CN108947895B - Compound with anticancer activity - Google Patents
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- CN108947895B CN108947895B CN201810962495.4A CN201810962495A CN108947895B CN 108947895 B CN108947895 B CN 108947895B CN 201810962495 A CN201810962495 A CN 201810962495A CN 108947895 B CN108947895 B CN 108947895B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
- C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
- C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D213/72—Nitrogen atoms
- C07D213/73—Unsubstituted amino or imino radicals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
Abstract
The present invention provides compounds of formula 1 and methods for their synthesis and use. Preferred compounds are potent inhibitors of c-Met protein kinase and are useful in the treatment of abnormal cell growth disorders, such as cancer.
Description
Technical Field
The present invention relates generally to novel chemical compounds and methods. More particularly, the invention provides aminoheteroaryl compounds having protein tyrosine kinase activity, and methods of synthesizing and using such compounds. Preferred compounds are c-Met inhibitors useful in the treatment of abnormal cell growth (e.g., cancer).
Background
Hepatocyte Growth Factor (HGF) receptor (c-MET or HGFR) Receptor Tyrosine Kinase (RTK) has been shown to be involved in tumorigenesis, tumor progression with enhanced cell motility and invasion, and Metastasis in many human cancers (see, e.g., Ma, p.c., Maulik, g., Christensen, J. & saligia, R. (2003 b); Cancer Metastasis Rev, 22, 309-25; Maulik, g., Shrikhande, a., Kijima, t., Ma, p.c., Morrison, P.T. & saligi, R. (2002 b); cytokinase Growth Factor Rev 13, 41-59). c-met (hgfr) can be activated by overexpression or mutation in various human cancers, including Small Cell Lung Cancer (SCLC) (Ma, p.c., Kijima, t., malulik, g., Fox, e.a., Sattler, m., Griffin, j.d., Johnson, B.E. & saligia, R. (2003 a.). Cancer Res, 63, 6272-.
c-MET is a receptor tyrosine kinase, which is encoded by the MET proto-oncogene and transduces the biological effects of Hepatocyte Growth Factor (HGF), also known as Scatter Factor (SF). Jiang et al, crit.rev.oncol.hematol.29: 209-248(1999). c-MET and HGF are expressed in many tissues, although their expression is usually mainly restricted to epithelial and mesenchymal derived cells, respectively. c-MET and HGF are required for normal mammalian development and have been shown to be important in cell migration, cell proliferation and survival, morphogenic differentiation, and the formation of 3-dimensional tubular structures (e.g., renal duct cells, glandular formation, etc.). In addition to its effect on epithelial cells, HGF/SF has been reported to be an angiogenic factor, and signaling of c-MET on endothelial cells can induce many cellular responses (proliferation, motility, invasion) necessary for angiogenesis.
The c-MET receptor has been shown to be expressed in many human cancers. c-MET and its ligand HGF have also been shown to be co-expressed at high levels in a variety of human cancers, particularly sarcomas. However, since this receptor and ligand are often expressed by different cell types, c-MET signaling is most often regulated by tumor-stroma (tumor-host) interactions. Furthermore, c-MET gene amplification, mutations and rearrangements have been found in a subfamily of human cancers. A population of germline mutants of activated c-MET kinase, susceptible to multiple renal tumors, and tumors in other tissues. Many studies have correlated the expression of c-MET and/or HGF/SF with the disease progression status of different cancer types (including lung, colon, breast, prostate, liver, pancreas, brain, kidney, ovary, stomach, skin and bone cancers). Furthermore, overexpression of c-MET or HGF has been shown to be associated with poor prognosis and disease progression in many major human cancers, including lung, liver, stomach, and breast. c-MET has also been directly implicated in cancer without successful treatment regimens, such as pancreatic cancer, glioma, and hepatocellular carcinoma.
Disclosure of Invention
The present invention provides a compound of formula I, or a pharmaceutically acceptable salt, hydrate or solvate thereof,
wherein Y is N or CR 7;
r1, R2 and R3 are the same or different and are selected from hydrogen, alkyl, alkylthio, alkoxy optionally substituted with fluorine, dialkylamino, piperidino, morpholino, halogen, phenylalkyl and phenylalkoxy;
r4 is selected from the group consisting of hydrogen, halogen, C6-12 aryl, 5-12 membered heteroaryl, C3-12 cycloalkyl, 3-12 membered heteroalicyclic, -CN, -NO2, C1-8 alkyl, C2-8 alkenyl, and C2-8 alkynyl; and each hydrogen atom in R4 is optionally substituted with one or more R6 groups;
r5 is hydrogen, halogen, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C3-12 cycloalkyl, C6-12 aryl, 3-12 membered heteroalicyclic, 5-12 membered heteroaryl, -NO2, -CN;
r6 is independently halogen, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C3-12 cycloalkyl, C6-12 aryl, 3-12 membered heteroalicyclic, 5-12 membered heteroaryl, -NO2, -CN, and the R6 groups on adjacent atoms may join together and form C6-12 aryl, 5-12 membered heteroaryl, C3-12 cycloalkyl or 3-12 membered heteroalicyclic;
r7 is hydrogen, halogen, C1-12 alkyl, C2-12 alkenyl, C2-12 alkynyl, C3-12 cycloalkyl, C6-12 aryl, 3-12 membered heteroalicyclic, or 5-12 membered heteroaryl.
In another particular aspect of this particular embodiment, R5 is hydrogen.
In another particular aspect of this embodiment, Y is N.
In another particular aspect of this embodiment, Y is N and R5 is hydrogen.
In another particular aspect of this particular embodiment, R4 is furan, thiophene, pyrrole, dihydropyrrole, tetrahydropyrrole, dioxolane, oxazole, thiazole, imidazole, dihydroimidazole, tetrahydroimidazole, pyrazole, dihydropyrazole, tetrahydropyrazole, isoxazole, isothiazole, oxadiazole, triazole, thiadiazole, pyran, pyridine, piperidine, dioxane, morpholine, dithiane, thiomorpholine, pyridazine, pyrimidine, pyrazine, piperazine, triazine, trithiacyclohexane, azetidine, or phenyl.
In another particular aspect of this particular embodiment, R1 is hydrogen.
In another particular aspect of this particular embodiment, R3 is hydrogen.
In another embodiment, the invention provides a pharmaceutical composition comprising any of the compounds of the invention, and a pharmaceutically acceptable carrier.
Preferred compounds of the invention include those having c-MET inhibitory activity as defined by any one or more of IC50, Ki or percent inhibition (% I). One skilled in the art can readily determine whether a compound has such activity by performing an appropriate assay, and a description of such an assay is provided in the examples section herein. In one embodiment, most preferred compounds have a c-MET Ki of less than 5 μ M, or less than 2 μ M, or less than 1 μ M, or less than 500nM or less than 200nM or less than 100 nM. In another particular embodiment, most preferably the compound has an inhibition of c-MET at 1 μ Μ of at least 10% or at least 20% or at least 30% or at least 40% or at least 50% or at least 60% or at least 70% or at least 80% or at least 90%. Methods for measuring c-MET/HGFR activity are described in the examples herein.
In another embodiment, the invention provides a method of treating abnormal cell growth in a mammal, including a human, comprising administering to the mammal any of the pharmaceutical compositions of the invention.
In any particular embodiment of the methods of the invention described herein, the abnormal cell growth is a cancer, including, but not limited to, lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intra-ocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, endometrial cancer, cervical cancer, vaginal cancer, Hodgkin's disease, esophageal cancer, small bowel cancer, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, chronic or acute leukemia, lymphobulbar lymphoma, bladder cancer, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, neoplasms of the Central Nervous System (CNS), neoplasms of the colon), and cancers of the urinary tract, Primary CNS lymphoma, spinal axis tumor, brain stem glioma, pituitary adenoma, or a combination of one or more of the foregoing cancers. In another embodiment of the method, the abnormal cell growth is a benign proliferative disease including, but not limited to, psoriasis, benign prostatic hypertrophy or restenosis.
In another embodiment, the invention provides a method of treating HGFR mediated diseases in a mammal, including a human, comprising administering to the mammal any of the pharmaceutical compositions of the invention.
Defining:
unless otherwise indicated, the following terms used in the present specification and claims have the meanings discussed below.
Alkyl refers to a saturated aliphatic hydrocarbon group, including straight and branched chain groups of 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms, more preferably 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 4 carbon atoms. Lower alkyl means in particular alkyl having 1 to 4 carbon atoms. Examples of alkyl groups include methyl, ethyl, propyl, 2-propyl, n-butyl, isobutyl, tert-butyl, pentyl, and the like. Alkyl groups may be substituted or unsubstituted. Typical substituents include cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl, thiocarbonyl, O-carbamoyl, N-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl, C-acylamino, N-acylamino, C-carboxy, O-carboxy, nitro, silyl, amino and-NRxRy, wherein Rx and Ry are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, carbonyl, acetyl, sulfonyl, trifluoromethanesulfonyl and combined five-or six-membered heteroalicyclic rings.
Cycloalkyl refers to a3 to 8 membered all carbon monocyclic ring, an all carbon 5-membered/6-membered or 6-membered/6-membered fused bicyclic ring, or a polycyclic fused ring (by fused ring system it is meant that each ring system in this system shares an adjacent pair of carbon atoms with every other ring in this system) group, wherein one or more rings may contain one or more double bonds, but such rings do not have a fully conjugated pi-electron system. Examples of cycloalkyl groups are, but are not limited to, cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexadiene, adamantane, cycloheptane, cycloheptatriene, and the like. Cycloalkyl groups may be substituted or unsubstituted. Typical substituents include alkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl, thiocarbonyl, C-carboxy, O-carbamoyl, N-carbamoyl, C-acylamino, N-acylamino, nitro, amino and-NRxRy, wherein Rx and Ry are as defined above. Illustrative examples of cycloalkyl groups are derived from (but not limited to) the following:
alkenyl refers to an alkyl group, as defined herein, comprising at least two carbon atoms and at least one carbon-carbon double bond. Representative examples include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 1-, 2-or 3-butenyl, and the like.
Alkynyl refers to an alkyl group as defined herein, comprising at least two carbon atoms and at least one carbon-carbon triple bond. Representative examples include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-, 2-or 3-butynyl, and the like.
Aryl means an all-carbon monocyclic or fused ring polycyclic group of 6 to 12 carbon atoms with an intact conjugated pi-electron system. Examples of aryl groups are, but not limited to, phenyl, naphthyl, and anthracenyl. Aryl groups may be substituted or unsubstituted. Typical substituents include halo, trihalomethyl, alkyl, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, nitro, carbonyl, thiocarbonyl, C-carboxy, O-carbamoyl, N-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl, C-acylamino, N-acylamino, sulfinyl, sulfonyl, amino and-NRxRy, wherein Rx and Ry are as defined above.
Heteroaryl means a monocyclic or fused ring of 5 to 12 ring atoms, containing one, two, three or four ring heteroatoms selected from N, O and S, the remaining ring atoms being C and, in addition, having an intact conjugated pi-electron system. Examples of unsubstituted heteroaryl groups are, but are not limited to, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline, purine, tetrazole, triazine, and carbazole. Heteroaryl groups may be substituted or unsubstituted. Typical substituents include alkyl, cycloalkyl, halo, trihalomethyl, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, nitro, carbonyl, thiocarbonyl, sulfonamido, C-carboxy, O-carboxy, sulfinyl, sulfonyl, O-carbamoyl, N-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl, C-acylamino, N-acylamino, amino and-NRxRy, wherein Rx and Ry are as defined above.
Heteroalicyclic or heterocyclic ring means a monocyclic or fused ring having 3 to 12 ring atoms in the ring, wherein one or two ring atoms are heteroatoms selected from N, O and S (O) n (wherein n is 0, 1 or 2), and the remaining ring atoms are C. Such rings may also have one or more double bonds. However, such rings do not have a fully conjugated pi-electron system.
The heterocyclic group may be substituted with one or two substituents independently selected from the group consisting of halo, lower alkyl substituted with carboxy, ester hydroxy or mono-or dialkylamino.
Hydroxyl refers to the-OH group.
Alkoxy refers to both-O- (alkyl) or-O- (unsubstituted cycloalkyl). Representative examples include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy, and the like.
Haloalkoxy means-O- (haloalkyl). Representative examples include, but are not limited to, trifluoromethoxy, tribromomethoxy, and the like.
Halo means fluoro, chloro, bromo or iodo, preferably fluoro or chloro.
Cyano means a-C.ident.N group.
Nitro means a-NO 2 group.
Haloalkyl means alkyl, preferably lower alkyl, which is substituted by one or more of the same or different halo atoms, e.g., -CH2Cl, -CF3, -CH2CF3, -CH2CCl3, and the like.
Hydroxyalkyl means alkyl, preferably lower alkyl, which is substituted by one, two or three hydroxy groups; such as hydroxymethyl, 1 or 2-hydroxyethyl, 1, 2-, 1, 3-or 2, 3-dihydroxypropyl, and the like.
Aralkyl means an alkyl group, preferably a lower alkyl group, which is substituted by an aryl group as defined above; such as-CH 2 phenyl, - (CH2)2 phenyl, - (CH2)3 phenyl, CH3CH (CH3) CH2 phenyl, and the like, and derivatives thereof.
Heteroarylalkyl means an alkyl group, preferably a lower alkyl group, which is substituted with a heteroaryl group; such as-CH 2 pyridyl, - (CH2)2 pyrimidinyl, - (CH2)3 imidazolyl, and the like, and derivatives thereof.
Monoalkylamino refers to the group-NHR, wherein R is alkyl or unsubstituted cycloalkyl; such as methylamino, (1-methylethyl) amino, cyclohexylamino, and the like.
Dialkylamino refers to the group-NRR, where each R is independently alkyl or unsubstituted cycloalkyl; dimethylamino, diethylamino, (1-methylethyl) -ethylamino, cyclohexylmethylamino, cyclopentylmethylamino and the like.
A pharmaceutical composition refers to a mixture of one or more compounds described herein, or a physiologically/pharmaceutically acceptable salt, solvate, hydrate or prodrug thereof, with other chemical ingredients such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate the administration of the compound to an organism.
As used herein, a physiologically/pharmaceutically acceptable carrier means that the carrier or diluent does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
Pharmaceutically acceptable excipients refer to inert substances that are added to a pharmaceutical composition to further aid in the administration of the compound. Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and starch types, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.
The term pharmaceutically acceptable salt refers to salts which retain the biological effectiveness and properties of the parent compound. Such salts include:
(1) acid addition salts obtainable by reaction of the free base of the parent compound with inorganic acids, such as hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid and perchloric acid, and the like, or with organic acids, such as acetic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, tartaric acid, citric acid, succinic acid or malonic acid, and the like; or
(2) When an acidic proton present in the parent compound is replaced by a metal ion, such as an alkali metal ion, an alkaline earth metal ion, or an aluminum ion; or a salt formed when coordinated with an organic base such as ethanolamine, diethanolamine, triethanolamine, butanetriethanolamine, N-methylglucamine, or the like.
Compared with the prior art, the compound provided by the invention has high anticancer activity.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to specific examples. The following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention.
The general synthetic scheme for the compounds of formula I of the present invention is:
wherein Y, R1-R5 are as defined in claim 1.
To an ice-cooled solution of compound a (1.0 molar equivalent) and anhydrous tetrahydrofuran (0.14M), sodium hydride (1.0 molar equivalent) was slowly added under a nitrogen atmosphere. After stirring for 30 minutes, compound B (1.0 molar equivalent) in tetrahydrofuran (0.56M) was added at a rapid dropwise rate through an addition funnel. Once the addition was complete, the ice bath was removed and the reaction was refluxed under nitrogen and monitored by reverse phase HPLC. After completion of the reaction, the reaction was cooled to room temperature, concentrated under reduced pressure, diluted with ethyl acetate and washed with brine. And (4) separating an organic layer, dehydrating and drying the organic layer by using anhydrous magnesium sulfate, and concentrating the dried organic layer under reduced pressure to obtain a crude product. And purifying the crude product by using a silica gel column chromatography to obtain the compound shown in the formula I.
Example 1
Preparation of 3- ((4-methoxy-3, 5-dimethylpyridin-2-yl) methoxy) -5-phenylpyridin-2-amine
Starting with the compound (4-methoxy-3, 5-dimethylpyridin-2-yl) methanol and 3-bromo-5-phenylpyridin-2-amine, compound 1, MS: 336.163[ M + H+],c-Met Ki:5.12nM
Example 2
Preparation of 5- (4-fluorophenyl) -3- (4-methoxy-3, 5-dimethylpyridin-2-yl) methoxy) pyridin-2-amine
Starting with the compound (4-methoxy-3, 5-dimethylpyridin-2-yl) methanol and 3-bromo-5- (4-fluorophenyl) pyridin-2-amine, compound 2 was prepared according to the general synthetic scheme, MS: 354.154[ M + H+],c-Met Ki:3.72nM
Example 3
Preparation of 5- (4-fluorophenyl) -3- ((4-methoxypyridin-2-yl) methoxy) pyridin-2-amine
Starting with the compound (4-methoxypyridin-2-yl) methanol and 3-bromo-5- (4-fluorophenyl) pyridin-2-amine, compound 3, MS: 326.123[ M + H+],c-Met Ki:4.55nM
Example 4
5- ((4-methoxy-3, 5-dimethylpyridin-2-yl) methoxy) -2-methyl-3, 4' -bipyridin-6-amine
Compound 5 was prepared according to the general synthetic scheme starting from compound (4-methoxy-3, 5-dimethylpyridin-2-yl) methanol and 5-bromo-2-methyl-3, 4' -bipyridin-6-amine (MS): 351.174[ M + H+],c-Met Ki:7.79nM
Example 5
Preparation of 5- ((3, 5-dimethyl-4- (2,2, 2-trifluoroethoxy) pyridin-2-yl) methoxy) -2-methyl-3, 4' -bipyridin-6-amine
Starting from the compound (3, 5-dimethyl-4- (2,2, 2-trifluoroethoxy) pyridin-2-yl) methanol and 5-bromo-2-methyl-3, 4' -bipyridin-6-amine, compound 6 was prepared according to the general synthetic scheme, MS: 419.162 c-Met Ki 6.32nM
Biological examples
It will be appreciated that in any particular series of compounds, a range of biological activities will be found. In its presently preferred aspects, the present invention relates to novel compounds capable of modulating, modulating and/or inhibiting the activity of protein kinases. The following assays can be employed to select compounds that exhibit the most desirable degree of activity.
Detection method
The following in vitro assays may be used to determine the activity and extent of action of various compounds of the invention on one or more PKs. Similar assays can be designed along the same route for any PK using techniques known in the art. Literature reference data is provided (Technikova-Dobrova Z, Sardanelli AM, Papa S FEBS Lett.1991Nov 4; 292: 69-72).
The general method is as follows: the compound and kinase detection reagent are introduced into the test well. Detection is initiated by addition of kinase. Enzyme inhibitors reduce the measured enzyme activity.
In a continuous coupled spectrophotometric assay, ADP is measured by time-dependent production of kinase by analyzing the rate of consumption of NADH by measuring the decrease in absorbance at 340 nm. When PK produces ADP, it is reconverted to ATP by reaction with pyruvate enolphosphate and pyruvate kinase. Pyruvate is also produced in this reaction. Pyruvate is then converted to lactate by reaction with lactate dehydrogenase, which simultaneously converts NADH to NAD. NADH has a measurable absorbance at 340 nm, whereas NAD is not.
The presently preferred method for performing a sequential coupled spectrophotometric assay for a particular PK is provided below. However, modifications of this method to determine the activity of compounds against other RTKs as well as CTK and STK are known within the knowledge of the person skilled in the art.
HGFR continuous coupling spectrophotometric detection
This assay analyzed the tyrosine kinase activity of HGFR for the Met-2 substrate peptide, a peptide derived from the HGFR activation loop.
Materials and reagents:
1. directory #14-526 of HGFR enzymes (Met, Activity) from Upstate
Met-2 peptide (HGFR activation loop) Ac-ARDMYDKEYYSVHNK (MW 1960). Dissolved in 200mM HEPES, pH7.5, in 10mM stock solution.
3.1MPEP (phosphoryl-enol pyruvate) in 200mM HEPES, pH7.5
4.100mM ADH (B-nicotinamide adenine dinucleotide, reduced form) in 200mM HEPES, pH7.5
5.4M MgCl2 (magnesium chloride) in ddH2In O
6.1M DTT (dithiothreitol) in 200mM HEPES, pH7.5
7.15 units/ml LDH (lactate dehydrogenase)
8.15 units/ml PK (pyruvate kinase)
9.5M NaCl, dissolved in ddH2In O
Tween-20 (protein grade) 10% solution
11.1M HEPES buffer: (N- [ 2-Hydroethyl)]piperazine-N- [ 2-ethanesulfonic acid]) Sodium salt. Dissolved in ddH2In O, the pH was adjusted to 7.5 to make the volume 1 liter. Filtration was carried out at 0.1 μm.
HPLC grade water; burdick and Jackson #365-4, 1X 4 liter (or equivalent)
13.100％DMSO(Sigma)
Costar #3880 Black clear Flat bottom half area plates for Ki determination and% inhibition
Costar #3359-96 well polypropylene plates, round bottom, for serial dilution
Costar # 3635-UV-plate, clear flat-bottom plate for% inhibition
Beckman DU-650 w/minicell holder
Beckman 4-position minicell cuvettes
The method comprises the following steps:
preparation of a Dilute Buffer (DB) for the enzyme (30 ml preparation is provided)
DB Final concentration of 2mM DTT, 25mM NaCl2, 5mM MgCl2, 0.01% Tween-20 and 50mM HEPES buffer, pH7.5.
2. 50mM HEPES was made up by adding 1.5 ml of 1M HEPES to 28.1 ml of ddH 2O. The remaining reagents were added. To a 50 ml conical vial, 60. mu.l of 1M DTT, 150. mu.l of 5M NaCl2, 150. mu.l of 1M MgCl2 and 30. mu.l of 10% Tween-20 were added to give a total volume of 30 ml.
3. Whirling for 5-10 seconds.
4. The DB liquid was taken out at 1 ml/tube and the tube was identified as "DB HGFR
5. Note that: this can be prepared and stored beforehand.
6. The unused aliquots were frozen in microcentrifuge tubes in a-20 ℃ freezer.
Preparation of the Compounds
1. For compound dilution plates, add 4 μ l of 10mM stock solution to plate lane 1 and bring the volume to 100 μ l with 100% DMSO.
2. A Precision (Precision)2000 dilution method was set up. Final concentration of 200. mu.M compound in 50% DMSO, 100mM HEPES (1: 2 serial dilution).
Preparation of coupling enzyme buffer:
1. final concentration in assay:
2. for 10 ml of reaction buffer, 10. mu.l of 1M PEP, 33. mu.l of 100mM NADH, 50. mu.l of 4M MgCl2, 20. mu.l of 1M DTT, 6. mu.l of 500mM ATP and 500. mu.l of 10mM Met-2 peptide were added to 100mM HEPES buffer pH7.5 and vortexed/mixed.
3. Enzyme, LDH and PK were added to the reaction mixture. Mixing was performed by gentle inversion.
Operating sample
1. Setting a spectrophotometer:
2. add 85 μ l of CE reaction mixture to each well of the test plate.
3. Add 5 μ l of diluted compound to wells of the test plate.
4. Add 5 μ l of 50% DMSO for negative control to the last straight row of the assay plate.
5. Mixing with multichannel pipettor or orbital shaker.
6. Preincubation was carried out at 37 ℃ for 10 minutes.
7. Add 10 μ l of 500nM HGFR to each well of the assay plate; the final HGFR concentration was 50nM in a total final volume of 100. mu.l.
8. The activity was measured at 340 nm at 37 ℃ for 10 minutes.
The following in vitro assays may be used to determine the activity and extent of action of various compounds of the invention on one or more PKs. Similar assays can be designed along the same route for any PK using techniques known in the art.
Several assays described herein were performed in an ELISA (enzyme-linked immunosorbent sandwich assay) format (Voller et al, 1980, "enzyme-linked immunosorbent assay", A clinical immunology Manual, 2 nd edition, Rose and Friedman, am. Soc. of Microbiology, Washington, D.C., p. 359-. The general method is as follows: the compound is introduced into cells expressing the test kinase, either naturally or recombinantly, over a selected time period, and then, if the test kinase is a receptor, a ligand known to activate the receptor is added. The cells were lysed and the lysate was transferred to the wells of an ELISA plate pre-coated with a specific antibody that would recognize the substrate of the enzymatic phosphorylation reaction. The cell lysate is washed free of non-substrate components and the amount of phosphorylation on the substrate is detected with an antibody that specifically recognizes phosphotyrosine, as compared to control cells that have not been contacted with the test compound.
The presently preferred method of performing ELISA experiments for specific PKs is provided below. However, modifications of such methods to determine the activity of compounds against other RTKs as well as CTK and STK are known within the knowledge of the person skilled in the art.
Other assays described herein measure the amount of DNA produced in response to activation of a test kinase, which is a common measure of proliferative response. The general method for this test is as follows: the compound is introduced into cells expressing the test kinase, either naturally or recombinantly, over a selected time period, and then, if the test kinase is a receptor, a ligand known to activate the receptor is added. After at least overnight incubation, DNA-labeling reagents, such as 5-bromodeoxyuridine (BrdU) or H3-thymidine, are added. The amount of labeled DNA is detected either as anti-BrdU antibody or by measuring radioactivity and compared to control cells not contacted with test compound.
MET transphosphorylation assay
This assay is used to measure phosphotyrosine content for poly (glutamate: tyrosine 4: 1) substrates as a means of identifying agonists/antagonists of met transphosphorylation of the substrate.
Materials and reagents:
corning 96-well ELISA plates, Corning catalog # 25805-96.
2. Poly (glu-tyr), 4: 1, Sigma catalog number; p0275.
PBS, Gibco catalog #450-1300EB
4.50mM HEPES
5. Blocking buffer: 25 g of bovine serum albumin, Sigma Cat. No. A-7888, was dissolved in 500 ml of PBS and filtered through a 4 micron filter.
6. Purified GST fusion protein containing Met kinase functional site, SUGEN
TBST buffer.
8.10% aqueous (MilliQue H2O) DMSO.
9.10mM aqueous (dH2O) adenosine-5' -triphosphate, Sigma catalog number A-5394.
10.2 diluted kinase buffer: for 100 ml, 10 ml of 1M HEPES, pH7.5, was mixed with 0.4 ml of 5% BSA/PBS, 0.2 ml of 0.1M sodium orthovanadate and 1 ml of 5M sodium chloride in 88.4 ml of dH 2O.
11.4 XATP reaction mix: for 10 ml, 0.4 ml of 1M manganese dichloride was mixed with 0.02 ml of 0.1MATP in 9.56 ml of dH 2O.
12.4X negative control mixture: for 10 ml, 0.4 ml of 1M manganese dichloride in 9.6 ml of dH2And mixing in O.
NUNC 96-well V-bottom polypropylene plate, catalog # S-72092 applied sciences
14.500mM EDTA。
15. Antibody diluted buffer: for 100 ml, 10 ml of 5% BSA/PBS, 0.5 ml of 5% Carnation quick-dissolving milk powder in PBS, and 0.1 ml of 0.1M sodium orthovanadate in 88.4 ml of TBST were mixed.
16. Rabbit polyclonal anti-phosphotyrosine antibody, SUGEN Corp
17. Goat anti-rabbit horseradish peroxidase conjugated antibody, Biosource
ABTS solution: for 1 liter, 19.21 g of citric acid and 35.49 g of Na2HPO4And 500 mg ABTS, with a sufficient amount of dH2O was mixed to make 1 liter.
ABTS/H2O 2: five minutes prior to use, 15 ml of ABST solution was mixed with 2. mu.l of H2O2And (4) mixing.
20.0.2M HCl
The method comprises the following steps:
1. ELISA plates were coated with 2. mu.g of poly (Glu-Tyr) in 100. mu.l of PBS and kept at 4 ℃ overnight.
2. The plate was blocked with 150 μ l of 5% BSA/PBS for 60 min.
3. The plate was washed twice with PBS and then once with 50mM Hepes buffer pH 7.4.
4. Add 50 μ l of diluted kinase to all wells (purified kinase diluted in kinase dilute buffer; final concentration should be 10 ng/well).
5. Add 25 μ l test compound (in 4% DMSO) or DMSO alone for control (4% in dH2O) to the plate.
6. The kinase/compound mixture was incubated for 15 minutes.
7. Add 25 μ l of 40mM MnCl2 to the negative control well.
8. Add 25 μ l ATP/MnCl2 mixture to all other wells (except negative control). Incubate for 5 minutes.
9. The reaction was stopped by adding 25. mu.l of 500mM EDTA.
10. Plate 3x was washed with TBST.
11. 100 microliters of rabbit polyclonal anti-Ptyr diluted 1: 10,000 in antibody diluted buffer was added to each well. Incubate at room temperature for one hour and shake.
12. Plate 3x was washed with TBST.
13. Biosource HRP conjugated anti-rabbit antibody was diluted 1: 6,000 in antibody diluent buffer. Add 100 μ l per well and incubate at room temperature for one hour with shaking.
14. Plate 1X was washed with PBS.
15. 100 microliter ABTS/H addition2O2Solution was added to each well.
16. If necessary, 100. mu.l of 0.2M HCl was added per well to stop the reaction from progressing.
17. Plates were read on a Dynatech MR7000 ELISA reader with test filter at 410nM and reference filter at 630 nM.
BrdU incorporation detection
The following assay uses cells designed to express selected receptors and then evaluates the effect of the compound of interest on ligand-induced DNA synthesis activity by determining BrdU incorporation into DNA.
The following materials, reagents and methods are generic for each of the BrdU incorporation assays described below. The variation in a particular assay is indicated.
General materials and reagents:
1. suitable ligands are described.
2. A suitably engineered cell.
BrdU labeling reagent: 10mM IN PBS, pH7.4(Roche molecular biochemicals, Indianapolis, IN).
FixDenat: fixative solution (Roche molecular biochemicals, Indiana polis, IN).
5. anti-BrdU-POD: mouse monoclonal antibodies conjugated to peroxidase (Chemicon, Temecula, CA).
TMB substrate solution: tetramethylbenzidine (TMB, immediately available, Roche molecular biochemicals, Indianapolis, IN).
PBS wash solution: 1XPBS, pH 7.4.
8. Bovine albumin (BSA), part V powder (Sigma chemical, USA).
The general method comprises the following steps:
1. cell lines were seeded in 96-well plates at 8000 cells/well in 10% CS, 2mM Gln in DMEM. The cell lines were cultured overnight at 37 ℃ in 5% CO 2.
After 2.24 hours, cells were washed with PBS and then serum depleted for 24 hours in serum free medium (0% CS in DMEM with 0.1% BSA).
3. On day 3, the appropriate ligand is added to the cells simultaneously with the test compound. Negative control wells received DMEM without serum with only 0.1% BSA; positive control cells received ligand but no test compound. Test compounds were prepared in 96-well plates in serum-free DMEM with ligands and serially diluted to provide 7 assay concentrations.
4. 18 hours after ligand activation, diluted BrdU-labelling reagent (1: 100 in DMEM, 0.1% BSA) was added and the cells were incubated with BrdU (final concentration 10. mu.M) for 1.5 hours.
5. After incubation with the labeling reagent, the medium was removed by decantation and the inverted plate was allowed to drain on paper towels. FixDenat solution (50. mu.l/well) was added and the plates were incubated at room temperature for 45 minutes on a plate shaker.
6. The FixDenat solution was removed by decantation and the inverted plate was drained on paper towels. Milk (5% dehydrated milk powder in PBS, 200 μ l/well) was added as blocking solution and the plates were incubated for 30 min at room temperature on a plate shaker.
7. The blocking solution was removed by decantation and the wells were washed once with PBS. anti-BrdU-POD solution (1: 200 diluted in PBS, 1% BSA, 50. mu.l/well) was added and the plates were incubated for 90 min at room temperature on a plate shaker.
8. The antibody conjugate was removed by decantation and the wells were washed 5 times with PBS and the plates were dried by inversion and tapping on paper towels.
9. TMB substrate solution (100 μ l/well) was added and incubated on a plate shaker at room temperature for 20 minutes until the color developed sufficiently for photometry detection.
10. The absorbance of the sample was measured at 410nm (in "two-wavelength" mode, with a filter reading at 490 nm as the reference wavelength) on a Dynatech ELISA plate reader.
HGF-induced BrdU incorporation detection
Materials and reagents:
1. recombinant human HGF (catalog No. 249-HG, R & D systems, USA).
BxPC-3 cells (ATCC CRL-1687).
The remaining materials and reagents were as described above.
The method comprises the following steps:
1. cells were seeded at 9000 cells/well in RPMI 10% FBS in 96-well plates. The cell lines were cultured overnight at 37 ℃ in 5% CO 2.
After 2.24 hours, cells were washed with PBS and then serum consumed in 100 μ l serum free medium (RPMI with 0.1% BSA) for 24 hours.
3. On day 3, 25 microliters containing ligand (prepared in RPMI with 0.1% BSA at 1 microgram/ml; final HGF concentration of 200 nanograms/ml) and test compound were added to the cells. Negative control wells received 25 microliters of RPMI without serum with only 0.1% BSA; positive control cells received ligand (HGF) but no test compound. Test compounds were prepared at 5-fold their final concentration in serum-free ligand-bearing RPMI in 96-well plates and serially diluted to obtain 7 assay concentrations. Typically, the highest final concentration of test compound is 100. mu.M, and a 1: 3 dilution is used (i.e., the final test compound concentration ranges from 0.137-100. mu.M).
4. After 18 hours of ligand activation, 12.5 microliters of diluted BrdU labeling reagent (1: 100 in RPMI, 0.1% BSA) was added to each well and the cells were incubated with BrdU (final concentration of 10. mu.M) for 1 hour.
5. The same as the general method.
6. The same as the general method.
7. The blocking solution was removed by decantation and the wells were washed once with PBS. anti-BrdU-POD solution (1: 100 diluted in PBS, 1% BSA) (100. mu.l/well) was added and the plates were incubated for 90 min at room temperature on a plate shaker.
8. The same as the general method.
9. The same as the general method.
10. The same as the general method.
Cellular HGFR autophosphorylation assay
A549 cells (ATCC) were used in this assay. Cells were seeded into 96-well plates in growth medium (RPMI + 10% FBS) and cultured overnight at 37 ℃ for adsorption. Cells were exposed to starvation medium (RPMI + 0.05% BSA). Dilutions of inhibitors were added to the plates and incubated at 37 ℃ for 1 hour. Cells were then stimulated for 15 minutes by the addition of 40 nanograms/ml HGF. Cells were washed once with 1mM Na3VO4 in HBSS and then lysed. The lysates were diluted with 1mM Na3VO4 in HBSS and transferred to 96-well goat anti-rabbit coated plates (Pierce) pre-coated with anti-HGFR antibody (Zymed laboratories). Plates were incubated overnight at 4 ℃ and washed seven times with 1% Tween 20 in PBS. HRP-PY20(Santa Cruz) was diluted and added to the plate over a 30 minute incubation period. Then, the plate was washed once more and TMB peroxidase substrate (Kirkegaard & Perry) was added and incubated for 10 minutes. Subsequently, the reaction was stopped by adding 0.09N H2SO 4. The plate was measured at OD-450 nm using a spectrophotometer. IC50 values were calculated by curve fitting using a four parameter analysis.
The compounds of the present invention were measured for HGFR inhibitory activity; data are shown in the examples. Ki data were obtained using HGFR sequential coupling spectrophotometric assay detection, whereas IC50 data were obtained using cellular HGFR autophosphorylation detection, both described above.
Claims (4)
1. A compound of the formula I, wherein,
wherein Y is CR7 ；
R1And R3Selected from hydrogen; r2Selected from methoxy;
R4is selected from phenyl, and R4Each hydrogen atom in (A) is optionally substituted by one or more R6Radical substitution, R6Selected from fluorine;
R5selected from hydrogen;
R7selected from hydrogen;
or a pharmaceutically acceptable salt thereof.
2. Use of a compound of claim 1, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for treating abnormal cell growth in a mammal.
3. The use of claim 2, wherein the abnormal cell growth is cancer.
4. A pharmaceutical composition comprising the compound of claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
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