CN105596330B - Application and drug of the virtual screening compound in preparing kinase inhibitor - Google Patents

Application and drug of the virtual screening compound in preparing kinase inhibitor Download PDF

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CN105596330B
CN105596330B CN201510923390.4A CN201510923390A CN105596330B CN 105596330 B CN105596330 B CN 105596330B CN 201510923390 A CN201510923390 A CN 201510923390A CN 105596330 B CN105596330 B CN 105596330B
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kinase
drug
compound
adp
formula
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CN105596330A (en
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张晶珠
陈宇综
陈上英
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Shenzhen Kun Jian Original New Drug Research Institute
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A61K31/4162—1,2-Diazoles condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The present invention provides the application and drug of a kind of virtual screening compound in preparing kinase inhibitor, which is with the compound of structure shown in Formulas I, Formula II, formula III or formula IV or its pharmaceutically acceptable salt, ester, solvate.It is verified by vitro kinase activity, above compound has excellent inhibitory activity for VEGFR2, FGFR1, HER2, SRC or Braf kinases.It can be used for the diagnosing and treating of kinase mediated disease.

Description

Application and drug of the virtual screening compound in preparing kinase inhibitor
Technical field
The present invention relates to drug fields, more particularly, to a kind of virtual screening compound in preparing kinase inhibitor Application, and using the virtual screening compound as the drug of active component, which can prevent and/or treat kinases Jie The disease led.
Background technology
The research and development of discovery and its drug with the relevant drug targets of mankind's major disease are the weights in targeted therapy research Want link.Protein kinase (protein kinase, PK) is one of intracellular maximum protein family, participates in cell growth, divides It splits and the various physiological processes such as apoptosis.Protein kinase plays the part of important role in eukaryocyte signal transduction.Protein kinase is A kind of phosphotransferase, effect is that the γ phosphates of ATP molecules are transferred on the specific amino acid residue of substrate protein, is made Substrate protein phosphorylation.Contain 518 protein kinase genes in human genome altogether, accounts for about the 1.7% of eukaryotic gene.Egg White abnormal kinase would generally cause many major diseases including cancer, diabetes, inflammation.More than the 400 kinds mankind Disease has direct or indirect relationship with protein kinase.Therefore protein kinase oneself become after g protein coupled receptor second Big drug therapy target.According to statistics, at present whole world drug grind or exploration project in about one third with protein kinase phase It closes.
Kinases inhibitor (protein kinase inhibitor, PKI) can be used for the treatment of a variety of diseases, wherein ATP- competitive protein kinase inhibitors are most studied, and this kind of inhibitor has been studied and has been developed into treatment Various Complex The new drug of disease.The research of ATP- competitive proteins kinase inhibition is concentrated mainly on setting for new framework types lead compound at present Meter and discovery and selective depressant and multiple target point inhibitor exploitation etc..Currently, Computer-Aided Drug Design (computer-aided drug design, CADD) is applied to albumen as the important technology and tool in medicament research and development In the research of kinase inhibitor, the tremendous development of this research field has been pushed.Quantitative structure activity relationship, molecular docking, pharmacophore, A variety of CADD methods such as the different kinds of molecules such as Blast search and molecular dynamics simulation analogy method and quantum chemistry are applied In the design and discovery of kinases inhibitor.
Therefore, by using computer assisted drug design techniques, simulation is carried out with function to protein kinase structures and is ground Study carefully, on the basis of fully understanding target active pocket three-dimensional structure, rational structural modification is proposed to micromolecular inhibitor, is ground Efficiently single-minded kinases inhibitor is sent out to be of great significance.
Invention content
The object of the present invention is to provide new application of the virtual screening compound in preparing kinase inhibitor, and provide one Kind drug.
Specifically, the present invention provides a kind of application of virtual screening compound in preparing kinase inhibitor, the virtual sieve It is with the compound of structure shown in Formulas I, Formula II, formula III or formula IV or its pharmaceutically acceptable salt, ester, molten to select compound Object is closed in agent；
Wherein, R1And R2Difference, and beR4、R5And R6It is each independently H, C1-C3's Alkyl or aryl；R3And R7It is each independently H, C1-C3Alkyl or be collectively formed saturated or unsaturated five membered hydrocarbon ring or Hexa-atomic hydrocarbon ring；
Preferably, in Formulas I, R4、R5And R6It is each independently H or C1-C3Alkyl；Alternatively, R4And R6One of be aryl, Other two group is H.
It is further preferred that Formulas I compound represented is following formula V, Formula IV, Formula VII or Formula VIII compound represented；
Heretofore described virtual screening is from Molport (5*109) and Vitas-M (1.2*109) compound library progress SVM is screened, and screens the compound with target spot identified below：HER2, VEGFR2, FGFR1, B-raf or SRC.Therefore, of the invention Described in kinases be preferably HER2, VEGFR2, FGFR1, B-raf or SRC.
The present invention also provides a kind of drug, which can prevent and/or treat kinase mediated disease, the work of the drug Property group is divided into the compound of structure shown in Formulas I, Formula II, formula III or formula IV or its pharmaceutically acceptable salt, ester, solvent Close object.The restriction with the compound of structure shown in Formulas I, Formula II, formula III or formula IV is same as described above, no longer superfluous herein It states.
In the present invention, the kinase mediated disease is selected from least one of following disease：Angiocardiopathy, inflammation, Breast cancer, liver cancer, cancer of pancreas, lung cancer, the cancer of the brain, oophoroma, uterine cancer, carcinoma of testis, cutaneum carcinoma, gastric cancer, nasopharyngeal carcinoma, colon cancer, Carcinoma of urinary bladder and the carcinoma of the rectum.Preferably at least one of breast cancer, liver cancer, lung cancer and carcinoma of urinary bladder.
According to the present invention, the drug can be made injection, tablet, pulvis, granule, capsule, oral solution, paste, The diversified forms such as creme；The drug of above-mentioned various dosage forms can be prepared according to the conventional method of pharmaceutical field.The drug can Pass through injection, injection, collunarium, eye drip, infiltration, absorption, method importing the body such as muscle, intradermal, skin physically or chemically mediated Under, vein, mucosal tissue；Or imported body after other material mixings or package.
When needs, one or more pharmaceutically acceptable carriers can also be added in said medicine；The load Body includes the diluent of pharmaceutical field routine, excipient, filler, adhesive, wetting agent, disintegrant, sorbefacient, surface Activating agent, absorption carrier, lubricant etc..
Other features and advantages of the present invention will be described in detail in subsequent specific embodiment part.
Description of the drawings
Exemplary embodiment of the invention is described in more detail in conjunction with the accompanying drawings.
Fig. 1 is 50 μM of ATP/ADP conversion ratio standard curves.
Fig. 2 is FGFR1 kinases titration curve (50 μM of ATP).
Specific implementation mode
Pass through ADP-GloTMKinase assay carries out vitro kinase inhibitors experiment, ADP-GloTMKinase assay principle：Swashing The ADP formed during enzyme reaction can be by fluoroscopic examination, to know its kinase activity.First, ADP-GloTMReagent can be eventually Only kinase reaction, remaining ATP are revoked.The ADP generated in reaction process is converted into ATP and immediately in Ultra-GloTM Fluorescence is shown under the conditions of Dual-Luciferase is existing, it is i.e. detectable by sepectrophotofluorometer.It is anti-that fluorescence signal represents kinases The ADP concentration that should be generated in the process and its corresponding kinase activity.
ADP-GloTMKinase assay due to can effect of the detection compound to a wide range of kinases, while high letter can also be detected The ADP of low concentration under number background, therefore it is suitable for vitro kinase inhibitors experiment.
ADP-Glo used in embodimentTMKinases is purchased from Promega companies.
1, preparation of samples
Dimethyl sulfoxide (DMSO) (DMSO) dissolving of sample certain volume is lyophilized, final concentration of 10mmol/L, refrigerator preserves standby With.
2, ATP converts ADP standard curve makings
With reference to the explanation of Promega, the ATP concentration in various kinase reactions, the standard for making each kinases are detected Curve.These curves represent the conversion ratio of ATP/ADP, and can intuitively show the corresponding concentration of ADP.Therefore, according to standard song Line can detect the ADP generated during kinase reaction.Concentration of the ATP in each kinase reaction is respectively：10μM(HER2) With 50 μM (VEGFR2, FGFR1, B-raf and SRC).The deionized water that certain volume is added in 10mM ATP and ADP mother liquors obtains 10 × ATP (ADP) solution.10 × the ATP and 10 × ADP of different volumes are made into 10 × ATP-ADP mixed liquors of various concentration.Table 1 Show the 10 × ATP-ADP mixed liquors for being equipped with various concentration needed for 100 μ L.5 × reaction buffer A, 0.01M DTT (final concentrations 200 μM), 4 × confactor (with reference to specific kinase reaction scheme) and deionized water are made into 4 × kinase buffer liquid.4 × kinases Buffer solution is further diluted to 1 × kinase buffer liquid with deionized water.10 × ATP-ADP mixed liquors are dilute with 1 × kinase buffer liquid It is interpreted into 1 × ATP-ADP mixed liquors.10 μ L developing solutions are added to 96 orifice plates, two multiple holes of each ATP-ADP concentration.Fluoroscopic examination With Perkin ElmerMultimode Plate Reader detections.Detect two multiple holes mean fluorescence readings meters ATP/ADP average conversions are calculated, corresponding chart is obtained.ATP/ADP conversion ratios standard curve when Fig. 1 is 50 μM.
Table 1
1 2 3 4 5 6 7 8 9 10 11 12
10×ATP(μL) 0 20 40 60 80 90 95 96 97 98 99 100
10×ADP(μL) 100 80 60 40 20 10 5 4 3 2 1 0
3, kinases titration and SB10 detections
Kinases is titrated with the ATP concentration needed for detection maximum quantity kinases, the kinases titrated is used for screening compound And IC50It measures.Here it is the generation required kinase concentrations of fluorescence in the kinases titration curve range of linearity, and generate enough letters Number/background ratio (10-20).10 × ATP solution is prepared in aforementioned manners, and 4 × kinase buffer liquid D adds 4% with 4 × kinase buffer liquid DMSO is prepared.4 × kinase buffer liquid D, 10 × ATP, 1mg/mL substrate is according to 1:1:2 ratio prepares Substrate cocktail.Kinases With 1 × kinase buffer liquid D, 2 times of gradient dilutions are at various concentration.4 μ L substrate mixtures and 6 μ L kinases are added 96 orifice plates and carry out ADP-GloTMKinase assay.Kinase buffer liquid D is as blank well, two multiple holes of each experimental group.According to kinases titration reading with Blank group ratio obtains titration logarithmic curve, and Fig. 2 is the titration curve by taking FGFR1 kinases as an example.
4, the kinase inhibiting activity of drug is detected
Mother liquid medicine is diluted to each concentration with 1 × kinase buffer liquid, takes 2 μ L that 96 orifice plates, negative control and blank is added 2 μ L kinase buffer liquids D are added in control group.Control wells and 4 μ L kinase solutions of the administration group of various concentration addition are separately added into each Blank control group is added in concentration administration group and negative control group, 4 μ L blank solutions, and finally 4 μ L ATP/ substrates are all added in all holes Mixed liquor.ADP-GloTMKinase assay repeats 2-3 times.
The enzyme activity accounting equation of each drug：
Inhibitor vigor calculates：
Inhibition vigor (%)=100%- enzyme activities (%)
It repeats to test when Drug inhibition rate >=50% or inconsistent multiple holes data, compound dosage is drawn by titration experiments Curve and calculating IC50。
Table 2 shows inhibitory activity of these compounds to each kinases of VEGFR2, FGFR1, HER2, Braf and SRC.Its In, staurosporine is control group.
Table 2
As can be seen from Table 2, the drug has the inhibitory activity of each kinases of VEGFR2, FGFR1, HER2, Braf and SRC There is preferable inhibition.For example, kinase inhibitions of the compound k22f (Formula V) to VEGFR2, FGFR1, HER2, Braf and SRC Activity value is respectively 1.5 μM, 6.24 μM, 7.98 μM, 15.2 μM and 9.32 μM.This result illustrates that compound k22f is one kind and has Multiple target point VEGFR2, FGFR1, HER2, SRC, Braf kinase inhibitor of foreground.Again it can be seen that compound from table 2 K23f (Formula IV) is a kind of VEGFR2 inhibitor；Compound k24f (Formula VII) is a kind of VEGFR2 inhibitor；Compound k26f (Formula VIII) is a kind of multiple target point SRC, VEGFR2 kinase inhibitor；Compound K 30 (Formula II) is a kind of multiple target point SRC, HER2 Kinase inhibitor；K32g (formula III) is a kind of multiple target point SRC, HER2 kinase inhibitor；K62 (formula IV) is a kind of multiple target point SRC, VEGFR2, Braf kinase inhibitor.
Various embodiments of the present invention are described above, above description is exemplary, and non-exclusive, and It is not limited to disclosed each embodiment.

Claims (1)

1. a kind of application of virtual screening compound in preparing kinase inhibitor, which is characterized in that the virtual screening compound For compound or its pharmaceutically acceptable salt with structure shown in Formula V；The kinases is HER2, VEGFR2, FGFR1, B- Raf or SRC；
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013144191A1 (en) * 2012-03-29 2013-10-03 Bayer Intellectual Property Gmbh Substituted 2-amino-3-cyanopyridines as inhibitors of sodium-calcium exchange and use thereof for cardiovascular diseases
CN104725362A (en) * 2010-04-30 2015-06-24 阿斯特克斯治疗有限公司 Pyrazolyl quinazoline kinase inhibitors

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104725362A (en) * 2010-04-30 2015-06-24 阿斯特克斯治疗有限公司 Pyrazolyl quinazoline kinase inhibitors
WO2013144191A1 (en) * 2012-03-29 2013-10-03 Bayer Intellectual Property Gmbh Substituted 2-amino-3-cyanopyridines as inhibitors of sodium-calcium exchange and use thereof for cardiovascular diseases

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
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Synthesis of New 3-( Pyridin-6-yl)pyrazolo[1,5-a]pyrimidines;Nadia Sobhy Ibrahim et al.;《Arch. Pharm.》;19871231;第87卷(第320期);487-491 *

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