CN103181918B - Application of fatty acid compound in preparation of medicines for preventing and treating liver cancer - Google Patents
Application of fatty acid compound in preparation of medicines for preventing and treating liver cancer Download PDFInfo
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Abstract
The invention relates to fatty acid compounds, and provides an application of a fatty acid compound in preparation of medicines for preventing and treating the liver cancer. Angelica is boiled by ethanol, flows back and then is filtered, and a filtrate is collected, so that an angelica extract is obtained; the obtained angelica extract is extracted, an obtained solution after the extraction is subjected to vacuum drying, and a crude angelica extract is obtained; and the crude angelica extract is subjected to column chromatography on silica gel and column chromatography on reverse phase silica gel, so that a compound 1, a compound 2 and a compound 3 are obtained respectively. The fatty acid compound can be prepared into a specific inhibitor of PTP-SHP2; through inhibiting PTP (protein tyrosine phosphatase), the anticancer function and the function of preventing and treating human diseases relevant to PTP-SHP are played; and the fatty acid compound can be widely applied to preparation of medicines for preventing and treating cancers.
Description
Technical field
The present invention relates to fatty acid compound, the particularly application of fatty acid compound in preparation prevention and Hepatoma therapy medicine.
Background technology
In recent years, be accompanied by the progress at full speed of pharmacy and life science, the various basic processes such as interaction of the signal transduction in malignant cell, apoptosis, angiogenesis and cell and extracellular matrix are just gradually illustrated.Using the key enzyme (as protein tyrosine kinase) of some intracellular signal transduction pathway relevant to tumor cell differentiation and proliferation as drug screening target spot; find that selectively acting is in efficient, the low toxicity of specific target site, the new antitumoral medicine of high specificity; be molecular targeted medicine (molecular targeted drugs) and antibody target medicine (antibody targeted drugs), become the research and development of current anticarcinogen important directions (1, Zhang Shige. the progress of neoplasm targeted therapy medicine and clinical evaluation. Chinese Hospitals medication is evaluated and is analyzed .2009; 9 (1): 4-7).
In the initial sum evolution of cancer, protein tyrosine phosphatase plays very important effect.And protein tyrosine phosphatase level depends on the balance between protein tyrosine kinase (PTKs) and Protein-tyrosine-phosphatase (PTPs).Protein tyrosine kinase (PTKs) is the important target spot of current generally acknowledged oncotherapy, plays very important effect in the treatment of cancer.Contrary with the effect of PTKs, PTPs can promote the growth of tumor.So; the discovery of PTPs inhibitor by the treatment of cancer is provided bright prospects (2, Scott LM; Chen LW; Daniel KG; Brooks WH; Guida WC, Lawrence HR, et al.Shp2protein tyrosine phosphatase inhibitor activity of estramustine phosphate and its triterpenoid analogs.Bioorg Med Chem Lett.2011; 21 (2): 730-733).
PTP-SHP2, is a kind of non-receptor protein tyrosine phosphatase, and it is by PTPN11 gene code, multiple organ wide expression in human body.2 SH2 domains that it is held by N, middle PTP domain and C-terminal form.In cancerous cell, SHP2 can be activated by some growth factor receptorses conventionally, and then regulates the phosphorylation of downstream albumen, Promote cell's growth.In the malignant tumor causing at proto-oncogene, PTP-SHP2 is activated conventionally.In a word, PTP-SHP2 brings into play positive regulating and controlling effect, Promote cell's growth in cell signal regulates.Along with the target treatment of cancer more and more obtains everybody approval, PTP-SHP2 also becomes the focus of everybody research.Nowadays, it has become the important target of the searching cancer therapy drug that people generally acknowledge.Some synthetic PTP-SHP2 specific inhibitors are also found; as phenylhydrazonopyrazolone sulfonate(3, Hellmuth K; Grosskopf S; Lum CT; Wurtele M; Roder N, von Kries JP, et al.Specific inhibitors of the protein tyrosine phosphatase Shp2identified by high-throughput docking.Proc Natl Acad Sci U S is A.2008; 105 (20): 7275-7280) and 8-hydroxy-7-(6-sulfonaphthalen-2-yl) diazenyl-quinoline-5-sulfonicacid (NSC-87877) (4, Chen L; Sung SS; Yip ML; Lawrence HR; Ren Y; Guida WC, et al.Discovery of a novel shp2protein tyrosine phosphatase inhibitor.Mol Pharmacol.2006; 70 (2): 562-570).China's Chinese medicine (TCM) is at the medicinal history of existing several thousand, and it has the various and complex structure double dominant of chemical composition, is the good source of finding PTP-SHP2 specific inhibitor, and this is that chemical synthetic drug cannot be equal to.
Caspases is relevant cysteine proteinase in one group of structure being present in cytosol, determine and at least have 11 kinds of caspase at present, wherein, caspase1 and caspase11, and may also have caspase4 to be considered to not participate in directly the transduction of apoptotic signal, they mainly participate in the activation of interleukin precursor; And caspase2, caspase8, caspase9 and caspase10 participate in apoptotic initial; That participate in apoptosis execution is caspase3, caspase6 and caspase7, wherein caspase3 and 7 has close substrate and inhibitor specificity, their PARP that degrades, DFF-45(DNA fragmentation factor-45), cause the inhibition that DNA repairs the degraded that starts DNA.Think at present apoptotic initiator (caspase2,8,9 and 10) and executor (caspase3; 6,7) between, exist upstream and downstream relation, initiator activation executor (5, Zhu is willing to; Ma Yong, the .Caspase of Chen Li army and apoptosis. People's Armed Police's medical college journal .2004; 13 (4): 346-348).
Caspase-3 plays irreplaceable effect in apoptosis, and topmost substrate is poly (ADP-ribose) polymerase PARP (poly (ADP-ribose) polymerase), and this enzyme is repaired with DNA, gene complete monitoring is relevant.When apoptosis starts, the PARP of 116kD is at Asp
216-Gly
217between by caspase-3, cut into 31kD and two fragments of 85kD, make two zinc fingerses of being combined with DNA in PARP separated with the catalysis region of c-terminus, the function of can not bringing into normal play.Result makes the Ca that affected by PARP negative regulation
2+/ Mg
2+the activity of dependency Cobra venom endonuclease increases, and the DNA between cracking nucleosome, causes apoptosis.Caspase-8 has FADD sample DED domain, and can be combined with FADD by DED domain, thus caspase-8 can contact in apoptotic process indirect and cell-membrane receptor, thereby cell membrane event is converted into cytoplasm event.Caspase-8 plays an important role in the apoptosis of death receptor mediation.And caspase-9 plays an important role to apoptosis in mitochondria pathway.
Summary of the invention
The object of the present invention is to provide the application of fatty acid compound in preparation prevention and Hepatoma therapy medicine.
Described fat acid compounds comprises:
Compound 1, compound 1 is grease, chemistry 10Z by name, 13Z-nonadecadienoic acid, molecular formula is C
19h
34o
2, molecular weight is 294, structural formula is:
Compound 2, compound 2 is grease, chemistry 8Z by name, 11Z-heptadecadienoic acid, molecular formula is C
17h
30o
2, molecular weight is 266, structural formula is:
Compound 3, compound 3 is grease, chemistry 14Z by name, 17Z-bis-undecandienoic acids, molecular formula is C
21h
38o
2, molecular weight is 322, structural formula is:
The preparation method of described fatty acid compound comprises the following steps:
1) use ethanol to carry out boiling reflux to the Radix Angelicae Dahuricae, filter, collect filtrate, obtain Radix Angelicae Dahuricae extracting solution;
2) gained Radix Angelicae Dahuricae extracting solution is extracted, extraction gained solution carries out vacuum drying, obtains Radix Angelicae Dahuricae crude extract;
3) Radix Angelicae Dahuricae crude extract is carried out to silica gel column chromatography, then carry out reversed-phase silica gel column chromatography, obtain respectively compound 1, compound 2, compound 3.
In step 1), the concentration of described ethanol is preferably 60%, and the ratio of described ethanol volume and Radix Angelicae Dahuricae quality is preferably 10L: 1kg, and wherein by volume, the Radix Angelicae Dahuricae is in mass for ethanol; The time of described boiling reflux can be 2～3h.
In step 2) in, the solvent of described extraction can be ethyl acetate etc., and the volume ratio of the solvent of described extraction and Radix Angelicae Dahuricae extracting solution is preferably 1: 1.
In step 3), the concrete steps of described silica gel column chromatography: Radix Angelicae Dahuricae crude extract is added to silica gel, the dry silica gel column chromatography sample that obtains; Silica gel column chromatography sample is placed in to silicagel column top, take volume ratio as 100: 0,99: 1,98: 2,95: 5,10: 1,5: 1, the chloroform-methanol solution of 0: 100 was eluant, carry out silicagel column gradient elution, the eluent of collecting chloroform-methanol volume ratio and be 99: 1 and 95: 5, evaporate to dryness, obtains Radix Angelicae Dahuricae column chromatography sample; Gained Radix Angelicae Dahuricae column chromatography sample obtains compound 1 through silica gel column chromatography and ODS column chromatography again, compound 2 and compound 3; The mobile phase of described reversed-phase silica gel column chromatography can be methanol-water or acetonitrile-aqueous solution etc.; The volume ratio of described methanol-water or acetonitrile-water can be (0.25～9): 1, be preferably (0.4～4): and 1, be preferably 1.85: 1.
Fatty acid compound of the present invention can be prepared into the specific inhibitor of PTP-SHP2, thereby brings into play the effect anticancer and human diseases that prevention is relevant to Protein-tyrosine-phosphatase PTP-SHP2 with treatment by Profilin tyrosine phosphatase.Described fatty acid compound has a wide range of applications in preparation prevention and treatment cancer drug.
Described medicine can be comprised of fatty acid compound of the present invention and pharmaceutically acceptable adjuvant.Medicine can be made a kind of dosage form in powder, tablet, capsule, pill, suppository, drop pill, enteric agents, injection, syrup, Emulsion, suspensoid, lozenge, unguentum, spray etc. according to conventional method.
Advantage of the present invention is as follows:
1) activity that 3 compounds in the present invention can specificity Profilin tyrosine phosphatase PTP-SHP2, can be used as the new inhibitor as Protein-tyrosine-phosphatase PTP-SHP2.
2) 3 compounds of the present invention are in the lower activity that gets final product remarkable Profilin tyrosine phosphatase PTP-SHP2 of concentration (17～45.2 μ mol/L), thereby affect its regulation and control to downstream albumen, illustrate these 3 compounds can anticancer propagation, have to become and take the potentiality of the new type anticancer medicine that PTP-SHP2 is target.
3) to the activity inhibition of PTP-SHP2, the increase with drug level strengthens 3 compounds of the present invention, within the scope of valid density, the activity of PTP-SHP2 is suppressed to show good dose-effect relationship.
4) 3 compounds of the present invention, except suppressing, the activity of PTP-SHP2, can also activate caspase by specificity, cause the cutting of PARP, promote the apoptosis of hepatoma carcinoma cell HepG2.
5) 3 compounds of the present invention can activate caspase8 and caspase9, and then cause that the increased activity of caspase3, useful effect concentration are 100 μ M, and be 10h effective acting time.。
6) 3 compounds of the present invention can cause the cutting of PARP, and present concentration and time gradient.Useful effect concentration is 50 μ M, and be 12h effective acting time.。
7) 3 compounds of the present invention is remarkable to growth of cancer cells inhibition, impels the death of hepatoma carcinoma cell, and effect obviously.
Accompanying drawing explanation
Fig. 1 is the specific inhibitory effect figure of 1 couple of PTP-SHP2 of compound.In Fig. 1, abscissa is compound 1 concentration, the activity that vertical coordinate is Protein-tyrosine-phosphatase, wherein ◆ represent PTP-SHP2, ▲ representing VHR, ■ represents HePTP, take is not 100% with the protein active of compound treatment.
Fig. 2 is the specific inhibitory effect figure of 2 couples of PTP-SHP2 of compound.In Fig. 2, abscissa is compound 2 concentration, the activity that vertical coordinate is Protein-tyrosine-phosphatase, wherein ◆ represent PTP-SHP2, ▲ representing VHR, ■ represents HePTP, take is not 100% with the protein active of compound treatment.
Fig. 3 is the specific inhibitory effect figure of 3 couples of PTP-SHP2 of compound.In Fig. 3, abscissa is compound 3 concentration, the activity that vertical coordinate is Protein-tyrosine-phosphatase, wherein ◆ represent PTP-SHP2, ▲ representing VHR, ■ represents HePTP, take is not 100% with the protein active of compound treatment.
Fig. 4 is the dissection figure of 3 couples of PARP of compound under variable concentrations.In Fig. 4,12.5,25,50 and 100 are respectively the concentration (μ M) of compound 3, and in contrast, compound 3 has caused the cutting of PARP in HepG2 to blank, and valid density is 50～100 μ M, and dose-effect relationship is good.
Fig. 5 is the dissection figure of 3 couples of PARP of compound under different time.In Fig. 5,3h, 6h and 12h are respectively the time that compound 3 acts on HepG2, and in contrast, compound 3 causes that be 12h the effective acting time of PARP cutting in HepG2 to blank.
Fig. 6 is 3 couples of caspase-3 of compound under variable concentrations, caspase-8, the activity influence figure of caspase-9.In Fig. 6, abscissa is drug level (μ M), and vertical coordinate is increased activity multiple.Wherein a is caspase-3, and b is caspase-8, and c is caspase-9; With compound 3, process HepG2 cell, concentration 0,50 and 100 μ M, the processing time is 10h; * P<0.05, shows to compare with blank group, and the activity of 100 μ M concentration group caspase-3 and caspase-9 obviously strengthens, significant difference.
The specific embodiment
The preparation of embodiment 1 fatty acid compound
1, prepare Radix Angelicae Dahuricae ethanol extract
1) take the dry Radix Angelicae Dahuricae root of 3kg, add 30L60% ethanol, stir, in extraction pot, after boiling reflux 2h, filter, collect filtrate;
2) filtering residue extracts 2 times with 60% ethanol boiling reflux again, filters, and collects filtrate, and 60% ethanol consumption is 30L/ time;
3) merge the filtrate of 3 times, by ethyl acetate, extract, each Solvent quantity is 30L, collects upper layer of extraction liquid;
4) underclad portion extracts 2 times by ethyl acetate again, merges 3 times upper layer of extraction liquid, and vacuum drying obtains 48.3g Radix Angelicae Dahuricae ethanol extract, and (numbering is designated as: AD).
2, silica gel column chromatography, ODS column chromatography, prepare monomeric compound
1) with chloroform, soak 200～300 order silica gel, stirring and evenly mixing, dress post, standby;
2) in 48.3g Radix Angelicae Dahuricae ethanol extract, add chloroform-methanol mixed solution, ultrasonicly make its dissolving; With 60～100 order silica gel mixed samples, grind evenly, dry, obtain silica gel column chromatography sample;
3) above-mentioned silica gel column chromatography sample is evenly contained in to silicagel column top, by chloroform-methanol system (100: 0,99: 1,98: 2,95: 5,10: 1,5: 1,0: 100), carries out gradient elution, each gradient elution 3600ml.Wherein, adsorbent silica gel is 15: 1 with the ratio of the weight of Radix Angelicae Dahuricae ethanol extract AD, and the column diameter of silicagel column is 1: 10 with the ratio of post height; According to TLC, analyze to merge and obtain 11 fractions (AD-A1, AD-A2, AD-B, AD-C1, AD-C2, AD-C3, AD-D, AD-E1, AD-E2, AD-F, AD-G);
4) fraction A D-A2 is carried out to silica gel column chromatography, mobile phase is cyclohexane extraction-ethyl acetate system (98: 2,19: 1,10: 1,5: 1,1: 1), each gradient elution 1500ml.Wherein, adsorbent silica gel is 20: 1 with the ratio of the weight of fraction A D-A2, and the column diameter of silicagel column is 1: 15 with the ratio of post height; According to TLC, analyze to merge and obtain 12 fractions (AD-A2-A, AD-A2-B, AD-A2-C, AD-A2-D, AD-A2-E, AD-A2-F, AD-A2-G, AD-A2-H, AD-A2-I, AD-A2-J, AD-A2-K, AD-A2-L).
Fraction A D-A2-E is carried out to ODS column chromatography, and mobile phase is methanol-water system (1: 1,13: 7,4: 1,19: 1,100: 0), each gradient elution 200ml.Wherein, adsorbent ODS is 25: 1 with the ratio of the weight of fraction A D-A2-E, and the column diameter of silicagel column is 1: 16 with the ratio of post height.According to ODS point plate analysis, merge and obtain monomeric compound 1(120mg).
Through Mass Spectrometric Identification, compound 1 is 10Z, 13Z-nonadecadienoic acid, and molecular formula is C
19h
34o
2, molecular weight is 294, grease.ESI-MS,m/z:295[M+H]
+,317[M+Na]
+,333[M+K]
+。
1h-NMR (400MHz, CDCl
3) δ: 5.44 (4H, m, H-10,11,13,14), 2.81 (2H, t, J=6.4Hz, H-12), 2.39 (2H, t, J=7.6Hz, H-2), 2.10 (4H, t, J=6.8Hz, H-9,15), 1.69 (2H, t, J=6.8Hz, H-3), 1.28～1.34 (16H, m, H-4～8,16,17,18), 0.92 (3H, t, J=6.8Hz, H-19).
13c NMR (101MHz, CDCl
3) δ: 180.1 (C-1), 130.1 (C-10), 130.0 (C-14), 128.1 (C-11), 127.9 (C-13), 34.1 (C-2), 31.8 (C-17), 29.6 (C-12), 29.3 (C-20), 29.0～29.6 (C-4～8,16), 27.2 (C-9,15), 25.6 (C-12), 24.6 (C-3), 22.6 (C-18), 14.0 (C-19). the known compound data consistent of reporting in above data and document, is defined as same compound.
Fraction A D-A2-F is carried out to ODS column chromatography, and mobile phase is methanol-water system (1: 1,3: 2,7: 3,17: 1,100: 0), each gradient elution 200ml.Wherein, adsorbent ODS is 23: 1 with the ratio of the weight of fraction A D-A2-F, and the column diameter of silicagel column is 1: 16 with the ratio of post height.According to ODS point plate analysis, merge and obtain monomeric compound 2(258mg).
Through Mass Spectrometric Identification, compound 2 is 8Z, 11Z-heptadecadienoic acid, and molecular formula is C
17h
30o
2, molecular weight is 266, grease.ESI-MS,m/z:265[M-H]
-。
1h-NMR (400MHz, CDCl
3) δ: 5.42 (4H, m, H-8,9,11,12), 2.80 (2H, t, J=6.4Hz, H-10), 2.37 (2H, t, J=7.6Hz, H-2), 2.09 (4H, t, J=6.8Hz, H-7,13), 1.64 (2H, t, J=6.8Hz, H-3), 1.27～1.32 (12H, m, H-4～6,14,15,16), 0.92 (3H, t, J=6.8Hz, H-23).
13c NMR (101MHz, CDCl
3) δ: 179.5 (C-1), 130.2 (C-9), 130.0 (C-12), 128.0 (C-8), 127.9 (C-11), 34.0 (C-2), 31.5 (C-15), 28.9～29.6 (C-4～6,14), 27.2 (C-7,13), 25.6 (C-10), 24.7 (C-3), 22.6 (C-16), 14.0 (C-17). the known compound data consistent of reporting in above data and document, is defined as same compound.
Fraction A D-A2-G is carried out to ODS column chromatography, and mobile phase is methanol-water system (2: 3,3: 2,4: 1,100: 0), each gradient elution 200ml.Wherein, adsorbent ODS is 15: 1 with the ratio of the weight of fraction A D-A2-F, and the column diameter of silicagel column is 1: 16 with the ratio of post height.According to ODS point plate analysis, merge and obtain monomeric compound 3(266mg).
Through Mass Spectrometric Identification compound 3, be 14Z, 17Z-bis-undecandienoic acids, molecular formula is C
21h
38o
2, molecular weight is 322, grease.ESI-MS,m/z:351[M+H]
+。
1h-NMR (400MHz, CDCl
3) δ: 5.43 (4H, m, H-14,15,17,18), 2.80 (2H, t, J=6.4Hz, H-16), 2.38 (2H, t, J=7.6Hz, H-2), 2.09 (4H, t, J=6.8Hz, H-13,19), 1.66 (2H, t, J=6.8Hz, H-3), 1.27～1.33 (24H, m, H-4～12,20,21,22), 0.92 (3H, t, J=6.8Hz, H-23).
13cNMR (101MHz, CDCl
3) δ: 179.9 (C-1), 130.2 (C-14), 130.0 (C-18), 128.0 (C-15), 127.9 (C-17), 34.1 (C-2), 31.5 (C-21), 29.0～29.6 (C-4～12,20), 27.2 (C-13,19), 25.6 (C-16), 24.7 (C-3), 22.6 (C-22), 14.0 (C-23). the known compound data consistent of reporting in above data and document, is defined as same compound.
Embodiment 2 fatty acid compound pharmacodynamic experiments
1. experiment material and reagent
(1) experimental cell: human liver cancer cell HepG2
(2) bacterial strain
pGEX4T1-PTP-SHP2 E.coli BL21
pGEX4T1-VHR E.coli BL21
pGEX4T1-HEPTP E.coli BL21
(3) Radix angelicae dahuricae is produced in medical material Zhejiang
(4) main agents
(5) culture medium
LB fluid medium:
1%（g/ml）Tryptone，0.5%（g/ml）Yeast Extract，1%（g/ml）NaCl
(6) the required buffer of GST purifying protein and reagent
PBS Bμffer：137mM NaCl,2.7mM KCl,10mM Na
2HPO
4,2mM KH
2PO
4
100mg/ml ampicillin
Protease inhibitor and inhibitors of phosphatases stock solution:
IPTG stock solution (0.8M)
Buffer W:25mM Tris-HCl pH7.5,150mM NaCl, 10mM beta-mercaptoethanol, 1mM EDTA
Buffer W1:25mM Tris-HCl pH7.5,150mM NaCl, Trion X-1001%, 10mM beta-mercaptoethanol
Buffer W2:50mM Tris-HCl pH8.0,150mM NaCl, 10mM beta-mercaptoethanol
Buffer E:50mM Tris-HCl pH8.0,10mM glutathion
(7) drug screening reactant liquor:
50mM Bis-Tris，2mM EDTA，50mM NaCl，2mM DTT pH6.5，1M NaOH，1M DTT
(8) Western blot and other experiments are used with solution preparation:
PBS buffer: 137mM NaCl, 2.7mM KCl, 10mM Na
2hPO
4, 2mM KH
2pO
4with hydrochloric acid, adjust pH to 7.4.
Giemsa dye liquor:
Jim Sa powder (Giemsa stain) l.0g
Glycerol (AR) 66ml
Methanol (AR) 66ml
Giemsa powder is put into mortar, first add a small amount of glycerol, be ground to without till granule, and then whole glycerol is poured into, put in 56 ℃ of incubators after 2h, add methanol, the dye liquor sealing preparing is preserved in brown bottle (being better than 0～4 ℃ of preservation most).
RIPA cell pyrolysis liquid: 50mM Tris-HCl(pH7.4); 150mM NaCl; 1mM EDTA; 1%Triton; 1% NaTDC; 0.1%SDS; 1mM PMSF; 5 μ g/ml Aprotenin; 5 μ g/ml Leupeptasin.
2. experimental technique and result
(1) specific inhibitory effect of fatty acid compound to Protein-tyrosine-phosphatase shp2
The high flux model discrimination of external protein tyrosine phosphatase inhibitor is: SHP2 is hydrolyzed p-nitrophenyl phosphoric acid (pNPP), generate the yellow compound of solubility, it has light absorption at 405nm place, so, by detecting the light absorption of hydrolysis substrate, can measure the activity of Protein-tyrosine-phosphatase.Based on this mechanism, applicant has set up the inhibitor high flux screening model of external Protein-tyrosine-phosphatase, for finding the novel specific inhibitor of natural product shp2.In order to observe the selectivity of compound to Protein-tyrosine-phosphatase, applicant has also carried out determination of activity to VHR and HePTP.Concrete operating procedure is as follows:
The activation of a.GST4B
1) from taking out GST4B in four degree refrigerators, jiggle evenly, getting 1.33ml mixed liquor, to put into 2ml centrifuge tube centrifugal, 1500rpm, 5min; Carefully remove supernatant.
2) with 10mlPBS washing GST4B pearl, centrifugal, 1500rpm, 5min; Remove supernatant.
3) repeating step 2).
4) the former GST4B of every 1.33ml add 1mlPBS shake up 4 ℃ standby.
B. the mensuration of Protein Separation and purification experimental technique and protein concentration
1) from culture dish picking monoclonal colony lift to 50ml LB(50mg/ml AMP) triangular flask in 37 ℃, incubated overnight in 250rpm shaking table.
2) from above-mentioned solution, take out 10ml bacterium liquid and transfer to that another is new for 200ml LB(50mg/ml AMP) triangular flask in 37 ℃, in 250rpm shaking table to bacterium liquid OD
600value approximately reaches 0.8.
3) treat that bacterium liquid is cooled to room temperature and adds IPTG derivant, make it final concentration and reach 0.1mM, in 25 ℃, in 225rpm shaking table, cultivate 16h.
4) collect bacterium liquid, centrifugal, 6000g, 4 ℃, 10min; Remove supernatant.
5) add the resuspended bacterium liquid precipitate of 10ml Buffer W, multigelation 3 times, each 15-30min.
6) measure and adjust bacterium liquid pH to 8.0 after add lysozyme to final concentration 1mg/ml, the standing 20min of room temperature.
7) excusing from death is on ice broken 60 times, each 8s.Add EDTA to final concentration 20mM, Triton X-100 is to content 0.5%, ice bath 20min.
8) centrifugal, 20000g, 4 ℃, 30min.Get in the new 50ml centrifuge tube of supernatant to, add activated good glutathione-Sepharose4B, the vertical 1h that mixes in 4 ℃ of refrigerators.
9) centrifugal above-mentioned mixed liquor, collects beads, uses respectively 8ml BufferW14 ℃ of vertical mixing, washing 4 times, each 10min; Centrifugal, 1000rpm1min; Collect beads; 8ml BufferW24 ℃ of vertical mixing, washing 1 time, each 10min.Centrifugal, 1,000rpm1min; Collect beads.
10) in centrifuge tube, add E4 ℃ of vertical mixing, washing of 1ml Buffer 3 times, each 30min; Centrifugal, 1000rpm1min; Collect supernatant stores to new centrifuge tube.
11) after Coomassie brilliant blue method is measured the protein concentration extracting, subpackage is stored in-70 ℃ of refrigerators, standby.
C. Purity
The destination protein that purification obtains is identified its size and purity by SDS-PAGE.
D. inhibitor screening experimental technique
1) high flux screening model:
25 μ l protein samples, (containing DTT final=1mM)
45 μ l reactant liquors (50mM Bis-Tris, 50mM NaCl, 2mM EDTA PH6.9)
10 μ l100mM compounds (compound final=10mM, DMSO final=1%)
20μl7.5mM p-NPP（final concentration=1.5mM）
Concrete steps are: testing compound and albumen react under room temperature 30 minutes, then add p-NPP after mixing, and mix, and react 60 minutes under room temperature, finally add the NaOH cessation reaction of 100 μ l1M, microplate reader, 405nm place reading.
2) mensuration of specificity effect
The inhibiting compound that has sifting out is measured the specificity inhibition of four kinds of PTPs albumen: take p-NPP as substrate, reaction system is 50mM Bis-Tris, 2mM EDTA, 50mM NaCl, 2mM DTT pH6.5 buffer, the consumption of four kinds of albumen is identical, YimolWei unit, drug level dilutes by gradient, 405nm reading.Finally make the inhibition curve (referring to Fig. 1～3) of medicine to 3 kinds of albumen.3 compounds are all inhibited to PTP-SHP2, and its IC50 is 17.2～45.2 μ M.
3) half-inhibition concentration (IC
50) mensuration
IC
50, half-inhibition concentration, is to make enzymatic activity be down to the inhibitor concentration of a former active half.Can be used for doing the judgment criteria that inhibitor suppresses capacity of water.With the inhibitor of variable concentrations and the enzyme-substrate reactions system under the same terms, interact, the reaction system contrast with inhibiting not by the recruitment of product within the unit interval, the curve of agent to the inhibition ability of enzyme that can be inhibited, selects suitable inhibitor concentration, just can obtain IC
50numerical value.
The selective inhibitory of table 1 fatty acid compound to PTP-SHP2
Experimental result shows that (referring to table 1) 3 compounds are all inhibited to PTP-SHP2, its IC
50be 17.2～45.2 μ M.But under same concentrations, the inhibitory action of PTP-SHP2 is obviously greater than to the inhibition to VHR and HePTP.This explanation, these 3 compounds have specificity to the inhibition of PTP-SHP2.
(2) compound 3 cuts liver cancer apoptosis reducing by PARP
Adopt Western blotting (Western Blotting) method to carry out protein level analysis, Western blotting (western blot), it is according to the method for certain albumen in the specific binding detection of complex sample of antigen-antibody.Western blot is a kind of new immune biochemical technology growing up in gel electrophoresis and solid-phase immunoassay technical foundation.Because immunoblotting (western blot) has the high resolution of SDS-PAGE and the high specific of solid-phase immunoassay and sensitivity, now become a kind of routine techniques of analysis of protein.Immunoblotting (western blot) is usually used in identifying certain albumen, and can carry out quantitative and semi-quantitative analysis to albumen.Concrete operating procedure is as follows:
A. cell culture
1) HepG2 human liver cancer cell is hatched in containing 10% hyclone, 100 Μ/ml penicillin, in the DMEM culture medium of 100 Μ/ml streptomycin, at 37 ℃, 5%CO
2in incubator, cultivate.
2) suck culture fluid, PBS washes 1 time, adds appropriate trypsinization liquid, standing 2-3min in incubator; Under microscope, observation of cell becomes bowlder, stops the Digestion of pancreatin by the DMEM culture medium containing serum, blows and beats Tissue Culture Dish, and cell suspension is sucked in 15ml centrifuge tube to centrifugal 800rpm, 2min with pipettor; Suck after supernatant, add containing 10% hyclone DMEM culture fluid, with liquid-transfering gun, blow out single cell suspension, HepG2 single cell suspension is divided and reaches 6 orifice plates, be put in 37 ℃ of incubators and cultivate.
B. cell is processed
Cell grow to account for greatly orifice plate floor space 80% time, suck culture fluid, add the DMEM culture medium that contains serum-free that 2ml is fresh, add respectively compound 3 or DMSO (blank group), mix, making its final concentration is 0,12.5,25,50,100 μ M/L cultivate 12h in incubator; Or final concentration is 100 μ M/L, in incubator, cultivate respectively 3h, 6h, 12h.
C. cell harvesting:
Suck the cell culture fluid containing medicine, with the PBS of 4 ℃ of pre-coolings, wash 2 times, every hole adds 1ml PBS, scrapes cell is scraped, and collect in 1.5ml centrifuge tube with cell.Centrifugal 4 ℃, 12000rpm, 25s.
D. lysis:
Remove supernatant, in each centrifuge tube, add 40 μ L RIPA cell pyrolysis liquids, cracking 30min on ice, therebetween, on agitator every 5min vibration 10s, then centrifugal 4 ℃, 12000rpm, 10min.Collect supernatant, obtain protein sample.
E. according to the conventional determination of protein concentration method of laboratory, measure the concentration of protein sample:
F.SDS-PAGE electrophoresis:
1) clean glass plate: a hands fastening glass plate, another hands dips in a liquid detergent and cleans gently.Two sides is all cleaned later and is rushed with tap water, then stands in basket and dry after clean with distilled water flushing.
2) encapsulating and loading:
A. after glass plate alignment, putting into folder clamps.Then vertical card is prepared encapsulating on the top of the shelf.
B. join 8% separation gel, add after TEMED and shake up immediately and get final product encapsulating.During encapsulating, available 10ml rifle is drawn 5ml glue and is emitted along glass, when glue face is raised to greenbelt intermediate line height.Then on glue, add one deck dehydrated alcohol, the gelling after fluid-tight faster.
C.15, after～20min, when having a fringence between ethanol and glue, illustrate that glue is solidifying.Wait again 3min that glue is fully solidified just can to remove photoresist upper strata ethanol and with filter paper, ethanol blotted.
D. by previous methods, join 4% concentrated glue, add after TEMED and shake up immediately and get final product encapsulating.Then remaining space is filled to concentrated glue inserts comb in concentrated glue.During encapsulating, also to make glue flow down in order to avoid in glue, have Bubble formation along glass plate.While inserting comb, to make comb maintenance level.While consolidating due to gelling, volume can shrink and reduce, thereby the loading volume of well is reduced, so will mend glue through the both sides of being everlasting in concentrating the solid process of gelling.Until after concentrated gelling is solid, two handss pinch respectively the both sides of comb and are extracted gently straight up.
E. water rinses concentrated glue, puts it in electrophoresis tank.
F. surveyed after protein content, the liquor capacity calculating containing 40 μ g albumen is applied sample amount.
The loading sample preparing is boiled to 10min in 110 ℃.Put-20 ℃ of preservations.
G. after adding enough electrophoresis liquid, start to prepare loading.With the adherent absorption sample of microsyringe, sample sucking-off is not wanted to inspiration bubble.Sample injector syringe needle is inserted to and in well, slowly adds sample.
3) electrophoresis:
Beginning is 80V when concentrated glue, after albumen Marker has separation, voltage is enlarged to 120V, and electrophoresis has just been run out of and can have been stopped electrophoresis to bromjophenol blue.
4) transferring film:
A. must wear glove when filter paper and film, because albumen on hand can polluted membrane.Before transferring film, pvdf membrane is dipped in to 10s in methanol, with tweezers, takes out, be placed on electricity and turn in liquid and wash several times.2 part of 5～6 metafiltration paper, 1 part of monolayer filter paper are soaked in to electricity and turn in liquid, stand-by.
B. clip is opened to the black one side maintenance level that makes.Pad a foam-rubber cushion in the above, with glass rod, roll back and forth several all over the bubble with inside rolling away.On mat, pad 5 metafiltration paper, fixedly filter paper is rolled bubble wherein with glass rod on the other hand on the other hand.
C. careful sled removes glue glass outer plate, and monolayer filter paper is laid in to Jiao Mianshang, removes gently an other glass plate, glue is lain on multi-layer filter paper, one side with the pvdf membrane just now soaking from glue face start slowly cover, note not having bubble.Then with a dropper soaking, from one side of film, start to catch up with bubble.Again the multi-layer filter paper having soaked is lain in above film, finally by one deck black sponge cover, roll several times and just can close clip.
D. clip is put into transfer groove groove, be made the black flour of the black flour of clip to groove, to groove red of the flour of folder.Pour about 1L transferring film liquid into, then this transferring film groove is placed in the basin of a filled with ice to 100v, 90min.
5) immunoreation:
A. sealing: by the room temperature sealing 1h in the TBST buffer of the skim milk containing 5% of the pvdf membrane after electrotransfer;
B. primary antibodie reaction: the pvdf membrane after sealing is encapsulated in hybridization bag, adds Tu Kang anti-PARP(Sigma company, concentration 1:1000), on 4 ℃ of shaking tables, spend the night;
C. two anti-reactions: pvdf membrane is shaken and washes 3 times in TBST buffer, each 5min.Add IgG (H+L) (goat antirabbit two is anti-, concentration 1:10000, Sigma company), incubated at room 1h.
6) chemiluminescence, develops, photographic fixing
Remove two and resist, with TBST buffer, wash film, room temperature is shaken and is washed three times, each 5min, and protein powder, is placed in preservative film upward, the A liquid of ECL chemiluminescence detection kit and B liquid is mixed with 1:1 (V/V), in darkroom, by 0.05-0.1mL/cm
2minim be added on film surface, hatch 1min, with filter paper, suck unnecessary liquid, seal after film, in darkroom, expose immediately, develop.
Experimental result shows: compound 3 is processed after HepG2 human liver cancer cell 12h, has caused the cutting of PARP; And under 100 μ mol/L concentration, when PARP cutting degree is obviously greater than 50 μ mol/L.So compound 3 can be induced the cutting of PARP in HepG2, and present good dose-effect relationship (referring to Fig. 4).Final concentration is that the compound 3 of 100 μ mol/L is processed after HepG2 human liver cancer cell, and during 6h, PARP cutting is also not obvious; And when 12h, show obvious PARP dissection.This explanation, in 3 couples of hepatoma carcinoma cell HepG2 of compound, the effect of PARP has time dependence (referring to Fig. 5).Therefore, compound 3 can cut approach cell death inducing by PARP, and has good concentration and time dependence, possesses good cancer therapy drug potentiality to be exploited.
(3) 3 couples of caspase-3 of compound, caspase-8, the activation of caspase-9
Caspases is relevant cysteine proteinase in one group of structure being present in cytosol of discovered in recent years, and in cell, synthetic caspase exists with the proenzyme state of non-activity, activatedly can carry out its function.Caspase8, caspase9 participates in apoptotic initial; Caspase3 participates in apoptosis and carries out.These 3 kinds of albumen have very important effect in apoptosis process, are also therefore the important indicators that apoptosis detects.
Caspase3/CPP32 can shear procaspase-2, procaspase-6, procaspase-7 and procaspase-9, PARP, gelsolin etc.Substrate DEVD-pNA is the coupling of caspase-3 sequence-specific polypeptide (acetyl-Asp-Glu-Val-Asp) and pNA (being p-nitroaniline), and after the caspase-3 shearing that substrate is activated, yellow group pNA is free out.PNA has obtained the maximum absorption at 405nm place, by detecting pNA, indirectly investigates the activity of caspase-3.The substrate of Caspase-8 and Caspase-9 is respectively IETD-pNA or LEHD-pNA, the same Caspase-3 of its active measuring principle.Concrete operation step is as follows:
1) cell culture
A. HepG2 human liver cancer cell is hatched in containing 10% hyclone, 100 Μ/ml penicillin, in the DMEM culture medium of 100 Μ/ml streptomycin, at 37 ℃, 5%CO
2in incubator, cultivate.
B. time, suck culture fluid, PBS washes 1 time, adds appropriate trypsinization liquid, standing 2-3min in incubator; Under microscope, observation of cell becomes bowlder, stops the Digestion of pancreatin by the DMEM culture medium containing serum, blows and beats Tissue Culture Dish, and cell suspension is sucked in 15mL centrifuge tube to centrifugal 800rpm, 2min with pipettor; Suck after supernatant, add containing 10% hyclone DMEM culture fluid, with liquid-transfering gun, blow out single cell suspension, above-mentioned HepG2 single cell suspension is divided and reaches the Tissue Culture Dish that diameter is 10cm, be put in incubator and cultivate.
2) cell is processed
Until cell grow to account for greatly orifice plate floor space 80% time, suck culture fluid, add the DMEM culture medium of the serum-free that 10ml is fresh, add respectively compound 3 or DMSO (blank group), mix, making its final concentration is 0,50,100 μ M/L cultivate 10h in incubator.
3) cell counting:
Suck the cell culture fluid containing medicine, with PBS, wash 2 times, by trypsinization (the same step 1) of digestion method.With cell counting count board counting, get 1-5 * 10
6individual cell in 1.5ml Ep pipe, centrifugal (4 ℃, 12000rpm, 25s), cell precipitation is for experiment below.
4) lysis:
To adding 50 μ L Cell Lysis Buffer (Caspase-3 ,-8, in-9Colorimetric Assay Kits) in each Ep pipe, vortex to be to mix, on ice cracking 10min.Centrifugal 4 ℃, 10000g, 1min.Collect supernatant in a new Ep pipe, be placed on ice (or put-80 ℃ standby).
5) press the conventional determination of protein concentration kit measurement of laboratory protein concentration:
6) get 50～200 μ g albumen in 96 orifice plates, with Cell Lysis Buffer, be diluted to 50 μ l.
7) add 50 μ l2 * Reaction Buffer (containing 10mM DTT), mix.
8) add 5 μ l4mM DEVD-pNA (substrate of caspase-3) or IETD-pNA (substrate of caspase-8) or LEHD-pNA (substrate of caspase-9), making its final concentration is 200 μ M, mixes, and puts 37 ℃ and hatches 1～2h.
9) by microplate reader, 400 or 405nm place, read OD value.
Compound 3 is processed after HepG2 human liver cancer cell 10h, and during 100 μ mol/L concentration, the activity of caspase-8 and caspase-9 has all strengthened, and caspase-9 enhancing degree is obviously greater than caspase-8; The enhancing of caspase-8 and caspase-9 has caused the activation (referring to Fig. 6) of caspase-3.When 50 μ mol/L, the activity of caspase is without obvious enhancing.This explanation, the activation of the dependent apoptosis caspase of compound 3 induction, and be mainly that mitochondria pathway by caspase-9 place plays a role.This experiment is clear and definite, and this compounds is brought into play the effect path of antitumor action by apoptosis.
(4) compound 3 suppresses clone's formation of hepatoma carcinoma cell
By Giemsa, dye and measure the growth inhibited effect of 3 pairs of HepG2 cells of compound.Giemsa dye liquor is comprised of reddish black and Yihong.The dyeing theory of Giemsa: the adsorption of existing physics, has again chemical affinity interaction.Various cell component chemical property are different, also different to the affinity of various dyestuffs.In nucleus, chromosome is acid, easily by dye liquor, is dyed blueness, and Cytoplasm dyes more shallow.Size that can observation of cell cluster with Giemsa staining and how many, thus the growth inhibited effect of compound to cancerous cell reflected.Concrete operation step is as follows:
1) cell culture
A. HepG2 human liver cancer cell is hatched in containing 10% hyclone, 100 Μ/ml penicillin, in the DMEM culture medium of 100 Μ/ml streptomycin, at 37 ℃, 5%CO
2in incubator, cultivate.
B. suck culture fluid, PBS washes 1 time, adds appropriate trypsinization liquid, standing 2-3min in incubator; Under microscope, observation of cell becomes bowlder, stops the Digestion of pancreatin by the DMEM culture medium containing serum, blows and beats Tissue Culture Dish, and cell suspension is sucked in 15mL centrifuge tube to centrifugal 800rpm, 2min with pipettor; Suck after supernatant, add containing 10% hyclone DMEM culture fluid, with liquid-transfering gun, blow out single cell suspension.HepG2 single cell suspension is divided and reaches 6 orifice plates, approximately 1 * 10
3/ hole.Be put in incubator and cultivate.
2) cell is processed
After 4～5 days, when cell cluster forms, suck culture fluid, add the DMEM culture medium that contains serum-free that 2ml is fresh, add respectively compound 3 or DMSO (blank group), mix, making its final concentration is 0,25,50 μ M/L cultivate 20h in incubator.
3) cell is fixed
Suck culture fluid, with PBS, wash 1 time, then use fixedly 15min of 4% paraformaldehyde (PFA).
4) Giemsa dyeing
Discard 4% paraformaldehyde, with PBS, wash 3 times.Add Giemsa dyeing 10min.
5) image acquisition
Discard Giemsa dyeing 10min, with PBS, wash 1 time.Canon collected by camera image.
Compound 3 can suppress the growth of HepG2 cell.Under 25 μ M concentration, 3 pairs of HepG2 cells of compound have certain growth inhibited effect; Under 50 μ M, this inhibitory action is very obvious.This explanation, in effective concentration range, the growth inhibited of 3 pairs of HepG2 cells of compound presents dose-effect relationship.This results suggest, this compounds that the compound 3 of take is representative significantly anticancer growth reaches antitumaous effect.
Embodiment 314Z, the preparation of 17Z-bis-undecandienoic acid tablets
Get 14Z, 17Z-bis-undecandienoic acid 1g, microcrystalline Cellulose 27g mix with magnesium stearate 2g, and it is 6mm that mixture is pressed into diameter with single punch tablet machine, the sheet that weight is 300mg, and in this tablet, every contains 14Z, 17Z-bis-undecandienoic acid 10mg.
Embodiment 4 prepares granule
Get 14Z, 17Z-bis-undecandienoic acid 1g mix with corn starch 29g, add water and make soft material, cross 12 mesh sieves and carry out pelletize, after being dried, obtain granule.In granule prepared by the present embodiment, in every 300mg, contain 14Z, 17Z-bis-undecandienoic acid 10mg.
Embodiment 5 prepares capsule
Get 14Z, 17Z-bis-undecandienoic acid 1g mix with lactose 27g, magnesium stearate 2g, with every 300mg, fill enteric coated capsule.In capsule prepared by the present embodiment, each capsule is containing 14Z, 17Z-bis-undecandienoic acid 10mg.
Claims (11)
1. the application of fatty acid compound in preparation prevention and Hepatoma therapy medicine, is characterized in that described fatty acid compound is:
Compound 1, compound 1 is grease, chemistry 10Z by name, 13Z-nonadecadienoic acid, molecular formula is C
19h
34o
2, molecular weight is 294, structural formula is:
Compound 2, compound 2 is grease, chemistry 8Z by name, 11Z-heptadecadienoic acid, molecular formula is C
17h
30o
2, molecular weight is 266, structural formula is:
2. the application of fatty acid compound as claimed in claim 1 in preparation prevention and Hepatoma therapy medicine, is characterized in that comprising the following steps the preparation method of described fatty acid compound:
1) use ethanol to carry out boiling reflux to the Radix Angelicae Dahuricae, filter, collect filtrate, obtain Radix Angelicae Dahuricae extracting solution;
2) gained Radix Angelicae Dahuricae extracting solution is extracted, extraction gained solution carries out vacuum drying, obtains Radix Angelicae Dahuricae crude extract;
3) Radix Angelicae Dahuricae crude extract is carried out to silica gel column chromatography, then carry out reversed-phase silica gel column chromatography, obtain respectively compound 1, compound 2.
3. the application of fatty acid compound as claimed in claim 2 in preparation prevention and Hepatoma therapy medicine, it is characterized in that in step 1) in, the concentration of described ethanol is 60%, the ratio of described ethanol volume and Radix Angelicae Dahuricae quality is 10L: 1kg, wherein by volume, the Radix Angelicae Dahuricae in mass for ethanol.
4. the application of fatty acid compound as claimed in claim 2 in preparation prevention and Hepatoma therapy medicine, is characterized in that in step 1) in, the time of described boiling reflux is 2～3h.
5. the application of fatty acid compound as claimed in claim 2 in preparation prevention and Hepatoma therapy medicine, is characterized in that in step 2) in, the solvent of described extraction is ethyl acetate.
6. the application of fatty acid compound as claimed in claim 5 in preparation prevention and Hepatoma therapy medicine, is characterized in that the ratio of the solvent of described extraction and the volume of Radix Angelicae Dahuricae extracting solution is 1: 1.
7. the application of fatty acid compound as claimed in claim 2 in preparation prevention and Hepatoma therapy medicine, is characterized in that in step 3) in, the concrete steps of described silica gel column chromatography: by Radix Angelicae Dahuricae crude extract, add silica gel, the dry silica gel column chromatography sample that obtains; Silica gel column chromatography sample is placed in to silicagel column top, the ratio of volume of take is 100: 0,99: 1, and 98: 2,95: 5,10: 1,5: 1, the chloroform-methanol solution of 0: 100 was eluant, carry out silicagel column gradient elution, the eluent of collecting chloroform-methanol volume ratio and be 99: 1 and 95: 5, evaporate to dryness, obtains Radix Angelicae Dahuricae column chromatography sample; Gained Radix Angelicae Dahuricae column chromatography sample obtains compound 1 through silica gel column chromatography and ODS column chromatography, compound 2 again.
8. the application of fatty acid compound as claimed in claim 2 in preparation prevention and Hepatoma therapy medicine, is characterized in that in step 3) in, the mobile phase of described reversed-phase silica gel column chromatography is methanol-water or acetonitrile-aqueous solution.
9. the application of fatty acid compound as claimed in claim 8 in preparation prevention and Hepatoma therapy medicine, the volume ratio that it is characterized in that described methanol-water or acetonitrile-water is (0.25～9): 1.
10. the application of fatty acid compound as claimed in claim 9 in preparation prevention and Hepatoma therapy medicine, the volume ratio that it is characterized in that described methanol-water or acetonitrile-water is (0.4～4): 1.
The application of 11. fatty acid compounds as claimed in claim 10 in preparation prevention and Hepatoma therapy medicine, the volume ratio that it is characterized in that described methanol-water or acetonitrile-water is 1.85: 1.
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| CA3051054A1 (en) | 2017-01-23 | 2018-07-26 | Revolution Medicines, Inc. | Pyridine compounds as allosteric shp2 inhibitors |
| MX2019008695A (en) | 2017-01-23 | 2019-09-11 | Revolution Medicines Inc | Bicyclic compounds as allosteric shp2 inhibitors. |
| WO2019051084A1 (en) | 2017-09-07 | 2019-03-14 | Revolution Medicines, Inc. | Shp2 inhibitor compositions and methods for treating cancer |
| RU2020115095A (en) | 2017-10-12 | 2021-11-12 | Революшн Медсинз, Инк. | PYRIDINE, PYRAZINE AND TRIAZINE COMPOUNDS AS ALLOSTERIC SHP2 INHIBITORS |
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| Standard Equivalent Chain Length Values of Monoenic and Polyenic (Methylene Interrupted) Fatty Acids;Karel Stransky, Tomas Jursik, Antonin Vitek;《Journal of High Resolution Chromatography》;19970331;第20卷;第143-158页 * |
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