CN101133325A - Organic compounds - Google Patents

Abstract

The invention relates to an assay for identifying an agent that modulates the interaction of a mRNA with a target protein, e.g. HuR and to organic compounds of formula (I); wherein R1, R2 and R3 are as defined above, and their use, e.g. as an inhibitor on the complex-formation of an ARE-containing mRNA and a target protein.

Description

Organic compound

Invention field

The present invention relates to organic compound, and relate to the determination method that is used to identify interactional material between regulating mRNA and target protein such as the ELAVL1 albumen.

Technical background

A large amount of expression of gene are controlled on messenger rna level.Especially, the early stage response gene that has a disease association with some stimulation from but the rapid answer of multiple target promoted because of post-transcriptional control mechanism usually.These processes are mainly by regulation protein that mRNA is worked or factor mediation.Suppress or regulate this class control said target mrna-protein interactions and represented attractive therapeutic intervention means.

In order to seek the low-molecular-weight depressor that suppresses the mRNA regulation and control, need a kind of determination method of monitoring target mRNA-protein-interacting, be in homogeneous solution, to carry out ideally.Described determination method should also be suitable for implementing in high flux screening (HTS) environment, and the present major part of high flux screening is usually based on fluoroscopic examination.

Relatively little protein, as mRNA stable protein HuR (36kD), with long mRNA or subfragrnent (281-1643 nucleotide, direct combination 100-500kD), can not detect based on the relative increase of size, because fluorescently-labeled RNA decays when compound forms.In view of this, can easily get rid of based on the spectroscopic methodology (for example fluorescence correlation spectroscopy (FCS), 2D-FIDA-anisotropy or other fluorescence polarization measurement methods) that rotates or translation detects diffusion time.

Determination method of the present invention has been represented a kind of new method of monitoring the mRNA-protein interaction in the homogeneous solution under real equilibrium condition.For example this method is used extremely sensitive Single Molecule Detection method, directly is suitable for HTS platform such as Evotec MarkII/III thus.Because described detection such as confocal detection have high sensitivity and degree of accuracy, so determination method of the present invention has been represented the attractive possibility that is different from conventional electrophoretic mobility shift assay, filter-binding assay or RNase protection analysis.It also will be flat-footed adapting to conventional macroscopical fluorescence intensity detection method (for example fluorescent plate reader), might not need to utilize confocal instruments.

Summary of the invention

One aspect of the present invention provides the determination method that is used to identify interactional material between regulating mRNA and the target protein, and described determination method comprises:

A) mRNA that provides length to be at least the mark of 100 nucleotide chooses wantonly the described material that is labeled as the change sensitivity in the mRNA environment is provided as homogeneous solution;

B) do not exist with the situation that has candidate compound under the mRNA that provides in target protein such as HuR and the step a) is contacted, described candidate compound expection will between described mRNA and described target protein are as HuR, can form in sufficiently long time of compound mRNA as described in the adjusting and as described in interaction between target protein such as the HuR;

C) detect the compound that forms in the step b);

D) whether the amount that is determined at formed compound under the situation that does not have or exist candidate compound in the step b) there are differences; With

E) candidate compound that will be measured to difference in step d) is elected the evaluation material as.

Measuring principle can be described as follows that (for example, as shown in fig. 1): mark mRNA for example, connects chemistry at 3 ' the terminal Cy3 mark of using by hydrazine aldehyde.In case target protein is bonded to the mRNA of mark, the quantum yield of then described mark such as Cy3 changes, and for example increase, and this change provides mRNA and the interactional readout of target protein.This effect does not obviously relate to protein and contacts with the direct of mark, and, even for target protein (as HuR) binding site away from its 3 ' end mark mRNA for, also can be observed repeatability.We infer that this possibility of result is because when forming compound with target protein at present, the RNA three-dimensional conformation changes, due to long-range effect generation translation, and (for example owing to the environmental sensitivity of mark such as Cy3, referring to Mujumdar R.B etc., Bioconj Chem.1993,4 (2) 105-11).The minimizing that forms because of the protein due to the potential inhibitor-mRNA compound will detect the decline into readout such as total fluorescence intensity, or the minimizing with material grains quantity of more macromolecule brightness.

In preferred embodiments, target protein is a HuR albumen.

MRNA can be for containing the mRNA of ARE, and comprise: inflammatory target for example comprises the ARE from TNF-α, IL-1 β, IL-2, IL-8, Cox-2, IL-4 or AT-R1, and comprises the target family of other ARE regulation and control.For example, proto-oncogene is as c-myc, c-jun or c-fos.

In another aspect of this invention, mRNA is selected from the sequence of coding IL-2, IL-1 β and TNF-α, or this class sequence contains the fragment of ARE.

In another aspect of this invention, mRNA has 100-500 nucleotide, the length of preferred 300 nucleotide.

Mark can be for routine, and biological example element or enzyme are as alkaline phosphatase (AP), horseradish peroxidase (HRP) or peroxidase (POD) or fluorescence molecule, for example fluorescent dye.Preferably be labeled as fluorescent dye, for example Cy3 or Cy5, for example Cy3.

In preferred one side of the present invention, be labeled as fluorescence labeling, for example Cy3 or Cy5.

Target protein can be known any protein in conjunction with mRNA, and wherein this class is in conjunction with causing the RNA three-dimensional conformation to change.In another aspect of this invention, target protein in conjunction with contain the mRNA of ARE ELAVL1 (=HuR).

In order to detect the compound of formation, can use detection means.This class detection means comprises those method commonly used, for example fluoroscopic examination mensurations in the mensuration field.Be used for the molecule that detection means of the present invention comprises identification marking mRNA.

For example, use one dimension intensity dependence confocal fluorescent detection method FIDA (fluorescence intensity distributional analysis), can be directly and with size independently mode, tracking target protein such as HuR and its said target mrna combines in homogeneous solution.

In another aspect of this invention, detect compound by measuring fluorescence intensity.

Choose wantonly and can from non-compound fraction, isolate the compound of formation.

For example, can be according to separating with the similar method of conventional method, for example chromatography, for example size exclusion chromatography.

Candidate compound comprises can measure its compound (storehouse) to mRNA and the interactional regulating action of target protein.Compound (storehouse) comprising: for example oligopeptides, polypeptide, protein, antibody, analogies, micromolecule, for example low molecular weight compound (LMW`s).

Material is interactional compound, for example material that detects between influence (inhibition) mRNA and the target protein in determination method step d) provided by the invention.

Material is one of candidate compound of selecting, and can comprise oligopeptides, polypeptide, protein, antibody, analogies, micromolecule, for example low molecular weight compound (LMW`s).Material comprises one or more materials, for example the combination of material.

The present invention provides the determination method of the present invention that is used for high flux screening on the other hand.

The present invention provides kit on the other hand, and it comprises:

The mRNA of-mark, for example fluorescently-labeled mRNA;

-target protein;

The instructions of this class kit of-use; With

-optional candidate compound.

This class kit provided by the invention may further include the entity component, comprises the control environment of testing sample, and the suitable means of for example measuring the effect of candidate compound in the testing sample.

The invention still further relates to the organic compound of identifying by above-mentioned Screening test method.

The present invention provides the compound of following formula on the other hand:

Wherein

R ₁Be (the C that is replaced by following group _1-4) alkyl: (the C that is not substituted or replaces _6-18) aryl or have 5 or 6 ring memberses and 1-4 is selected from the heteroatomic heterocyclic radical of N, O and S;

(the C of R2 for being replaced by following group _1-4) alkyl: hydroxyl, carboxyl, amino or the (C that is not substituted or replaces _6-18) aryl, or R ₂Be (the C that is not substituted or replaces _6-18) aryl; And

R ₃Be (the C that is not substituted or replaces _6-18) aryl, or have 5 or 6 ring memberses and 1-4 heteroatomic heterocyclic radical that is selected from N, O and S.

In aspect of formula I compound is preferred, R ₁Be (the C that is replaced by following group _1-3) alkyl: be not substituted the phenyl that replaces or have 5 ring memberses and N as heteroatomic heterocyclic radical;

R ₂Be (the C that is replaced by following group _1-2) alkyl: hydroxyl, carboxyl, amino or the phenyl that is not substituted or replaces, or R ₂Be the phenyl that is not substituted or replaces; And

R ₃Be the phenyl that is not substituted or replaces, or have 5 or 6 ring memberses and N as heteroatomic heterocyclic radical.

Another of formula I compound preferred aspect, R ₁Be the methyl that is replaced by p-methylphenyl, or the n-pro-pyl that is replaced by the 1-pyrrolidin-2-one, R ₂For by the methyl of carboxyl substituted, the methyl that is replaced by p-methylphenyl, the methyl that is replaced by the 1H-indol-3-yl, the ethyl that is replaced by hydroxyl, the ethyl that is replaced by amino or p-methylphenyl, and

R ₃Compound for following formula:

Wherein

R ₄Be 1-piperidines or 1-(to amino carbonyl)-piperidines,

R ₅Be methoxy ethyl, benzyl or (p-methoxyphenyl)-ethyl; Or

The compound of following formula:

Wherein

R ₆For to a phenyl or a pyridine; And

R ₇Be the methyl that is replaced by m-methoxyphenyl or 1-amino carbonyl-2-hydroxypropyl.

If do not define in addition in the literary composition, alkyl comprises (C so _1-8) alkyl, for example (C _1-4) alkyl.Aryl comprises (C _6-18) aryl, for example phenyl.Heterocyclic radical comprises 5 or 6 yuan of rings, has 1-4 and is selected from S, O and N, for example heteroatoms of N; For example piperidines, pyridine and pyrrolidine, it is optional to condense (anellated) with another ring (being), for example condenses with phenyl ring; Or for example with heterocyclic fused.Alkyl, aryl and heterocyclic radical comprise alkyl, aryl or the heterocyclic radical that is not substituted or replaces, and are for example replaced by group conventional in the organic chemistry.Amino comprises the amine that is substituted and replaces, for example alkyl amine and two alkyl amine.

In the compound of formula I, each substituting group of definition can be preferred substituted separately, for example the substituting group of definition independently of one another.

The present invention provides the compound of formula I on the other hand:

Wherein

A) R ₁Be the n-pro-pyl that is replaced by the 1-pyrrolidin-2-one, R ₂Be the ethyl that is replaced by amino; And R ₃Compound for following formula:

B) R ₁Be the methyl that is replaced by p-methylphenyl, R ₂Be the ethyl that is replaced by amino; And R ₃Compound for following formula:

C) R ₁Be the methyl that p-methylphenyl replaces, R ₂Be the methyl of quilt to amino methyl-phenyl replacement, and R ₃For as b) in the compound of definition;

D) R ₁Be the n-pro-pyl that is replaced by the 1-pyrrolidin-2-one, R ₂Be the ethyl that is replaced by hydroxyl, and R ₃Compound for following formula:

E) R ₁Be the n-pro-pyl that is replaced by the 1-pyrrolidin-2-one, R ₂For by the methyl of carboxyl substituted, and R ₃Compound for following formula:

F) R ₁Be the n-pro-pyl that is replaced by the 1-pyrrolidin-2-one, R ₂Be the methyl that is replaced by the 1H-indol-3-yl, and R ₃Compound for following formula:

G) R ₁Be the n-pro-pyl that is replaced by the 1-pyrrolidin-2-one, R ₂Be the methyl that is replaced by p-methylphenyl, and R ₃For as f) in the compound of definition.

Compound provided by the invention is named as " (according to) compound of the present invention " hereinafter.Compound of the present invention comprises any type of compound, free form for example, salt form, solvate forms and salt and solvate forms.

The present invention provides the The compounds of this invention of salt form on the other hand.

This class salt preferably includes officinal salt, but, can comprise pharmaceutically unacceptable salt, for example is used for preparation/separation/purification purpose.

The salt of The compounds of this invention comprises slaine or acid-addition salts.Slaine comprises: for example alkaline metal or alkali salt; Acid-addition salts comprises the salt that formula I compound and acid form, and described acid is for example hydrogen fumaric acid (hydrogen fumaric acid), fumaric acid, naphthalene-1,5-sulfonic acid, hydrochloric acid, deuterium chloric acid; Preferred hydrochloric acid.

The compound of free form of the present invention can be changed into the compound of corresponding salt form, vice versa.The The compounds of this invention of free form or salt form and solvate forms can be changed into the respective compound of the salt form of free form or non-solvent form, vice versa.

Compound of the present invention can exist with the form of pure isomeride or its potpourri; For example optical isomer, diastereo-isomerism, suitable/trans isomer.Compound of the present invention can for example comprise unsymmetrical carbon, and thus can be with enantiomter or diastereo-isomerism and composition thereof, and for example the form of racemate exists.Any unsymmetrical carbon all can be with (R), (S) or (R, S) configuration, preferred (R) or (S) configuration existence.

Under the situation that is suitable for, can according to, for example obtain pure isomeride with the similar mode separating isomerism of conventional method body potpourri.The present invention includes the The compounds of this invention of any isomeric forms and any isomer mixture form.

The present invention also comprises the dynamic isomer of formula I compound, under the situation that can have dynamic isomer.

The present invention provides the method that is used for production formula I compound on the other hand, wherein

-R ₃Be the compound of formula III, the method comprising the steps of:

R wherein ₁, R ₂, R ₆And R ₇As above-mentioned definition, thereby obtain the compound of formula I, and separate the formula I compound that from reaction mixture, obtains; Or

-R ₃Be the compound of formula II, the method comprising the steps of:

R wherein ₁, R ₂, R ₄And R ₅As above-mentioned definition, thereby obtain the compound of formula I, and separate the formula I compound that from reaction mixture, obtains.

In the intermediate (raw material) of formula III a, IIIb, IIa, IIb or IIc, functional group if present, optional can be the form of protected form or salt, if there is salt forming group.Protecting group is optional to be existed, can remove in suitable stage, for example according to the similar method of conventional method.

The formula I compound that so obtains can be changed into another kind of formula I compound, for example or with the formula I compound that free form obtains can be converted to the salt of formula I compound, vice versa.

The intermediate of formula III a, IIIb, IIa, IIb or IIc (raw material) known or can according to, for example with similar method of conventional method or preparation as described herein.

Under the situation that is suitable for, can be for example according to, for example with the similar method of conventional method, or for example the method that describes in detail of this paper prepares any compound as herein described, the intermediate of compound for example of the present invention and formula III a, IIIb, IIa, IIb or IIc.

The present invention provides the purposes of The compounds of this invention as inhibitor on the other hand, is used to suppress to contain the formation of compound between the mRNA of ARE and target protein such as the HuR albumen.

Aspect preferred, the mRNA that contains ARE is selected from IL-2, IL-1 β and TNF-α.

Compound of the present invention for example comprises the compound of formula I showing pharmacological activity, thus useful as drug.

The present invention provides the application of The compounds of this invention as medicine on the other hand.

With regard to medicinal application, compound of the present invention comprises one or more, preferred a kind of compound of the present invention, for example two or more combination of compounds of the present invention.

The present invention provides pharmaceutical composition on the other hand, it comprises the compound and at least a drug excipient of invention, for example Shi Yi carrier and/or thinning agent, for example comprise filling agent, bonding agent, disintegrant, flowing regulator, lubricant, carbohydrate and sweetener, aromatic, antiseptic, stabilizing agent, wetting agent and/or emulsifying agent, solubilizer, be used to regulate the salt of osmotic pressure, and/or buffering agent.

The present invention provides pharmaceutical composition of the present invention on the other hand, and it also comprises the other drug active substance.

For example, can according to the similar method of conventional method, for example prepare this based composition by mixing, granulation, dressing, dissolving or desivac.For example, unit dosage forms can contain the 0.5mg that has an appointment to about 1000mg, as 1mg about 500mg extremely.

The present invention provides The compounds of this invention in medicine that preparation the is used for the treatment of illness purposes in the pharmaceutical composition for example on the other hand, described illness has the cause of disease relevant with the generation that is selected from following material: cell factor, growth factor, proto-oncogene or virus protein, preferred described material is selected from IL-1, IL-2, IL-3, IL-4, IL-8, GM-CSF, TNF-α, VEGF, AT-R1, Cox-2, c-fos and c-myc.

The present invention provides sanatory method on the other hand, described illness has the cause of disease relevant with the generation that is selected from following material: cell factor, growth factor, proto-oncogene or virus protein, preferred described material is selected from IL-1, IL-2, IL-3, IL-4, IL-8, GM-CSF, TNF-α, VEGF, AT-R1, Cox-2, c-fos and c-myc, and described methods of treatment comprises the The compounds of this invention of the experimenter that these class treatment needs are arranged being used effective dose; For example with the form of pharmaceutical composition.

Treatment comprises treatment and prevention.

With regard to this class treatment, appropriate dosage changes according to for example chemical property of used The compounds of this invention and the character and the different of the order of severity of pharmacokinetic data, host's individuality, administering mode and treatment disease certainly.Yet, generally speaking, for bigger mammal, people for example, in order to obtain gratifying effect, indication dosage range every day is about 0.01g The compounds of this invention of about 1.0g extremely; For example, advantageously every day, divided dose was used nearly 4 times.

Can give compound of the present invention by any conventional route, for example administration in the intestines for example comprises nose, sucks, rectum, oral administration; Parenterai administration for example comprises intravenous, intramuscular, subcutaneous administration; Or topical, for example comprise epidermis, interior, the interior administration of tracheae of nose; For example with the dressing or the form of coating tablet, capsule, Injectable solution or suspension not,, creme, gel, paste, can suck the form of pulvis, foaming agent, tincture, lipstick, drops, spray or the form of suppository for example with the form of ampoule, bottle.

Can give compound of the present invention, for example acid-addition salts or slaine with the form of officinal salt; Or free form; The form of optional solvents thing.The compound of salt form of the present invention shows and free form of the present invention, the active grade that the compound of optional solvents thing form is identical.

Compound of the present invention can be separately or is used for medicinal treatment of the present invention with one or more other drugs are active compound combined.

Drug combination comprises: fixing combination, and wherein two or more pharmaceutically active substances are in identical preparation; Kit, wherein two or more pharmaceutically active substances in independent preparation for example attach the instructions of using jointly as same packing and selling; And free combination, packaged drug active substance separately wherein, but provided simultaneously or instructions that preface is used to use.

Description of drawings

Fig. 1: measuring principle (diagram)

(herein: 3 ' end mark), then the quantum yield of environmental sensitivity mark (as Cy) increases in case protein bound is to (as the Cy3-mark) mRNA of mark.This increase can detect the increase into suitable readout, for example according to the increase of the molecule brightness of FIDA algorithm, or for example is the increase of the total fluorescence intensity that uses whole average readout.For the protein binding site in the mRNA sequence that is in mark (as the Cy3 mark) near-end (A) or far-end (B), observed this effect with being equal to.

Fig. 2: representative Cy3FIDA measures mRNA-protein bound curve

Shown HuR _F1The interactional representative binding curve of 3 ' UTR (=untranslated district) with 3 ' the terminal Cy3 mark of IL-2 (A), IL-1 β (B) and TNF-α (C).According to as the Eq.1 that hereinafter provides among the embodiment measure dissociation constant (Kd).In order to get rid of the non-specific interaction of HuR and Cy3, use free dye (D) as negative control.The brightness of Cy3 molecule is using BSA (=bovine serum albumin(BSA)) still to remain unchanged during protein (E) titration IL-23 ' UTR in contrast.The concentration of the RNA of 3 ' terminal Cy3 mark is 0.5nM in all experiments.The 5S RNA concentration with 100nM in all samples that does not contain any HuR binding site exists, as non-specific competition RNA.Yet, under situation about existing, recorded binding curve much at one (TNF-α 3 ' UTR, (F) shown in) without any competition RNA.

Fig. 3: the rna transcription thing shown in Fig. 2 in conjunction with experiment

3 ' UTR in-vitro transcription thing (GenBank accession number NM_000589, NM_000576 and NM_000594 with Cy3 labelling human IL-2, IL-1 β and TNF-α; Be respectively 707-1035,897-1490 and 872-1568 position) 3 ' end.Although HuR binding site (shown in the blueness) end-labelled with 3 ' (sequence) proximity is different, when protein bound, observed to consistance the increase of Cy3 quantum yield.

In the following example, all temperature are degree centigrade and not calibration.

Use following abbreviation:

The BSA bovine serum albumin(BSA)

The Cy3 fluorescent dye

The FCS fluorescence correlation spectroscopy

The distributional analysis of FIDA fluorescence intensity

HuR _F1Total length HuR albumen

HuR _1,2HuR _F1Truncated variant

The OD optical density

The PBS phosphate-buffered saline

The RP-HPLC reversed-phased high performace liquid chromatographic

The rt room temperature

The confocal nano scanning of CONA

The DMF dimethyl formamide

HuR ₁₂The truncated variant of HuR

TMR 5 ' carboxyl tetramethyl rhodamine

The rt room temperature

Embodiment:

A) shaker test

Use the Cy3 (Amersham Biosciences) of hydrazides activation, according to standard scheme (Qin PZ etc. for example, Methods 1999; 18 (1): 60-70) mRNA to in-vitro transcription carries out 3 ' end mark.RNA by RP-HPLC purifying mark.By 1: 1 stoichiometry of UV/VIS spectroscopic methodology control.Measuring damping fluid (PBS, 0.1% (w/v) Pluronic F-127,5mM MgCl ₂) in 80 ° with the mRNA heat denatured 2 minutes of Cy3-mark and by with-0.13 ° of s ^-1Gradient be cooled to room temperature and carry out refolding.The final concentration of labeled rna is 0.5nM in every duplicate samples, and it can guarantee mean value＜1 of the fluorescent grain in the confocal volume under following imposing a condition.Based on deriving from the accurate concentration that amounts of particles that parallel FCS estimates and confocal volume size are measured every duplicate samples, this is to draw by the parameter of adjusting the some scattering function.Along with reorganization HuR _F1Or HuR _1,2The fluorescently-labeled RNA of increase titration of concentration.Under real equilibrium condition, by use 1D-FIDA measure molecule brightness monitor the formation of HuR-mRNA compound (Kask P. etc. for example, Introduction to thetheory of fluorescence intensity distribution analysis.FLUORESCENCECORRELATION SPECTROSCOPY:THEORY AND APPLICATIONS 65PG 396-409.2001[Figures]-409; Kask P. etc., Fluorescence-intensitydistribution analysis and its application in biomolecular detectiontechnology.Proceedings of the National Academy of Sciences of the UnitedStates of America 96[24], 13756-13761.23-11-1999).Perhaps, can use conventional whole average detection method, for example the dull and stereotyped reader of fluorescence is measured the Cy3 fluorescence intensity.

Carrying out FIDA in room temperature (constant in 23.5 °) in the microtiter plate (Whatman) at the bottom of 96 holes on EvotecOAI PickoScreen 3 instruments or the 384 hole glass measures.Apparatus preparation based on Olympus inverted microscope IX70 has 2 fluorescence detectors, the dichroic mirror in fluorescence excitation path.HeNe laser (λ=543nm, laser energy=478 μ W) is used for fluorescence excitation.Use has the excitation laser of the interference barrier filter blocking-up of OD=5 from light detection path.Utilize the Cy3 or the TMR (c=0.5nM) that measure in the damping fluid to adjust confocal pinhole (70 μ m).To averaging of the 1D-FIDA signal of 20 continuous coverage values (each 10 seconds).Use FIDA Algorithm Analysis raw data to extract:

(a) the mean molecule brightness suitable with conventional ensemble average measured value; Or

(b) has the equilibrium concentration (free mRNA is to compound) of each component of corresponding molecule brightness.

By non-linear least square regression fit molecule brightness data (GraFit 5.0.3, Erithacus software, London) so that extract equilibrium dissociation constant K in conjunction with equational accurate algebraic method with what derive from the mass action law _d, be described below:

(a) average steady state signal q depends on dissociation constant Kd determined (1: 1) compound and forms degree:

Equation 1

[mRNA wherein ₀]: the total concentration of RNA, [HuR ₀]: the total concentration of HuR, q _Min: the molecule brightness of free RNA, q _Max: the molecule brightness of RNA-HuR compound, q: specifying HuR ₀And RNA ₀The mean molecule brightness of homeostasis under the concentration;

(b) dissociation constant K _dThe determined free equilibrium concentration that reaches the mRNA of conjugated protein (is respectively [mRNA _Free] and [mRNAHuR], directly draw by the FIDA analysis):

Equation 2

The interactional representative binding curve of each said target mrna of HuR and Cy3 mark is (all data of listing are the mean value of 20 FIDA measured values, and at least three independently experiments of representative) as shown in Figure 2.

Say that briefly this determination method provides the means of (control) mRNA protein-protein interaction in the new monitoring homogeneous solution.This determination method has made up the advantage of real equilibrium condition and high detection sensitivity and degree of accuracy, and is suitable for interactional potential inhibitor of screening or correctives in high flux screening.Because used the disease association target of a large amount of post-transcriptional controls, described determination method will can be used as the treatment intervention means of cancer, inflammation, virus or anaphylactia based on new RNA target-seeking means.

B) analytical approach:

A) HPLC:, use Abimed (D-Langenfeld) the HPLC system that forms by 2 pump unit 306, dynamic mixing chamber assembly 811C, pressure measurement assembly 805, UV-detecting device UV/VIS 155 and automatic injector 234 in order to analyze separation.(3 μ m separate on 120 * 2mm) the analytical column GromSil150ODS-5ST that produces at Grom (D-Herrenberg).Use H ₂O/0,1%TFA (v/v) (eluent A) and acetonitrile/0,1%TFA (v/v) (eluent B) gradient, flow velocity 0.4mL/ minute.Measure the UV track in λ=214 and λ=305nm place respectively.Provide degree of purity of production based on the peak area of measuring at λ=214nm place.

B) ES-MS: use to have Waters (D-Eschborn 515 assembling pump (60 μ L/ branches such as flow velocity such as degree of grade, acetonitrile/water 1: 1, contain 0,1% formic acid) and Micromass (Altrinchan/UK) the Quattro II triple quadrupole mass spectrometer of Abimed (D-Langenfeld) Gilson 232X automatic sampler carry out ES-MS-and analyze.

C) LC-MS: use Waters-Micromass (D-Eschborn) the ZQ mass spectrometer that has HPLC-system 2790Alliance HT separation assembly and 996 diode array detector to carry out LC-MS-and analyze.Analytical column GromSil120ODS-5ST (3 μ m, 60 * 2mm) of using Grom (D-Herrenberg) to produce.Use H ₂O/0,1%TFA (v/v) (eluent A) and acetonitrile/0,1%TFA (v/v) (eluent B) gradient, flow velocity 0.6mL/ minute.

D) preparation HPLC: separate in order to be prepared type, use by pump unit 322, UV detecting device UV/VIS 151 (Abimed (D-Langenfeld) the HPLC system that λ=305nm) and Gilson 215 liquid processors are formed.(5 μ m separate on 125 * 25mm) the preparative column Purospher RP18 that produces at Merck (D-Darmstadt).Use H ₂O/0,1%TFA (v/v) (eluent A) and acetonitrile/0,1%TFA (v/v) (eluent B) gradient, flow velocity 30mL/ minute.

Embodiment 1-3:

According to scheme 1 synthetic compound 1-3.

A) have the last sample of the solid support of linking group

With DMF (4 * 20mL) pre-wash 4.2g TentaGel ^TMS NH ₂-resin 1 (applied sample amount=0.25mmol/g).With the 4-bromomethyl-3-nitrobenzoic acid 2 of 0.82g and the HOBt*H of 0.482g ₂O is dissolved in the DCM solution of 60mL 8%DMF (v/v).The DIC that adds 487 μ L.After 30 minutes, the solution that obtains is changed over to the TentaGel of pre-wash in stirring at room ^TMS NH ₂On-the resin 1.With the potpourri that obtains in room temperature jolting 19 hours.Filter out the resin 3 of acquisition, with DMF (6 * 50mL), DCM (6 * 50mL) and MeOH (6 * 50mL) wash, and drying under reduced pressure.Finish by negative Kaiser ninhydrin test confirmatory reaction.

B) resin 3 and primary amine class 4a, the reaction of 4b

Be in to 3.18g and add 2.133g 1-(3-aminopropyl)-pyrrolidin-2-one (4a) in the resin 3 among the 20mL DMSO.Be in the 4-methylbenzylamine (4b) that adds 0.73g in the resin 3 among the 10mL DMSO to 1.27g.With the potpourri that obtains in room temperature jolting 3 hours.Filter out the resin 5a and the 5b of acquisition, with DMSO (3 * 6mL), DMF (6 * 6mL), DCM (6 * 6mL), MeOH (6 * 6mL) washings, and drying under reduced pressure.The chloranil test is positive.

C) resin 5a-b goes up the coupling of protected amino acid 6a-f and the cracking of N (α) protecting group (Fmoc)

5a is divided into 5 equal portions with the 0.65g resin.5b is divided into 2 equal portions with the 0.65g resin.Amino acid 6a-f (6a:N (α)-Fmoc-N (γ)-Boc-L-2,4-DAB, 2 times with each N-protected of 0.45mmol; 6b:Fmoc-(OTrt)-L-homoserine; 6c:Fmoc-Asp (OtBu)-OH; 6d:Fmoc-Trp (Boc)-OH; 6e:Fmoc-L-4-MePhe-OH; 6f:Fmoc-L-(4-Boc-aminomethyl) Phe-OH is dissolved in 5mL DMF, 93 μ L DIC and 92mg HOBt*H separately ₂Among the O.With the solution that obtains stirring at room 30 minutes.The solution of the activation 6a that obtains is divided into 2 equal portions.These 2 equal portions are changed over to respectively in resin 5a and the resin 5b sample aliquot, obtain protected resin 7a of side chain and 8a.The 6b-e solution of the activation that obtains is changed in the remaining resin 5a sample aliquot, obtain protected resin 7b-e.The solution that will contain 6f changes in second sample aliquot of resin 5b, obtains protected resin 8b.After 16.5 hours, filter out the resin of acquisition in the room temperature jolting, and with DMF (9 * 10mL), DCM (6 * 10mL) and MeOH (6 * 10mL) washing.Complete protected resin 7a-e of drying under reduced pressure and 8a-b.All chloranil tests are all negative.

With piperidines DMF (400mL, 1/1, v/v) storage liquid sample aliquot (5mL) join among protected resin 7a-e of each 0.66g and the 8a-b, with cracking N (α)-Fmoc protecting group.With the potpourri jolting that obtains 30 minutes.Filter out resin 7a-e, the 8a-b of the deprotection of acquisition, and use repeatedly DMF (9 * 25mL), DCM (6 * 25mL) and MeOH (6 * 25mL) washing.The resin that drying under reduced pressure obtains.The Kaiser ninhydrin test is positive.

D) contain compound 14a-c synthetic of symmetrical dicarboxylic acid substructure

The resin 7a of Fmoc deprotection and 8a-b go up the coupling of symmetrical dicarboxylic acid 9a and 9b

With symmetrical dicarboxylic acid 9 (9a: pyridine-2,6-dioctyl phthalate; 9b: terephthalic acid (TPA), 2 times; Each 1.5mmol) is dissolved in 115mg HOBt*H ₂In the 5mL DMF solution of O, 485mg DIEA and 95mg DIC.The solution of the 9a that obtains joined among the resin 7a and obtain resin 10a.The 2 equal portions 9b solution that obtain are joined respectively among resin 8a and the 8b and obtain resin 10b and 10c.After 20 hours, filter out the resin 10a-c of acquisition in the room temperature jolting, with DMF (9 * 20mL), DCM (6 * 10mL), Anaesthetie Ether (6 * 10mL) washings, and drying under reduced pressure.The Kaiser ninhydrin test of all resin sample is all negative.

E) coupling of the dicarboxylic acid monoamides 10a-c of resin-bonded and amino acid amide 11 and primary amine 12

The 5mL nmp solution of 339mg trifluoroacetic acid pentafluorophenyl esters (pentafluorophenyl trifluoroacetate) and 242 μ L pyridines is joined among each 650mg resin 10a-c.With the potpourri that obtains in room temperature jolting 2.5 hours.Filter out the resin of acquisition, and (10 * 5mL) wash with NMP.The L-threonine amide hydrochloride (11) of 298mg (O-Trt) protection is dissolved in the 3.0mL nmp solution of anhydrous HOBt of 20.3mg and 3mmol DIEA, and joins among the preactivated resin 10a of 650mg and obtain resin 13a.182mg 3-methoxybenzylamine (12) is dissolved in anhydrous HOBt of 40.6mg and 1.5mmol DIEA) the 6.0mg nmp solution in.The solution that obtains is divided into 2 equal portions, they are joined respectively among preactivated resin 10b and the 10c and obtains resin 13b and 13c.With the potpourri that obtains in room temperature jolting 14.5 hours.Filter out the resin of acquisition, with NMP (6 * 10mL), DMF (6 * 10mL), DCM (6 * 10mL) and MeOH (6 * 10mL) wash, and drying under reduced pressure.

F) cracking of Boc-protecting group on amino acid 6 side chains

DCM (v/v) solution of 5mL 50%TFA is joined among each resin 13a-c of 650mg.With the potpourri that obtains in room temperature jolting 2 hours.Filter out the resin of acquisition, with the DCM solution of 20%TFA (v/v, 3 * 5mL), DMF (6 * 10mL), DCM (6 * 10mL) and anhydrous EtOH (6 * 10mL) washings, and drying under reduced pressure.

G) cracking of final compound 14a-c from the resin 13a-c

With 5mL MeOH+1%TFA v/v) join among each resin 13a-c of deprotection.Place plastic carrier to be used for photodissociation resiniferous vial.NEC Blacklight T5, FL8Bl, the Stratagene of 8W-lamp, UV Strataliner be equipped with ^TMStir in the 366nm place on 1800 and carried out photodissociation 90 minutes.In the photodestruciton process, use the forced air cooling chamber.Filter out the resin material of acquisition, and (2 * 5mL) wash with DCM.Evaporating solvent from the filtrate that obtains.Can slightly carry material to this by preparation HPLC and further carry out purifying.

Embodiment 1:

Obtain pyridine-2,6-dioctyl phthalate 2-(3-amino-1-[3-(2-oxo-pyrrolidine-1-yl)-propyl group carbamyl]-propyl group }-acid amides) 6-[(1-carbamyl-2-hydroxyl-propyl group)-acid amides].

MW=491.5552 retention time: 3.8 minutes LC-MS[M ⁺H] ⁺: 492

Embodiment 2:

Obtain N-[3-amino-1-(4-methyl-benzyl carbamyl)-propyl group]-N '-(3-methoxyl-benzyl)-terephthalamide.

MW=488.592 retention time: 8.29 minutes LC-MS[M ⁺H] ⁺: 489

Embodiment 3:

Obtain N-[2-(4-aminomethyl-phenyl)-1-(4-methyl-benzyl carbamyl)-ethyl]-N '-(3-methoxyl-benzyl)-terephthalamide.

MW=564.69 retention time: 8.46 minutes LC-MS[M ⁺H] ⁺: 565

Embodiment 4-7:

According to scheme 2 synthetic compound 4-7.

A) acetoacetylization of resin 7b-e

The resin 7b-e of N (α) deprotection is put into the 5mL syringe.359mg N-hydroxy-succinamide base acetoacetic ester 15 is dissolved in 17.0mL DCM.Add 310mg DIEA, and the aliquot that 4mL should store liquid is changed in each syringe of each resin 7b-e of 650mg.After 5 hours, filter out the acetoacetyl resin 16a-d of acquisition in the room temperature jolting, with DCM (3 * 5mL), DMF (6 * 5mL), DCM (6 * 5mL) and MeOH (6 * 5mL) washing and the drying.The Kaiser ninhydrin test is negative.

B) reaction of resin 16a-d and primary amine 17a-c (formation of enamine ketone)

With the storage liquid of 5mL THF and TMOF (1/1, v/v) join among each resin 16a-d of 650mg.Add each primary amine 17a-c of 1.5mmol (17a: with the 2-methoxyethyl amine of resin 16a reaction; 17b: with 2-(3-the methoxyphenyl)-ethamine of resin 16b reaction; 2 times of 17c: respectively with the benzylamine of resin 16c and 16d reaction), and in room temperature with the potpourri jolting that obtains 18 hours.Filter out the resin 18a-d of acquisition, with THF/TMOF (1/1, v/v) (3 * 6mL), DMF (6 * 6mL), DCM (6 * 6mL) and MeOH (6 * 6mL) washing and the drying.

C) from the enamine ketone 18a-d and 1 of resin-bonded, 4-two bromo-2,3-diacetyl 19 synthetic pyrroles

With 230mg 2,6-two-tert .-butylpyridine and 292mg 1,4-two bromo-2,3-diacetyl 19 are dissolved in 24mL two  alkane and obtain storing liquid.The aliquot (6mL/ resin) of the storage liquid that obtains is joined among each resin 18a-d of 650mg.With the potpourri that obtains in room temperature jolting 1.5 hours.Filter out the resin 20a-d of acquisition, with two  alkane (6 * 6mL), DMF (6 * 6mL), THF (6 * 6mL) and DCM (6 * 6mL) washing.

D) use secondary amine 21a and 21b to replace the acetyl bromide-pyrroles 20a-d of resin-bonded

(4 * 6mL) join among each resin 20a-d of 650mg with each secondary amine 21 of 192mg (21a: with the piperidines-4-formamide of 128mg resin 20a reaction, 21b: with the piperidines of 128g resin 20b-d reaction) and DMSO.The potpourri that obtains shaken in room temperature broadcast 15.5 hours.Filter out the resin 22a-d of acquisition, with DMSO (3 * 1mL), DMF (6 * 1mL), DCM (6 * 1mL) and MeOH (6 * 1mL) washing and the drying.

E) protecting group cracking (resin 22a-c) from amino acid 6 side chains

The DCM solution (v/v) of 5mL 20%TFA is joined among each resin 22a-c so that remove R ²Side chain protective group (Trt, t-Bu, Boc).With the potpourri that obtains in room temperature jolting 1 hour.Filter out the resin 22a-c of the deprotection of acquisition, with the DCM solution of 20%TFA (v/v, 3 * 1mL), DMF (6 * 2mL), DCM (6 * 2mL), EtOH (6 * 2mL) and ether (3 * 2mL) washings and drying.

F) final compound 23a-d goes up cracking from resin 22a-d

5mL acidic methanol and 1%v/v TFA are joined among the resin 22a-c and the resin 22d without the TFA processing of each deprotection.Vial is placed plastic carrier.NECBlacklight T5 is being equipped with, FL8Bl, the Stratagene of 8W-lamp ^UV Strataliner ^TMOn 1800 stir and 366nm irradiation under photodissociation 90 minutes.In light-cracking process, use the forced air cooling chamber.Filter out the resin material of acquisition and use DCM (2 * 5mL) washings.Solvent in the merging filtrate that evaporation obtains.Can further carry out purifying to the material of slightly carrying of compound by preparation HPLC.

Embodiment 4:

Obtain 1-{2-[4-{3-hydroxyl-1-[3-(2-oxo-pyrrolidine-1-yl)-propyl group carbamyl]-the propyl group carbamyl }-1-(2-methoxyl-ethyl)-5-methyl isophthalic acid H-pyrroles-2-yl]-2-oxo-ethyl }-piperidines-4-benzoic acid amides.

MW=576.699 retention time: 2.24 minutes LC-MS[M ⁺H] ⁺: 577

Embodiment 5:

Obtain 3-{[1-[2-(3-methoxyl-phenyl)-ethyl]-2-methyl-5-(2-piperidines-1-base-acetyl group)-1H-pyrroles-3-carbonyl]-amino }-N-[3-(2-oxo-pyrrolidine-1-yl)-propyl group]-succinic acid.

MW=623.756 retention time: 7.59 minutes LC-MS[M ⁺H] ⁺: 624

Embodiment 6:

Acquisition 1-benzyl-2-methyl-5-(2-piperidines-1-base-acetyl group)-1H-pyrroles-3-formic acid 2-(1H-indol-3-yl)-1-[3-(2-oxo-pyrrolidine-1-yl)-third carbamyl]-ethyl }-acid amides.

MW=650.828 retention time: 8.248 minutes LC-MS[M ⁺H] ⁺: 651

Embodiment 7

Acquisition 1-benzyl-2-methyl-5-(2-piperidines-1-base-acetyl group)-1H-pyrroles-3-formic acid 1-[3-(2-oxo-pyrrolidine-1-yl)-third carbamyl]-2-p-methylphenyl-ethyl }-acid amides.

MW=625.818 retention time: 8.44 minutes LC-MS[M ⁺H] ⁺: 626

Embodiment 8: inhibition is in conjunction with the evaluation of the micromolecular inhibitor of the mRNA stability regulation and control of HuR

In order to identify the micromolecular inhibitor of inhibition, carried out three kinds of different complementary HT screenings in conjunction with the mRNA stability regulation and control of HuR:

(1) uses and to be in the cell reporter-gene assays that IL-2 or TNF-α are rich in the firefly luciferase under the control of AU element.

Design the stable cytoactive avirulence inhibitor of mRNA that this determination method is used to suppress to identify the ARE mediation.Use the inhibitor that hits that test is confirmed in the control test of luciferase CDS under IL-2 or the control of TNF-α promoter, to get rid of the compound that on transcriptional level, works.As definition, the compound that hits of evaluation disturbs the back of transcribing of short life ARE mRNA to stablize on certain level along the ARE path.

(2) use confocal fluctuation spectroscopic methodology in-vitro screening HuR-ARE inhibitor.

This determination method is used 2D-FIDA monitoring HuR ₁₂Be the HuR truncated variant and ARE RNA the combining in solution of TMR mark.Expectation compounds identified in this screening is passed through in conjunction with ARE or HuR ₁₂And disturb HuR-ARE to discern.

(3) CONA of use HuR.

Use CONA to the screening of the combinatorial libraries on pearl high-affinity HuR bond.Gathering the pearl of hitting and behind the bond on the cracking pearl, compound structure is being decoded from the solid support by μ HPLC-MS.Test is synthetic again in solution hits combining of compound and HuR.As PRELIMINARY RESULTS, compounds identified has been represented high-affinity HuR bond.They can be the inhibitor of HuR activity arbitrarily on function, described HuR activity comprises ARE identification, caryoplasm shuttles back and forth and prevent the ARE dependence degraded of mRNA.

Based on y=(r-r _Min)/((r-r _Min)+Q (r _Max-r)), by the RNA fraction y of anisotropy data computation combination, the anisotropy measured of r ≡ wherein, r _Min, r _MaxRespectively ≡ is free and in conjunction with the anisotropy of the RNA of HuR, Q ≡ quencher.Use the nonlinear curve of the KMath match among the Mathematica 5.0.0, suppose 1: 1: 1 competitive inhibition of stoichiometry, measure dissociation constant Kd.。

Table 1.HuR inhibitor

Compound	Kd(HuR f1)
		Embodiment 3	0.092±0.022μM
Embodiment 4	0.103±0.020μM
		Embodiment 5	0.104±0.014μM
Embodiment 6	0.143±0.027μM

Based in conjunction with HuR _F1The dissociation constant of competitive measuring compound of ARE RNA

Claims (21)

1. identify the determination method of interactional material between regulating mRNA and the target protein, comprising:

A) mRNA that provides length to be at least the mark of 100 nucleotide chooses wantonly the described material that is labeled as the change sensitivity in the mRNA environment is provided as homogeneous solution;

B) do not exist with the situation that has candidate compound under the mRNA that provides in target protein and the step a) is contacted, the interaction in the sufficiently long time of described candidate compound expection will between described mRNA and described target protein, can formation compound between described mRNA of adjusting and the described target protein;

C) detect the compound that forms in the step b);

D) whether the amount that is determined at formed compound under the situation that does not have or exist candidate compound in the step b) there are differences; With

E) candidate compound that will be measured to difference in step d) is elected the evaluation material as.

2. the described determination method of claim 1, wherein be labeled as fluorescent dye, as Cy3 or Cy5.

3. each described determination method in the claim 1 or 2 wherein detects compound by measuring fluorescence intensity.

4. each described determination method in the claim 1 to 3, wherein said target protein is a HuR albumen.

5. each described determination method in the claim 1 to 4, wherein mRNA is selected from IL-2, IL-1 β and TNF-α.

6. each described determination method in the claim 1 to 5, wherein mRNA length is 100 to 500 nucleotide, preferred 300 nucleotide.

7. each described determination method in the claim 1 to 6 is used for high flux screening.

8. kit, it comprises:

The mRNA of-mark, for example fluorescently-labeled mRNA;

-target protein;

The instructions of this class kit of-use; With

-optional candidate compound.

9. the compound of following formula I:

Wherein

R ₁Be (the C that is replaced by following group _1-4) alkyl: (the C that is not substituted or replaces _6-18) aryl or have 5 or 6 ring memberses and 1-4 is selected from the heteroatomic heterocyclic radical of N, O and S;

R ₂Be (the C that is replaced by following group _1-4) alkyl: hydroxyl, carboxyl, amino or the (C that is not substituted or replaces _6-18) aryl, or R ₂Be (the C that is not substituted or replaces _6-18) aryl; And

R ₃Be (the c that is not substituted or replaces _6-18) aryl, or have 5 or 6 ring memberses and 1-4 heteroatomic heterocyclic radical that is selected from N, O and S.

10. the compound of claim 9, wherein

R ₁Be (the C that is replaced by following group _1-3) alkyl: be not substituted the phenyl that replaces or have 5 ring memberses and N as heteroatomic heterocyclic radical;

R ₂Be (the C that is replaced by following group _1-2) alkyl: hydroxyl, carboxyl, amino or the phenyl that is not substituted or replaces, or R ₂Be the phenyl that is not substituted or replaces; And

R ₃Be the phenyl that is not substituted or replaces, or have 5 or 6 ring memberses and N as heteroatomic heterocyclic radical.

11. the compound of each formula I in claim 9 or 10, wherein

R ₁Be methyl that is replaced by p-methylphenyl or the n-pro-pyl that is replaced by the 1-pyrrolidin-2-one;

R ₂The methyl that replaces for the methyl that replaces by the methyl of carboxyl substituted, by p-methylphenyl, by the 1H-indol-3-yl, the ethyl that is replaced by hydroxyl, the ethyl that is replaced by amino or p-methylphenyl; And

R ₃Compound for Formula Il:

Wherein

R ₄Be 1-piperidines or 1-(to amino carbonyl)-piperidines;

R ₅Be methoxy ethyl, benzyl or (p-methoxyphenyl)-ethyl; Or

The compound of Formula Il I

Wherein

R ₆For to a phenyl or a pyridine; And

R ₇Be the methyl that is replaced by m-methoxyphenyl or 1-amino carbonyl-2-hydroxypropyl.

12. the compound of following formula I:

Wherein

A) R ₁Be the n-pro-pyl that is replaced by the 1-pyrrolidin-2-one, R ₂Be the ethyl that is replaced by amino; And R ₃Compound for following formula:

B) R ₁Be the methyl that is replaced by p-methylphenyl, R ₂Be the ethyl that is replaced by amino; And R ₃Compound for following formula:

C) R ₁Be the methyl that is replaced by p-methylphenyl, R ₂Be the methyl of quilt to amino methyl-phenyl replacement, and R ₃For as b) in the compound of definition;

D) R ₁Be the n-pro-pyl that is replaced by the 1-pyrrolidin-2-one, R ₂Be the ethyl that is replaced by hydroxyl, and R ₃Compound for following formula:

E) R ₁Be the n-pro-pyl that is replaced by the 1-pyrrolidin-2-one, R ₂For by the methyl of carboxyl substituted, and R ₃Compound for following formula:

F) R ₁Be the n-pro-pyl that is replaced by the 1-pyrrolidin-2-one, R ₂Be the methyl that is replaced by the 1H-indol-3-yl, and R ₃Compound for following formula:

G) R ₁Be the n-pro-pyl that is replaced by the 1-pyrrolidin-2-one, R ₂Be the methyl that is replaced by p-methylphenyl, and R ₃For as f) in the compound of definition.

13. each compound in the claim 9 to 12 is the form of salt.

14. each compound is as the purposes of inhibitor in the claim 9 to 13, described inhibitor suppresses to contain mRNA and the formation of the compound between the target protein of ARE.

15. the described purposes of claim 14, wherein target protein is HuR.

16. each compound is as the purposes of medicine in the claim 9 to 13.

17. the purposes of each compound in the sanatory medicine of preparation in the claim 9 to 13, described illness has the cause of disease relevant with the generation that is selected from following material: cell factor, growth factor, proto-oncogene or virus protein are preferably selected from IL-1, IL-2, IL-3, IL-4, IL-8, GM-CSF, TNF-α, VEGF, AT-R1, Cox-2, c-fos and c-myc.

18. pharmaceutical composition comprises in the claim 9 to 13 each compound and at least a drug excipient.

19. the pharmaceutical composition of claim 18 also comprises the other drug active substance.

20. sanatory method, described illness has the cause of disease relevant with the generation that is selected from following material: cell factor, growth factor, proto-oncogene or virus protein, be preferably selected from IL-1, IL-2, IL-3, IL-4, IL-8, GM-CSF, TNF-α, VEGF, AT-R1, Cox-2, c-fos and c-myc, described methods of treatment comprises the compound of the experimenter that these class needs are arranged being used in the claim 9 to 13 of effective dose each.

21. the method for claim 20, wherein simultaneously or preface be used in the co-administered claim 9 to 13 in ground each compound and another kind of pharmaceutically active substance.

Images