CN101890010B - Application of derivative of pyridine carboxamide in preparation of anti-tumor medicaments - Google Patents
Application of derivative of pyridine carboxamide in preparation of anti-tumor medicaments Download PDFInfo
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- CN101890010B CN101890010B CN2010101750149A CN201010175014A CN101890010B CN 101890010 B CN101890010 B CN 101890010B CN 2010101750149 A CN2010101750149 A CN 2010101750149A CN 201010175014 A CN201010175014 A CN 201010175014A CN 101890010 B CN101890010 B CN 101890010B
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Abstract
The invention belongs to the field of genetic engineering and chemistry, and relates to the application of a derivative of pyridine carboxamide in the preparation of anti-tumor medicaments. The invention provides the application of a small-molecular compound N2,N5-di[2-(3,4-dimethoxyphenyl)ethyl]-2,5-pyridine carboxamide in the preparation of the anti-tumor medicaments. The compound can remarkably suppress the multiplication of tumor cells. The medicaments prepared from the compound for a cellular target CYPJ are difficult for patients to form medicament resistance, and have evident effect on the tumor cells of high-expression CYPJ proteins. Therefore, the small-molecular compound of the invention is developed as a novel anti-tumor medicament, has evident tumor suppression effect and high specificity, and provides a novel way and means for the treatment and cure of tumors.
Description
Technical field
The present invention relates to chemical field and field of medicaments, relate to the application of a kind of derivative of pyridine carboxamide in the preparation antitumor drug.Be specifically related to N
2, N
5-two [2-(3, the 4-dimethoxy phenyl) ethyls]-2, the application of 5-ascorbyl palmitate in the preparation antitumor drug.
Background technology
Cyclophilin Cycliphilins (CyPs) is the intracellular protein of widespread distribution, in plant, antibacterial and mammal, all exists, and has high conservative property, is found as the cell receptor of ciclosporin A at first.Ciclosporin A (CyclosporinA; CsA) be from fungus metabolite, to separate 11 the amino acid whose ring type polypeptides that contain that obtain; Be a kind of immunosuppressant that is used for organ transplantation and autoimmune disease, be widely used in clinically that annual sales amount is more than 5,000,000,000 dollars.Because CsA is used for having shown various toxic and side effects since clinical, the influence of patient and graft survival rate is more and more caused people's attention, the substitute that many scientific research persons are striving to find with CsA effect is similar, toxic and side effects is low.At present CsA has also begun to be used in the middle of the oncotherapy, and mainly is to use with collocation such as other cancer therapy drug such as paclitaxel, amycin, vincristine, strengthen these medicines anti-tumor activity (Clin Cancer Res.11,2320-2326).Because cyclosporin A also is simultaneously very effective immunosuppressant, unavoidably can bring the immunosuppressant side effect when therefore being used for these treatment of diseases.Overcome main two kinds of immunosuppressant method, the one, the immunosuppressant group of cyclosporin A is modified, perhaps from fungus, extract non-immunosuppressant cyclosporin derivatives; Another kind method is according to the proteic structure of CYP, screens or design new non-peptide type small molecular inhibitor.
CyPs is distributed widely in from microorganism to mammiferous all kinds of species.Cyclophilin A (CyPA) is first member in the CyP family that at first from the thymocyte cell of cattle, finds, and verifies that it has PPIase activity (Science, 226,544-547; Nature 337,473-478).Existing (Proc.Nat.Acad.Sci.U.S.A.88, the 9483-9487 of reporting of three dimensional structure of the complex that three dimensional structure and it of reorganization human T-cell's cyclophilin A (hCyPA) and CsA, tetrapeptide and some dipeptides form; J.Mol.Biol.228,539-550; J.Mol.Biol.234,1119-1130.; Nature, 353,276-279; Proc.Natl.Acad.Sci.U.S.A.88,3324-3328; Biochemistry, 35,7362-7368).The structure of the complex of CyPA and HIV related protein is also reported (Cell, 87,1285-1294; Structure 5,139-146).Other members of CyP family and CyPA have sequence homology and the similar characteristic of three dimensional structure (Proc.Natl.Acad.Sci.U.S.A.90,11850-11854; Proc.Natl.Acad.Sci.U.S.A.91,5183-5186; J.Mol.Biol.331,45-56).
CyPJ is the newcomer of CyP family, the long 1.25Kb of CYP-J cDNA, and 161 aminoacid of encoding, separation and evaluation from people's tire brain cDNA library (Cytogenet.Cell Genet.92,231-236).On aminoacid sequence, has 72% homology with nematicide (C.elegans.) CYP10.Its concrete nucleotide and aminoacid sequence see the gene report for AF146799 of the gene number of landing on the NCBI website for details.CYPJ albumen can promote the cloning efficiency of HCC, can promote the propagation of duplicating of HCC material, and can promote the hepatocarcinoma growth of tumour cell, therefore, can be used as the target of screening anti-liver cancer drug agent.
Malignant tumor has become one of human lethal most important reason, but commercially available antitumor drug all has various untoward reaction at present, therefore, seeks the big focus that novel anti-tumor medicine with low side effects becomes biomedicine field.
Summary of the invention
The purpose of this invention is to provide the application of a kind of derivative of pyridine carboxamide in the preparation antitumor drug, be specifically related to N
2, N
5-two [2-(3, the 4-dimethoxy phenyl) ethyls]-2, the application of 5-ascorbyl palmitate in the preparation antitumor drug.
Described antitumor drug is injection or tablet.Described antitumor drug is medicines resistant to liver cancer or medicament for resisting cervical cancer.
The present invention also provides a kind of method that suppresses tumor cell proliferation, is about to N
2, N
5-two [2-(3, the 4-dimethoxy phenyl) ethyls]-2, the 5-ascorbyl palmitate adds in the culture fluid of tumor cell.
Described tumor cell can be HCC or cervical cancer cell etc.Specifically, described tumor cell is the tumor cell of QGY cell or SK-Hepl cell for example, perhaps the cervical cancer cell of Hela cell for example.
The concrete grammar that adds FD7 can adopt the method that is adopted among the embodiment 4, perhaps other conventional operation.
N of the present invention
2, N
5-two [2-(3, the 4-dimethoxy phenyl) ethyls]-2, the 5-ascorbyl palmitate (is N
～2
～, N
～5
～-bis [2-(3,4-dimethoxyphenyl) ethyl]-2,5-pyridinedicarboxamide), abbreviate FD7 in the present invention as.
N of the present invention
2, N
5-two [2-(3, the 4-dimethoxy phenyl) ethyls]-2,5-ascorbyl palmitate, its molecular weight are 493.56, structural formula is:
Its relevant information is following:
Compound I d:AK-968/13026076
MDL number: MFCD01835541
It is a kind of CypJ inhibitor that previous research for FD7 of the present invention mainly concentrates on it, and the BIAcore molecule is done the appearance checking mutually, and it can combine its balance-dissociation constant KD (M): 4.64 * 10 with CypJ
-5Secondly, (α-chymotrypsin-coupled enzymic assay) records N of the present invention through the alpha-chymotrypsin activation measurement
2, N
5-two [2-(3, the 4-dimethoxy phenyl) ethyls]-2,5-ascorbyl palmitate, its enzyme are lived and are suppressed IC
50Value (μ M) is 56.3 ± 2.67.
Further research shows that CypJ micromolecular inhibitor FD7 of the present invention has inhibitory action to growth of tumour cell.
The present invention has measured the kill and wound situation of FD7 to HCC QGY cell, and the result shows that FD7 has lethality to a certain degree to tumor cell SK-Hepl and Hela cell.In addition, FD7 can also effectively reduce the cloning efficiency of QGY cell.
Therefore, FD7 of the present invention can suppress the active or expression of CYPJ, for the high tumor cytotoxicity better effects if of CYPJ expression.
Micromolecular compound of the present invention can adopt the method for preparing preparation of various routines.For example, adopt the method for artificial chemosynthesis.
Utilize micromolecular compound of the present invention,, can filter out with FD7 interactional material takes place, like receptor, inhibitor or antagonist etc. through various conventional screening techniques.
The present invention and inhibitor, antagonist etc. when in treatment, using (administration), can provide different effects.Usually, can these materials are formulated in nontoxic, the inert and pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although pH value can be with being changed to some extent by preparation Substance Properties and disease to be treated.The pharmaceutical composition for preparing can carry out administration through conventional route, comprising (but being not limited to): intramuscular, intraperitoneal, subcutaneous, Intradermal or topical.
With people FD7 of the present invention is example, can be with itself and suitable pharmaceutically acceptable carrier coupling.This type pharmaceutical composition contains chemical compound and the pharmaceutically acceptable carrier or the excipient of treating effective dose.This type carrier comprises (but being not limited to): saline, buffer, glucose, water, glycerol, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.People FD7 of the present invention can be made into the injection form, for example prepares through conventional method with normal saline or the aqueous solution that contains glucose and other adjuvant.Pharmaceutical composition such as tablet and capsule can prepare through conventional method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of active component is the treatment effective dose, for example every day about 1 microgram/kg body weight-Yue 5 mg/kg body weight.In addition, FD7 of the present invention also can use with the other treatment agent.
When people FD7 of the present invention is used as medicine; Can the FD7 of treatment effective dose be applied to mammal; Wherein should treat effective dose usually at least about 10 micrograms/kg body weight; And in most of the cases be no more than about 8 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage is factor such as considered route of administration, patient health situation also, and these all are within the skilled practitioners skill.
The invention provides micromolecular compound N
2, N
5-two [2-(3, the 4-dimethoxy phenyl) ethyls]-2, the application of 5-ascorbyl palmitate in the preparation antitumor drug.This chemical compound can obviously suppress the propagation of tumor cell.FD7 is the medicine to cell target spot CYPJ design, is difficult for forming drug resistance, and, obvious especially for the proteic tumor cell effect of high expressed CYPJ.Therefore, micromolecular compound of the present invention is developed as new antitumor drug, and tumor killing effect is obvious, and specificity is good.Therefore, FD7 of the present invention can be used as new antitumor drug and develops, and for treating and curing tumor a kind of new approach and means is provided.
The specific embodiment
The virtual screening of embodiment 1CypJ micromolecular inhibitor
In PDB protein structure data base, retrieved the proteic X-ray diffraction crystal of human CYPA structure (PDB code: 1CWA).This structure is the compound crystal structure of CYPA and its natural inhibitor cyclosporin A (CsA).From this structure, confirmed the avtive spot of CYPA, and confirmed in the avtive spot, can be by some key amino acids site that CsA suppressed.According to CYPJ and the highly homologous sequence of CYPA, we and Chinese Academy of Sciences's Shanghai medicine are cooperated, and have set up the 3D structural model, and when after this albumen crystallization and structure elucidation, have also obtained confirmation (CYPJ PDB code: 1XYH).To the CypJ avtive spot, some micromolecule data bases are screened.The micromolecule data base who is used to screen mainly comprises SPECS and CNPD.Screened FD7 of the present invention at last.The The whole calculations process is on Chinese Academy of Sciences's Shanghai medicine institute 64CPU-SGI ORIGN3800 computer and ultra calculation center, Shanghai 392CPU-martial prowess I supercomputer, to carry out.
Embodiment 2 utilizes the BIAcore molecule to make appearance checking virtual screening result mutually
The BIAcore molecule is based on surface plasma resonance technology as appearance mutually and realizes following the tracks of the interaction between biomolecule, need not any label, has therefore guaranteed the verity of experimental result to greatest extent.During experiment, biological molecules of interest (CypJ albumen) is fixed on the sensing chip surface, then micromolecular compound is dissolved in solvent and flows through chip surface.Monitor can real-time tracking detects combine, the dissociate variation of whole process of molecule and chip surface biological molecules of interest in the solution.Through the binding data of BIAcore, N of the present invention
2, N
5-two [2-(3, the 4-dimethoxy phenyl) ethyls]-2, the bonded balance of 5-ascorbyl palmitate and CypJ-dissociation constant KD (M): 4.64 * 10
-5
Embodiment 3 utilizes enzyme to live experiment proof micromolecular compound to the CypJ enzyme ability that suppresses alive
It is a lot of to measure the active method of CyP, but the most frequently used with alpha-chymotrypsin activation measurement (α-chymotrypsin-coupled enzymic assay).Its principle is the oligopeptide substrate that contains proline; Be in balance like N-succinyl-Ala-Ala-Pro-Phe paranitroanilinum (N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide) its cis, transconfiguration in solution; This substrate generation cis-trans isomerism effect of CyP ability catalysis, promptly the catalysis proline is become trans by cis; When it was in transconfiguration, C end p-nitroanilide was discharged pigment group paranitroanilinum by the alpha-chymotrypsin cracking, can learn that in the variation of 390nm METHOD FOR CONTINUOUS DETERMINATION light absorption value the PPIase of CyP is active.
The present invention with the reaction that do not add micromolecular inhibitor as control reaction, measure the micromolecule part under variable concentrations to the enzyme suppression ratio of reaction of living, thereby calculate the IC of micromolecule part
50Value.N of the present invention
2, N
5-two [2-(3, the 4-dimethoxy phenyl) ethyls]-2, its enzyme of 5-ascorbyl palmitate (Interchim company) are lived and are suppressed IC
50Value (μ M) is 56.3 ± 2.67.
Embodiment 4MTS method is measured the growth inhibited effect of FD7 to tumor cell
Human hepatoma cell strain SK-Hepl cell (ATCC company) 3 * 10
3/ hole is seeded to 96 orifice plates, cultivates to make it adherent back adding FD7 (Interchim company) in 24 hours, establishes 6 Concentraton gradient, and each concentration is established 3 multiple holes.Cell is at 37 ℃, 5%CO
2Cultivate after 72 hours under the condition; Outwell culture fluid; Measure cell survival rate with MTS test kit (Promega company); Method of testing is: cell is washed one time with serum-free medium, added the MTS chromophoric solution (adding 2ml solution 1 and 100 μ l solution 2 in the 10ml serum-free medium, abundant mixings) for preparing in advance according to the amount of 100 μ l/well.Do not have the hole of cell to be made as this bottom outlet with one, absorb in order to the bias light of calibration solution.Putting into cell culture incubator to cell continues to cultivate 2～4 hours; Read absorbance value (reference wavelength 630-700nm with ELIASA then; Measure wavelength 490nm), calculate cell survival rate, to measure the numerical value of hole absorbance value/control wells absorbance value as cell survival rate.
Result: added N
2, N
5-two [2-(3, the 4-dimethoxy phenyl) ethyls]-2, behind the 5-ascorbyl palmitate, the quantity of SK-Hepl cell is about 63% of contrast.
Use the same method and handle the Hela cell, the quantity of Hela cell is about 70% of contrast as a result.
Embodiment 5FD7 is to the influence of cloning efficiency
1000/ware of QGY cell (ATCC company) is inoculated in the 60mm Tissue Culture Dish, and one group adds FD7, and another group adds DMSO as contrast.Continuous culture 15-20 days time, liquid is taken the circumstances into consideration to change in the centre.Wash once with room temperature 1 * PBS, 4 ℃ of methanol that add the 2ml pre-cooling are 10min fixedly.Discard the fixedly methanol of usefulness, wash once, add the GIMESA stain with 1 * PBS of pre-cooling, room temperature dyeing 10min, clear water is washed 3 times, and removal floating color is taken pictures, is counted.
Confirm clone's size according to cell number,, be called middle clone between 100-200 cell, be called little clone between 50-100 cell greater than big clone of being called of 200 cells.Difference between the big-and-middle clone repeats 2 each three wares and averages, and adds behind the FD7 the formed clone's digital display of QGY cell work and is less than and adds formed clone's number behind the DMSO.These results suggest, FD7 can suppress cell clone and form.
Claims (3)
1.N
2, N
5-two [2-(3, the 4-dimethoxy phenyl) ethyls]-2, the application of 5-ascorbyl palmitate in the preparation antitumor drug is characterized in that described antitumor drug is a medicament for resisting cervical cancer.
2. application as claimed in claim 1 is characterized in that, this antitumor drug is injection or tablet.
3.N
2, N
5-two [2-(3, the 4-dimethoxy phenyl) ethyls]-2, the application of 5-ascorbyl palmitate in the preparation antitumor drug is characterized in that, with N
2, N
5-two [2-(3, the 4-dimethoxy phenyl) ethyls]-2, the 5-ascorbyl palmitate adds in the culture fluid of tumor cell, and described tumor cell is the Hela cell.
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Non-Patent Citations (4)
| Title |
|---|
| 第64页第1-8行，图2-7，第70页第1-8行，表2-2. |
| 第83页第1-2行，第7页第1-2行 |
| 陈帅.《人脯氨酸异构酶CYPJ的功能探讨及其与CYPA的小分子抑制剂筛选》.《中国博士学位论文全文数据库 基础科学辑》.2007,(第06期),第1页第10-12行，第6页最后一行 |
| 陈帅.《人脯氨酸异构酶CYPJ的功能探讨及其与CYPA的小分子抑制剂筛选》.《中国博士学位论文全文数据库 基础科学辑》.2007,(第06期),第1页第10-12行,第6页最后一行,第83页第1-2行,第7页第1-2行,第64页第1-8行,图2-7,第70页第1-8行,表2-2. * |
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