable than other compounds, the survival rate of AMD3100 is minimum, but
Its lethal time ratio m2018C is late.As shown in figure 13, same concentration, three times volume, that is, 0.3ml, the death of AMD3100
Rate was begun to ramp up from the tenth day, and m2018C security performance is worst, followed by m2018A, and safety is slightly higher, m2018B safety
Performance is better than other compounds.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from of the invention
Many other change and modification can be made in the case where spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
Claims (7)
1. laurel diamine derivative is preparing the application in the drug for treating breast cancer, which is characterized in that the laurel two
Amine derivative is selected from such as at least one of Formulas I, Formula II and formula III compound represented:
2. laurel diamine derivative is preparing the application in the antagonist for inhibiting breast cancer cell CXCR4 to express, feature
Be, the laurel diamine derivative in Formulas I as claimed in claim 1, Formula II and formula III compound represented at least one
Kind.
3. laurel diamine derivative according to claim 2 is short of money for inhibiting breast cancer cell CXCR4 to express in preparation
Application in anti-agent, which is characterized in that the CXCR4 expression includes that the CXCR4 of CXCR4 agonist induction is expressed.
4. laurel diamine derivative according to claim 3 is short of money for inhibiting breast cancer cell CXCR4 to express in preparation
Application in anti-agent, which is characterized in that the CXCR4 agonist is SDF-1.
5. laurel diamine derivative according to claim 3 is short of money for inhibiting breast cancer cell CXCR4 to express in preparation
Application in anti-agent, which is characterized in that the laurel diamine derivative by specifically Competitive assays CXCR4 agonist with
The combination of CXCR4 inhibits CXCR4 to express.
6. laurel diamine derivative is preparing the application in the inhibitor for inhibiting breast cancer cell to shift, which is characterized in that
The laurel diamine derivative is selected from least one of Formulas I as claimed in claim 1, Formula II and formula III compound represented.
7. laurel diamine derivative according to claim 6 is preparing answering in the inhibitor for inhibiting cancer metastasis
With, which is characterized in that the breast cancer cell transfer includes the cancer metastasis of SDF-1 induction.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0472093A1 (en) * | 1990-08-22 | 1992-02-26 | BASF Aktiengesellschaft | Bis guanidines and fungicides containing same |
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2018
- 2018-09-05 CN CN201811034130.1A patent/CN109223776A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0472093A1 (en) * | 1990-08-22 | 1992-02-26 | BASF Aktiengesellschaft | Bis guanidines and fungicides containing same |
Non-Patent Citations (3)
| Title |
|---|
| CHRISTOPHE DARDONVILLE等: "Bisguanidine, Bis(2-aminoimidazoline), and Polyamine Derivatives as Potent and Selective Chemotherapeutic Agents against Trypanosoma brucei rhodesiense. Synthesis and in Vitro Evaluation", 《J. MED. CHEM.》 * |
| CHRISTOPHE DARDONVILLE等: "I2-Imidazoline Binding Site Affinity of a Structurally Different Type of Ligands", 《BIOORGANIC & MEDICINAL CHEMISTRY》 * |
| ROSANNA FILOSA等: "N, N′ (4,5-Dihydro-1H-imidazol-2-yl)3-aza-1,10-decanediamine and N, N′(4,5-dihydro-1H-imidazol-2-yl)3-aza-1,10-dodecane-diamine antagonize cell proliferation as selective ligands towards topoisomerase II", 《JOURNAL OF PHARMACY AND PHARMACOLOGY》 * |
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