CN107141287A - The pyrido-pyrimidines of 2- imines -5- ketone group -2,5- dihydros -1-H- two - Google Patents
The pyrido-pyrimidines of 2- imines -5- ketone group -2,5- dihydros -1-H- two Download PDFInfo
- Publication number
- CN107141287A CN107141287A CN201610116185.1A CN201610116185A CN107141287A CN 107141287 A CN107141287 A CN 107141287A CN 201610116185 A CN201610116185 A CN 201610116185A CN 107141287 A CN107141287 A CN 107141287A
- Authority
- CN
- China
- Prior art keywords
- group
- substituent
- compound
- spop
- unsubstituted
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 0 CC(N(CCCN1CCOCC1)C(C(C(NC1CCCCC1)=O)=C1)=N)=C1C(N(C=CC=C1)C1=CC*)=O Chemical compound CC(N(CCCN1CCOCC1)C(C(C(NC1CCCCC1)=O)=C1)=N)=C1C(N(C=CC=C1)C1=CC*)=O 0.000 description 1
- BSDCNFHOXLSPOE-UHFFFAOYSA-N CN(C(C(C(NC1CCCCC1)=O)=C1)=N)C(N=C2N3C=CC=C2)=C1C3=O Chemical compound CN(C(C(C(NC1CCCCC1)=O)=C1)=N)C(N=C2N3C=CC=C2)=C1C3=O BSDCNFHOXLSPOE-UHFFFAOYSA-N 0.000 description 1
- RDFUVQAEICJUIP-UHFFFAOYSA-N N=C(C(C(N1C2CCCCC2)=N)=CC2=C1N=C(C=CC=C1)N1C2=O)NCCc1ccccc1 Chemical compound N=C(C(C(N1C2CCCCC2)=N)=CC2=C1N=C(C=CC=C1)N1C2=O)NCCc1ccccc1 RDFUVQAEICJUIP-UHFFFAOYSA-N 0.000 description 1
- WXIJFGMGCVIYRR-UHFFFAOYSA-N N=C(C(C(NCCCN1CCOCC1)=O)=C1)N(CCc2ccccc2)C(N=C2N3C=CC=C2)=C1C3=O Chemical compound N=C(C(C(NCCCN1CCOCC1)=O)=C1)N(CCc2ccccc2)C(N=C2N3C=CC=C2)=C1C3=O WXIJFGMGCVIYRR-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
- C07D471/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains three hetero rings
- C07D471/14—Ortho-condensed systems
Abstract
The invention provides a kind of pyrido [3,4 c of 2,5 dihydro of 2 imines, 5 ketone group, 1 H bis-:1 ', 2 ' f] pyrimidines synthesis and application thereof, specifically, the invention discloses with the compound shown in below formula (I), and its pharmaceutically acceptable salt or pharmaceutically acceptable solvate, wherein the definition of each group is as noted in the discussion.The compound of the present invention can be as the inhibitor using SPOP as target spot, or prepares the medicine for the treatment of and/or prevention using SPOP as target spot disease (such as kidney etc.).
Description
Technical field
The invention discloses with the pyridos of 2- imines -5- ketone group -2,5- dihydros -1-H- two shown in below formula (I)
[3,4-c:1 ', 2 '-f] pyrimidines and its pharmaceutically acceptable salt or pharmaceutically acceptable solvate,
It can treat the disease using SPOP as target spot, such as kidney etc. as the inhibitor using SPOP as target spot.
Background technology
Clear-cell carcinoma (Renal cell carcinoma, RCC), abbreviation kidney is the common malignant tumour of urinary system,
Account for the 2-3% of adult malignancies.Different according to histological type, kidney is broadly divided into clear cell carcinoma, and papillary carcinoma dislikes color
Cell cancer and Collecting duct carcinoma etc., wherein clear cell carcinoma of kidney (Clear cell renal cell carcinoma, ccRCC) are
The most common histological type of kidney, accounts for kidney and always falls ill the 80% of number.It is metastatic tumor that 30% is up in kidney, and remaining
There are about half can be developing progressively metastatic tumor, and the death of the annual kidney in the whole world is more than 100,000.
SPOP (Speckle-type POZ protein) belongs to MATH/BTB protein families, is used as E3 ubiquitin ligases
CUL-3 linkers, play a significant role in the ubiquitination of regulation substrate protein white matter and degraded, such as Daxx, Pdx1,
PIPKIIb, SRC-3 etc..It has now been found that there is SPOP protein abnormal expressions in kinds of tumor cells, show SPOP in maintenance
The growth and development of normal cell plays an important role.Liu Jiang in 2009 etc. discloses SPOP mistakes in hyaline cell JEG-3
Amount expression, causes people using SPOP as therapy target, the interest applied to targeted therapy clear cell carcinoma.
Hypoxia inducible factor HIF is the nucleoprotein with transcriptional activity, with quite extensive target gene spectrum, to a variety of cancers
Cell tachyauxesis plays an important role.Liu Jiang etc. has found that the hypoxia inducible factor HIF of overactivity can be with transcriptional control SPOP
Expression, and make nucleoprotein SPOP in renal carcinoma tissue overexpression and location of mistake in cytoplasm.With nuclear location SPOP's
Apoptotic function is different, and plasmotype SPOP can accelerate cell to breed.Plasmotype SPOP can be with tumor suppressor PTEN, ERK phosphorus
Sour enzyme, Daxx and Gli2 are combined, and it is degraded by ubiquitination path, so as to cause kidney to produce.Knock out energy after SPOP
Specificity kills clear cell carcinoma of kidney, but smaller to normal impact cell.
SPOP gives a clue as potential molecular probe or drug targets, while being also that Diagnosis of Renal Cell Carcinoma and treatment are provided newly
Theoretical foundation.In summary, this area, which is still needed, will develop the compound with SPOP inhibitory activity.
The content of the invention
In order to solve the above-mentioned technical problem, it is an object of the present invention to provide a kind of 2- imines -5- ketone groups -2,5- bis-
Pyrido [the 3,4-c of hydrogen -1-H- two:1 ', 2 '-f] pyrimidines and its pharmaceutically acceptable salt or pharmaceutically can be with
The solvate of receiving.
It is a further object to provide the pyridines of 2- imines -5- ketone group -2,5- dihydros -1-H- two according to the present invention
And [3,4-c:1 ', 2 '-f] pyrimidines and its pharmaceutically acceptable salt or pharmaceutically acceptable solvate
Application in SPOP inhibitor is prepared.
It is also another object of the present invention to provide the pyridines of 2- imines -5- ketone group -2,5- dihydros -1-H- two according to the present invention
And [3,4-c:1 ', 2 '-f] pyrimidines and its pharmaceutically acceptable salt or pharmaceutically acceptable solvate
It is used to treat the application in the medicine of the disease related to SPOP preparing, is especially preparing for treating in the medicine of kidney
Application.
It is yet another object of the invention to provide comprising therapeutically effective amount be selected from according to the present invention 2- imines -5- ketone groups -
Pyrido [the 3,4-c of 2,5- dihydros -1-H- two:1 ', 2 '-f] pyrimidines, its pharmaceutically acceptable salt or pharmaceutically
One or more pharmaceutical compositions as active component in acceptable solvate or its mixture.
It is yet another object of the invention to provide a kind of method for treating the disease (kidney etc.) related to SPOP, methods described
Including the pyridos of 2- imines -5- ketone group -2,5- dihydros -1-H- two being selected from according to the present invention to patient's drug treatment effective dose
[3,4-c:1 ', 2 '-f] pyrimidines, its pharmaceutically acceptable salt or pharmaceutically acceptable solvate or
One or more in its mixture of person are used as active component.
There is provided 2- imines -5- ketone group -2,5- dihydros -1- shown in a kind of below formula (I) for the first aspect of the present invention
Pyrido [the 3,4-c of H- bis-:1', 2'-f] pyrimidines, or its pharmaceutically acceptable salt or solvate：
Wherein,
X and Y are each independently nothing, or with 1, the substitution or unsubstituted of 2,3,4,5,6,7,8,9 or 10 carbon atoms
Alkylidene or alkylidene-O-；Wherein, described alkylidene is the alkylidene of straight or branched；
R1For positioned atOne or more H or C on ring1-C4Alkyl；
R2、R3It is each independently selected from the following group：Hydrogen, halogen, hydroxyl, nitro, amino, cyano group, carbonyl, substitution or unsubstituted
C2-C12Alkenyl, substitution or unsubstituted C2-C12Alkynyl, carboxyl, substitution or unsubstituted C1-C12Alkoxy, substitution or unsubstituted C1-
C12Alkylamino radical, substitution or unsubstituted C3-C9Cycloalkyl, substituted or unsubstituted C2-C9Non- heteroaryl, substituted or unsubstituted 3~
12 circle heterocycles, substituted or unsubstituted C6-C12Aryl, substituted or unsubstituted C4-C12Heteroaryl；
M is selected from the group：NH, O or S；
Wherein, the substituent that one or more hydrogen atoms that described substitution refers on group are selected from the following group is replaced：Halogen
Element, hydroxyl, nitro, amino, cyano group, carbonyl, carboxyl, C1-C6Alkyl, C1-C6Alkoxy, C1-C6Alkylamino radical, hydroxyl C1-C6Alkane
Base, amino C1-C6Alkyl, carbonyl C1-C6Alkyl, C3-C9Cycloalkyl, C6-C12Aryl, 3~12 circle heterocycles, C4-C12Heteroaryl.
In another preference, X and Y are each independently nothing, or with 1,2,3,4,5,6,7,8,9 or 10 carbon atoms
Unsubstituted straight-chain alkyl-sub；
R2、R3It is each independently selected from the following group：Hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, nitro, cyano group, carbonyl, carboxyl, amino,
Or the substituted or unsubstituted group being selected from the group：Methoxyl group, ethyoxyl, propoxyl group, different oxygen propyl group, n-butoxy, tertiary fourth oxygen
Base, N- (N, N- dimethyl) substituent, N- (N, N- diethyl) substituent, N- (N, N- dipropyl) substituent, the N- (fourths of N, N- bis-
Base) substituent, N- (N- methyl-N ethyls) substituent, N- (N- Methyl-N-propyls) substituent, N- (N- methyl-N- butyl) take
Dai Ji, N- (N- ethyl base-N- propyl group) substituent, N- (N- ethyl-N- butyl) substituent, N- (N- propyl group-N- butyl) substitutions
Base, cyclopropyl, cyclobutyl, cyclopenta, cyclohexyl, tetrahydrofuran base, tetrahydro-thienyl, nafoxidine base, THP trtrahydropyranyl, piperazine
Piperidinyl, morpholinyl, piperazinyl, phenyl, naphthyl, pyridine radicals, furyl, pyrrole radicals, thienyl, indyl, quinolyl, imidazoles
Base, or benzimidazolyl.
In another preference, X and Y are each independently nothing, or with 1, the substitution of 2,3 or 4 carbon atoms or do not take
The alkylidene or alkylidene-O- in generation；
R1Selected from hydrogen, methyl, ethyl, propyl group, isopropyl, cyclopropyl, normal-butyl or the tert-butyl group；
R2It is selected from the group：Hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, acetenyl, nitro, cyano group, carboxyl, methoxyl group, ethyoxyl, third
Epoxide, cyclohexyl epoxide, N- (N, N- dimethyl) substituent, N- (N, N- diethyl) substituent, N- (N, N- dipropyl) substitutions
Base, N- (N, N- dibutyl) substituent, N- (N- methyl-N ethyls) substituent, N- (N- Methyl-N-propyls) substituent, N- (N-
Methyl-N- butyl) substituent, N- (N- ethyl-N- propyl group) substituent, N- (N- ethyl-N- butyl) substituent, N- (N- propyl group-
N- butyl) substituent, cyclopropyl, cyclobutyl, cyclopenta, cyclohexyl, suberyl, 2,3 or 4- methylcyclohexyls, 2,3 or 4- second
Butylcyclohexyl, 2,3 or 4- hydroxy-cyclohexyls, 2,3 or 4- carboxycyclohexyls, 2,3 or 4- cyanocyclohexanoic bases, 2,3 or 4- amino rings
Hexyl, 2 or 3- tetrahydrofuran bases, 2 or 3- tetrahydro-thienyls, N, 2 or 3- nafoxidine bases, 2,3 or 4- THP trtrahydropyranyls, N, 2,
3 or 4- piperidyls, N, 2 or morpholinyl, N or 2- piperazinyls, N, 2 or 3- (N methyl piperazine base), N, 2 or 3- (N- benzyl piperazines
Piperazine base) phenyl, 2,3 or 4- aminomethyl phenyls, 2,3 or 4- methoxyphenyls, 2,3 or 4- aminophenyls, 2,3 or 4- cyano-phenyls,
2,3 or 4- carboxyl phenyls, 2,3 or 4- nitrobenzophenones, 2,3 or 4- hydroxy phenyls, naphthyl, 2 or 3- furyls, 2 or 3- thienyls,
N, 2 or 3- pyrrole radicals, 2,3 or 4- pyridine radicals, N, 2 or 3- (N- morpholinyls) phenyl, indyl.
In another preference, described X, Y, R1、R2、R3It is right that particular compound institute in embodiment is each independently with M
The group answered.
In another preference, described R1Selected from hydrogen or methyl.
In another preference, described R1For methyl.
In another preference, described R1For positioned atA methyl on ring.
In another preference, R2- X- is the group being selected from the group：Methyl, ethyl, propyl group, cyclohexyl or N, 2 or 3-
(N- morpholinyls) phenyl；
Or X be with 1, the alkylidene of 2,3 carbon atoms, and R2For the group being selected from the group：N- (N, N- diethyl) takes
Dai Ji, N, 2 or morpholinyl, phenyl, 2,3 or 4- aminomethyl phenyls, 2,3 or 4- fluorophenyls, 2,3 or 4- methoxyphenyls, 2,3 or
4- hydroxy phenyls, 2 or 3- furyls, 2 or 3- thienyls, indyl.
There is provided a kind of formula (I) compound as described in the first aspect of the invention, or its pharmacy for the second aspect of the present invention
The purposes of upper acceptable salt or solvate, for preparing SPOP target spot inhibitor, or for preparing treatment and/or preventing
Using SPOP as the disease of target spot.
In another preference, described formula (I) compound be used to suppress SPOP activity.
In another preference, described is suppressed to Reverse transcriptase.
There is provided a kind of formula (I) compound as described in the first aspect of the invention, or its pharmacy for the third aspect of the present invention
The purposes of upper acceptable salt or solvate, the proliferation activity for suppressing kidney cancer cell.
In another preference, described disease is selected from the group：Kidney, carcinoma of endometrium, germinoma.
The fourth aspect of the present invention includes therapeutically effective amount there is provided a kind of pharmaceutical composition, described pharmaceutical composition
Compound as described in the first aspect of the invention, and/or its pharmaceutically acceptable salt or pharmaceutically acceptable is molten
Agent compound is used as active component, and pharmaceutically acceptable carrier.
There is provided a kind of preparation side of formula (I) compound as described in the first aspect of the invention for the fifth aspect of the present invention
Method, methods described includes step：
In atent solvent, reacted with Formulas I a compounds and Formulas I b compounds, obtain compound of formula I.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment)
It can be combined with each other between each technical characteristic of body description, so as to constitute new or preferred technical scheme.As space is limited, exist
This no longer tires out one by one states.
Brief description of the drawings
Accompanying drawing 1 shows the pyrido [3,4-c of cell proliferation experiment checking 2- imines -5- ketone group -2,5- dihydros -1-H- two:
1 ', 2 '-f] pyrimidines are to kidney cancer cell A498 inhibitory activity；
Accompanying drawing 2 shows the pyrido [3,4-c of cell proliferation experiment checking 2- imines -5- ketone group -2,5- dihydros -1-H- two:
1 ', 2 '-f] pyrimidines are to the inhibitory activity of 6 plants of renal carcinoma cell lines and 1 plant of normal kidney proximal tubular cell line；
The display protein immunoblotting experiment of accompanying drawing 3 (Western blot) checking 2- imines -5- ketone group -2,5- dihydros -
Pyrido [the 3,4-c of 1-H- bis-:1 ', 2 '-f] pyrimidines are to the inhibitory activity of SPOP downstream signaling pathways；
Accompanying drawing 4 shows the pyridos of co-immunoprecipitation experiment (Co-IP) checking 2- imines -5- ketone group -2,5- dihydros -1-H- two
[3,4-c:1 ', 2 '-f] the intracellular Reverse transcriptase SPOP of pyrimidines and substrate interaction；
Ubiquitination experiment (In Vivo Ubiquitination Assay) checking 2- imines -5- ketone in the display body of accompanying drawing 5
Pyrido [the 3,4-c of base -2,5- dihydros -1-H- two:1 ', 2 '-f] the intracellular suppression SPOP of pyrimidines is to the ubiquitin of substrate
Change is acted on；
The pyridines of the display surface plasma resonance of accompanying drawing 6 experiment (SPR) checking 2- imines -5- ketone group -2,5- dihydros -1-H- two
And [3,4-c:1 ', 2 '-f] pyrimidines and SPOP protein-interactings；
Accompanying drawing 7 shows the pyrido [3,4- of fluorescence polarization experiment (FP) checking 2- imines -5- ketone group -2,5- dihydros -1-H- two
c:1 ', 2 '-f] pyrimidines and SPOP protein-interactings；
Accompanying drawing 8 shows normal nude mice body weight and organ weight and slices of organs Coloration experiment checking 2- imines -5- ketone group -2,
Pyrido [the 3,4-c of 5- dihydros -1-H- two:1 ', 2 '-f] pyrimidines are smaller on the influence of the body weight and organ of normal nude mice.
Accompanying drawing 9 shows the pyridos of compound plasma intracellular metabolite experimental verification 2- imines -5- ketone group -2,5- dihydros -1-H- two
[3,4-c:1 ', 2 '-f] pyrimidines have good metabolic stability.
Pyridos of tumor formation in nude mice checking 2- imines -5- ketone group -2,5- dihydros -1-H- two in the display body of accompanying drawing 10 [3,
4-c:1 ', 2 '-f] pyrimidines are to the inhibitory action of nude mice by subcutaneous tumour growth.
Embodiment
The present inventor's in-depth study by long-term, being prepared for a class has the compound of structure shown in Formulas I, and finds
It has SPOP inhibitory activity.And described compound is in extremely low concentration (IC50Value can as little as≤50nmol/L) under, i.e., to SPOP
Activity produces inhibitory action, and inhibitory activity is quite excellent, thus can be used for the treatment disease related to SPOP activity or expression quantity
Disease, such as kidney.Based on above-mentioned discovery, inventor completes the present invention.
Term
As used herein, term " C1-6Alkyl " refers to the straight or branched alkyl with 1~6 carbon atom, such as methyl,
Ethyl, propyl group, isopropyl, butyl, isobutyl group, sec-butyl, the tert-butyl group, or similar group.The statement of shape such as " C1-C6 " is intended to bag
The corresponding group with 1,2,3,4,5 or 6 carbon atom is included, for example, " C1-6Alkyl " refer to 1,2,3
The alkyl of individual, 4,5 or 6 carbon atoms, " C2-9Non- heteroaryl " refer to 2,3,4,5,6,7,8,9
The non-heteroaryl of individual carbon atom.
As used herein, term " C1-C6Alkoxy " finger-type is as " having straight or branched alkyl-oxygen of 1~6 carbon atom
The substituent of base " structure, such as ethyoxyl, propoxyl group, butoxy, or similar group.
As used herein, term " C1-C6Alkylamino radical " finger-type is as " having straight or branched alkyl-amine of 1~6 carbon atom
The substituent of base " structure, such as ethylamino-, Propylamino, dimethylamino, or similar group.
The alkyl that term " alkylidene " refers to as described above loses the group formed after a hydrogen atom, for example-
CH2-、-CH2-CH2-, or similar group.
Term " C2-C12Alkenyl " refers to the group that the alkene with 2-12 carbon atom loses a hydrogen atom formation, including
Vinyl, acrylic, n-butene base, or similar group.
Term " C3-C9Cycloalkyl " refers to the cycloalkyl with 3-9 carbon atom, such as cyclopropane, hexamethylene.
Term " C6-C12Aryl " refers to 6-12 carbon atom, and does not have heteroatomic aromatic substituent, such as benzene on skeleton
Base, or similar group.
Term " C4-C12Heteroaryl " refers to 4-12 carbon atom, and with one or more miscellaneous selected from O, S, N or P
The unsaturation ring system substituent of atom, such as pyridine radicals, thienyl, or similar group.
Term " 3~12 circle heterocycles ", which refers to, has one or more miscellaneous originals selected from O, S, N or P in the ring system with 3-12 members
The saturation ring system substituent of son, such as piperidyl, pyrrole radicals, or similar group.
Term " halogen " refers to F, Cl, Br and I.
In the present invention, term " containing ", "comprising" or " comprising " represent that various composition can be applied to the mixed of the present invention together
In compound or composition.Therefore, term " mainly by ... constitute " and " consist of " are included in term " containing ".
In the present invention, term " pharmaceutically acceptable " composition refers to suitable for people and/or animal without excessive bad pair
React (such as toxicity, stimulation and allergy), that is, have rational benefit/risk than material.
In the present invention, term " effective dose " refers to therapeutic agent treatment, alleviates or prevent the amount of target disease or situation, or table
Reveal the amount of detectable treatment or prevention effect.Accurate effective dose for a certain object depends on the build of the object and is good for
The combination of therapeutic agent and/or therapeutic agent that health situation, the nature and extent of illness and selection are given.Therefore, standard is preassigned
True effective dose is useless.However, for certain given situation, the effective dose being determined with normal experiment, is faced
Bed doctor can interpolate that out.
Herein, except special instruction part, one or more hydrogen atoms that term " substitution " refers on group are selected from down
The substituent substitution of group：Halogen, unsubstituted or halo C1-C6 alkyl, unsubstituted or halo C2-C6 acyl groups, it is unsubstituted or
The C1-C6 alkyl-hydroxyls of halo.
Unless stated otherwise, in the present invention, the compound occurred is intended to including all possible optical isomer,
Such as the mixture (i.e. racemic modification) of the compound of single chiral, or various different chipal compounds.All chemical combination of the present invention
Among thing, each asymmetric carbon atom can be optionally R configurations or S configurations, or R configurations and S configurations mixture.
As used herein, term " the compounds of this invention " refers to the compound shown in Formulas I.The term also includes and Formulas I chemical combination
Various crystalline forms, pharmaceutically acceptable salt, hydrate or the solvate of thing.
As used herein, term " pharmaceutically acceptable salt " refers to the suitable use that the compounds of this invention is formed with acid or alkali
Make the salt of medicine.Pharmaceutically acceptable salt includes inorganic salts and organic salt.The preferred salt of one class is the compounds of this invention and acid
The salt of formation.The acid for suitably forming salt includes but is not limited to：Hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid etc. are inorganic
Acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, butanedioic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid,
Picric acid, methanesulfonic acid, benzene methanesulfonic acid, the organic acid such as benzene sulfonic acid；And the acidic amino acid such as aspartic acid, glutamic acid.
Compound of formula I and its preparation
The invention provides the pyridos of 2- imines -5- ketone group -2,5- dihydros -1-H- two shown in a kind of below formula (I)
[3,4-c:1', 2'-f] pyrimidines, or its pharmaceutically acceptable salt or solvate：
Wherein, each group is as defined above described in text.
It is preferred that the compound be a kind of compound in following compounds：
Pharmaceutical composition and application process
Because the compounds of this invention has the excellent inhibitory activity to SPOP, therefore the compounds of this invention and its various crystalline substances
Type, pharmaceutically acceptable inorganic or organic salt, hydrate or solvate, and containing the compounds of this invention be chief active
The pharmaceutical composition of composition can be used for treating, prevent and alleviating by the disease related to SPOP activity or expression quantity.According to existing
There is technology, the compounds of this invention can be used for treating following disease：Kidney, clear cell carcinoma etc..
The pharmaceutical composition of the present invention comprising the compounds of this invention in the range of safe and effective amount or its be pharmacologically subjected to
Salt and pharmacologically acceptable excipient or carrier.Wherein " safe and effective amount " is referred to：The amount of compound is enough substantially
Improve the state of an illness, and be unlikely to produce serious side effect.Generally, pharmaceutical composition contains 1-2000mg the compounds of this invention/agent,
More preferably, containing 5-200mg the compounds of this invention/agent.It is preferred that described is " one " for a capsule or tablet.
" pharmaceutically acceptable carrier " is referred to：One or more biocompatible solids or liquid filler or jello
Matter, they are suitable for people and used and it is necessary to have enough purity and sufficiently low toxicity." compatibility " is referred to herein as combined
In thing each component energy and the compound of the present invention and they between mutually admix, and significantly reduce the drug effect of compound.Medicine
Acceptable carrier part example has cellulose and its derivates (such as sodium carboxymethylcellulose, ethyl cellulose sodium, fibre on
Plain acetic acid esters of dimension etc.), gelatin, talcum, kollag (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oil (such as soya-bean oil, sesame
Sesame oil, peanut oil, olive oil etc.), polyalcohol (such as propane diols, glycerine, mannitol, sorbierite), emulsifying agent ( )、
Wetting agent (such as lauryl sodium sulfate), colouring agent, flavor enhancement, stabilizer, antioxidant, preservative, apirogen water.
The method of application of the compounds of this invention or pharmaceutical composition is not particularly limited, and representational method of application includes
(but being not limited to)：Orally, in knurl, rectum, parenteral (intravenous, intramuscular is subcutaneous) and local administration.
Solid dosage forms for oral administration includes capsule, tablet, pill, powder and granule.In these solid formulations
In type, reactive compound is mixed with least one conventional inert excipients (or carrier), such as sodium citrate or Dicalcium Phosphate, or with
Following compositions are mixed：(a) filler or bulking agent, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid；(b) bond
Agent, for example, hydroxymethyl cellulose, alginates, gelatin, PVP, sucrose and Arabic gum；(c) NMF, example
Such as, glycerine；(d) disintegrant, for example, agar, calcium carbonate, farina or tapioca, alginic acid, some composition silicates,
And sodium carbonate；(e) retarding solvent, such as paraffin；(f) absorbsion accelerator, for example, quaternary ammonium compound；(g) wetting agent, such as spermaceti
Alcohol and glycerin monostearate；(h) adsorbent, for example, kaolin；Lubricant, for example, talcum, calcium stearate, tristearin (i)
Sour magnesium, solid polyethylene glycol, lauryl sodium sulfate, or its mixture.In capsule, tablet and pill, formulation can also be included
Buffer.
Solid dosage forms such as tablet, sugar-pill, capsule, pill and granule can using be coated and shell material prepare, such as casing and
Other materials well known in the art.They can include reactive compound or compound in opacifying agent, also, this composition
Release can discharge in certain part in a delayed fashion in alimentary canal.The example of adoptable embedding component is polymeric material
And Wax.If necessary, reactive compound also can be with one or more formation microencapsulation forms in above-mentioned excipient.
Liquid formulation for oral administration includes pharmaceutically acceptable emulsion, solution, suspension, syrup or tincture.
Except active ingredient beyond the region of objective existence, liquid dosage form can include the inert diluent routinely used in this area, and such as water or other solvents increase
Solvent and emulsifying agent, example knows, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propane diols, 1,3-BDO, dimethyl formyl
The mixture of amine and oil, particularly cottonseed oil, peanut oil, maize germ, olive oil, castor oil and sesame oil or these materials
Deng.
In addition to these inert diluents, composition can also include auxiliary agent, such as wetting agent, emulsifying agent and suspending agent, sweet taste
Agent, tender taste agent and spices.
Except active ingredient beyond the region of objective existence, suspension can include suspending agent, for example, ethoxylation isooctadecane alcohol, polyoxyethylene
Mixture of sorbierite and Isosorbide Dinitrate, microcrystalline cellulose, aluminium methoxide and agar or these materials etc..
For parenteral injection composition can comprising physiologically acceptable sterile, aqueous or anhydrous solution, dispersion liquid,
Suspension or emulsion, and for being dissolved into the aseptic powdery of sterile Injectable solution or dispersion liquid again.It is suitable aqueous and
Nonaqueous carrier, diluent, solvent or excipient include water, ethanol, polyalcohol and its suitable mixture.
Formulation for the local the compounds of this invention being administered includes ointment, powder, patch, propellant and inhalant.
Active component aseptically with physiologically acceptable carrier and any preservative, buffer, or if necessary may need
Propellant be mixed together.
The compounds of this invention can be administered alone, or with other pharmaceutically acceptable compound administering drug combinations.
It is the mammal that the compounds of this invention of safe and effective amount is applicable to treatment during using pharmaceutical composition
(such as people), wherein dosage is the effective dosage pharmaceutically thought when applying, for the people of 60kg body weight, day is to medicament
Amount is usually 1~2000mg, preferably 5~500mg.Certainly, specific dosage be also contemplated that method of administration, patient health situation etc. because
Element, within the scope of these are all skilled practitioners technical ability.
Main advantages of the present invention include：
1. there is provided a kind of compound shown in formula I.
2. there is provided a kind of novel SPOP inhibitor of structure and its preparation and application, described inhibitor is in extremely low concentration
All kinds of SPOP activity can be suppressed down.
3. treat the pharmaceutical composition with SPOP activity related diseases there is provided a class.
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.The experimental method of unreceipted actual conditions in the following example, generally according to conventional strip
Part, or according to the condition proposed by manufacturer.Unless otherwise indicated, otherwise percentage and number are calculated by weight.
With reference to specific embodiment, the present invention is further elaborated.These embodiments are only in order at the mesh of explanation
, rather than limitation the scope of the present invention and essence.
The source of following examples medicine:DMEM/HG culture mediums are purchased from gibco, and 10% hyclone is purchased from life
Technology, penicillin, streptomysin and tetrazolium bromide are purchased from Shanghai Sheng Gong bioengineering Co., Ltd, prepare reagent in embodiment
Purchased from Chemical Reagent Co., Ltd., Sinopharm Group and lark prestige Science and Technology Ltd..SPF grades of BALD/c null Female nude mices purchase to
Shanghai Slac Experimental Animal Co., Ltd. simultaneously raises in SPF grades of Animal Houses of Shanghai medicine institute, the care of animal and using have followed
Chinese Academy of Sciences's Shanghai medicine institute IACUC policies and guideline.
Prepare embodiment
Pyrido [the 3,4-c of 2- imines -5- ketone group -2,5- dihydros -1-H- two of the present invention:1 ', 2 '-f] miazines
Compound and its pharmaceutically acceptable salt or pharmaceutically acceptable solvate can be prepared by following route：
Below with compound N-(3- (4- morpholinyls) propyl group) -1- (2- phenylethyls) -2- imines -10- methyl -5- ketone groups
Two pyridine [4,3-b:1 ', 2 '-f] and pyrimidine -3- formamides exemplified by brief description 2- imines -5- ketone group -2,5- dihydros -1-H- two
Pyrido [3,4-c:1 ', 2 '-f] pyrimidines synthesis.
Step 1:By 10.81g (100mmol) 2- amino -3- picolines and 24.03g (150mmol) diethyl malonate
Adding in 250mL eggplants type bottle, 200 DEG C of reaction 2h, course of reaction has a large amount of solid products to generate.Reaction is cooled to room after terminating
Temperature, adds 200ml alcohol refluxs and stays overnight, be cooled to room temperature, filter, obtain 13g products, yield 76%.
1H NMR (400MHz, DMSO) δ 8.79 (dd, J=7.2,1.6Hz, 1H), 7.83 (d, J=6.9Hz, 1H), 7.17
(t, J=7.0Hz, 1H), 5.40 (s, 1H), 2.42 (s, 3H)
Step 2:200mL (calcium hydride water removal) DMF (DMF) is added into 500mL eggplants type bottle, ice bath is cold
But, 13mL (140mmol) POCl3 (P (O) Cl is then slowly added dropwise3), continue under ice bath to react 1h, 7.05g is added portionwise
(40mmol) 2- hydroxy-4-hydroxymethyl base -9- picolines [1,2- α]-pyridine amine, add rear ice bath reaction 1h, react at room temperature 2h, with
95 DEG C of reaction 2h, after reaction terminates in addition 200mL frozen water, react 1h, filtering, solid is beaten with ethanol, is filtered, and is dried afterwards,
Obtain 6.2g products, yield 70%.
1H NMR(400MHz,CDCl3) δ 10.43 (s, 1H), 9.12 (ddd, J=7.0,1.6,0.8Hz, 1H), 7.94
(ddd, J=7.1,1.7,0.9Hz, 1H), 7.35 (t, J=7.0Hz, 1H), 2.66 (s, 3H)
Step 3:222.6mg (1mmol) 2- chloro- 3- aldehyde radicals -4- carbonyls -4H-9- picolines [1,2- α] pyrimidine is added
In 5mL absolute ethyl alcohols, 484.7mg (4mmol) phenyl ethylamine is added dropwise, 12h is reacted at room temperature, reaction is filtered after terminating, solid ethanol
Fully washing, obtains 320.2mg faint yellow solids, yield 78%.
1H NMR(400MHz,CDCl3) δ 10.69 (s, 1H), 8.86 (s, 1H), 8.79 (d, J=7.0Hz, 1H), 7.47
(d, J=6.7Hz, 1H), 7.43-7.05 (m, 10H), 6.77 (t, J=7.0Hz, 1H), 3.90-3.71 (m, 4H), 2.91 (t, J
=7.4Hz, 2H), 2.86 (t, J=7.3Hz, 2H), 2.47 (s, 3H)
Step 4：1.44g (10mmol) N- (2- aminopropyls) morpholines and 1.36g (1.2mmol) cyan-acetic ester are added
Enter in 25mL eggplant-shape bottles, react 48h, reaction is washed three times after terminating with absolute ether, is dried, is put into -20 DEG C of refrigerators, obtains
1.7g faint yellow solids, yield 80%.
1H NMR(400MHz,CDCl3) δ 7.90 (s, 1H), 3.78-3.75 (m, 4H), 3.40 (dd, J=11.5,5.8Hz,
2H), (ddd, J=12.2,7.6,3.7Hz, the 2H) of 3.35 (s, 2H), 2.52-2.48 (m, 6H), 1.72
Step 5:By 108mg (0.5mmol) 9- methyl -2- (2- phenylethylamines base) -3- (2- phenethyls formimino group) pyrrole
Pyridine [1,2-a] and pyrimidin-4-one and 103.5mg (0.55mmol) 2- cyano group-N- (3- (4- morpholinyls) propyl group) acetamide are added
In 2mL chloroforms, back flow reaction 8h, reaction adds ether after terminating, and filters, and solid is recrystallized with absolute ethyl alcohol, obtains about 60mg
Product, yield 32%.
1H NMR(400MHz,CDCl3) δ 8.88 (d, J=6.8Hz, 1H), 7.65 (dt, J=6.9,1.4Hz, 1H),
7.35-7.27 (m, 4H), 7.24 (d, J=4.8Hz, 1H), 7.01 (t, J=7.0Hz, 1H), 4.62 (s, 2H), 3.72 (t, J=
4.7Hz, 4H), 3.52 (q, J=6.2Hz, 2H), 3.05 (t, J=8.1Hz, 2H), 2.59 (s, 3H), 2.49 (d, J=6.6Hz,
5H), 1.82 (q, J=6.7Hz, 3H)
Other compound of formula I can be prepared by similar method using corresponding raw material.
Pyrido [the 3,4-c of 2- imines -5- ketone group -2,5- dihydros -1-H- two:1 ', 2 '-f] miazines class compound nuclear-magnetism
Data are listed in the table below.
It is below the pyrido [3,4-c of 2- imines -5- ketone group -2,5- dihydros -1-H- two shown in logical formula (I):1’,2’-f]
Pyrimidines suppress SPOP activity experiments.
EXPERIMENTAL EXAMPLE one：Cell proliferation experiment (MTT) is to the pyridos of 2- imines -5- ketone group -2,5- dihydros -1-H- two
[3,4-c:1 ', 2 '-f] pyrimidines suppression SPOP activity ratings
Renal cancer cell line A498 with containing 10% hyclone nutrient solution (DMEM/HG culture mediums, 100U penicillin,
100U streptomysins, 10% hyclone) concentration is made into for 5 × 104-1×105Individual/mL cell suspensions.In 96 porocyte culture plates
100 μ L cell suspensions, 37 DEG C and 5%CO are inoculated with per hole2Under the conditions of overnight incubation to adherent growth, be subsequently added concentration gradient table
Compound in 1, continues to cultivate after 68h, 10 μ L tetrazolium bromides solution (5mg/mL) is added per hole, 4h is cultivated for.Culture knot
Shu Hou, inhales abandon culture medium in hole completely, 150 μ L dimethyl sulfoxides (DMSO) are added per hole, vibrate 10min, crystal is fully melted.
ELIASA determines the light absorption value of 490nm wavelength, records result and calculates cell survival rate.Using compound concentration as abscissa, carefully
Born of the same parents' survival rate is mapped for ordinate, and IC is obtained by the software nonlinear regression and fitting curves of GraphPad Prism 5.050Value.2-
Pyrido [the 3,4-c of imines -5- ketone group -2,5- dihydros -1-H- two:1 ', 2 '-f] pyrimidines IC50Value is listed in table 1.From table
It can see in 1, the pyridos [3,4-c of 2- imines -5- ketone group -2,5- dihydros -1-H- two:1 ', 2 '-f] pyrimidines can
To suppress SPOP activity.
Pyrido [the 3,4-c of 1 2- imines -5- ketone group -2,5- dihydros -1-H- of table two:1 ', 2 '-f] pyrimidines IC50
Value
EXPERIMENTAL EXAMPLE two：Pyrido [the 3,4- of cell proliferation experiment checking 2- imines -5- ketone group -2,5- dihydros -1-H- two
c:1 ', 2 '-f] pyrimidines are to the inhibitory activity of 6 plants of renal carcinoma cell lines and 1 plant of normal kidney proximal tubular cell line
Experiment is with the pyrido [3,4-c of 2- imines -5- ketone group -2,5- dihydros -1-H- two:1 ', 2 '-f] pyrimidines
In SPOP-B-30 and SPOP-B-88 exemplified by.
The renal carcinoma cell line that this experiment is selected includes A498, Caki-2, Ketr-3,769-P, OS-RC-2 and 786-O, just
Normal renal cells is HK-2 cells.Cell containing 10% (vol/vol) hyclone, 100U/ml penicillin and
The DMEM/HG culture mediums (A498, Ketr-3) or McCoy ' s5a culture mediums (Caki-2) or RPMI1640 of 100ng/ml streptomysins
Culture medium (786-O, 769-P, OS-RC-2) or DMEM/F121:Cultivated in 1 culture medium (HK-2).Cell proliferation experiment embodiment party
Method such as EXPERIMENTAL EXAMPLE one.As can be seen that SPOP-B-30 and SPOP-B-8 can significantly inhibit kidney cancer cell from accompanying drawing 2
Proliferation activity, but it is unobvious to normal renal cells HK-2 (non-tumor cell) inhibitory activity.
EXPERIMENTAL EXAMPLE three：Protein immunoblotting method experiment (Western blot) checking 2- imines -5- ketone groups -2,5-
Pyrido [the 3,4-c of dihydro -1-H- two:1 ', 2 '-f] pyrimidines are to the inhibitory activity of SPOP downstream signaling pathways
Experiment is with the pyrido [3,4-c of 2- imines -5- ketone group -2,5- dihydros -1-H- two:1 ', 2 '-f] pyrimidines
In SPOP-B-30 and SPOP-B-88 exemplified by.
By 2mL A498 cell lines re-suspension liquid (5 × 104-1×105Individual/mL) add in 6 porocyte culture plates, incubated overnight
To cell attachment state.Concentration gradient SPOP-B-30 and SPOP-B-88 are added, continues to cultivate 10h.After culture terminates, training is abandoned in suction
Base is supported, phosphate buffer is washed 3 times, is added 150 μ L RIPA cell pyrolysis liquids and 1mM phenylmethylsulfonyl fluorides (PMSF), is put on ice
Crack 30min.Lysate is scraped with cell to 1.5mL centrifuge tubes, 15000g centrifugations 20min at 4 DEG C, supernatant is transferred to
1.5mL centrifuge tubes, Supernatant protein concentration is determined with BCA methods.100 μ L of supernatant liquid are taken to add 25 μ 5 × sample-loading buffers of L, 100
DEG C heating 10min, 12000g centrifugation 1min.According to concentration adjustment applied sample amount is determined, polyacrylamide denaturing electrophoretic (SDS- is carried out
PAGE).The protein band of separation goes to nitrocellulose membrane (NC films) by electrophoretic blotting, is then closed by 5% skim milk,
Primary antibody (rabbit source PTEN antibody) is incubated, the washing of TBST buffer solutions is incubated secondary antibody (the goat-anti rabbit of horseradish peroxidase HRP marks
Antibody), the washing of TBST buffer solutions lights development step to detect protein content finally by Enhanced chemiluminescence (ECL).From attached
Can significantly it find out in Fig. 3, with the increase of SPOP-B-30 and SPOP-B-8 concentration, SPOP substrate PTEN and DUSP7 content increase
Plus, downstream signaling pathway bioactive molecule AKT and ERK1/2 phosphorylation weaken, and illustrate that SPOP-B-30 and SPOP-B-8 can be with
Suppress SPOP activity, and then increase suppression cancer substrate protein PTEN and DUSP7 content.
EXPERIMENTAL EXAMPLE four：The pyrroles of co-immunoprecipitation experiment (Co-IP) checking 2- imines -5- ketone group -2,5- dihydros -1-H- two
Pyridine simultaneously [3,4-c:1 ', 2 '-f] the intracellular Reverse transcriptase SPOP of pyrimidines and substrate interaction
Experiment is with the pyrido [3,4-c of 2- imines -5- ketone group -2,5- dihydros -1-H- two:1 ', 2 '-f] pyrimidines
In SPOP-B-30 and SPOP-B-88 exemplified by.
HEK293 cells, density about 30%-50% are planted in 60mm culture dishes.Next day transfects corresponding plasmid myc-
PTEN, Flag-SPOP.40h is transfected, each group sucks culture medium after adding corresponding amount micromolecular compound, 8h, with the PBS of precooling
Wash twice.The 200 μ l RIPA cell pyrolysis liquids in the addition precooling of every ware cell after remnants PBS are blotted, with the cell of precooling
Scrape and scrape off cell from culture dish, draw suspension into EP pipes, 10-30min is placed on ice.Under the conditions of 4 DEG C, 12000rpm
10min is centrifuged, supernatant is transferred into new EP manages, BCA standard measure protein concentrations.The 30 total eggs of μ l are reserved after leveling protein concentration
In vain, 5 × sample-loading buffer is added, boiling water boiling is denatured for 5 minutes.Each sample takes the agarose beads suspensions of 20 μ l antibody couplings,
Washed with PBST three times, finally carry out 1 by volume:1 is resuspended.Add 30 μ l beads re-suspension liquids in each sample, then add 300 μ l
RIPA lysates, 4 DEG C of rotations are incubated 4h or stayed overnight.5000g is centrifuged 1 minute at 4 DEG C, removes supernatant.Add 500 μ l PBST bufferings
Liquid, 5000g is centrifuged 1 minute at 4 DEG C of rotation washings 10 minutes, 4 DEG C, removes supernatant.Repetition is washed three times.Supernatant is removed in last time centrifugation
Afterwards, the supernatant participated in only is blotted as far as possible, is then added in 30 μ l 2 × albumen sample-loading buffers, boiling water and is denatured 5 minutes.After denaturation
Sample at room temperature 8000g centrifuge, draw supernatant carry out SDS-PAGE electrophoresis, be careful not to be drawn onto beads.Can be with from accompanying drawing 4
It is be combined with each other, it is evident that SPOP-B-30 and SPOP-B-88 can suppress SPOP and substrate PTEN in the cell.
EXPERIMENTAL EXAMPLE five：Internal ubiquitination experiment (In Vivo Ubiquitination Assay) checking 2- imines-
Pyrido [the 3,4-c of 5- ketone group -2,5- dihydros -1-H- two:1 ', 2 '-f] the intracellular suppression SPOP of pyrimidines is to substrate
Ubiquitination is acted on
Experiment is with the pyrido [3,4-c of 2- imines -5- ketone group -2,5- dihydros -1-H- two:1 ', 2 '-f] pyrimidines
In SPOP-B-30 and SPOP-B-88 exemplified by.
HEK293 cells, density about 30%-50% are planted in 60mm culture dishes.Next day is by myc-PTEN, Flag-SPOP
And HA-Ub difference cotransfection HEK cells.40h after transfection, each group, which is added, adds 10 μM after corresponding amount micromolecular compound, 4h
MG132 culture medium continues to cultivate 4h.After culture terminates, culture medium is removed, culture dish is placed on ice chest, with the PBS of precooling
Wash the denatured lysis liquid (1%SDS for once adding 100 μ l precoolings afterwards；0.5mM EDTA；1mM DTT；50mM Tris,pH
7.5).Quick scraped with cell scrapes cell into 1.5ml EP pipes from culture dish, is denatured 5min at 100 DEG C immediately.Denaturation terminates
Lysate is placed in cooled on ice afterwards, the RIPA lysates for being eventually adding 900 μ l are diluted.With ultrasonically treated after having diluted,
Ultrasound condition is：Amplitude 10%, interval 10s switches, total ultrasonic time 1 minute.12000g centrifuges 5min at 4 DEG C, and supernatant is turned
Move on in new EP pipes.According to protein quantification result leveling sample room protein concentration.Reserved 100 μ l are as on input, plus albumen
100 DEG C of denaturation 5min of sample buffer solution.Remaining lysate carries out immunoprecipitation with the agarose beads of myc antibody couplings, last
Eluted product carries out Western blotting with HA antibody.The result of accompanying drawing 5 is, it is apparent that SPOP-B-30 and SPOP-B-88 can be
Intracellular suppression SPOP is acted on substrate PTEN ubiquitination.
EXPERIMENTAL EXAMPLE six：Surface plasma resonance experiment (SPR) checking 2- imines -5- ketone group -2,5- dihydros -1-H- two
Pyrido [3,4-c:1 ', 2 '-f] pyrimidines and SPOP protein-interactings
Experiment is with the pyrido [3,4-c of 2- imines -5- ketone group -2,5- dihydros -1-H- two:1 ', 2 '-f] pyrimidines
In SPOP-B-30 and SPOP-B-88 exemplified by.
The SPOP of fresh purifyingMATHAlbumen (purity ＞ 95%, concentration 5mg/mL) is dilute with 10mM sodium acetate solutions (pH4.5)
Release to 0.1mg/mL.By BIAcore T200 (GE companies) using the amino coupled program of standard by SPOPMATHAlbumen coupling
To CM5 chips.CM5 chips balance 2h with HBS-EP buffer solutions before experiment.SPOP-B-30 and SPOP-B-88 is slow with HBS-EP
Fliud flushing gradient dilution, balances 60s with 30 μ L/min flow velocitys, with reference to 120s, then rinses 120s.The response curve of gained passes through
BIA DAS (GE companies) is fitted, and obtains corresponding KDValue.From accompanying drawing 6, it is apparent that SPOP-B-30 and
SPOP-B-88 and SPOPMATHAlbumen has certain combination.
EXPERIMENTAL EXAMPLE seven：The pyridos of fluorescence polarization experiment (FP) checking 2- imines -5- ketone group -2,5- dihydros -1-H- two
[3,4-c:1 ', 2 '-f] pyrimidines and SPOP protein-interactings
Experiment is with the pyrido [3,4-c of 2- imines -5- ketone group -2,5- dihydros -1-H- two:1 ', 2 '-f] pyrimidines
In SPOP-B-30 and SPOP-B-88 exemplified by.
Fluorescence polarization (Fluorescence polarization, FP) is tested in multi-function microplate reader (PerkinElmer
Company) on complete.INSTRUMENT MODEL is EnVision Multilabel Plate Reader.Experiment fluorescein isothiocynate
(FITC) polypeptide of mark is as spike substrate, and sequence is FITC-LACDEVTSTTSSSTA (synthesis of gill biochemical corp).
2 μM of SPOP are added in 100 μ L hydroxyethyl piperazines second sulphur acid buffer (50mM, pH 7.5) buffer solutionsMATH, 20nM polypeptides bottom
Thing, concentration gradient SPOP-B-30 and SPOP-B-88,4 DEG C are incubated 1h, detect its fluorescence polarization value.Exciting light used and transmitting light
Respectively 480nm and 535nm.In triplicate, experimental data used uses the software analysis of GraphPad Prism 5.0 for experiment.From
In accompanying drawing 7 as can be seen that SPOP-B-30 and SPOP-B-88 with SPOP protein-interactings.
EXPERIMENTAL EXAMPLE eight：Pyrido [the 3,4- of mouse animal experiment checking 2- imines -5- ketone group -2,5- dihydros -1-H- two
c:1 ', 2 '-f] pyrimidines toxicity
Experiment is with the pyrido [3,4-c of 2- imines -5- ketone group -2,5- dihydros -1-H- two:1 ', 2 '-f] pyrimidines
In SPOP-B-30 and SPOP-B-88 exemplified by.
Compound toxicity in vivo experiment 6 to 8 week old BALB/c and ICR mouse used are dynamic purchased from Shanghai Si Laike experiments respectively
Western pul Bi Kai experimental animals Co., Ltd of thing Co., Ltd (SLAC) and Shanghai.Random packet, every group 3.Abdomen once a day
Chamber injection solvent, 80mg/kg, 120mg/kg compounds, and weigh in and Survey Operations state.Posterior orbit blood sampling in 6-7 days, enters
Row analysis of Hematology Changes and Plasma Biochemical detection.Internal organ (heart, liver, spleen, lung and kidney) are taken to carry out after mouse anesthesia sudden death
HE is dyed and pathological section detection.Accompanying drawing 8 is as can be seen that compound injection concentration is that 80mg/kg is safe concentration.
EXPERIMENTAL EXAMPLE nine：Pyrido [the 3,4- of mouse animal experiment detection 2- imines -5- ketone group -2,5- dihydros -1-H- two
c:1 ', 2 '-f] the interior metabolism of pyrimidines body
Experiment is with the pyrido [3,4-c of 2- imines -5- ketone group -2,5- dihydros -1-H- two:1 ', 2 '-f] pyrimidines
In SPOP-B-30 exemplified by.
4-6 week old BALB/c mouses are grouped at random, every group 3.Compound uses physiological saline, 0.5%2- benzyl carbinols,
1.5% Tween-80 is prepared suspension as solvent and is injected intraperitoneally, and is 10mg/kg to compound concentration.Respectively to chemical combination
0.25,0.5,1,2,4,6,10 and 24 hours click through end of line venous blood collection, every blood sampling 0.15mL after thing.Blood sample is used
EDTA anti-freezings, centrifuging and taking supernatant carries out SPOP-B-30 compounds contents in LC-MS/MS methods detection serum.Can from accompanying drawing 9
It is 1070ng/ml, half-life period is 6.25 hours to calculate after blood concentration highest appears in administration 0.5 hour.
EXPERIMENTAL EXAMPLE ten：Pyrido [the 3,4- of mouse animal experiment checking 2- imines -5- ketone group -2,5- dihydros -1-H- two
c:1 ', 2 '-f] pyrimidines activity
A498 cells are with DMEM/HG+10% hyclone culture mediums in 5%CO2, 37 DEG C of CMC models.Pancreatin is collected to disappear
A498 cells after change, cell suspension, cell number to 10 are prepared with without hyclone culture medium8Cells/ml concentration.Take -20
DEG C preserve matrigel (Martrigel, BD company) dissolved naturally at 4 DEG C, cell suspension：Matrigel Martrigel 3:1(v/
V) 100 μ L (10 are injected at once after mixing7Individual cell) it is subcutaneous to 6 week old nude mice right fore armpits.Observe tumour growth situation simultaneously
With vernier caliper measurement length of tumor (a millimeters) and width (b millimeters), according to v=a*b2/ 2 calculate gross tumor volume, treat tumour body
Product about 125mm3Shi Jinhang is grouped, 40 4 groups, every group 10 of experiment nude mices point.Compound SPOP-B-30 solvent (physiology
Salt solution, 0.5%2- benzyl carbinols, 1.5% Tween-80) to 5mg/mL, SPOP-B-88 physiological saline solutions to 5mg/mL.Daily
It is injected intraperitoneally according to 0,40,60,80mg/kg amount, and every 3 days carry out a weighing body weight and measurement of tumor, carry out corresponding
Nursing and record.Control group and experimental group tumour growth situation are observed, stops administration within 25 days or so, it is sudden to carry out anesthesia to nude mice
Dead and dissected, every group of heart, liver, spleen, lung, kidney for taking 2 nude mices respectively carries out tissue HE dyeing observations.Simultaneously
Isolate tumour to be weighed, taken pictures and liquid nitrogen cryopreservation, the tumor tissues of preservation are used for protein immunoblot detection tissue PTEN
Amount.
It can be seen from figure 10 that the gross tumor volume of administration SPOP-B-30 and SPOP-B-88 mouse is significantly less than only
The mouse of injection solvent, from the protein immunoblot detection of tumor tissues, can equally observe SPOP contents in tissue
Substantially reduction.Zoopery shows that SPOP-B-30 and SPOP-B-88 can slow down the growth of tumour by suppressing SPOP.
All documents referred in the present invention are all incorporated as reference in this application, independent just as each document
It is incorporated as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read, those skilled in the art can
To be made various changes or modifications to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited
Enclose.
Claims (10)
1. pyrido [the 3,4-c of 2- imines -5- ketone group -2,5- dihydros -1-H- two shown in a kind of below formula (I):1',2'-f]
Pyrimidines, or its pharmaceutically acceptable salt or solvate：
Wherein,
X and Y are each independently nothing, or with 1, the substituted or unsubstituted Asia of 2,3,4,5,6,7,8,9 or 10 carbon atoms
Alkyl or alkylidene-O-；Wherein, described alkylidene is the alkylidene of straight or branched；
R1For positioned atOne or more H or C on ring1-C4Alkyl；
R2、R3It is each independently selected from the following group：Hydrogen, halogen, hydroxyl, nitro, amino, cyano group, carbonyl, substitution or unsubstituted C2-C12
Alkenyl, substitution or unsubstituted C2-C12Alkynyl, carboxyl, substitution or unsubstituted C1-C12Alkoxy, substitution or unsubstituted C1-C12Alkanamine
Base, substitution or unsubstituted C3-C9Cycloalkyl, substituted or unsubstituted C2-C9Non- heteroaryl, substituted or unsubstituted 3~12 yuan it is miscellaneous
Ring, substituted or unsubstituted C6-C12Aryl, substituted or unsubstituted C4-C12Heteroaryl；
M is selected from the group：NH, O or S；
Wherein, the substituent that one or more hydrogen atoms that described substitution refers on group are selected from the following group is replaced：Halogen, hydroxyl
Base, nitro, amino, cyano group, carbonyl, carboxyl, C1-C6Alkyl, C1-C6Alkoxy, C1-C6Alkylamino radical, hydroxyl C1-C6Alkyl, amino
C1-C6Alkyl, carbonyl C1-C6Alkyl, C3-C9Cycloalkyl, C6-C12Aryl, 3~12 circle heterocycles, C4-C12Heteroaryl.
2. formula (I) compound as claimed in claim 1, or its pharmaceutically acceptable salt or solvate, its feature exists
In,
X and Y are each independently nothing, or with 1, the unsubstituted straight chain alkylene of 2,3,4,5,6,7,8,9 or 10 carbon atoms
Base or alkylidene-O-；
R2、R3It is each independently selected from the following group：Hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, nitro, cyano group, carbonyl, carboxyl, amino, or take
Generation or the unsubstituted group being selected from the group：Methoxyl group, ethyoxyl, propoxyl group, different oxygen propyl group, n-butoxy, tert-butoxy, N-
(N, N- dimethyl) substituent, N- (N, N- diethyl) substituent, N- (N, N- dipropyl) substituent, N- (N, N- dibutyl) take
Dai Ji, N- (N- methyl-N ethyls) substituent, N- (N- Methyl-N-propyls) substituent, N- (N- methyl-N- butyl) substituent,
N- (N- ethyl base-N- propyl group) substituent, N- (N- ethyl-N- butyl) substituent, N- (N- propyl group-N- butyl) substituent, ring
Propyl group, cyclobutyl, cyclopenta, cyclohexyl, tetrahydrofuran base, tetrahydro-thienyl, nafoxidine base, THP trtrahydropyranyl, piperidyl,
Morpholinyl, piperazinyl, phenyl, naphthyl, pyridine radicals, furyl, pyrrole radicals, thienyl, indyl, quinolyl, imidazole radicals, or benzene
And imidazole radicals.
3. formula (I) compound as claimed in claim 1, or its pharmaceutically acceptable salt or solvate, its feature exists
In,
X and Y are each independently nothing, or with 1, the substituted or unsubstituted alkylidene of 2,3 or 4 carbon atoms or alkylidene-
O-；
R1Selected from hydrogen, methyl, ethyl, propyl group, isopropyl, cyclopropyl, normal-butyl or the tert-butyl group；
R2It is selected from the group：Hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, acetenyl, nitro, cyano group, carboxyl, methoxyl group, ethyoxyl, propoxyl group,
Cyclohexyl epoxide, N- (N, N- dimethyl) substituent, N- (N, N- diethyl) substituent, N- (N, N- dipropyl) substituent, N-
(N, N- dibutyl) substituent, N- (N- methyl-N ethyls) substituent, N- (N- Methyl-N-propyls) substituent, N- (N- methyl-
N- butyl) substituent, N- (N- ethyl-N- propyl group) substituent, N- (N- ethyl-N- butyl) substituent, N- (N- propyl group-N- fourths
Base) substituent, cyclopropyl, cyclobutyl, cyclopenta, cyclohexyl, suberyl, 2,3 or 4- methylcyclohexyls, 2,3 or 4- ethyl rings
Hexyl, 2,3 or 4- hydroxy-cyclohexyls, 2,3 or 4- carboxycyclohexyls, 2,3 or 4- cyanocyclohexanoic bases, 2,3 or 4- aminocyclohexyls,
2 or 3- tetrahydrofuran bases, 2 or 3- tetrahydro-thienyls, N, 2 or 3- nafoxidine bases, 2,3 or 4- THP trtrahydropyranyls, N, 2,3 or 4-
Piperidyl, N, 2 or morpholinyl, N or 2- piperazinyls, N, 2 or 3- (N methyl piperazine base), N, 2 or 3- (N-benzyl piperazine base)
Phenyl, 2,3 or 4- aminomethyl phenyls, 2,3 or 4- methoxyphenyls, 2,3 or 4- aminophenyls, 2,3 or 4- cyano-phenyls, 2,3 or
4- carboxyl phenyls, 2,3 or 4- nitrobenzophenones, 2,3 or 4- hydroxy phenyls, naphthyl, 2 or 3- furyls, 2 or 3- thienyls, N, 2 or
3- pyrrole radicals, 2,3 or 4- pyridine radicals, N, 2 or 3- (N- morpholinyls) phenyl, indyl.
4. formula (I) compound as claimed in claim 1, or its pharmaceutically acceptable salt or solvate, its feature exists
In R2- X- is the group being selected from the group：Methyl, ethyl, propyl group, cyclohexyl or N, 2 or 3- (N- morpholinyls) phenyl；
Or X be with 1, the alkylidene of 2,3 carbon atoms, and R2For the group being selected from the group：N- (N, N- diethyl) substituent,
N, 2 or morpholinyl, phenyl, 2,3 or 4- aminomethyl phenyls, 2,3 or 4- fluorophenyls, 2,3 or 4- methoxyphenyls, 2,3 or 4- hydroxyls
Base phenyl, 2 or 3- furyls, 2 or 3- thienyls, indyl.
5. formula (I) compound as claimed in claim 1, or its pharmaceutically acceptable salt or solvate, its feature exists
In the compound is a kind of compound in following compounds：
6. formula (I) compound as described in claim any one of 1-5, or its pharmaceutically acceptable salt or solvate
Purposes, it is characterised in that for preparing SPOP target spot inhibitor, or for preparing treatment and/or preventing using SPOP as target spot
Disease.
7. formula (I) compound as described in claim any one of 1-5, or its pharmaceutically acceptable salt or solvate
Purposes, it is characterised in that the proliferation activity for suppressing kidney cancer cell.
8. purposes as claimed in claim 6, it is characterised in that described disease is selected from the group：Kidney, carcinoma of endometrium, life
Cell colonization tumour.
9. a kind of pharmaceutical composition, it is characterised in that described pharmaceutical composition include therapeutically effective amount such as claim 1-5
Compound described in any one, and/or its pharmaceutically acceptable salt or pharmaceutically acceptable solvate is used as work
Property composition, and pharmaceutically acceptable carrier.
10. the preparation method of formula (I) compound as described in claim 1-5 is any, it is characterised in that including step：
In atent solvent, reacted with Formulas I a compounds and Formulas I b compounds, obtain compound of formula I.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610116185.1A CN107141287B (en) | 2016-03-01 | 2016-03-01 | 2-imine-5-keto-2, 5-dihydro-1-H-dipyridopyrimidine compounds |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610116185.1A CN107141287B (en) | 2016-03-01 | 2016-03-01 | 2-imine-5-keto-2, 5-dihydro-1-H-dipyridopyrimidine compounds |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107141287A true CN107141287A (en) | 2017-09-08 |
| CN107141287B CN107141287B (en) | 2021-10-26 |
Family
ID=59783373
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610116185.1A Active CN107141287B (en) | 2016-03-01 | 2016-03-01 | 2-imine-5-keto-2, 5-dihydro-1-H-dipyridopyrimidine compounds |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107141287B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107954933A (en) * | 2017-10-19 | 2018-04-24 | 浙江大学 | A kind of new 2- azabicyclics [4.2.0] octane compound and its synthesis and application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015061280A1 (en) * | 2013-10-21 | 2015-04-30 | Alevere Medical Corporation | Fused heterocyclic organic compounds, pharmaceutical compositions, and medical uses thereof |
| WO2016020892A1 (en) * | 2014-08-07 | 2016-02-11 | University Of Cape Town | Small molecule inhibitors for cancer therapy |
-
2016
- 2016-03-01 CN CN201610116185.1A patent/CN107141287B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015061280A1 (en) * | 2013-10-21 | 2015-04-30 | Alevere Medical Corporation | Fused heterocyclic organic compounds, pharmaceutical compositions, and medical uses thereof |
| WO2016020892A1 (en) * | 2014-08-07 | 2016-02-11 | University Of Cape Town | Small molecule inhibitors for cancer therapy |
Non-Patent Citations (6)
| Title |
|---|
| DORJBAL DORJSUREN ET AL.: "Diverse Small Molecule Inhibitors of Human Apurinic/Apyrimidinic Endonuclease APE1 Identified from a Screen of a Large Public Collection", 《PLOS ONE》 * |
| LATECA S. GLASS ET AL.: "Semi-automated high-throughput fluorescent intercalator displacement-based discovery of cytotoxic DNA binding agents from a large compound library", 《BIOORGANIC & MEDICINAL CHEMISTRY LETTERS》 * |
| REGISTRY: "1794159-07-1", 《STN COLUMN》 * |
| REGISTRY: "370573-01-6", 《STN COLUMN》 * |
| REGISTRY: "879947-97-4", 《STN COLUMN》 * |
| SLIVCHUK, S. R. ET AL.: "Cyclization of condensation products of 5-(acylamino)-4-aryl-2-thiazoleacetonitriles with aromatic aldehydes", 《ZHURNAL ORGANICHNOI TA FARMATSEVTICHNOI KHIMII》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107954933A (en) * | 2017-10-19 | 2018-04-24 | 浙江大学 | A kind of new 2- azabicyclics [4.2.0] octane compound and its synthesis and application |
| CN107954933B (en) * | 2017-10-19 | 2020-10-23 | 浙江大学 | 2-azabicyclo [4.2.0] octane compounds and synthesis and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107141287B (en) | 2021-10-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP1163243B1 (en) | Calanolides for inhibiting btk | |
| CN107213466B (en) | A kind of column aromatic hydrocarbons compound, preparation method, pharmaceutical composition and purposes | |
| CN107889488A (en) | Imidazoles ketone group quinoline and its purposes as ATM kinase inhibitors | |
| CN104203242B (en) | Substituted quinolines are used as bruton's tyrosine kinase inhibitor | |
| CN103974955B (en) | Pyrimido-pyridazinone compound and application thereof | |
| CN107286077A (en) | A kind of selective C-KIT kinase inhibitors | |
| CN106928206A (en) | Aldehyde radical class compound and its preparation method and purposes | |
| CN106573906A (en) | Piperidine-dione derivatives | |
| PT2588457E (en) | Pyrazoloquinoline derivatives as dna-pk inhibitors | |
| CN106831824A (en) | Pyrrolopyridines and its application containing naphthyridones structure | |
| CN108969522A (en) | Purposes of the N- benzyl tryptamines ketones derivant as tryptophan dioxygenase (TDO) inhibitor | |
| CN109475559A (en) | Inhibit the novel quinazoline quinoline ketone derivatives of PI3K and the pharmaceutical composition containing it | |
| CN106831725A (en) | The quinazoline compounds and its application of quinoline containing indoline and similar structures | |
| CN105189486A (en) | Conjugate of benzofuranone and indole or azaindole, and preparation and uses thereof | |
| CN106083704B (en) | Application of-two aryl methylene-N- cyclopropyl piperidine -4- ketone compounds of 3,5- (E) as Hsp90 inhibitor | |
| CN104161759B (en) | The anticancer usage of anagrelide and its derivative | |
| CN106565685A (en) | Tubulin inhibitor | |
| CN102066362A (en) | Dihydroindolinone derivatives | |
| CN107141287A (en) | The pyrido-pyrimidines of 2- imines -5- ketone group -2,5- dihydros -1-H- two | |
| JP6239103B2 (en) | Substance having tyrosine kinase inhibitory activity and preparation method and use thereof | |
| CN110152001A (en) | Purposes of small molecule compound and combinations thereof | |
| CN105130960B (en) | 1,3,5- triazine derivatives and its application | |
| CN102268014B (en) | Condensed heteroaryl derivative and its preparation method and use | |
| CN103965107A (en) | 2-aryl-substituted quinoline compounds and application thereof | |
| IL298864A (en) | Alkenyl pyrimidine compound, preparation method therefor, and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |