CN102552228B - Application of vitamin K in preparation of pyruvate kinase inhibitor - Google Patents
Application of vitamin K in preparation of pyruvate kinase inhibitor Download PDFInfo
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- CN102552228B CN102552228B CN 201010615924 CN201010615924A CN102552228B CN 102552228 B CN102552228 B CN 102552228B CN 201010615924 CN201010615924 CN 201010615924 CN 201010615924 A CN201010615924 A CN 201010615924A CN 102552228 B CN102552228 B CN 102552228B
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Abstract
The invention provides two inhibitors of M2 type pyruvate kinase (PKM2)-vitamin K type compounds (vitamin K3 and vitamin K5). The currently reported PKM2 inhibitor which belongs to small molecular compounds is Compound 3 only. The effect of the vitamin K on inhabitation of the PKM2 is found to be higher than that of the Compound 3. Therefore, as an inhibitor of pyruvic acid, the vitamin K has important application value. Two new pyruvate kinase inhibitors-vitamin K3 and vitamin K5 are provided, provide basis for selection of new medicaments and have important application prospect.
Description
(1) technical field
The present invention relates generally to the application of vitamin K compounds (vitamin K3, K5) in the preparation pyruvate kinase inhibitor.
(2) background technology
Two kinds of vitamin K compounds (vitamin K3, K5) that the present invention relates generally to are the naphthoquinone compound of synthetic, and its structural formula is as follows:
Pyruvate kinase is a crucial rate-limiting enzyme in the glycolysis, plays pivotal role in energy metabolism.The M2 type pyruvate kinase that the present invention relates to (being called for short PKM2) mainly is present in individual early development and the tumor tissues.The PKM2 inhibitor of having reported at present seldom, wherein micromolecular compound inhibitor only is compound 3 one examples (Vander Heiden MG, Christofk HR, Schuman E, Subtelny AO, Sharfi H, Harlow EE et al.Biochem Pharmacol2010,79,1118.), its amount (being IC50) that suppresses the work of 50% enzyme is 50 μ M.
Up to the present, also there is not the vitamin K compounds to suppress the report of Pyruvate kinase activity.
(3) summary of the invention
The purpose of this invention is to provide the new application of vitamin K in the preparation pyruvate kinase inhibitor.
The technical solution used in the present invention is: under the room temperature condition, the PKM2 of chemical compound and restructuring is hatched half an hour, can determine the effect that the medicine inhibitory enzyme is lived by the difference that detects matched group (only adding drug solvent DMSO) and each experimental group (interpolation medicine) PKM2 enzymatic activity.
Described vitamin K comprises vitamin K3 and vitamin K5.
Described pyruvate kinase is PKM2.
The PKM2 inhibitor of report has Compound 3 (molecular structure is as follows) at present, and the present invention finds that vitamin K3 and vitamin K5 suppress the effect of M2 type pyruvate kinase than Compound the last 3.Therefore, vitamin K3 and vitamin K5 itself have important using value as the inhibitor of acetone acid.
Beneficial effect of the present invention is mainly reflected in: two kinds of new pyruvate kinase inhibitors---vitamin K3 and vitamin K5 are provided, for new medicament screen provides the foundation, have had the important application prospect.
(4) specific embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Experiment material:
Vitamin K3 is available from Sigma company, and vitamin K5 is available from Wako company.
Restructuring PKM2: gene is cloned from breast cancer cell MCF-7, gene integration is arrived among the plasmid pQE-30 (available from Qiagen company), be transformed among the escherichia coli XL1 Blue (available from Qiagen company), check order through Invitrogen company, the ncbi database comparison, the gene that clones is PKM2 gene (SEQ ID NO.1) really, go out PKM2 albumen by IPTG (available from Gibco company) abduction delivering, use out PKM2 albumen (SEQ ID NO.2) of Ni-Sepharose chromatographic column (available from GE company) purification.
β-NADH, ADP and phosphoenolpyruvate (PEP) are available from Roche company;
Tris, HCl, KCl, MgCl are available from sky day company.
Dimethyl sulfoxine (DMSO), lactic acid dehydrogenase (LDH) are available from Sigma company.
Compound 3 is synthetic voluntarily, and synthetic method is as follows: add successively 5-aminosalicylic acid (5mmol) in the 25ml round-bottomed flask, 2,5-acetyl butyryl (6mmol), p-methyl benzenesulfonic acid (0.05mmol), toluene 10ml, reflux 1h.After reaction finished, unnecessary toluene was removed in decompression, gets the crude product column chromatography, and eluent is petroleum ether: the mixed solvent of ethyl acetate=1: 1, productive rate is about 70%.Product petroleum ether and re-crystallizing in ethyl acetate.Reaction equation:
Synthetic Compound 3 character: Orange solid, mp.167-169 ℃,
1H-NMR (CDCl
3) δ 10.45 (s, 1H), 7.83 (d, J=2.4Hz, 1H), 7.42-7.39 (m, 1H), 7.13 (d, J=8.8Hz, 1H), 5.93 (s, 2H), 2.06 (s, 6H) ppm; MS (ESI) m/z 232.0 ([M+H]
+).The product that the Mass Spectrometer Method proof is synthesized is Compound 3.
Experimental technique:
PKM2 is active in lactic acid dehydrogenase (LDH) coupled reaction system (Vander Heiden MG, Christofk HR, Schuman E, Subtelny AO, Sharfi H, Harlow EE et al.BiochemPharmacol 2010,79,1118.) detect, enzyme reaction solution comprises 50mM Tris-HCl (pH 7.5), 100mM KCl, 10mM MgCl
20.6mM PEP, 0.9mM ADP, 0.12mM β-NADH, 4.8units/ml LDH is in 20 minutes, judge the vigor of PKM2 catalytic action by the detection substrate β-minimizing of NADH content, known substrate β-NADH by ultraviolet excitation after, can launch 340nm fluorescence, the amount that 340nm fluorescence reduces is directly proportional with the amount of the minimizing of β-NADH content.PKM2 catalytic reaction formula is as follows:
The compound solution that 1 μ l variable concentrations is got in this experiment and 9 μ l concentration are that the PKM2 protein solution of 50pg/ μ l mixes, after hatching 30 minutes under the room temperature, join in the above-mentioned enzyme reaction solution of 125 μ l, detect 340nm fluorescent absorption amount, judge under the variable concentrations compound effects, the catalytic activity of PKM2 is when recycling software SigmaPlot 10.0 calculating inhibition 50% enzyme is lived, the concentration of vitamin K, i.e. IC50.This experiment has detected the activity of Compound 3 inhibition PKM2 simultaneously, and under same experiment condition, the effect and the Compound 3 that vitamin K are suppressed PKM2 contrast.
Experimental result:
Experimental result sees Table 1, and vitamin K3 is different with the IC50 that K5 suppresses PKM2, but all is lower than the IC50 (37.4 μ M) of Compound 3 inhibition PKM2 under the same experiment condition.Known IC50 is lower, and the dosage of the required inhibitor of inhibitory enzyme activity is less, and namely inhibitor effect is better.The present invention illustrates that the effect of these 2 kinds of vitamin Ks inhibition PKM2 far above the inhibitor Compound 3 of having reported for work, is the inhibitor of very effective PKM2.
Table 1: vitamin K3 and K5 and Compound 3 suppress the IC50 of PKM2
| Title | IC50(μM) |
| Vitamin K3 | 13 |
| Vitamin K5 | 10.1 |
| Compound 3 | 37.4 |
SEQUENCE LISTING
＜110〉Zhejiang University
＜120〉application of vitamin K in the preparation pyruvate kinase inhibitor
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Claims (1)
1. the application of vitamin K in the preparation pyruvate kinase inhibitor, it is characterized in that: described vitamin K is vitamin K3 or vitamin K5, described pyruvate kinase is M2 type pyruvate kinase.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010615924 CN102552228B (en) | 2010-12-30 | 2010-12-30 | Application of vitamin K in preparation of pyruvate kinase inhibitor |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010615924 CN102552228B (en) | 2010-12-30 | 2010-12-30 | Application of vitamin K in preparation of pyruvate kinase inhibitor |
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| Publication Number | Publication Date |
|---|---|
| CN102552228A CN102552228A (en) | 2012-07-11 |
| CN102552228B true CN102552228B (en) | 2013-09-18 |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101247800A (en) * | 2005-08-26 | 2008-08-20 | 明治乳业株式会社 | Anti-allergic agent |
| CN101830792A (en) * | 2010-05-10 | 2010-09-15 | 苏州康正生物医药有限公司 | Vitamin K3 derivatives serving as IDO inhibitor |
-
2010
- 2010-12-30 CN CN 201010615924 patent/CN102552228B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101247800A (en) * | 2005-08-26 | 2008-08-20 | 明治乳业株式会社 | Anti-allergic agent |
| CN101830792A (en) * | 2010-05-10 | 2010-09-15 | 苏州康正生物医药有限公司 | Vitamin K3 derivatives serving as IDO inhibitor |
Non-Patent Citations (2)
| Title |
|---|
| Identification of small molecule inhibitors of pyruvate kinase M2;Vander Heiden MG,et al;《Biochemical Pharmacology》;20091211;第79卷(第8期);1118-1124 * |
| Vander Heiden MG,et al.Identification of small molecule inhibitors of pyruvate kinase M2.《Biochemical Pharmacology》.2009,第79卷(第8期),1118-1124. |
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| CN102552228A (en) | 2012-07-11 |
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