CN105213362A - A kind of application of polyphenol compound - Google Patents

A kind of application of polyphenol compound Download PDF

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CN105213362A
CN105213362A CN201410310204.5A CN201410310204A CN105213362A CN 105213362 A CN105213362 A CN 105213362A CN 201410310204 A CN201410310204 A CN 201410310204A CN 105213362 A CN105213362 A CN 105213362A
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cancer
carcinoma
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mtorc1
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康经武
张艳梅
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Shanghai Institute of Organic Chemistry of CAS
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Shanghai Institute of Organic Chemistry of CAS
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Abstract

The invention discloses a kind of application of polyphenol compound.Disclosed by the inventionly can be used for preparing mTOR inhibitors such as formula the polyphenol compound shown in I, especially mTORC1 inhibitor.Polyphenol compound disclosed by the invention is preparing the application prevented and/or treated in cancer, metabolism disturbance syndrome, neurodegenerative diseases or anti-inflammatory drugs.Described disease comprises the disease that this area is prevented and/or treated by the catalytic activity of the enzyme suppressing mTORC1, preferred cancer, metabolism disturbance syndrome, neurodegenerative diseases or inflammation.Polyphenol compound of the present invention effectively can suppress the catalytic activity of the enzyme of mTOR, shows good curative effect to cancer, metabolism disturbance syndrome, neurodegenerative diseases or inflammation.

Description

A kind of application of polyphenol compound
Technical field
The present invention relates to a kind of application of polyphenol compound.
Background technology
Mammal rapamycin target protein (mTOR) is a kind of serine/threonine protein kitase of atypia high conservative, be otherwise known as the associated proteins (FRAP) of FKBP12 (FKBP12)-rapamycin complex, or the target proteins of rapamycin and FKBP12 (RAFT), belongs to Phosphoinoside kinase related protein kinase enzyme family (Phosphatidylinositol3-kinase (PI3K)-likekinase (PIKK) superfamilyofkinases).In cell, mainly through forming mammal rapamycin target protein complex 1 (mTORC1) albumen composition different with mammal rapamycin target protein complex 2 (mTORC2) two kinds of functions, play its physiologically active.MTORC1 comprises mTOR, Raptor, Deptor, PRAS40 and mLST8, and mTORC2 comprises mTOR, Riptor, Deptor, Protor, mSin1 and mLST8.The major function of mTORC1 is synthesis and the cell cycle progression of Function protein matter, and mTORC2 then plays a significant role in actin cytoskeleton tissue and cell survival.
MTOR is an effector molecule of PI3K/Akt/mTOR signal path, and it regulates maincenter as metabolism and growth important in cell, participates in the cellular physiological processes that cell differentiation and survival, protein synthesis, apoptosis, cell autophagy etc. are important.The regulation and control of mTOR signal path are abnormal to be occurred closely related with tumor, by the transduction suppressing mTOR path effectively can block various somatomedin abnormal signal, thus suppress generation, the development of cancer, mTOR has become the important target of Therapeutic cancer, therefore, the screening of its inhibitor is seemed particularly important.
The inhibitor of mTOR mainly contains two classes, one class is allosteric inbibitor, mainly rapamycin and analog thereof, by forming complex with FKBP12, targeting is attached to the non-phosphorylating catalytic site territory of mTOR and suppresses the formation of its kinase activity and mTOR complex again, tumor cell is arrested in the G1 phase, thus inhibition tumor cell grows and blocks its cell proliferation and even impels apoptosis.Another kind of is the micromolecular inhibitor of ATP competitive type, belongs to micromolecule ATP analog, can with the kinase domain of ATP competition binding mTOR, thus suppress kinase activity and the autophosphorylation of mTOR, suppress the activity of mTORC1 and mTORC2 simultaneously.
According to " Zhang, Y.J.DrugDiscovToday2011,325-331 and Benjamin, D.NatRevDrugDiscov2011,868-880 " report, the framing structure of mTOR inhibitors is macrolides compound and/or nitrogen-containing heterocycle compound.Wherein, macrolides compound belongs to allosteric inbibitor, mainly rapamycin and analog thereof, although this compounds has been successfully applied to the treatment of tumor as medicine, but antitumor spectra is narrow, be only used to some cancer such as treatment lymphatic cancer, renal carcinoma and carcinoma of endometrium, simultaneously, such inhibitor also can activate the survival pathway of mTOR dependence thus cause therapeutic effect bad, shows Drug resistance clinically.And the micromolecular inhibitor of ATP competitive type mainly nitrogen-containing heterocycle compound, but the ATP competitive type inhibitor specificity of part nitrogen-containing heterocycle compound is lower, easily produces cytotoxicity to normal cell; And in clinical experiment, cytoactive is weak, blood drug level is low.In sum, find that new mTOR inhibitors is subject to the extensive concern of academia and pharmaceutical industry.
Summary of the invention
Technical problem to be solved by this invention is narrow in order to overcome the existing inhibitor antitumor spectra for the mTOR of oncotherapy, has drug resistance; Specificity is lower, easily produces cytotoxicity to normal cell; The defects such as in clinical experiment, cytoactive is weak, and blood drug level is low, and provide a kind of application of polyphenol compound.Polyphenol compound of the present invention can be used for preparing mTOR inhibitors, especially mTORC1 inhibitor.Polyphenol compound of the present invention also can be used for preparing the medicine preventing and/or treating cancer, metabolism disturbance syndrome, neurodegenerative diseases or inflammation.Polyphenol compound of the present invention effectively can suppress the catalytic activity of the enzyme of mTOR, and its minimal inhibitory concentration is nanomole level, shows good curative effect to cancer, metabolism disturbance syndrome, neurodegenerative diseases or inflammation.
The invention provides and a kind ofly preparing the application in mTOR inhibitors such as formula the polyphenol compound shown in I, its stereoisomer, tautomer, hydrate or pharmaceutically acceptable salt;
Wherein, R 1, R 2, R 3, R 4, R 5, R 6, R 7, R 8, R 9, R 10, R 11, R 12, R 13, R 14and R 15be hydrogen, halogen (preferred fluorine, chlorine, bromine or iodine), substituted or unsubstituted C independently of one another 1~ C 20alkyl, substituted or unsubstituted C 3~ C 20cycloalkyl, substituted or unsubstituted C 2~ C 20thiazolinyl, substituted or unsubstituted C 3~ C 20cycloalkenyl group, substituted or unsubstituted C 2~ C 20alkynyl, substituted or unsubstituted C 5~ C 20aryl, substituted or unsubstituted C 2~ C 20heteroaryl, substituted or unsubstituted C 2~ C 20heterocyclic radical ,-cyano group, nitro, or r 16and R 17be hydrogen, substituted or unsubstituted C independently of one another 1~ C 20alkyl, substituted or unsubstituted C 3~ C 20cycloalkyl, substituted or unsubstituted C 2~ C 20thiazolinyl, substituted or unsubstituted C 3~ C 20cycloalkenyl group, substituted or unsubstituted C 2~ C 20alkynyl, substituted or unsubstituted C 5~ C 20aryl, substituted or unsubstituted C 2~ C 20heteroaryl, or, substituted or unsubstituted C 2~ C 20heterocyclic radical;
Described " the C of replacement 1~ C 20alkyl ", the described " C of replacement 3~ C 20cycloalkyl ", the described " C of replacement 2~ C 20thiazolinyl ", the described " C of replacement 3~ C 20cycloalkenyl group ", the described " C of replacement 2~ C 20alkynyl ", the described " C of replacement 5~ C 20aryl ", the described " C of replacement 2~ C 20heteroaryl ", and the described " C of replacement 2~ C 20heterocyclic radical " described in " replacement " refer to and to be replaced by one or more substituents: C 1~ C 4alkyl (described C 1~ C 4alkyl be preferably methyl, ethyl, n-pro-pyl, isopropyl, normal-butyl, isobutyl group or the tert-butyl group), C 1~ C 4alkoxyl (described C 1~ C 4alkoxyl be preferably methoxyl group, ethyoxyl, positive propoxy, isopropoxy, n-butoxy, isobutoxy or tert-butoxy), halogen (described halogen preferably for fluorine, chlorine, bromine or iodine), hydroxyl or amino, when substituent group is multiple, described substituent group is identical or different;
represent singly-bound or double bond; represent singly-bound or nothing;
R 1, R 2, R 3, R 4, R 5, R 6, R 7, R 8, R 9, R 10, R 11, R 12, R 13and R 14in at least three be hydroxyl.
Described " substituted or unsubstituted C 1~ C 20alkyl " be preferably substituted or unsubstituted C 1~ C 10alkyl, be more preferably substituted or unsubstituted C 1~ C 4alkyl.Described substituted or unsubstituted C 1~ C 4alkyl be preferably substituted or unsubstituted methyl, substituted or unsubstituted ethyl, substituted or unsubstituted n-pro-pyl, substituted or unsubstituted isopropyl, substituted or unsubstituted normal-butyl, substituted or unsubstituted isobutyl group, or, the substituted or unsubstituted tert-butyl group.
Described " substituted or unsubstituted C 3~ C 20cycloalkyl " be preferably substituted or unsubstituted C 3~ C 10cycloalkyl, be more preferably substituted or unsubstituted C 3~ C 6cycloalkyl.Described substituted or unsubstituted C 3~ C 6cycloalkyl be preferably substituted or unsubstituted cyclopropyl, substituted or unsubstituted cyclobutyl, substituted or unsubstituted cyclopenta, or, substituted or unsubstituted cyclohexyl.Described " substituted or unsubstituted C 2~ C 20thiazolinyl " be preferably substituted or unsubstituted C 2~ C 10thiazolinyl, be more preferably substituted or unsubstituted C 2~ C 4thiazolinyl.Described substituted or unsubstituted C 2~ C 4thiazolinyl be preferably substituted or unsubstituted vinyl, substituted or unsubstituted acrylic, substituted or unsubstituted pi-allyl, substituted or unsubstituted 1-butylene, 2-butylene, or, substituted or unsubstituted isobutene..
Described " substituted or unsubstituted C 3~ C 20cycloalkenyl group " be preferably substituted or unsubstituted C 3~ C 10cycloalkenyl group, be more preferably substituted or unsubstituted C 3~ C 6cycloalkenyl group.Described substituted or unsubstituted C 3~ C 6cycloalkenyl group be preferably substituted or unsubstituted cyclopropanyl, substituted or unsubstituted cyclobutane base, substituted or unsubstituted cyclopentenyl, or, substituted or unsubstituted cyclobutane base.
Described " substituted or unsubstituted C 2~ C 20alkynyl " be preferably substituted or unsubstituted C 2~ C 10alkynyl, be more preferably substituted or unsubstituted C 2~ C 4alkynyl.Described substituted or unsubstituted C 2~ C 4alkynyl be preferably substituted or unsubstituted acetenyl, substituted or unsubstituted propinyl or, substituted or unsubstituted 2-butyne base.
Described " substituted or unsubstituted C 5~ C 20aryl " be preferably substituted or unsubstituted C 5~ C 10aryl.Described substituted or unsubstituted C 5~ C 10aryl be preferably substituted or unsubstituted phenyl, or, substituted or unsubstituted naphthyl.
Described " substituted or unsubstituted C 2~ C 20heteroaryl " preferably refer to that hetero atom is N, O or S, hetero atom number is the substituted or unsubstituted C of 1 ~ 4 2~ C 20heteroaryl.It is described that " hetero atom is N, O or S, and hetero atom number is the substituted or unsubstituted C of 1 ~ 4 2~ C 20heteroaryl " preferably refer to that hetero atom is N, O or S, hetero atom number is the C of 1 or 2 2~ C 6substituted or unsubstituted heteroaryl.Described substituted or unsubstituted C 2~ C 6heteroaryl be preferably substituted or unsubstituted pyrrole radicals, substituted or unsubstituted furyl, substituted or unsubstituted thienyl, or, substituted or unsubstituted pyrimidine radicals.
Described " substituted or unsubstituted C 2~ C 20heterocyclic radical " preferably refer to that hetero atom is N, O or S, hetero atom number is the substituted or unsubstituted C of 1 ~ 4 2~ C 20heterocyclic radical.It is described that " hetero atom is N, O or S, and hetero atom number is the substituted or unsubstituted C of 1 ~ 4 2~ C 20heterocyclic radical " preferably refer to that hetero atom is N or O, hetero atom number is the C of 1 or 2 2~ C 6substituted or unsubstituted heterocyclic radical.Described substituted or unsubstituted C 2~ C 6heterocyclic radical be preferably substituted or unsubstituted nafoxidine base, substituted or unsubstituted tetrahydrofuran base, or, substituted or unsubstituted THP trtrahydropyranyl.
Preferably, such as formula in the polyphenol compound shown in I, its stereoisomer, tautomer, hydrate or pharmaceutically acceptable salt,
R 2, R 3, R 6, R 7, R 11and R 12be hydroxyl, R 1, R 4, R 5, R 8, R 9, R 10, R 13and R 14be hydrogen; R 15for carboxyl; represent double bond; indicate without;
Or, R 2, R 3, R 7, R 11and R 12be hydroxyl, R 1, R 4, R 5, R 8, R 9, R 10, R 13and R 14be hydrogen; R 15for carboxyl; represent singly-bound; represent singly-bound; R 6represention oxygen atom; It is following arbitrary compound:
Described mTOR inhibitors is preferably mTORC1 inhibitor.
Present invention also offers a kind of described application prevented and/or treated in cancer, metabolism disturbance syndrome, neurodegenerative diseases or anti-inflammatory drugs in preparation such as formula the polyphenol compound shown in I, its stereoisomer, tautomer, hydrate or pharmaceutically acceptable salt.Described disease comprises the disease that this area prevents and/or treats by suppressing the enzyme of mTORC1, the preferred cancer of the present invention, metabolism disturbance syndrome, neurodegenerative diseases or inflammation are (see Cornu, M.Curropingenetdev2013,23 (1), 53-62. and Dazert, E.CurrOpinCellBiol2011,744-755).
Described cancer preferably comprises breast carcinoma, pulmonary carcinoma, colon cancer, renal cell carcinoma, nonsmall-cell lung cancer, pancreas neuroendocrine carcinoma, stomach esophageal carcinoma, hepatocarcinoma, T cell lymphatic cancer, carcinoma of prostate, carcinoma of testis, genitourinary cancer, ovarian cancer, cervical cancer, glioblastoma, thyroid carcinoma, skin carcinoma, bladder cancer, cancer of pancreas, melanoma, colorectal cancer, rectal cancer, leukemia, hepatocarcinoma, carcinoma of small intestine, colorectal cancer, brain and central nervous system's cancer or cancer of bile ducts.
Described metabolism disturbance syndrome preferably comprises hypertension, diabetes, coronary heart disease, apoplexy, obesity or accelerates atherosclerotic vascular disease.
Described neurodegenerative diseases preferably comprises Alzheimer, Parkinson's disease, Heng Tingdunshi disease, amyotrophic lateral sclerosis, cerebral atrophy, multiple sclerosis or spinal muscular atrophy.
Described inflammation preferably comprises rheumatoid arthritis, psoriasis, lymphadenitis, hepatitis, pneumonia or nephritis.
Present invention also offers a kind of pharmaceutical composition, it comprises described such as formula the polyphenol compound shown in I, its stereoisomer, tautomer, hydrate or pharmaceutically acceptable salt and physiology thereof or pharmaceutically acceptable carrier.Wherein, the described quality such as formula the polyphenol compound shown in I, its stereoisomer, tautomer, hydrate or pharmaceutically acceptable salt is for more than treatment effective dose, and be generally 0.01% ~ 99%, described percentage ratio refers to mass percent.Described carrier can be the physiology of any appropriate or pharmaceutically acceptable excipient substance.Described excipient substance can be the excipient substance that this area routine is suitable for, and is preferably one or more in thickening agent, filler, diluent and pharmaceutical carrier.Described thickening agent is preferably glucosan or derivatives thereof.Described pharmaceutical carrier is preferably gelatine microsphere and/or liposome.
Present invention also offers a kind of aforementioned pharmaceutical compositions and prepare the application in mTOR inhibitors.
Described mTOR inhibitors is preferably mTORC1 inhibitor.
Present invention also offers a kind of aforementioned pharmaceutical compositions and prepare the application prevented and/or treated in cancer, metabolism disturbance syndrome, neurodegenerative diseases or anti-inflammatory drugs.Described disease comprises the disease that this area prevents and/or treats by suppressing the enzyme of mTORC1, the preferred cancer of the present invention, metabolism disturbance syndrome, neurodegenerative diseases or inflammation.
Described cancer preferably comprises breast carcinoma, pulmonary carcinoma, colon cancer, renal cell carcinoma, nonsmall-cell lung cancer, pancreas neuroendocrine carcinoma, stomach esophageal carcinoma, hepatocarcinoma, T cell lymphatic cancer, carcinoma of prostate, carcinoma of testis, genitourinary cancer, ovarian cancer, cervical cancer, glioblastoma, thyroid carcinoma, skin carcinoma, bladder cancer, cancer of pancreas, melanoma, colorectal cancer, rectal cancer, leukemia, hepatocarcinoma, carcinoma of small intestine, colorectal cancer, brain and central nervous system's cancer or cancer of bile ducts.
Described metabolism disturbance syndrome preferably comprises hypertension, diabetes, coronary heart disease, apoplexy, obesity or accelerates atherosclerotic vascular disease.
Described neurodegenerative diseases preferably comprises Alzheimer, Parkinson's disease, Heng Tingdunshi disease, amyotrophic lateral sclerosis, cerebral atrophy, multiple sclerosis or spinal muscular atrophy.
Described inflammation preferably comprises rheumatoid arthritis, psoriasis, lymphadenitis, hepatitis, pneumonia or nephritis.
Described medicine can be any applicable regular dosage form.Described medicine can only using such as formula the polyphenol compound shown in I, its stereoisomer, tautomer, hydrate or pharmaceutically acceptable salt as sole active agent, also can also containing except such as formula shown in I such as formula other active component except the polyphenol compound shown in I, its stereoisomer, tautomer, hydrate or pharmaceutically acceptable salt.Other described active component is for not having harmful effect can combine applicable other active component (as antagonism etc.), preferably for having the medicine preventing and/or treating cancer, metabolism disturbance syndrome, neurodegenerative diseases or inflammation such as formula the polyphenol compound shown in I, its stereoisomer, tautomer, hydrate or pharmaceutically acceptable salt.
In the present invention, described such as formula the polyphenol compound shown in I, its stereoisomer, tautomer, hydrate or pharmaceutically acceptable salt can be that the preparation method of this area routine obtains such as formula the polyphenol compound shown in I, its stereoisomer, tautomer, hydrate or pharmaceutically acceptable salt, or commercially available such as formula the polyphenol compound shown in I, its stereoisomer, tautomer, hydrate or pharmaceutically acceptable salt (purity: HPLC >=98%).
Without prejudice to the field on the basis of common sense, above-mentioned each optimum condition, can combination in any, obtains the preferred embodiments of the invention.
Agents useful for same of the present invention and raw material are all commercially.
Positive progressive effect of the present invention is:
Present invention finds a kind of novelty teabag such as formula the polyphenol compound shown in I, i.e. the purposes of above-claimed cpd in preparation mTORC1 inhibitor, above-mentioned polyphenol compound effectively can suppress the enzymatic activity of mTORC1.Utilize and contain such as formula the Drug therapy cancer of the polyphenol compound shown in I as active fraction preparation, metabolism disorder syndrome, nerve degenerative disease or inflammation, all can obtain significant curative effect.
Accompanying drawing explanation
Figure 1A is that the laser-induced fluorescence (LIF) of 2 μMs of mTORC1 peptide substrate systems absorbs chromatogram.
Figure 1B adds 31.1nMmTORC1 albumen in 2 μMs of mTORC1 peptide substrate systems, and the laser-induced fluorescence (LIF) of 25 DEG C of reactions kinase reaction solution of gained after 20 minutes absorbs chromatogram.
Fig. 1 C for add 0.5 μM of salvianolic acid A in mTORC1 catalytic substrate peptide phosphorylation reaction system, and the laser-induced fluorescence (LIF) of 25 DEG C of reactions kinase reaction solution of gained after 20 minutes absorbs chromatogram.
Fig. 1 D for add 0.5 μM of salvianolic acid C in mTORC1 catalytic substrate peptide phosphorylation reaction system, and the laser-induced fluorescence (LIF) of 25 DEG C of reactions kinase reaction solution of gained after 20 minutes absorbs chromatogram.
Fig. 2 A is the uv absorption chromatogram of the kinase reaction solution of gained after mTORC1 catalytic substrate peptide phosphorylation reaction.
Fig. 2 B is the fluorescent absorption chromatogram of the kinase reaction solution of gained after mTORC1 catalytic substrate peptide phosphorylation reaction.
Fig. 2 C is the BasePeak chromatogram of the kinase reaction solution of gained after mTORC1 catalytic substrate peptide phosphorylation reaction.
Fig. 2 D is that in the kinase reaction solution of gained after mTORC1 catalytic substrate peptide phosphorylation reaction, substrate extracts flow chromatography figure.
Fig. 2 E is that in the kinase reaction solution of gained after mTORC1 catalytic substrate peptide phosphorylation reaction, product extracts flow chromatography figure.
Fig. 3 is the mass spectrum of mTORC1 peptide substrate.
Fig. 4 is the product mass spectra figure of mTORC1 catalytic substrate peptide phosphorylation reaction.
Fig. 5 is the second order ms figure of the product of mTORC1 catalytic substrate peptide phosphorylation reaction.
Fig. 6 is the suppression curve of salvianolic acid A to mTORC1 catalytic substrate peptide phosphorylation reaction activity.
Fig. 7 is the suppression curve of salvianolic acid C to mTORC1 catalytic substrate peptide phosphorylation reaction activity.
Detailed description of the invention
Mode below by embodiment further illustrates the present invention, but does not therefore limit the present invention among described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example, conventionally and condition, or selects according to catalogue.
Embodiment 1
(sequence of described peptide substrate is as shown in SEQ ID NO:1 for peptide substrate STTPGGTLFSTTPG, mTOR kinases method of testing see PerkinElmer company), by gill, biochemical (Shanghai) Co., Ltd. synthesizes this peptide substrate, and CF (5-FAM) labelling is carried out to its N end, so that the sensitivity that fluoroscopic examination and raising detect, the peptide segment structure of gained peptide substrate is such as formula shown in II:
The mTORC1 used in embodiment purchased from sigma reagent company limited, product article No.: SRP0364; Salvianolic acid A purchased from Shanghai Yuan Ye Reagent Company, product article No.: YY90055; Salvianolic acid C purchased from Shanghai Yuan Ye Reagent Company, product article No.: YY90623.
Preparation enzyme reaction buffer solution 25mL:50mM4-hydroxyethyl piperazine ethanesulfonic acid (Hepes), pH value is adjusted to 7.5 by 4MNaOH, 2mM1,4-dithiothreitol dithio, 10mM magnesium chloride, 1mM ethylene glycol bis (2-amino-ethyl ether) tetraacethyl, 0.01% (v/v, lower same) polysorbas20 and 3mM manganese chloride.
2 μ L enzyme reaction buffer solution are used to dissolve peptide substrate (containing 5 × 10 -8m fluorescein sodium, as interior mark, improves quantitative accuracy) and mTORC1.Wherein, the concentration of peptide substrate is 2 μMs, and the concentration of mTORC1 is 31.1nM.
Preparation is utilized to have the capillary electrophoresis apparatus of laser induced fluorescence detector (CE) to be separated the reaction system of 31.1nMmTORC1 and 2 μM of mTORC1 peptide substrate, and investigate and there is not polyphenol compound (namely there is not salvianolic acid A or salvianolic acid C) (adding 0.25% dimethyl sulfoxide in contrast) and the difference adding this reaction system after 0.5 μM of polyphenol compound (salvianolic acid A or salvianolic acid C, 0.25% dmso solution polyphenol compound) respectively in reaction system.
Experiment instrument is P/ACEMDQ capillary electrophoresis apparatus (BeckmanCoulter, CA, USA), is furnished with laser induced fluorescence detector, argon laser source, excitation wavelength 488nm, emission wavelength 520nm; Data are by being equipped with the computer acquisition of 32KaratSoftware; Fused-silica capillary column specification is 50 μm of I.D. (370 μm of O.D.) × 30cm (effective column length 19.5cm, PolymicroTechnologies, AZ, USA).As not having special indicating, the deposition condition of all analyses is all: column temperature 25 DEG C, opposite ends hydrodynamic injection.
Result is as shown in Figure 1A ~ 1D, and wherein Figure 1A is the laser-induced fluorescence (LIF) absorption chromatogram of 2 μMs of mTORC1 peptide substrates.Figure 1B adds 31.1nMmTORC1 in 2 μMs of mTORC1 peptide substrate systems, and the laser-induced fluorescence (LIF) of 25 DEG C of reactions kinase reaction solution of gained after 20 minutes absorbs chromatogram.Fig. 1 C adds 0.5 μM of salvianolic acid A in the reaction system of 31.1nMmTORC1 and 2 μM of mTORC1 peptide substrate, and the laser-induced fluorescence (LIF) of 25 DEG C of reactions kinase reaction solution of gained after 20 minutes absorbs chromatogram.Fig. 1 D adds 0.5 μM of salvianolic acid C in the reaction system of 31.1nMmTORC1 and 2 μM of mTORC1 peptide substrate, and the laser-induced fluorescence (LIF) of 25 DEG C of reactions kinase reaction solution of gained after 20 minutes absorbs chromatogram.
Result shows in the reaction system comprising 31.1nMmTORC1 and 2 μM of mTORC1 peptide substrate, add polyphenol compound (salvianolic acid A or salvianolic acid C) respectively, can effectively suppress mTORC1 to the phosphorylation reaction of peptide substrate.The peptide substrate phosphorylation reaction that mTORC1 catalysis 5-FAM modifies is as follows:
In order to prove that laser-induced fluorescence (LIF) absorbs the ownership at each peak in chromatogram further, prove the generation of Phosphorylated products, we utilize the technology of high performance liquid chromatography (HPLC)-UV-detector (UV)-fluorescence detector (FLD) and Electrospray ion trap mass spectrometry (ESI/MS) to test mTORC1 catalytic substrate peptide phosphorylation reaction, confirm the existence of Phosphorylated products peptide further.
Testing conditions is: Agilent 1260 liquid chromatographic system, is furnished with UV-detector (UV) and fluorescence detector (FLD); Chromatographic column is AgilentZORBAXEclipseXDB-C18 post (2.1mm × 150mm, 3.5-Micron); UV determined wavelength is 220nm; FLD excitation wavelength is 488nm, and emission wavelength is 520nm; Sample size 5 μ L; Flow velocity 0.3mL/min; Column temperature 30 DEG C.Mobile phase A and B are respectively water and acetonitrile, and both are all containing 0.1% formic acid (v/v, lower same).Adopt gradient elution, be specially: 0-20 minute, 20%-40%B; 20-25 minute, 40%-100%B.HPLC and LCQ-Fleet ion trap mass spectrometry (ThermoScientific, CA) coupling, all mass spectrums obtain all in the positive-ion mode; Spray voltage is 4.5kV; Capillary voltage is 30V; Capillary temperature is 320 DEG C.Testing result is as shown in Fig. 2 A ~ 2E, Fig. 3, Fig. 4 and Fig. 5, and Fig. 2 A is the uv absorption chromatogram of the kinase reaction solution of gained after mTORC1 catalytic substrate peptide phosphorylation reaction.Fig. 2 B is the fluorescent absorption chromatogram of the kinase reaction solution of gained after mTORC1 catalytic substrate peptide phosphorylation reaction.Fig. 2 C is the BasePeak chromatogram of the kinase reaction solution of gained after mTORC1 catalytic substrate peptide phosphorylation reaction.Fig. 2 D is that in the kinase reaction solution of gained after mTORC1 catalytic substrate peptide phosphorylation reaction, substrate extracts flow chromatography figure.Fig. 2 E is that in the kinase reaction solution of gained after mTORC1 catalytic substrate peptide phosphorylation reaction, product extracts flow chromatography figure.Fig. 3 is the mass spectrum of the peptide substrate of mTORC1.Fig. 4 is the mass spectrum of mTORC1 catalytic substrate peptide phosphorylation reaction products therefrom.Fig. 5 is the second order ms figure of mTORC1 catalytic substrate peptide phosphorylation reaction products therefrom.In Fig. 3, peptide substrate molecular ion peak theoretical value is 1681.71, and measured value is 841.65 ([M+2H] 2+).In Fig. 4, Phosphorylated products molecular ion peak theoretical value is 1759.67, and measured value is 881.30 ([M+2H] 2+).The above results shows that mTORC1 can the phosphorylation reaction of effective its peptide substrate of catalysis.Secondary fragment ion pair substrate phosphorylation site according to mTORC1 catalytic substrate peptide phosphorylation reaction products therefrom is confirmed, from the information of Fig. 5 fragment ion, the site of peptide substrate generation phosphorylation can only be 12nd amino acids in short peptide sequence-threonine.Wherein, b ion and y ion refer to polypeptide ion MS/MS collision indoor dissociate through inert gas collide time, peptide chain amido link place fracture produce ion, through such process formed N hold ion be b ion, C hold ion be y ion.
When inhibitory action research is carried out to the catalytic activity of enzyme, need the polyphenol compound (salvianolic acid A or salvianolic acid C) adding variable concentrations in reaction system respectively, carry out phosphorylation reaction, polyphenol compound (salvianolic acid A or salvianolic acid C) toward in reaction system to add concentration as shown in table 1:
The interpolation concentration of table 1 polyphenol compound
Experimental technique is: new capillaries post is used 0.1MNaOH solution-treated 30 minutes under 30psi pressure condition, then rinses 10 minutes respectively with ultra-pure water, back-ground electolyte respectively.3 minutes are respectively rinsed with 1MNaOH, ultra-pure water, back-ground electolyte under being interposed between 30psi pressure condition between each run.Sample is sample introduction 5s under 0.2psi condition; Separation voltage is-15kV; Column temperature maintains 25 DEG C.Dissociating buffer is 100mMHepes, and pH value is adjusted to 7.5 by NaOH, and fluorescein sodium is marked with correction sample size in being.
The activity of mTORC1 is determined by the generation detecting Phosphorylated products peptide, namely by the mensuration of Phosphorylated products peptide calibration peak area (calibration peak area and Phosphorylated products peptide and interior target peak area ratio), and can the activity of quantitative assay mTORC1.For inhibit activities research or inhibitor screening, nanomole is joined in reaction solution respectively to the above-mentioned polyphenol compound of micro-molar concentration (salvianolic acid A or salvianolic acid C), the inhibit activities of above-claimed cpd can by compared with blank (namely not adding polyphenol compound), minimizing according to Phosphorylated products peptide peak area is determined, the suppression percentage ratio of above-claimed cpd can pass through following formulae discovery:
I % = ( 1 - A x A 0 ) × 100 % .
Wherein, I% suppresses percent; A xand A obe the peak area of Phosphorylated products peptide when adding inhibitor and do not add any inhibitor respectively, get the IC of half-inhibition concentration that when inhibitory action percentage ratio is 50%, corresponding inhibitor concentration obtains and inhibitor 50value.
Through inspection determine, when the peptide substrate concentration of mTORC1 is 2 μMs, when mTORC1 concentration is 31.1nM and 46.7nM, incubation time in 0-20 minute, product formation and incubation time proportional; When mTORC1 concentration is 31.1nM, when incubation time is 20 minutes, substrate conversion efficiency is no more than 10%, meets the initial velocity condition of enzyme kinetics.Also inspection determines that the reaction efficiency of mTORC1 catalytic substrate peptide phosphorylation reaction at 25 DEG C is higher than 37 DEG C simultaneously.Final selected reaction system is: the concentration of the peptide substrate of mTORC1 is 2 μMs, and the concentration of mTORC1 is 31.1nM, and incubation time is 20 minutes, is no more than 10% condition carries out following inhibitor sifting work at the conversion ratio of substrate.
According to shown in table 1, the polyphenol compound (salvianolic acid A or salvianolic acid C) of variable concentrations is joined in above-mentioned reaction system respectively and carries out enzymatic reaction (in reaction system, the concentration of the peptide substrate of mTORC1 is 2 μMs, the concentration of mTORC1 is 31.1nM, incubation time is 20 minutes), detect polyphenol compound (salvianolic acid A or salvianolic acid C) to the suppression percentage ratio of mTORC1 activity according to above-mentioned high performance capillary electrophoresis, testing result is respectively as shown in table 2 and table 3.
The salvianolic acid A of table 2 variable concentrations suppresses percentage ratio to mTORC1 activity
Salvianolic acid A concentration (nmol/L) 0.05 0.5 5 50 125 375 500 1×10 3 1×10 4
Suppress percentage ratio (%) 0 0 0 0 40 79 95 100 100
The salvianolic acid C of table 3 variable concentrations suppresses percentage ratio to mTORC1 activity
By the suppression percentage result of above-mentioned salvianolic acid A, universal data processing software Origin8.0 non-linear fitting method is utilized to process, obtain the suppression curve of salvianolic acid A to mTORC1, wherein salvianolic acid A to the suppression curve of mTORC1 catalytic substrate peptide phosphorylation reaction activity as shown in Figure 6.By the suppression percentage result of above-mentioned salvianolic acid C, universal data processing software Origin8.0 non-linear fitting method is utilized to process, obtain the suppression curve of salvianolic acid C to mTORC1, wherein salvianolic acid C to the suppression curve of mTORC1 catalytic substrate peptide phosphorylation reaction activity as shown in Figure 7.
According to above-mentioned testing result and suppression curve, calculate the half-inhibition concentration IC of salvianolic acid A or salvianolic acid C 50value is respectively: the IC of salvianolic acid A 50value is 150nM, the IC of salvianolic acid C 50value is 165nM, and experimental result shows: salvianolic acid A and salvianolic acid C can suppress mTORC1 active to the phosphorylation reaction of peptide substrate effectively, have extremely strong mTORC1 inhibit activities, can be used in preparation mTORC1 inhibitor.
Comparative example 1
According to the identical method of embodiment 1, the salvianolic acid B of variable concentrations, rosmarinic acid, alkannic acid, danshensu and protocatechualdehyde are joined respectively in the identical reaction system of embodiment 1 and carry out enzymatic reaction (in reaction system, the concentration of the peptide substrate of mTORC1 is 2 μMs, the concentration of mTORC1 is 31.1nM, incubation time is 20 minutes), detect salvianolic acid B, rosmarinic acid, alkannic acid, danshensu and protocatechualdehyde to the suppression percentage ratio of mTORC1 activity according to the high performance capillary electrophoresis in embodiment 1.
Experimental result shows that these 5 kinds of compounds are when concentration is 1.25 μMs, to the catalytic activity of the enzyme of mTORC1 without any inhibitory action.

Claims (10)

1. preparing the application in mammal rapamycin target protein inhibitor such as formula the polyphenol compound shown in I, its stereoisomer, tautomer, hydrate or pharmaceutically acceptable salt for one kind;
Wherein, R 2, R 3, R 6, R 7, R 11and R 12be hydroxyl, R 1, R 4, R 5, R 8, R 9, R 10, R 13and R 14be hydrogen; R 15for carboxyl; represent double bond; indicate without;
Or, R 2, R 3, R 7, R 11and R 12be hydroxyl, R 1, R 4, R 5, R 8, R 9, R 10, R 13and R 14be hydrogen; R 15for carboxyl; represent singly-bound; represent singly-bound; R 6for oxygen atom;
Described is following arbitrary compound such as formula the compound shown in I;
2. as claimed in claim 1 such as formula the application in preparation mammal rapamycin target protein inhibitor of the polyphenol compound shown in I, its stereoisomer, tautomer, hydrate or pharmaceutically acceptable salt, it is characterized in that, described mammal rapamycin target protein inhibitor is mammal rapamycin target protein complex 1 inhibitor.
3. preparing such as formula the polyphenol compound shown in I, its stereoisomer, tautomer, hydrate or pharmaceutically acceptable salt the application prevented and/or treated in cancer, metabolism disturbance syndrome, neurodegenerative diseases or anti-inflammatory drugs as claimed in claim 1 for one kind.
4. apply as claimed in claim 3, it is characterized in that, described cancer comprises breast carcinoma, pulmonary carcinoma, colon cancer, renal cell carcinoma, nonsmall-cell lung cancer, pancreas neuroendocrine carcinoma, stomach esophageal carcinoma, hepatocarcinoma, T cell lymphatic cancer, carcinoma of prostate, carcinoma of testis, genitourinary cancer, ovarian cancer, cervical cancer, glioblastoma, thyroid carcinoma, skin carcinoma, bladder cancer, cancer of pancreas, melanoma, colorectal cancer, rectal cancer, leukemia, hepatocarcinoma, carcinoma of small intestine, colorectal cancer, brain and central nervous system's cancer or cancer of bile ducts;
And/or described metabolism disturbance syndrome comprises hypertension, diabetes, coronary heart disease, apoplexy, obesity or accelerates atherosclerotic vascular disease;
And/or described neurodegenerative diseases comprises Alzheimer, Parkinson's disease, Heng Tingdunshi disease, amyotrophic lateral sclerosis, cerebral atrophy, multiple sclerosis or spinal muscular atrophy;
And/or described inflammation comprises rheumatoid arthritis, psoriasis, lymphadenitis, hepatitis, pneumonia or nephritis.
5. a pharmaceutical composition, it comprises as claimed in claim 1 such as formula the polyphenol compound shown in I, its stereoisomer, tautomer, hydrate or pharmaceutically acceptable salt, and physiology or pharmaceutically acceptable carrier.
6. pharmaceutical composition as claimed in claim 5, it is characterized in that, described carrier is the physiology of any appropriate or pharmaceutically acceptable excipient substance; Described excipient substance is one or more in thickening agent, filler, diluent and pharmaceutical carrier.
7. the application of the pharmaceutical composition as described in claim 5 or 6 in preparation mammal rapamycin target protein inhibitor.
8. the application of pharmaceutical composition as claimed in claim 7 in preparation mammal rapamycin target protein inhibitor, it is characterized in that, described mammal rapamycin target protein inhibitor is mammal rapamycin target protein complex 1 inhibitor.
9. the pharmaceutical composition as described in claim 5 or 6 is preparing the application prevented and/or treated in cancer, metabolism disturbance syndrome, neurodegenerative diseases or anti-inflammatory drugs.
10. apply as claimed in claim 9, it is characterized in that, described cancer comprises breast carcinoma, pulmonary carcinoma, colon cancer, renal cell carcinoma, nonsmall-cell lung cancer, pancreas neuroendocrine carcinoma, stomach esophageal carcinoma, hepatocarcinoma, T cell lymphatic cancer, carcinoma of prostate, carcinoma of testis, genitourinary cancer, ovarian cancer, cervical cancer, glioblastoma, thyroid carcinoma, skin carcinoma, bladder cancer, cancer of pancreas, melanoma, colorectal cancer, rectal cancer, leukemia, hepatocarcinoma, carcinoma of small intestine, colorectal cancer, brain and central nervous system's cancer or cancer of bile ducts;
And/or described metabolism disturbance syndrome comprises hypertension, diabetes, coronary heart disease, apoplexy, obesity or accelerates atherosclerotic vascular disease;
And/or described neurodegenerative diseases comprises Alzheimer, Parkinson's disease, Heng Tingdunshi disease, amyotrophic lateral sclerosis, cerebral atrophy, multiple sclerosis or spinal muscular atrophy;
And/or described inflammation comprises rheumatoid arthritis, psoriasis, lymphadenitis, hepatitis, pneumonia or nephritis.
CN201410310204.5A 2014-07-01 2014-07-01 A kind of application of polyphenol compound Pending CN105213362A (en)

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CN109071425A (en) * 2016-03-23 2018-12-21 陈裕仁 Farnesyl transferase inhibitor and application thereof
KR20190135263A (en) * 2018-05-28 2019-12-06 충남대학교산학협력단 Composition comprising salvianolic acid A for preventing or treating for inflammatory skin diseases
CN111053764A (en) * 2020-01-15 2020-04-24 吉林农业大学 Application of salvianolic acid A in preparation of medicine for treating methicillin-resistant staphylococcus aureus infectious pneumonia
CN114224879A (en) * 2022-02-28 2022-03-25 深圳市人民医院 Application of salvianolic acid A in preparing anti-esophageal cancer drugs and drugs for increasing sensitivity of radiotherapy and chemotherapy
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Publication number Priority date Publication date Assignee Title
CN105534973A (en) * 2016-02-20 2016-05-04 通化华夏药业有限责任公司 Salvianolic acid C serving as dipeptidyl peptidase IV inhibitor and preparation method and application
CN109071425A (en) * 2016-03-23 2018-12-21 陈裕仁 Farnesyl transferase inhibitor and application thereof
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KR20190135263A (en) * 2018-05-28 2019-12-06 충남대학교산학협력단 Composition comprising salvianolic acid A for preventing or treating for inflammatory skin diseases
KR102085592B1 (en) * 2018-05-28 2020-03-06 충남대학교산학협력단 Composition comprising salvianolic acid A for preventing or treating for inflammatory skin diseases
CN111053764A (en) * 2020-01-15 2020-04-24 吉林农业大学 Application of salvianolic acid A in preparation of medicine for treating methicillin-resistant staphylococcus aureus infectious pneumonia
WO2022272043A1 (en) * 2021-06-24 2022-12-29 Research Institute At Nationwide Children's Hospital , Inc. Salvianoic acid or adenosine dialdehyde for treating fagcioscapulohumeral muscular dystrophy (fshd)
CN114224879A (en) * 2022-02-28 2022-03-25 深圳市人民医院 Application of salvianolic acid A in preparing anti-esophageal cancer drugs and drugs for increasing sensitivity of radiotherapy and chemotherapy

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