CN103214479A - Pyrazole naphthylurea tyrosine kinase inhibitor and its application - Google Patents
Pyrazole naphthylurea tyrosine kinase inhibitor and its application Download PDFInfo
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- IOFANXQXRYYKAR-UHFFFAOYSA-N Cc1cc(NC(Nc(cc2)c(cccc3)c3c2-c2c(c(N)n[nH]3)c3ncc2)=O)ccc1F Chemical compound Cc1cc(NC(Nc(cc2)c(cccc3)c3c2-c2c(c(N)n[nH]3)c3ncc2)=O)ccc1F IOFANXQXRYYKAR-UHFFFAOYSA-N 0.000 description 1
- QZASRQHPMJJQCB-UHFFFAOYSA-N Nc1n[nH]c2c1c(-c(cc1)c(cccc3)c3c1NC(Nc1cccc(C(F)(F)F)c1)=O)ccn2 Chemical compound Nc1n[nH]c2c1c(-c(cc1)c(cccc3)c3c1NC(Nc1cccc(C(F)(F)F)c1)=O)ccn2 QZASRQHPMJJQCB-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention relates to a pyrazole naphthylurea tyrosine kinase inhibitor. The pyrazole naphthylurea tyrosine kinase inhibitor is compounds having structures represented by general formula I, and medicinal salts thereof. The compounds have tyrosine kinase inhibition activities, and can be used in medicines for treating tyrosine kinase mediated diseases or illnesses as a tyrosine kinase inhibitor.
Description
Technical field
The invention belongs to the tyrosine kinase inhibitor field, be specifically related to pyrazoles naphthalene ureas tyrosine-kinase enzyme inhibitor and application thereof.
Background technology
Tyrosylprotein kinase is the protein that a class has tyrosine kinase activity, they can catalysis phosphate on the ATP transfer on the tyrosine residues of many key proteins, make it that phosphorylation take place, thereby activate the signal transduction pathway in downstream.Protein tyrosine kinase has occupied crucial status in intracellular signal transduction pathway, regulating a series of physiological and biochemical procedures such as growth in the cell paste, differentiation, death.The imbalance of protein tyrosine kinase function then can cause a series of disease in the organism, comprising tumour and eye disease.
The generation development of many tumours and transfer and tumor neovasculature generation all have extremely close getting in touch with the unconventionality expression of Tyrosylprotein kinase.Particularly some tyrosine kinase receptor has unconventionality expression at the cell of solid tumor, vascular endothelial growth factor receptor (vascular endothelial cell growth factor wherein, VEGFR) in many tumour cells and tumor vascular endothelial cell, all be high expression level, platelet derived growth factor receptor (platelet-derived growth factor, PDGFR) unconventionality expression in the inoblast in tumor stroma.What its part and acceptor formed self secretes generation and the development that loop is participated in tumour cell directly, and for example, vascular endothelial growth factor receptor (VEGFR) is present in melanoma; Platelet derived growth factor receptor (PDGFR) is present in glioma; Stem cell factor acceptor (c-KIT) is present in small cell lung cancer etc.In addition, similar loop is also in meningioma, neuroendocrine tumor, and ovarian cancer exists in prostate cancer and the carcinoma of the pancreas.The generation development of this loop and tumour has extremely confidential relation; Stem cell factor (Kit) is stem cell factor acceptor (SCFR, part c-Kit).The hemopoietic function of KIT/SCFR and body, the growth of the growth of mastocyte and Cajal mesenchymal cell is closely related.Discover that KIT/SCFR exists more than 30 kind of function gain mutation form, they are that the direct inducement of development takes place many tumours.In addition, exist KIT/SCFR autocrine loop in 70% small cell lung cancer patient, having of this loop is beneficial to tumour in the growth that does not rely on somatomedin.
In addition, the new vessel that the generation of noumenal tumour, development and transfer all depend on tumour generates, and it provides essential nutrition and oxygen for growth of tumor.Tumor-blood-vessel growth (tumor angiogenesis) is infiltration, the migration of tumour cell, the significant process of propagation.Wherein vascular endothelial growth factor receptor family (VEGFR) and Platelet Derived Growth Factor Receptor Family (PDGFR) have direct relation with the generation development and the tumor neovasculature generation of tumour.Vascular endothelial growth factor (VEGF) is as the strongest known blood vessel permeate agent and the mitotic division source of endothelial-cell specific, and in the propagation of endotheliocyte, migration and blood vessel play an important role in making up.The vascularization degree of its expression level and tumor tissues presents tangible positive correlation.VEGF makes it Tyrosylprotein kinase generation phosphorylation by acceptor VEGFR-1 that acts on high-affinity on the endotheliocyte and KDR to bring into play its biological action, and both have the unlike signal transduction pathway.Wherein, KDR plays a part crucial in growth of tumor, transfer and tumor neogenetic blood vessels form.Thr6 PDGF BB (PDGF) and its acceptor (PDGFR) relate to the pathogenesis of kinds of tumors and play an important role in vasculogenesis.Thr6 PDGF BB (PDGF) shows its cell biological effect via its acceptor (PDGFR).Pericyte and vascular smooth muscle cell proliferation, the migration integrity of keeping vessel wall of PDGFR by regulating vessel wall promotes tumor neovasculature formation.And, promote tumor growth by the microenvironment that changes in the tumour.
Because the generation development of the unconventionality expression of Tyrosylprotein kinase and tumour and transfer and tumor neovasculature generation have extremely close getting in touch, therefore, be that the medicament research and development of target spot has become the focus that antitumor drug is in the world studied with the Tyrosylprotein kinase.Especially be that target spot suppresses nutrition supply and the migratory route that tumor vessel forms, blocks tumour with the new vessel, prevent tumor growth and shift the New Policy that has become present treatment tumour.And KDR or PDGFR receptor abnormality are expressed in and play keying action in the new vessel forming process of tumour, become the target spot of ideal antitumor drug treatment their the sixth of the twelve Earthly Branches.And, two main antitumor drugs that suppress KDR and PDGFR receptor tyrosine kinase of nearest drugs approved by FDA, Xarelto (Sorafenib) or Sutent (SU11248) have fully confirmed the antineoplaston effect of curative effect height and few side effects clinically the sixth of the twelve Earthly Branches.But most of antitumor drugs still are cytotoxic drug on the Chinese market at present, because its nonselective tumour cell that promptly acts on also kills normal human body cell simultaneously, its drug selectivity is poor, and drug effect is remarkable inadequately, and toxic side effects is big.
In addition, modal clinically ophthalmology blinding disease senile macular degeneration SMD and ret diab all have pathologic choroid or retinal neovascularization in becoming, and still do not have effective methods of treatment at present.Reach scientific research clinically and prove that fully unusual KDR expresses and the interior signal conduction of cell plays an important role the sixth of the twelve Earthly Branches in pathologic choroid or retinal neovascularization forming process.KDR is by with after its ligands specific VEGF combines, and receptor tyrosine kinase generation autophosphorylation has been induced signal conduction in a series of cell, thereby causes propagation, migration and the vascularization of vascular endothelial cell.Therefore, suppress KDR Tyrosylprotein kinase phosphorylation and can block signal conduction in the KDR inductive abnormal cells effectively, reduced pathologic choroid or retinal neovascularization and formed, thereby the choroid or the retinal hemorrhage that have prevented to bring out, scabbed and fibrosis causes eyesight seriously to lose because of new vessel.For example, the Lucentis or the Macugen medicine (two are VEGF antibody) of approval are used for the treatment of senile macular degeneration SMD to U.S. FDA in the recent period.And clinical data proves that intraocular injection Lucentis or Macugen can prevent that effectively senile macular degeneration SMD patient's eyesight from further descending by signal conducting energy in the inhibition VEGF/KDR expression institute inductive abnormal cells the sixth of the twelve Earthly Branches.In addition, PDGFR also participates in the formation of new vessel, suppress PDGFR receptor tyrosine kinase phosphorylation, can make the pericyte of pathologic choroid or retinal neovascularization wall that self apoptosis takes place, cause to form that pathologic new vessel generation degeneration changes, atrophy the sixth of the twelve Earthly Branches, from so that improve eyesight effectively.Therefore, with control VEGF/KDR and or the conduction of PDGF/PDGFR unconventionality expression and signal be that the drug research of target spot has become the New Policy that current treatment senile macular degeneration SMD and ret diab become.But up to now, still do not have receptor tyrosine kinase inhibitors and be applied to treating on clinical the patient that senile macular degeneration SMD or ret diab become.
Summary of the invention
Technical problem to be solved by this invention provides a kind ofly to be had Tyrosylprotein kinase and suppresses active pyrazoles naphthalene ureas tyrosine kinase inhibitor and application thereof, it can be used for during tyrosine kinase activity suppresses, further can be used for treating malignant tumour and senile macular degeneration SMD pathology and ret diab become and the medicine of ophthalmic diseasess such as neovascular glaucoma in application.
For solving above technical problem, the present invention takes following technical scheme:
Pyrazoles naphthalene ureas tyrosine kinase inhibitor has described compound of following general formula I and pharmaceutical salts thereof:
In the above-mentioned general formula I:
R
1, R
2For independently, identical or inequality be selected from a kind of in the following groups:
H, OH, F, Br, Cl, I, CN, NO
2, NH
2, CH
3, CF
3, OCF
3, OCH
3, OCH
2CH
3, (CH
2)
nCH
3, (CH
2)
nNH
2, (CH
2)
nOH, (CH
2)
nSH, C (=O) (CH
2)
nCH
3, C (=O) NH (CH
2)
nCH
3, C (=O) O (CH
2)
nCH
3, NHC (=O) (CH
2)
nCH
3, OC (=O) (CH
2)
nCH
3, NHCH
3, NHCH
2CH
3, n=1-5 wherein;
Pharmacologically acceptable salt described in the above-mentioned general formula I includes but not limited to hydrochloride, phosphoric acid salt, vitriol, acetate, maleate, benzene sulfonate, toluenesulfonate, fumarate, tartrate etc.
The preparation method of the compound that general formula I is represented is: make naphthylamines boric acid ester (1, buy) and isocyanic ester (2, buy) under the normal temperature in methylene dichloride condensation reaction make intermediate naphthyl-phenyl carbamide compound (3) (reaction formula 1).If can not directly buy isocyanic ester, naphthyl-phenyl carbamide compound (3) also can be reacted under the same conditions by isocyanic ester boric acid ester (1 ', buy) and substituted aniline (2 ', buy) according to raw material and obtain (seeing reaction formula 2).Naphthyl-phenyl carbamide compound (3) carries out the Suzuki condensation reaction, makes the target product general formula I with 4-iodo-1H-pyrazolo [3,4-B] pyridine-3-amine (4) again.Synthetic route is:
Reaction formula 1
Reaction formula 2
Above-claimed cpd 4,4-iodo-1H-pyrazolo [3,4-B] pyridine-3-amine is known compound.According to delivering document preparation method preparation, concrete preparation method sees: WO 2006/077319A1, SUBSTITUTED PYRAZOLOPYRIDINES, COMPOSITIONS CONTAINING THEM, METHOD FOR THE PRODUCTION THEREOF, AND THEIR USE.
Compound 2, compound 3 and compound 2 ' middle R
1, R
2Implication identical with general formula I.
In the such scheme, representational compound has the compound that formula II, formula III, formula IV, formula V or formula VI represent in the general formula I.
Medicinal compositions comprises described compound of general formula I and pharmaceutically acceptable carrier.
The prodrug of above-mentioned any described compound.
Above-mentioned any described compound and pharmacologically acceptable salt thereof are being treated by the tyrosine kinase mediated disease or the purposes of the medicine in the illness.
Above-mentioned any described compound and pharmacologically acceptable salt thereof are in the treatment malignant tumour and with the application in the medicine of the ophthalmic diseases of pathologic new vessel.Above-mentioned malignant tumour includes but not limited to kidney, liver cancer, colorectal carcinoma, gastrointestinal stromal tumor, lung cancer, mammary cancer, carcinoma of the pancreas, neurospongioma, fibrosarcoma, ovarian cancer and prostate cancer etc.Above-mentioned ophthalmic diseases comprises senile macular degeneration SMD pathology and ophthalmic diseasess such as ret diab change and neovascular glaucoma.
Description of drawings
Fig. 1 is the phosphorylation inhibiting rate of respective instance compound to the KDR Tyrosylprotein kinase;
Fig. 2 and Fig. 3 are the phosphorylation inhibiting rate of respective instance compound to PDGF beta receptor Tyrosylprotein kinase;
Fig. 4 is the phosphorylation inhibiting rate of respective instance compound to the c-Kit Tyrosylprotein kinase.
Embodiment
Embodiment 1: instantiation compound II-VI's is synthetic
Instantiation compound II
1-[4-(3-Amino-1H-pyrazolo[3,4-b] pyridin-4-yl)-naphthalen-1-yl]-3-m-tolyl-ur ea (II), 1-[4-(3-amino-1H pyrazolo [3,4B] pyridine 4 bases) naphthalene 1-yl]-3-toluene-urea
The preparation method: with compound naphthylamines boric acid ester (1, purchase in Aldrich, 2.7g 0.01mol) is dissolved in methylene dichloride (20mL), after gained solution is cooled to 0 ℃, add tolyl isocyanic ester between compound (6, purchase in Aldrich, 1.6g, 0.012mol).The gained reaction mixture under agitation rises to room temperature gradually and continues and at room temperature stirred 18 hours.On Rotary Evaporators, remove and desolvate.The gained mixture adds the mixed solvent of 15mL methylene dichloride/normal hexane (1/3), and stirring was left standstill one hour after half an hour.The white solid of separating out filters and is dry, gets compound naphthyl-phenyl carbamide compound 7 (2.8g, 81%).
(0.6g, 0.0017mol) (press the document preparation, 0.36g 0.0014mol) is suspended in dioxan (9mL) and the water (3mL) compound 7, adds K with compound 4
3PO
4(0.6g, 0.0028mol), catalyst P dCl
2DppfCH
2Cl
2(2mol%).The gained reaction mixture stirred 6 hours under 90 ℃ of temperature.On Rotary Evaporators, remove and desolvate.The gained mixture is purifying (elutriant: the methylene dichloride of 3% methyl alcohol) get pale solid II (0.31g, 53%) on silicagel column.
Concrete synthetic route (reaction formula 3) as follows:
Reaction formula 3
Molecular formula: C
24H
20N
6O
Ultimate analysis (%, C
24H
20N
6O ° of 1H
2O), calculated value is: C67.59, H5.20, N19.71; Measured value: C67.61, H4.93, N19.69;
1HNMR(DMSO，400MHz)：δ12.33(s，1H)；9.10(s，1H)；8.97(s，1H)；8.48(d，1H，J＝4.8Hz)；8.29(d，1H，J＝8.4Hz)；8.24(d，1H，J＝8.0)；7.68(d，1H，J＝7.2)；7.58-7.55(m，3H)；7.40(s，1H)；7.33(d，1H，J＝8.0)；7.23(t，1H，J＝7.6Hz)；6.96(d，1H，J＝4.0Hz)；6.84(d，1H，J＝7.6)；4.11(s，2H)；2.31(s，3H)；
13CNMR(DMSO，400MHz)：δ152.812(C)，152.655(C)，148.733(CH)，147.257(C)，142.687(C)，139.581(C)，138.053(C)，135.335(C)，131.215(C)，128.817(C)，128.717(CH)，127.056(CH)，126.828(C H)，126.059(CH)，125.802(CH)，125.620(C)，122.745(CH)，121.810(CH)，118.763(CH)，116.328(CH)，116.247(CH)，115.421(CH)，104.864(C)，21.243(CH3)。
High-pressure liquid phase/mass spectrum: LC/MS (+APCI) m/z[(M+H)
+], calculated value: 409.2, measured value: 409.9.
Following instantiation compound III-VI all prepares according to the method for instantiation compound II.
The example compound III
1-[4-(3-Amino-1H-pyrazolo[3,4-b] pyridin-4-yl)-naphthalen-1-yl]-3-(2-fluoro-5-methyl-phenyl)-urea, 1-[4-(3-amino-1H-pyrazolo [3,4-B] pyridin-4-yl) naphthalene-1-yl]-3-(2-fluoro-5-aminomethyl phenyl) urea
Molecular formula: C
24H
19FN
6O;
Ultimate analysis (%, C
24H
19FN
6O ° of 0.2TFA ° of 1.5H
2O), calculated value is: C61.53, H4.70, N17.64; Measured value is: C61.50, H4.29, N17.55;
HNMR(DMSO，400MHz)δ12.41(s，1H)；9.35(s，1H)；9.11(s，1H)；8.49(d，1H，J＝4.4Hz)；8.27(d，1H，J＝8.0Hz)；8.14(d，1H，J＝8.4)；7.73-7.71(m，1H)；7.67-7.50(m，5H)；7.22-7.13(m，1H)；6.97(d，1H，J＝4.8Hz)；6.85-6.82(m，2H)；2.40(s，3H)；
13CNMR(DMSO，400MHz)：δ152.52(C)，151.50(C)，149.13(CH)，148.52(C)，146.98(C)，135.211(C)，133.57(C)，131.16(C)，128.75(C)，127.15(C)，127.07(CH)，126.89(CH)，126.12(CH)，125.79(CH)，125.51(CH)，122.78(C)，122.71(CH)，121.81(CH)，120.96(CH)，116.31(CH)，116.11(CH)，114.65(CH)，105.03(C)，20.78(CH3)；
High-pressure liquid phase/mass spectrum: LC/MS (+APCI) m/zcalcd.forC
24H
19FN
6O[(M+H)
+], calculated value: 427.2, measured value: 427.0.
The example compound IV
1-[4-(3-Amino-1H-pyrazolo[3,4-b] pyridin-4-yl)-naphthalen-1-yl]-3-(3-trifluoromethyl-phenyl)-urea, 1-[4-(3-amino-1H-pyrazolo [3,4-B] pyridin-4-yl) naphthalene, the 1-yl]-3-(3-trifluoromethyl) urea
Molecular formula: C
24H
17F
3N
6O
Ultimate analysis (%, C
24H
17F
3N
6O1.2H
2O): calculated value is: C59.55, H4.04, N17.36; Measured value is: C59.31, H4.32, N17.07;
1HNMR(DMSO，400MHz)：δ12.35(s，1H)；9.50(s，1H)；9.07(s，1H)；8.48(d，1H，J＝4.4)；8.27(d，1H，J＝8.0)；8.17(d，1H，J＝8.0)；8.11(s，1H)；7.70-7.50(m，6H)；7.36(d，1H，J＝7.6)；6.96(d，1H，J＝4.4)；3.50(s，2H)；
13CNMR(DMSO，400MHz)：δ152.95(C)，152.38(C)，148.56(CH)，147.02(C)，143.15(C)，140.56(C)，135.00(C)，131.20(C)，130.10(C)，131.01(C)，129.78(CH)，129.47(CH)，127.02(CH)，126.92(CH)，125.77(CH)，125.57(C)，122.86(CH)，121.97(CH)，118.20(CH)，117.21(CH)，116.30(CH)，114.18(CH)，104.97(C)，17.20(CF3)；
High-pressure liquid phase/mass spectrum LC/MS (+APCI) m/z[(M+H)
+], 463.1, found463.9.
Instantiation compound V
1-[4-(3-Amino-1H-pyrazolo[3,4-b] pyridin-4-yl)-naphthalen-1-yl]-3-(3-hydroxy-5-methyl-phen yl)-urea, 1-[4-(3-amino-1H-pyrazolo [3,4-B] pyridin-4-yl) naphthalene-1-yl]-3-(3-hydroxy-5-methyl base phenyl) urea
Molecular formula: C
24H
20N
6O
2
Ultimate analysis (%, C
24H
20N
6O20.5TFA2.2H
2O): calculated value: C57.62, H4.82, N16.13; Measured value: C57.13, H4.32, N15.81.
1HNMR(DMSO，400MHz)：δ12.30(s，1H)；9.22(s，1H)；9.01(s，1H)；8.49(d，1H，J＝2.4)；8.28-8.21(m2H)；7.69-7.47(m，4H)；7.21(s，1H)；6.97(s，1H)；6.89(s，1H)；6.74(s，1H)；6.23(s，1H)，3.89(s，2H)，2.51(s，3H).
13CNMR(DMSO，400MHz)：δ157.69(C)，152.65(C)，151.89(CH)，148.23(C)，146.67(C)，140.43(C)，138.87(C)，135.66(C)，131.11(C)，128.24(C)，127.18(CH)，126.88(CH)，126.06(CH)，125.74(CH)，125.55(CH)，121.83(C)，116.29(CH)，115.94(CH)，110.0(CH)，109.69(CH)，105.26(CH)，102.62(CH)，21.33(CH3).
High-pressure liquid phase/mass spectrum LC/MS (+APCI) m/z[(M+H)
+], 425.2, measured value: 425.0.
The example compound VI
1-[4-(3-Amino-1H-pyrazolo[3,4-b] pyridin-4-yl)-naphthalen-1-yl]-3-(4-fluoro-3-methyl-phenyl)-urea, 1-[4-(3-amino-1H-pyrazolo [3,4-B] pyridin-4-yl) naphthalene-1-yl]-3-(4-fluoro-3-aminomethyl phenyl) urea
Molecular formula: C
24H
19FN
6O
Ultimate analysis (%, C
24H
19FN
6O1.2H
2O) calculated value: C64.33, H4.81, N18.76; Measured value: C64.05, H4.53, N18.47.
1HNMR(DMSO，400MHz)δ12.32(s，1H)；9.11(s，1H)；8.96(s，1H)；8.47(d，1H，J＝4.4Hz)；8.28(d，1H，J＝8.4Hz)；8.20(d，1H，J＝8.0)；7.69(dd，1H，J＝7.2)；7.59-7.33(m，5H)；7.11(dd，1H，J＝8.6)；6.95(d，1H，J＝4.8Hz)；4.10(s，2H)；2.25(s，3H).
13CNMR(DMSO，400MHz)：δ157.24(C)，154.88(C)，152.93(CH)，152.64(C)，148.73(C)，147.23(C)，142.69(C)，135.64(C)，135.30(C)，131.21(CH)，128.88(CH)，127.04(CH)，126.83(CH)，126.07(CH)，125.78(C)，124.39(CH)，121.86(CH)，121.24(CH)，121.20(CH)，117.40(CH)，116.38(CH)，115.10(C)，104.84(CH)，20.78(CH3).
High-pressure liquid phase/mass spectrum LC/MS (+APCI) m/z[(M+H)
+], 427.2, measured value: 427.0.
Embodiment 2 pharmacological testings
Compound is to KDR and PDGFR-β and c-Kit receptor tyrosine kinase activity inhibition in vitro tests
(1) experimental technique
Enzyme-linked immunosorbent assay (Enzyme-LinkedImmunosorbentAssay, ELISA)
Key instrument
The wavelengthtunable orifice plate microplate reader Molecular DevicesSPECTRAMAX190 that declines.
Main agents
Given the test agent: a series of instantiation compound II-VI of the foregoing description 1, face the time spent to be diluted to desired concn with solvent DMSO; Control group: Sorafenib or Su11248;
Tyrosylprotein kinase VEGFR2 (KDR), c-Kit expresses for utilizing insect baculovirus expression system, obtains with Ni-NTA post affinity purification, and meets requirement of experiment after testing;
Tyrosylprotein kinase PDGFR β is available from Millipore company;
Kinase reaction substrate Poly (Glu, Tyr is 4:1) available from Sigma company;
The monoclonal antibody PY99 of anti-phosphorylated tyrosine is available from SantaCruz company;
The IgG of horseradish peroxidase-labeled sheep anti mouse is available from Calbiochem company;
ATP, DTT, OPD are available from Amresco company;
Enzyme plate is available from Corning company;
Su11248, Sorafenib are available from LC LabORATORIES company;
Other reagent is homemade.
Experimental procedure
(1) (4:1) PBS with no potassium ion is diluted to 20 μ g/ml to enzyme reaction substrate Poly for Glu, Tyr, and coated elisa plate is put 37 ℃ of reaction 12-16h, discards liquid in the hole.
(2) T-PBS washes plate three times, each 10min.
(3) dry enzyme plate in 37 ℃ of baking ovens.
(4) in wrapping, add given the test agent by good enzyme plate hole:
Face with preceding given the test agent and be mixed with 10 with DMSO earlier
-2The storage liquid of M is diluted to desired concn with reaction buffer again, adds in the experimental port, makes it reach corresponding final concentration in 100 μ l reaction systems.Set up the positive control hole simultaneously.Deposit in-20 ℃ after the packing of residue storage liquid.
(5) add ATP and tried Tyrosylprotein kinase:
Adding reaches the Tyrosylprotein kinase that tried with the reaction buffer dilution with the ATP solution (ATP final concentration 5 μ M) of reaction buffer dilution.The reaction system cumulative volume is 100 μ l.Set up negative control hole and no enzyme control wells simultaneously.
(6) reaction system is placed in the wet box, 37 ℃ of shaking table lucifuge reaction 1h, reaction finishes back T-PBS and washes plate three times.
(7) add antibody PY99100 μ l/ hole, 37 ℃ of shaking table reaction 30min.T-PBS washes plate three times.
(8) the IgG100 μ l/ hole of the sheep anti mouse of adding horseradish peroxidase-labeled, 37 ℃ of shaking tables reaction 30min.T-PBS washes plate three times.
(9) add OPD colour developing liquid 100 μ l/ holes, room temperature lucifuge reaction 1-10min.
(10) add 2M H
2SO
450 μ l stopped reactions are surveyed A with the wavelengthtunable orifice plate microplate reader Molecular Devices SPECTRAMAX190 that declines
490Value.
(11) inhibiting rate of sample is tried to achieve by following formula:
(2) experimental result
Representative instance Compound I I-VI is to the KDR Tyrosylprotein kinase, and the activity of PDGFR-β Tyrosylprotein kinase and c-Kit Tyrosylprotein kinase suppresses the result and sees Table 1-table 3 and Fig. 1-Fig. 3 respectively:
Table 1 compound suppresses the KDR tyrosine kinase activity
| Instantiation compound concentration (nM) | IC 50Mean value (nM) | The SD value |
| II | 7.7 | 1.0 |
| III | 15.5 | 6.4 |
| IV | 22.0 | 5.6 |
| V | 19.0 | 3.1 |
| VI | 22.4 | 9.4 |
| Sorafenib (positive controls) | 13.4 | 5.5 |
Table 2 compound suppresses PDGFR-β tyrosine kinase activity
| Instantiation compound concentration (nM) | IC 50Mean value (nM) | The SD value |
| II | 14.3 | 5.5 |
| III | 2.2 | 1.3 |
| IV | 28.4 | 9.3 |
| V | 8.2 | 2.5 |
| VI | 20.2 | 2.0 |
| Sorafenib (positive controls) | 41.0 | 4.4 |
Table 3 compound suppresses the c-Kit tyrosine kinase activity
| Instantiation compound concentration (nM) | IC 50Mean value (nM) | The SD value |
| II | 20.6 | 2.8 |
| III | 25.5 | 8.1 |
| SU11248 (positive controls) | 50.2 | 3.2 |
Above-mentioned pharmacological experiment is all implemented to finish by Shanghai Pharmaceutical Inst., Chinese Academy of Sciences.IC
50± SD value and inhibiting rate are the mean value of testing from 3-4 different time respectively; Wherein, the sample of each test repeats twice at least in each test.
Confirm to draw from top experimental result: pyrazoles naphthalene ureas compound of the present invention all can suppress KDR and PDGFR-β and c-Kit receptor tyrosine kinase phosphorylation significantly in the nanomolar concentration level.Wherein typical compound II-VI is to the IC of KDR tyrosine kinase activity inhibition
50Scope is 7.7-22.4nM, and PDGFR-β tyrosine kinase activity is suppressed IC
50Scope is 2.2-28.4nM, and positive controls Sorafenib suppresses IC to KDR or PDGFR-β tyrosine kinase activity
50Be respectively 13.4nM or 41.0nM.With Sorafenib relatively, be better than on the pharmacological effect that The compounds of this invention suppresses KDR or PDGFR-β tyrosine kinase activity or the activity of close Sorafenib; Typical compound II and III all can significantly suppress c-Kit Tyrosylprotein kinase phosphorylation, and it suppresses active IC
50Be starkly lower than SU11248.
More than experiment shows: the alternative target particular proteins-receptor tyrosine kinase of pyrazoles naphthalene ureas compound of the present invention, be novel many target spots tyrosine kinase inhibitor, can be used on treatment by in the tyrosine kinase mediated disease or the medicine in the illness.It can be by KDR and the PDGFR acceptor that acts on tumour cell, the phosphorylation that suppresses receptor tyrosine kinase by selectivity effectively, block signal conduction in its unusual cell, reach the effect that suppresses growth of tumour cell propagation and shift, can form by stoping tumor neogenetic blood vessels simultaneously, block required blood and the nutritive substance of tumor growth and supplied with, caused cancer cell death, suppressed generation and the development and the transfer of tumour indirectly.Remove this, because of KDR and PDGFR receptor expression mainly are confined to the vascular endothelial cell and the cells of vascular wall of breeding, so target vascular therapy endothelial growth factor receptor and platelet derived growth factor receptor treatment tumour have selectivity height, high specificity and low toxin for cytotoxic drug.In addition, pyrazoles naphthalene ureas compound of the present invention is used in the medicine that treatment becomes with the senile macular degeneration SMD of pathologic new vessel or ret diab, it can be by the PDGFR-beta receptor tyrosine phosphorylation on the pericyte film of KDR receptor kinase phosphorylation and vessel wall on the endothelial cell membrane that suppresses lesion pathologic new vessel, thus prevented choroid that due to illness rational new vessel growth causes or retinal edema, hemorrhage and scab due to patient's vision descend.
Claims (7)
1. pyrazoles naphthalene ureas tyrosine kinase inhibitor has described compound of following general formula I and pharmacologically acceptable salt thereof:
In the above-mentioned general formula I:
R
1, R
2For independently, identical or inequality be selected from a kind of in the following groups:
H, OH, F, Br, Cl, I, CN, NO
2, NH
2, CH
3, CF
3, OCF
3, OCH
3, OCH
2CH
3, (CH
2)
nCH
3, (C H
2)
nNH
2, (CH
2)
nOH, (CH
2)
nSH, C (=O) (CH
2)
nCH
3, C (=O) NH (CH
2)
nCH
3, C (=O) O (CH
2)
nCH
3, NHC (=O) (CH
2)
nCH
3, OC (=O) (CH
2)
nCH
3, NHCH
3, NHCH
2CH
3, n=1-5 wherein.
2. pyrazoles naphthalene ureas tyrosine kinase inhibitor according to claim 1 includes but not limited to following instantiation compound:
3. compound according to claim 1 and pharmaceutical salts thereof, described pharmacologically acceptable salt includes but not limited to hydrochloride, phosphoric acid salt, vitriol, acetate, maleate, benzene sulfonate, toluenesulfonate, fumarate, tartrate etc.
4. medicinal compositions, it is characterized in that: it comprises described compound of general formula I and pharmaceutically acceptable carrier.
5. the prodrug of compound according to claim 1.
6. above-mentioned any compound according to claim 1 and pharmacologically acceptable salt thereof are being treated by the tyrosine kinase mediated disease or the purposes of the medicine in the illness.
7. purposes according to claim 6 is characterized in that: described by in tyrosine kinase mediated disease or the illness for malignant tumour and with the ophthalmic diseases of pathologic new vessel.
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Cited By (1)
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| CN103524421A (en) * | 2013-09-29 | 2014-01-22 | 镇江蓝德特药业科技有限公司 | Novel naphthoyl urea derivate and medical application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103524421A (en) * | 2013-09-29 | 2014-01-22 | 镇江蓝德特药业科技有限公司 | Novel naphthoyl urea derivate and medical application thereof |
| CN103524421B (en) * | 2013-09-29 | 2015-04-01 | 镇江蓝德特药业科技有限公司 | Novel naphthoyl urea derivate and medical application thereof |
| WO2015043342A1 (en) * | 2013-09-29 | 2015-04-02 | 镇江蓝德特药业科技有限公司 | Novel a-naphthylurea derivative and medical application of same |
| JP2016531936A (en) * | 2013-09-29 | 2016-10-13 | レイディアント ファーマ アンド テクノロジー カンパニー、リミテッドＲａｄｉａｎｔ Ｐｈａｒｍａ ＆ Ｔｅｃｈ． Ｃｏ．， Ｌｔｄ． | Naphthylurea derivatives and their medical applications |
| US9469639B2 (en) | 2013-09-29 | 2016-10-18 | Radiant Pharma & Tech. Co., Ltd. | Naphthylurea derivatives and medical applications thereof |
| AU2014328190B2 (en) * | 2013-09-29 | 2017-02-16 | Suzhou Raymon Pharmaceuticals Company, Ltd. | Novel a-Naphthylurea derivative and medical application of same |
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