CN102276444B - Fatty acid compounds and preparation method and application thereof - Google Patents

Abstract

The invention relates to fatty acid compounds and provides the fatty acid compounds and a preparation method and application thereof. The preparation method comprises the following steps of: boiling and refluxing angelica dahurica by using ethanol, filtering, and collecting filtrate to obtain angelica dahurica extracting solution; extracting the obtained angelica dahurica extracting solution, andperforming vacuum drying on solution obtained through extraction to obtain a crude angelica dahurica extract; and performing silica gel column chromatography on the crude angelica dahurica extract, and performing reversed phase silica gel column chromatography to obtain a compound 1, a compound 2 and a compound 3 respectively. The fatty acid compounds can be prepared into specific inhibitors for inhibiting protein tyrosine phosphatase-Src homology domain-containing protein tyrosine phosphatase 2 (PTP-SHP2), and achieve the effects of resisting cancer as well as preventing and treating PTP-SHP2-related human diseases by inhibiting PTP. The fatty acid compounds are widely applied to the preparation of medicines for preventing and treating cancer.

Description

Fatty acid compound and preparation method thereof and application

Technical field

The present invention relates to fatty acid compound, particularly fatty acid compound and preparation method thereof and application.

Background technology

In recent years, be accompanied by the progress at full speed of pharmacy and life science, signal transduction, apoptosis, vasculogenesis and the cell in the malignant cell and the various primary processes such as interaction of extracellular matrix are just gradually illustrated.With the key enzyme (as protein tyrosine kinase) of some intracellular signal transduction pathway relevant with the tumour cell differentiation and proliferation as the 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. the Chinese Hospitals medication is estimated and is analyzed .2009; 9 (1): 4-7).

In the initial sum evolution of cancer, protein tyrosine phosphatase plays important effect.And the protein tyrosine phosphatase level depends on the balance between protein tyrosine kinase (PTKs) and the Protein-tyrosine-phosphatase (PTPs).Protein tyrosine kinase (PTKs) is the important target spot of current oncotherapy of generally acknowledging, plays important effect in treatment for cancer.Opposite with the effect of PTKs, PTPs can promote growth of tumor.So, the discovery of PTPs inhibitor will to treatment for cancer provide bright prospects (2, Scott LM, Chen LW, Daniel KG, Brooks WH, Guida WC, Lawrence HR, et al.Shp2 protein 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 the PTPN11 genes encoding, multiple organ wide expression in human body.It is by 2 SH2 structural domains of N end, and middle PTP structural domain and C-terminal are formed.In cancer cells, SHP2 can be activated by some growth factor receptorses usually, and then regulates the phosphorylation of downstream albumen, promotes the cell growth.In the malignant tumour that proto-oncogene causes, PTP-SHP2 is activated usually.In a word, PTP-SHP2 is the positive regulating and controlling effect of performance in cell signal is regulated, and promotes the cell growth.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 also are 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 Shp2 identified 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 ofa novel shp2 protein tyrosine phosphatase inhibitor.Mol Pharmacol.2006; 70 (2): 562-570).China's traditional Chinese medicine (TCM) is at the medicinal history that has several thousand, and it has the various and complex structure double dominant of chemical ingredients, is the good source of seeking the PTP-SHP2 specific inhibitor, and this is that chemical synthetic drug can't be equal to.

Caspases is L-Cysteine HCL Anhydrous relevant on one group of structure that is present in the cytosol, 11 kinds of caspase have been determined to exist at least at present, wherein, caspase 1 and caspase 11, and may also have caspase 4 to be considered to not participate in directly the transduction of apoptotic signal, they mainly participate in the activation of interleukin precursor; And caspase 2, caspase 8, and it is apoptotic initial that caspase 9 and caspase10 participate in; That participate in the apoptosis execution then is caspase 3, caspase 6 and caspase 7, wherein caspase 3 and 7 has close substrate and inhibitor specificity, their PARP that degrades, DFF-45 (DNA fragmentation factor-45) causes the inhibition of DNA reparation and the degraded of startup DNA.(caspase 3 to think apoptotic initiator (caspase2,8,9 and 10) and executive at present, 6,7) exist upstream and downstream relation between, i.e. initiator activation executive (5, Zhu is willing to, Ma Yong, the .Caspase of Chen Li army and apoptosis. the journal .2004 of People's Armed Police medical college; 13 (4): 346-348).

Caspase-3 plays irreplaceable effect in apoptosis, topmost substrate is poly (ADP-ribose) polysaccharase PARP (poly (ADP-ribose) polymerase), and this enzyme is repaired with DNA, the gene complete monitoring is relevant.When apoptosis started, the PARP of 116kD was at Asp ²¹⁶-Gly ²¹⁷Between cut into 31kD and two fragments of 85kD by caspase-3, make two zinc fingerses of being combined with DNA among the PARP separate the function of can not bringing into normal play with the catalysis region of carboxyl terminal.The result makes the Ca that influenced by the PARP negative regulation ²⁺/ Mg ²⁺The activity of dependency endonuclease increases, and the DNA between the cracking nucleosome causes apoptosis.Caspase-8 has FADD sample DED structural domain, and can be combined with FADD by the DED structural domain, thus caspase-8 can contact in apoptotic process indirect and cell-membrane receptor, thereby the cytolemma event is converted into the 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 the plastosome approach.

Summary of the invention

The object of the present invention is to provide fatty acid compound and preparation method thereof and application.

Described fat acid compounds comprises:

Compound 1, compound 1 is oily matter, chemistry 10Z by name, the 13Z-nonadecadienoic acid, molecular formula is C ₁₉H ₃₄O ₂, molecular weight is 294, structural formula is:

Compound 2, compound 2 is oily matter, chemistry 8Z by name, the 11Z-heptadecadienoic acid, molecular formula is C ₁₇H ₃₀O ₂, molecular weight is 266, structural formula is:

Compound 3, compound 3 is oily matter, chemistry 14Z by name, 17Z-two undecandienoic acids, molecular formula is C ₂₁H ₃₈O ₂, molecular weight is 322, structural formula is:

The preparation method of described fatty acid compound may further comprise the steps:

1) uses ethanol that the root of Dahurain angelica is carried out boiling reflux, filter, collect filtrate, namely get root of Dahurain angelica extracting solution;

2) gained root of Dahurain angelica extracting solution is extracted, extraction gained solution carries out vacuum-drying, namely gets root of Dahurain angelica crude extract;

3) root of Dahurain angelica crude extract is carried out silica gel column chromatography, carry out reversed-phase silica gel column chromatography again, get compound 1, compound 2, compound 3 respectively.

In step 1), described concentration of ethanol is preferably 60%, and the ratio of described ethanol volume and root of Dahurain angelica quality is preferably 10L: 1kg, and ethanol by volume wherein, the root of Dahurain angelica is in mass; 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 root of Dahurain angelica extracting solution is preferably 1: 1.

In step 3), the concrete steps of described silica gel column chromatography: root of Dahurain angelica crude extract is added silica gel, and drying obtains the silica gel column chromatography sample; The silica gel column chromatography sample is placed the silicagel column top, be 100: 0 with volume ratio, 99: 1,98: 2,95: 5,10: 1,5: 1,0: 100 chloroform-methanol solution was eluent, carry out the silicagel column gradient elution, collect the chloroform-methanol volume ratio and be the elutriant of 99: 1 and 95: 5, evaporate to dryness namely gets root of Dahurain angelica column chromatography sample; Gained root of Dahurain angelica column chromatography sample namely gets compound 1 through silica gel column chromatography and ODS column chromatography again, compound 2 and compound 3; The moving 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 with Protein-tyrosine-phosphatase PTP-SHP2 with treatment by the arrestin tyrosine phosphatase.Described fatty acid compound has widely in preparation prevention and treatment cancer drug to be used.

Described medicine can be made up of fatty acid compound of the present invention and acceptable accessories.Medicine can be made a kind of formulation in powder, tablet, capsule, pill, suppository, pill, enteric agents, injection, syrup, emulsion, suspensoid, lozenge, paste, the sprays etc. according to ordinary method.

Advantage of the present invention is as follows:

1) activity that 3 compounds among the present invention can specificity arrestin tyrosine phosphatase PTP-SHP2 can be used as the new inhibitor as Protein-tyrosine-phosphatase PTP-SHP2.

2) 3 compounds of the present invention get final product the activity of remarkable arrestin tyrosine phosphatase PTP-SHP2 down in concentration (17～45.2 μ mol/L), thereby influence it to the regulation and control of downstream albumen, illustrate these 3 compounds can anticancer propagation, have the potentiality that become with PTP-SHP2 the new type anticancer medicine that is target.

3) increase with drug level strengthens 3 compounds of the present invention to the activity inhibition of PTP-SHP2, and the activity to PTP-SHP2 in the effective concentration scope suppresses to show good dose-effect relationship.

4) 3 compounds of the present invention cause the cutting of PARP except can specificity suppressing can also activate caspase the activity of PTP-SHP2, promote the apoptosis of liver cancer cell HepG2.

5) 3 compounds of the present invention can activate caspase 8 and caspase 9, and then cause that the increased activity of caspase 3, 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 the growth of cancer cells inhibition, impels the death of liver cancer cell, and effect obviously.

Description of drawings

Fig. 1 is the specific inhibitory effect figure of 1 couple of PTP-SHP2 of compound.In Fig. 1, X-coordinate is compound 1 concentration, and ordinate zou is the activity of Protein-tyrosine-phosphatase, wherein ◆ represent PTP-SHP2, ▲ representing VHR, ■ represents HePTP, is 100% with the protein-active with compound treatment not.

Fig. 2 is the specific inhibitory effect figure of 2 couples of PTP-SHP2 of compound.In Fig. 2, X-coordinate is compound 2 concentration, and ordinate zou is the activity of Protein-tyrosine-phosphatase, wherein ◆ represent PTP-SHP2, ▲ representing VHR, ■ represents HePTP, is 100% with the protein-active with compound treatment not.

Fig. 3 is the specific inhibitory effect figure of 3 couples of PTP-SHP2 of compound.In Fig. 3, X-coordinate is compound 3 concentration, and ordinate zou is the activity of Protein-tyrosine-phosphatase, wherein ◆ represent PTP-SHP2, ▲ representing VHR, ■ represents HePTP, is 100% with the protein-active with compound treatment not.

Fig. 4 is the cutting action figure of 3 couples of PARP of compound under the different concns.In Fig. 4,12.5,25,50 and 100 are respectively the concentration (μ M) of compound 3, blank in contrast, compound 3 has caused the cutting of PARP among the HepG2, effective concentration is 50～100 μ M, dose-effect relationship is good.

Fig. 5 is the cutting action figure of 3 couples of PARP of compound under the different time.In Fig. 5,3h, 6h and 12h are respectively the time that compound 3 acts on HepG2, blank in contrast, compound 3 causes that be 12h the effective acting time of the cutting of PARP among the HepG2.

Fig. 6 is 3 couples of caspase-3 of compound under the different concns, caspase-8, the activity influence figure of caspase-9.In Fig. 6, X-coordinate is drug level (μ M), and ordinate zou is the increased activity multiple.Wherein a is caspase-3, and b is caspase-8, and c is caspase-9; Handle the HepG2 cell with compound 3, concentration 0,50 and 100 μ M, the treatment time is 10h; * P＜0.05 shows with the blank group and compares, and the active of 100 μ M concentration group caspase-3 and caspase-9 obviously strengthens significant difference.

Embodiment

The preparation of embodiment 1 fatty acid compound

1, preparation root of Dahurain angelica alcohol extract

1) take by weighing the dry root of Dahurain angelica root of 3kg, add 30L 60% ethanol, stir, boiling reflux 2h after-filtration in extractor is collected filtrate;

2) filter residue extracts 2 times with 60% ethanol boiling reflux again, filters, and collects filtrate, and 60% ethanol consumption is 30L/ time;

3) merge 3 times filtrate, extract with ethyl acetate, each extraction agent consumption is 30L, collects upper layer of extraction liquid;

4) underclad portion extracts 2 times with ethyl acetate again, merges 3 times upper layer of extraction liquid, and vacuum-drying obtains 48.3g root of Dahurain angelica alcohol extract, and (numbering is designated as: AD).

2, silica gel column chromatography, ODS column chromatography, the preparation monomeric compound

1) soak 200～300 order silica gel with chloroform, stirring and evenly mixing, the dress post, standby;

2) add the chloroform-methanol mixing solutions in the 48.3g root of Dahurain angelica alcohol extract, ultrasonicly make its dissolving; With 60～100 order silica gel mixed samples, to grind evenly, drying namely gets the silica gel column chromatography sample;

3) above-mentioned silica gel column chromatography sample evenly is contained in the silicagel column top, carries out gradient elution, each gradient elution 3600ml with chloroform-methanol system (100: 0,99: 1,98: 2,95: 5,10: 1,5: 1,0: 100).Wherein, sorbent material silica gel is 15: 1 with the ratio of the weight of root of Dahurain angelica alcohol extract AD, and the column diameter of silicagel column is 1: 10 with the ratio of post height; According to TLC analyze to merge obtain 11 cuts (AD-A1, AD-A2, AD-B, AD-C1, AD-C2, AD-C3, AD-D, AD-El, AD-E2, AD-F, AD-G);

4) fraction A D-A2 is carried out silica gel column chromatography, moving phase is hexanaphthene-ethyl acetate system (98: 2,19: 1,10: 1,5: 1,1: 1), each gradient elution 1500ml.Wherein, sorbent material 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 obtain 12 cuts (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 the ODS column chromatography, and moving phase is methanol-water system (1: 1,13: 7,4: 1,19: 1,100: 0), each gradient elution 200ml.Wherein, sorbent material 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, merging obtains monomeric compound 1 (120mg).

Identify that through mass spectrum compound 1 is 10Z, the 13Z-nonadecadienoic acid, molecular formula is C ₁₉H ₃₄O ₂, molecular weight is 294, oily matter.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 the document is defined as same compound.

Fraction A D-A2-F is carried out the ODS column chromatography, and moving phase is methanol-water system (1: 1,3: 2,7: 3,17: 1,100: 0), each gradient elution 200ml.Wherein, sorbent material 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, merging obtains monomeric compound 2 (258mg).

Identify that through mass spectrum compound 2 is 8Z, the 11Z-heptadecadienoic acid, molecular formula is C ₁₇H ₃₀O ₂, molecular weight is 266, oily matter.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 the document is defined as same compound.

Fraction A D-A2-G is carried out the ODS column chromatography, and moving phase is methanol-water system (2: 3,3: 2,4: 1,100: 0), each gradient elution 200ml.Wherein, sorbent material 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, merging obtains monomeric compound 3 (266mg).

Be 14Z through mass spectrum authenticating compound 3,17Z-two undecandienoic acids, molecular formula is C ₂₁H ₃₈O ₂, molecular weight is 322, oily matter.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 the 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 medicinal material Zhejiang

(4) main agents

(5) substratum

The LB liquid nutrient medium:

1％(g/ml)Tryptone，0.5％(g/ml)Yeast Extract，1％(g/ml)NaCl

(6) the required damping fluid of GST purifying protein and reagent

PBS B μ ffer:137mM NaCl, 2.7mM KCl, 10mM Na ₂HPO ₄, 2mM KH ₂PO ₄The 100mg/ml penbritin

Proteinase 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, the 10mM gsh

(7) drug screening reaction solution:

50mM Bis-Tris，2mM EDTA，50mM NaCl，2mM DTT pH6.5，1M NaOH，1M DTT

(8) Western blot and other experiment is used prepares with solution:

PBS damping fluid: 137mM NaCl, 2.7mM KCl, 10mM Na ₂HPO ₄, 2mM KH ₂PO ₄Transfer pH to 7.4 with hydrochloric acid.

The Giemsa dye liquor:

Jim Sa powder (Giemsa stain) 1.0g

Glycerine (AR) 66ml

Methyl alcohol (AR) 66ml

The Giemsa powder is put into mortar, add earlier a small amount of glycerine, be ground to till the no particle, and then whole glycerine are poured into, put in 56 ℃ of incubators behind the 2h, add methyl alcohol, with in the dye liquor sealing preservation brown bottle for preparing (being better than 0～4 ℃ of preservation most).

RIPA cell pyrolysis liquid: 50mM Tris-HCl (pH7.4); 150mM NaCl; 1mM EDTA; 1%Triton; 1% Sodium desoxycholate; 0.1%SDS; 1mM PMSF; 5 μ g/ml Aprotenin; 5 μ g/ml Leupeptasin.

2. experimental technique and result

(1) fatty acid compound is to the specific inhibitory effect of Protein-tyrosine-phosphatase shp2

The high-throughput model discrimination of external inhibitors of protein tyrosine phosphatase is: SHP2 hydrolysis p-nitrophenyl phosphoric acid (pNPP), generate the yellow compound of solubility, it has photoabsorption at the 405nm place, so, can measure the activity of Protein-tyrosine-phosphatase by the photoabsorption that detects hydrolysis substrate.Based on this mechanism, the applicant has set up the inhibitor high flux screening model of external Protein-tyrosine-phosphatase, is used for seeking the novel specific inhibitor of natural product shp2.In order to observe compound to the selectivity of Protein-tyrosine-phosphatase, the applicant has also carried out determination of activity to VHR and HePTP.Concrete operation steps is as follows:

The activation of a.GST4B

1) take out GST4B in the four degree refrigerators and jiggle evenly, getting the 1.33ml mixed solution, to put into the 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 albumen sepn and purifying experimental technique and protein concentration

1) picking mono-clonal colony lift to the triangular flask of 50ml LB (50mg/ml AMP) 37 ℃ from culture dish, incubated overnight in the 250rpm shaking table.

2) from above-mentioned solution, take out 10ml bacterium liquid and transfer in another new triangular flask that contains 200ml LB (50mg/ml AMP) in 37 ℃, in the 250rpm shaking table to bacterium liquid OD ₆₀₀Value approximately reaches 0.8.

3) treat that bacterium liquid is cooled to room temperature and adds the IPTG inductor, make it final concentration and reach 0.1mM, in 25 ℃, cultivate 16h in the 225rpm shaking table.

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) measurement and accent bacterium liquid pH to 8.0 back add N,O-Diacetylmuramidase to final concentration 1mg/ml, and room temperature leaves standstill 20min.

7) excusing from death on ice is 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-Sepharose 4B, the vertical 1h that mixes in 4 ℃ of refrigerators.

9) centrifugal above-mentioned mixed solution is collected beads, uses 4 ℃ of vertical mixing, washings of 8ml BufferW1 4 times respectively, each 10min; Centrifugal, 1000rpm 1min; Collect beads; 4 ℃ of vertical mixing, washings of 8ml BufferW2 1 time, each 10min.Centrifugal, 1,000rpm 1min; Collect beads.

10) in centrifuge tube, add 4 ℃ of vertical mixing, washings of 1ml Buffer E 3 times, each 30min; Centrifugal, 1000rpm 1min; Collecting supernatant stores to new centrifuge tube.

11) packing was stored in-70 ℃ of refrigerators after the Xylene Brilliant Cyanine G method was measured the protein concentration that extracts, and is standby.

C. purity is identified

The target protein that purifying 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 reaction solutions (50mM Bis-Tris, 50mM NaCl, 2mM EDTA PH 6.9)

10 μ l 100mM compounds (compound final=10mM, DMSO final=1%)

20μl 7.5mM p-NPP(final concentration＝1.5mM)

Concrete steps are: after testing compound and albumen mixed, reaction was 30 minutes under the room temperature, adds p-NPP again, mixing, and reaction is 60 minutes under the room temperature, adds the NaOH termination reaction of 100 μ l 1M at last, microplate reader, 405nm place reading.

2) mensuration of specificity effect

The inhibiting compound that has that sifts out is measured the specificity inhibition of four kinds of PTPs albumen: be substrate with p-NPP, reaction system is 50mM Bis-Tris, 2mM EDTA, 50mM NaCl, 2mM DTT pH6.5 damping fluid, the consumption of four kinds of albumen is identical, is unit with mol, drug level dilutes by gradient, the 405nm reading.Make medicine at last to the inhibition curve (referring to Fig. 1～3) of 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, namely half-inhibition concentration is to make enzymic activity be down to the inhibitor concentration of a former active half.Can be used for doing the judgment criteria that inhibitor suppresses the ability size.Interact with the inhibitor of different concns and the enzyme-substrate reactions system under the same terms, in the unit time with the reaction system contrast of the increasing amount of product with inhibiting not, can be inhibited agent to the curve of the inhibition ability of enzyme, select suitable inhibitor concentration, just can obtain IC ₅₀Numerical value.

Table 1 fatty acid compound is to the selective inhibitory of PTP-SHP2

Experimental result shows that (referring to table 1) 3 compounds are all inhibited to PTP-SHP2, its IC ₅₀Be 172～452 μ M.But under the same concentrations, to the restraining effect of PTP-SHP2 obviously greater than the inhibition to VHR and HePTP.This explanation, these 3 compounds have specificity to the inhibition of PTP-SHP2.

(2) compound 3 is by PARP cutting liver cancer apoptosis reducing

Adopt western blotting (Western Blotting) method to carry out the protein level analysis, western blotting (western blot), it is according to the specificity of the antigen-antibody method in conjunction with certain albumen in the detection of complex sample.Western blot is a kind of new immune biochemical technology that grows up in gel electrophoresis and solid-phase immunoassay technical foundation.Because immunoblotting (western blot) has high specific and the susceptibility of very high resolution and the solid-phase immunoassay of SDS-PAGE, has 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 qualitative and semi-quantitative analysis to albumen.Concrete operation steps is as follows:

A. cell cultures

1) the HepG2 human liver cancer cell is hatched in containing 10% foetal calf serum, 100M/ml penicillin is in the DMEM substratum of 100M/ml Streptomycin sulphate, at 37 ℃, 5%CO ₂Cultivate in the incubator.

2) suction goes nutrient solution, PBS to wash 1 time, adds an amount of trysinization liquid, leaves standstill 2-3min in incubator; The microscopically observation of cell becomes bowlder, with the digestion of the DMEM substratum termination pancreatin that contains serum, blows and beats Tissue Culture Dish with pipettor, and cell suspension is sucked in the 15ml centrifuge tube centrifugal 800rpm, 2min; After supernatant liquor is removed in suction, add and contain 10% foetal calf serum DMEM nutrient solution, blow out single cell suspension with liquid-transfering gun, HepG2 single cell suspension branch is reached 6 orifice plates, be put in 37 ℃ of incubators and cultivate.

B. cell is handled

Cell is long to be inhaled and removes nutrient solution by 80% o'clock that accounts for the orifice plate floorage greatly, added the fresh DMEM substratum that contains serum-free of 2ml, added compound 3 or DMSO (blank group) respectively, mixing, making its final concentration is 0,12.5,25,50,100 μ M/L cultivate 12h in incubator; Perhaps final concentration is 100 μ M/L, cultivates 3h, 6h, 12h in incubator respectively.

C. cell harvesting:

Suction removes to contain the cell culture fluid of medicine, washes 2 times with the PBS of 4 ℃ of precoolings, and every hole adds 1ml PBS, scrapes with cell cell is scraped, and collects in the 1.5ml centrifuge tube.Centrifugal 4 ℃, 12000rpm, 25s.

D. lysis:

Remove supernatant liquor, add 40 μ L RIPA cell pyrolysis liquids in each centrifuge tube, cracking 30min on ice, therebetween, on vibrator every 5min vibration 10s, centrifugal 4 ℃ then, 12000rpm, 10min.Collect supernatant liquor, namely get protein sample.

E. measure the concentration of protein sample according to the conventional determination of protein concentration method in laboratory:

The f.SDS-PAGE electrophoresis:

1) clean sheet glass: a hand fastening sheet glass, the another hand dips in a liquid detergent and cleans gently.The two sides is all cleaned and is dashed with tap water later, stands in the basket after clean with distilled water flushing again and dries.

2) encapsulating and last sample:

A. putting into folder after the sheet glass alignment tightens.Vertical card is prepared encapsulating on the top of the shelf then.

B. join 8% separation gel, shake up immediately behind the adding TEMED and get final product encapsulating.During encapsulating, available 10ml rifle is drawn 5ml glue and is emitted along glass, treats to get final product when the glue face is raised to greenbelt mid-line height.Add one deck dehydrated alcohol then on the glue, the gelling after the fluid-tight faster.

C.15～20min after, when a refracted ray is arranged between ethanol and the glue, illustrate that glue has coagulated.Wait 3min that glue is fully solidified just can to remove photoresist upper strata ethanol again and with filter paper ethanol is blotted.

D. join 4% concentrated glue by previous methods, add to shake up immediately behind the TEMED and get final product encapsulating.Remaining space is filled concentrated glue to be inserted comb in the concentrated glue then.Glue is flowed down in order to avoid there is bubble to produce in the glue along sheet glass.To make comb maintenance level when inserting comb.Because volume can shrink and reduce when gelling was solid, thereby the last sample volume of well is reduced, so in concentrating the solid process of gelling, will mend glue through the both sides of being everlasting.By the time after concentrating gelling admittedly, two hands pinch the both sides of comb respectively and gently it are extracted straight up.

E. water washes and concentrates glue, puts it in the electrophoresis chamber.

F. after surveying finished white content, calculate the liquor capacity that contains 40 μ g albumen and be applied sample amount.

The last all product that prepare are boiled 10min in 110 ℃.Put-20 ℃ of preservations.

G. begin to prepare sample after adding enough electrophoresis liquid.With the adherent absorption sample of microsyringe, the inspiration bubble is not wanted in the sample sucking-off.The sample injector syringe needle is inserted to the slow sample that adds in the well.

3) electrophoresis:

Beginning is used 80V when concentrating glue, treat after albumen Marker has separation voltage to be enlarged to 120V, and electrophoresis has just been run out of to bromjophenol blue can stop electrophoresis.

4) change film:

A. must wear gloves when filter paper and film, because albumen on hand can polluted membrane.Change before the film, pvdf membrane is dipped in 10s in the methyl alcohol, take out with tweezers, be placed in the electricity commentaries on classics liquid and wash several times.2 part of 5～6 metafiltration paper, 1 part of individual layer filter paper are soaked in the electricity commentaries on classics liquid, stand-by.

B. clip is opened the black one side maintenance level that makes.Fill up a sponge pad in the above, roll several times back and forth to roll away the bubble of the inside with glass rod.At mat pad 5 metafiltration paper, on the other hand fixedly filter paper proficiency is rolled wherein bubble with glass rod.

C. careful sled removes glue glass outer plate, and individual layer filter paper is tiled in Jiao Mianshang, removes an other sheet glass gently, glue is lain on the multilayer filter paper, one side with the pvdf membrane that soaked just now from the glue face begin slowly cover, note not having bubble.Begin to catch up with bubble with a dropper that soaks from one side of film then.To soak the multilayer filter paper of getting well again and lie in above the film, and at last with on one deck black sponge cover, roll several times and just can close clip.

D. clip is put into the transfer groove groove, be made the black flour of clip to the black flour of groove, the fine flour of folder is to red of groove.Pouring about 1L into changes film liquid, then this is changeed the film groove and is placed in the basin of a filled with ice 100v, 90min.

5) immune response:

A. sealing: with the room temperature sealing 1h in the TBST damping fluid that contains 5% skimmed milk of the pvdf membrane behind the electrotransfer;

B. primary antibodie reaction: the pvdf membrane after will sealing is encapsulated in the hybridization bag, adds the anti-anti-PARP of rabbit (Sigma company, concentration 1: 1000), spends the night on 4 ℃ of shaking tables;

C. two anti-reactions: pvdf membrane shaken in the TBST damping fluid wash 3 times, each 5min.Add IgG (H+L) (goat antirabbit two is anti-, concentration 1: 10000, Sigma company), incubated at room 1h.

6) photographic fixing is developed in chemoluminescence

Remove two anti-ly, wash film with the TBST damping fluid, room temperature is shaken and is given a baby a bath on the third day after its birth time, 5min at every turn, and protein powder places preservative film up, and A liquid and the B liquid of ECL chemiluminescence detection kit is mixed with 1: 1 (V/V), in the darkroom, presses 0.05-0.1mL/cm ²Minim be added on the film surface, hatch 1min, inhale with filter paper and remove unnecessary liquid, seal film after, in the darkroom, expose immediately, develop.

Experimental result shows: compound 3 has caused the cutting of PARP after handling HepG2 human liver cancer cell 12h; And under 100 μ mol/L concentration, when PARP cuts degree obviously greater than 50 μ mol/L.So compound 3 can be induced the cutting of PARP among the HepG2, and present good dose-effect relationship (referring to Fig. 4).Final concentration is after the compound 3 of 100 μ mol/L is handled the HepG2 human liver cancer cell, and during 6h, the PARP cutting is also not obvious; And when 12h, show tangible PARP cutting action.This explanation, the effect of PARP has time-dependent manner (referring to Fig. 5) among 3 couples of liver cancer cell HepG2 of compound.Therefore, compound 3 can cut the approach cell death inducing by PARP, and has good concentration and time-dependent manner, 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 L-Cysteine HCL Anhydrous relevant on one group of structure that is present in the cytosol of discovered in recent years, and synthetic caspase exists with the proenzyme state of non-activity in the cell, activatedly can carry out its function.Caspase 8, and caspase 9 participates in apoptotic initial; Caspase 3 participates in apoptosis and carries out.These 3 kinds of albumen have important effect in apoptosis process, therefore also be the important indicator that apoptosis detects.

Caspase 3/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 dissociates out.PNA has obtained the maximum absorption at the 405nm place, by detecting pNA, investigates the activity of caspase-3 indirectly.The substrate of Caspase-8 and Caspase-9 is respectively IETD-pNA or LEHD-pNA, the same Caspase-3 of its active measuring principle.The concrete operations step is as follows:

1) cell cultures

A. the HepG2 human liver cancer cell is hatched in containing 10% foetal calf serum, 100M/ml penicillin is in the DMEM substratum of 100M/ml Streptomycin sulphate, at 37 ℃, 5%CO ₂Cultivate in the incubator.

B. inhale the time and go nutrient solution, PBS to wash 1 time, add an amount of trysinization liquid, in incubator, leave standstill 2-3min; The microscopically observation of cell becomes bowlder, with the digestion of the DMEM substratum termination pancreatin that contains serum, blows and beats Tissue Culture Dish with pipettor, and cell suspension is sucked in the 15mL centrifuge tube centrifugal 800rpm, 2min; After supernatant liquor is removed in suction, add and contain 10% foetal calf serum DMEM nutrient solution, blow out single cell suspension with liquid-transfering gun, above-mentioned HepG2 single cell suspension branch is reached the Tissue Culture Dish that diameter is 10cm, be put in the incubator and cultivate.

2) cell is handled

Treat cell length by 80% o'clock that accounts for the orifice plate floorage greatly, nutrient solution is removed in suction, adds the DMEM substratum of the fresh serum-free of 10ml, add compound 3 or DMSO (blank group) respectively, mixing, making its final concentration is 0,50,100 μ M/L cultivate 10h in incubator.

3) cell counting:

Suction removes to contain the cell culture fluid of medicine, washes 2 times with PBS, with trysinization (the same step 1) of digestion method.With the cell counting count board counting, get 1-5 * 10 ⁶Individual cell in 1.5ml Ep pipe, centrifugal (4 ℃, 12000rpm, 25s), cell precipitation is used for following experiment.

4) lysis:

Add 50 μ L Cell Lysis Buffer (Caspase-3 ,-8, in-9Colorimetric Assay the Kits) in each Ep pipe, vortex is with mixing, on ice cracking 10min.Centrifugal 4 ℃, 10000g, 1min.Collect supernatant liquor in a new Ep pipe, be placed on ice (or put-80 ℃ standby).

5) by laboratory conventional determination of protein concentration kit measurement protein concentration:

6) get 50～200 μ g albumen in 96 orifice plates, be diluted to 50 μ l with Cell Lysis Buffer.

7) add 50 μ l, 2 * Reaction Buffer (containing 10mM DTT), mixing.

8) add 5 μ l 4mM 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, and mixing is put 37 ℃ and hatched 1～2h.

9) use microplate reader, 400 or the 405nm place, read the OD value.

After compound 3 was handled HepG2 human liver cancer cell 10h, during 100 μ mol/L concentration, the activity of caspase-8 and caspase-9 had all strengthened, and caspase-9 strengthens degree 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 does not have obvious enhancing.This explanation, apoptosis that compound 3 is induced relies on the activation of caspase, and mainly is that plastosome approach by the caspase-9 place plays a role.This experiment is clear and definite this compounds is by the effect path of apoptosis performance antitumor action.

(4) compound 3 suppresses clone's formation of liver cancer cell

Measure the growth-inhibiting effect of 3 pairs of HepG2 cells of compound by Giemsa dyeing.The Giemsa dye liquor is made up of reddish black and Yihong.The dyeing theory of Giemsa: the adsorption of existing physics has chemical affinity interaction again.Various cellular constituent chemical property differences, also different to the avidity of various dyestuffs.Karyomit(e) is acid in the nucleus, is easily dyed blueness by dye liquor, and tenuigenin then dyes more shallow.With the Giemsa staining can the observation of cell cluster size and what, thereby reflect that compound is to the growth-inhibiting effect of cancer cells.The concrete operations step is as follows:

1) cell cultures

A. the HepG2 human liver cancer cell is hatched in containing 10% foetal calf serum, 100M/ml penicillin is in the DMEM substratum of 100M/ml Streptomycin sulphate, at 37 ℃, 5%CO ₂Cultivate in the incubator.

B. inhale and go nutrient solution, PBS to wash 1 time, add an amount of trysinization liquid, in incubator, leave standstill 2-3min; The microscopically observation of cell becomes bowlder, with the digestion of the DMEM substratum termination pancreatin that contains serum, blows and beats Tissue Culture Dish with pipettor, and cell suspension is sucked in the 15mL centrifuge tube centrifugal 800rpm, 2min; After supernatant liquor is removed in suction, add and contain 10% foetal calf serum DMEM nutrient solution, blow out single cell suspension with liquid-transfering gun.HepG2 single cell suspension branch is reached 6 orifice plates, about 1 * 10 ³/ hole.Be put in the incubator and cultivate.

2) cell is handled

After 4～5 days, when the cell cluster forms, inhale and remove nutrient solution, add the fresh DMEM substratum that contains serum-free of 2ml, add compound 3 or DMSO (blank group) respectively, mixing, making its final concentration is 0,25,50 μ M/L, cultivates 20h in incubator.

3) cell fixation

Nutrient solution is removed in suction, washes 1 time with PBS, uses fixedly 15min of 4% Paraformaldehyde 96 (PFA) then.

4) Giemsa dyeing

Discard 4% Paraformaldehyde 96, wash 3 times with PBS.Add Giemsa dyeing 10min.

5) IMAQ

Discard Giemsa dyeing 10min, wash 1 time with PBS.The Canon camera is gathered 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-inhibiting effect; Under 50 μ M, this restraining effect is very obvious.This explanation, in effective concentration range, the growth-inhibiting of 3 pairs of HepG2 cells of compound presents dose-effect relationship.This results suggest, with compound 3 be representative this compounds significantly anticancer growth reach antitumous effect.

Embodiment 314Z, the preparation of 17Z-two undecandienoic acid tablets

Get 14Z, 17Z-two undecandienoic acid 1g, Microcrystalline Cellulose 27g mix with Magnesium Stearate 2g, and it is 6mm that mixture is pressed into diameter with Singlepunchtabletpress, and weight is the sheet of 300mg, and every contains 14Z in this tablet, 17Z-two undecandienoic acid 10mg.

Embodiment 4 preparation granules

Get 14Z, 17Z-two undecandienoic acid 1g mix with W-Gum 29g, add water and make softwood, cross 12 mesh sieves and carry out granulation, obtain granule after the drying.Contain 14Z, 17Z-two undecandienoic acid 10mg among every 300mg in the granule of present embodiment preparation.

Embodiment 5 preparation capsules

Get 14Z, 17Z-two undecandienoic acid 1g mix with lactose 27g, Magnesium Stearate 2g, fill enteric coated capsule with every 300mg.In the capsule of present embodiment preparation, each capsule contains 14Z, 17Z-two undecandienoic acid 10mg.

Claims (5)

1. the preparation method of fatty acid compound is characterized in that described fatty acid compound is:

Compound 1, compound 1 is oily matter, chemistry 10Z by name, the 13Z-nonadecadienoic acid, molecular formula is C ₁₉H ₃₄O ₂, molecular weight is 294, structural formula is:

Compound 2, compound 2 is oily matter, chemistry 8Z by name, the 11Z-heptadecadienoic acid, molecular formula is C ₁₇H ₃₀O ₂, molecular weight is 266, structural formula is:

Compound 3, compound 3 is oily matter, chemistry 14Z by name, 17Z-two undecandienoic acids, molecular formula is C ₂₁H ₃₈O ₂, molecular weight is 322, structural formula is:

Described preparation method may further comprise the steps:

1) uses ethanol that the root of Dahurain angelica is carried out boiling reflux, filter, collect filtrate, namely get root of Dahurain angelica extracting solution;

2) gained root of Dahurain angelica extracting solution is extracted, extraction gained solution carries out vacuum-drying, namely gets root of Dahurain angelica crude extract; The solvent of described extraction is ethyl acetate; The solvent of described extraction is 1: 1 with the ratio of the volume of root of Dahurain angelica extracting solution;

3) root of Dahurain angelica crude extract is carried out silica gel column chromatography, carry out reversed-phase silica gel column chromatography again, get compound 1 respectively, compound 2, compound 3, the concrete steps of described silica gel column chromatography: with root of Dahurain angelica crude extract, add silica gel, drying obtains the silica gel column chromatography sample; The silica gel column chromatography sample is placed the silicagel column top, ratio with volume is 100: 0,99: 1, and 98: 2,95: 5,10: 1,5: 1,0: 100 chloroform-methanol solution was eluent, carry out the silicagel column gradient elution, collect the chloroform-methanol volume ratio and be the elutriant of 99: 1 and 95: 5, evaporate to dryness namely gets root of Dahurain angelica column chromatography sample; Gained root of Dahurain angelica column chromatography sample namely gets compound 1 through silica gel column chromatography and ODS column chromatography again, compound 2 and compound 3; The moving phase of described reversed-phase silica gel column chromatography is methanol-water or acetonitrile-aqueous solution; The volume ratio of described methanol-water or acetonitrile-water is (0.25～9): 1.

2. the preparation method of fatty acid compound as claimed in claim 1 is characterized in that in step 1), and described concentration of ethanol is 60%, and the ratio of described ethanol volume and root of Dahurain angelica quality is 10L: 1kg, ethanol by volume wherein, and the root of Dahurain angelica is in mass.

3. the preparation method of fatty acid compound as claimed in claim 1 is characterized in that in step 1), and the time of described boiling reflux is 2～3h.

4. the preparation method of fatty acid compound as claimed in claim 1 is characterized in that the volume ratio of described methanol-water or acetonitrile-water is (0.4～4): 1.

5. the preparation method of fatty acid compound as claimed in claim 4, the volume ratio that it is characterized in that described methanol-water or acetonitrile-water is 1.85: 1.

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