CN101514191A - Methods of treating or preventing autoimmune diseases with 2,4-pyrimidinediamine compounds - Google Patents

Methods of treating or preventing autoimmune diseases with 2,4-pyrimidinediamine compounds Download PDF

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CN101514191A
CN101514191A CNA2009100067710A CN200910006771A CN101514191A CN 101514191 A CN101514191 A CN 101514191A CN A2009100067710 A CNA2009100067710 A CN A2009100067710A CN 200910006771 A CN200910006771 A CN 200910006771A CN 101514191 A CN101514191 A CN 101514191A
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cell
group
base
enzyme
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R·辛格
A·阿加德
D·G·帕颜
J·克罗格
H·凯姆
C·赛尔维恩
H·李
S·伯汉米迪帕提
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Rigel Pharmaceuticals Inc
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Abstract

The present invention provides 2,4-pyrimidinediamine compounds that inhibit the IgE and/or IgG receptor signaling cascades that lead to the release of chemical mediators, intermediates and methods of synthesizing the compounds and methods of using the compounds in a variety of contexts, including in the treatment and prevention of diseases characterized by, caused by or associated with the release of chemical mediators via degranulation and other processes effected by activation of the IgE and/or IgG receptor signaling cascades.

Description

With 2, the method for treatment of 4-pyrimidinediamine compounds or prevention autoimmune disease
The application is that international application no is that PCT/US2003/024087, international filing date are on July 29th, 2003, the application number that enters the China national stage is CN03821120.3, denomination of invention is divided an application for the Chinese patent application of " with 2, the treatment of 4-pyrimidinediamine compounds or prevent the method for autoimmune disease ".
The mutual reference of related application
The application's case obtains the rights and interests of following application case according to 35 piece of 119 (e) bar of United States Code specified requirement, that is: applying for the sequence number on July 29th, 2002 is 60/399,673 application case, to apply for the sequence number on January 31st, 2003 be 60/443,949 application case and the sequence number that applies on March 6th, 2003 are 60/452,339 application case.
Technical field under the invention
What the present invention discussed in general is 2, the 4-pyrimidinediamine compounds, the medicinal components that comprises this compound, intermediate, make the synthetic method of this compound, use the method for this compound and composition in all cases, for example with its treatment or prevention autoimmune disease and/or method related indication with it.
The background of invention
Fc acceptor crosslinked, for example the high-affinity receptor of the high-affinity receptor of IgE (Fc ε RI) and/or IgG (Fc γ RI) activates a signal cascade in mastocyte, basophilic leukocyte and other immunocyte, and this cascade causes producing the release of the chemical mediator of many adverse events.For example, this crosslinked pre-formation medium (for example histamine) of I class (immediately) anaphylactic hypersensitivity that causes discharges via the storage location of degranulation from granule.It also causes the synthetic of other medium (comprising leukotriene, prostaglandin(PG) and platelet activating factor (PAFs)) and discharges, and these media are played an important role in inflammatory response.The extra media that is synthesized when the Fc acceptor is crosslinked and discharges comprises cytokine and nitrogen protoxide.
The activated signal cascade comprises a large amount of cell proteins by Fc acceptor (for example Fc ε RI and Fc γ RI) is crosslinked.Signal propagation agent is tyrosine-kinase Enzyme in its most important cell.And, participate in Fc ε RI with/or the important tyrosine-kinase Enzyme of the crosslinked relevant signal transduction path of Fc γ RI acceptor and other signal transduction cascade be the sharp Enzyme of Syk (referring to people such as Valent, 2002, Intl.J.Hematol.75 (4): the summary of 257-362).
Fc ε RI and the crosslinked medium that discharges of Fc γ RI acceptor are the reasons that causes many adverse events, perhaps play an important role therein, and it will be in demand therefore playing inhibiting compound to the signal cascade that causes this type of release.And, because swashing Enzyme, Syk in these and other receptor signal cascade, plays the part of pivotal player, can play inhibiting compound to the sharp Enzyme of Syk equally also is that people thirst for obtaining.
Summary of the invention
On the one hand, it is novel 2 to the invention provides, the 4-pyrimidinediamine compounds, this compounds, just as discussed in more detail below shown in, have numerous biological activitys.This compounds comprises usually and has 2 of following array structure and numbering convention, 4-pyrimidinediamine " core ":
Compound of the present invention is substituted in C2 nitrogen (N2) position, forms secondary amine, also can locate with one of upper/lower positions or many places are further replaced, and these positions are: C4 nitrogen (N4) position, C5 position and C6 position.When being substituted in the N4 position, substituting group forms secondary amine.At the substituting group of N2, and at any substituting group of another position, its characteristic and physics and chemical property scope can be very extensive.For example, (respectively) substituting group can be branched-chain alkyl, straight chained alkyl or cycloalkyl; Branched heteroalkyl groups, the assorted alkyl of straight chain or the assorted alkyl of ring; Monocycle or polyaromatic; Monocycle or polyheteroaromatic; Or the combination of these groups.These substituted radicals can further be replaced, and its more detailed description is as mentioned below.
N2 and N4 substituting group can be connected directly to its each nitrogen-atoms, perhaps can separate via linker and its each nitrogen-atoms, and linker can be identical or different.Linker character can have very big-difference, can comprise can be used for a spacer molecule part and the atom of another part or almost any combination of group.For example, linker can be non-cyclic hydrocarbon bridge (as the saturated or unsaturated alkyl (alkyleno) of stretching, for example stretch methyl, stretch ethyl, stretch vinyl, stretch propyl group, stretch third (1) base, stretch butyl, (1) thiazolinyl of stretching fourth, stretch fourth (2) thiazolinyl, stretch fourth (1,3) dialkylene and similar substance), monocycle or bridged polycyclic hydrocarbon are (for example: (1,2) stretch phenyl, (2,3) stretch naphthyl and similar substance), simple non-ring hetero atom or assorted alkane two Ji Qiao (for example :-O-,-S-,-S-O-,-NH-,-PH-,-C (O)-,-C (O) NH-,-S (O)-,-S (O) 2-,-S (O) NH-,-S (O) 2NH-,-O-CH 2-,-CH 2-O-CH 2-,-O-CH=CH-CH 2-and similar substance), the combination of monocycle or polyheteroaromatic bridge (for example: (3,4) are stretched furyl, stretch pyridyl, stretch thienyl, stretch piperidyl, stretch piperazinyl, stretch pyrazine pyridine base, stretched Pyrrolizidine base and similar substance) or such bridge.
Substituting group in N2, N4, C5 and/or C6 position, and also selectable linker can further be replaced by one or more identical or different substituting group.The character of these substituted radicals can have very big-difference.Suitably the limiting examples of substituted radical comprises branched-chain alkyl; straight chained alkyl or cycloalkyl; monocycle or polyaromatic; branched heteroalkyl groups; assorted alkyl of straight chain or the assorted alkyl of ring; monocycle or polyheteroaromatic; halogen; the side chain alkylhalide group; straight chain alkylhalide group or ring alkylhalide group; hydroxyl; oxo group; the thioketones base; branched alkoxy; straight chain alkoxyl group or cycloalkyloxy; side chain halogen alkoxyl group; straight chain halogen alkoxyl group or ring halogen alkoxyl group; trifluoromethoxy; monocycle or encircle aryloxy more; monocycle or encircle heteroaryloxy more; ether; alcohol; sulfide; thioether; sulfenyl (mercaptan); imines; azo-group; trinitride; amine (one-level; secondary and three grades); nitrile (any isomer); cyanate (any isomer); thiocyanate-(any isomer); nitroso-group; nitro; azo-group; sulfoxide (sulfoxides); alkylsulfonyl; sulfonic acid; sulphamide; sulphonamide; the sulphonamide ester; aldehyde; ketone; carboxylic acid; ester; acid amides; amidine; carbonamidine (formadines); amino acid; acetylene; amido formate; lactone; lactan; glucosides; the glucose acyl (gluconurides), the combination of sulfone, ketal, acetal, thioketal, oxime, careless amino acid, careless amino acid ester etc. and these groups.Substituting group with reactive functional group can be through protection or without protection, as on record in the industry.
In an illustrated example, the present invention's 2, the 4-pyrimidinediamine compounds is the compound according to structural formula (I):
Figure A20091000677100071
Comprise its salt, hydrate, solvate and N-oxide compound, wherein:
L 1And L 2Can respectively be selected from a direct key and a linker;
R 2Be selected from following group, that is: also can be by one or more identical or different R 8The base replacement (C1-C6) alkyl, also can be by one or more identical or different R 8The base replacement (C3-C8) cycloalkyl, also can be by one or more identical or different R 8The base replacement cyclohexyl, also can be by one or more identical or different R 8The base replacement 3-8 person encircle assorted alkyl, also can be by one or more identical or different R 8The base replacement (C5-C15) aryl, also can be by one or more identical or different R 8The base replacement phenyl and also can be by one or more identical or different R 85-15 person's ring heteroaryl of base replacement;
R 4Be selected from following group, that is: hydrogen, also can be by one or more identical or different R 8The base replacement (C1-C6) alkyl, also can be by one or more identical or different R 8The base replacement (C3-C8) cycloalkyl, also can be by one or more identical or different R 8The base replacement cyclohexyl, also can be by one or more identical or different R 8The base replacement 3-8 person encircle assorted alkyl, also can be by one or more identical or different R 8The base replacement (C5-C15) aryl, also can be by one or more identical or different R 8The base replacement phenyl and also can be by one or more identical or different R 85-15 person's ring heteroaryl of base replacement;
R 5Be selected from following group, that is: R 6, also can be by one or more identical or different R 8The base replacement (C1-C6) alkyl, also can be by one or more identical or different R 8The base replacement (C1-C4) alkyl group (alkanyl), also can be by one or more identical or different R 8The base replacement (C2-C4) thiazolinyl and also can be by one or more identical or different R 8(C2-C4) alkynyl of base replacement;
Each R 6Respectively be selected from following group, that is: hydrogen, electronegativity base ,-OR d,-SR d, (C1-C3) halogen alkoxyl group, (C1-C3) perhalogeno alkoxyl group ,-NR cR c, halogen, (C1-C3) alkylhalide group, (C1-C3) perhaloalkyl radical ,-CF 3,-CH 2CF 3,-CF 2CF 3,-CN ,-NC ,-OCN ,-SCN ,-NO ,-NO 2,-N 3,-S (O) R d,-S (O) 2R d,-S (O) 2OR d,-S (O) NR cR c-S (O) 2NR cR c,-OS (O) R d,-OS (O) 2R d,-OS (O) 2OR d,-OS (O) NR cR c,-OS (O) 2NR cR c,-C (O) R d,-C (O) OR d,-C (O) NR cR c,-C (NH) NR cR c,-OC (O) R d-SC (O) R d,-OC (O) OR d,-SC (O) OR d,-OC (O) NR cR c,-SC (O) NR cR c,-OC (NH) NR cR c,-SC (NH) NR cNHC ,-[(O)] nR dNHC ,-[(O)] nOR dNHC ,-[(O)] nNR cR cWith-[NHC (NH)] nNR cR c, also can be by one or more identical or different R 8The base replacement (C5-C10) aryl, also can be by one or more identical or different R 8The base replacement phenyl, also can be by one or more identical or different R 8The base replacement (C6-C16) aralkyl, also can be by one or more identical or different R 8The base replacement 5-10 person's ring heteroaryl and also can be by one or more identical or different R 8The 6-16 person of base replacement encircles heteroaralkyl;
R 8Be selected from following group, that is: R a, R b, by one or more identical or different R aOr R bThe R of replacement a, by one or more identical or different R aOr R bReplace it-OR a,-B (OR a) 2,-B (NR cR c) 2,-(CH 2) m-R b,-(CHR a) m-R b,-O-(CH 2) m-R b,-S-(CH 2) m-R b,-O-CHR aR b,-O-CR a(R b) 2,-O-(CHR a) m-R b,-O-(CH 2) m-CH[(CH 2) mR b] R b,-S-(CHR a) m-R b,-C (O) NH-(CH 2) m-R b,-C (O) NH-(CHR a) m-R b,-O-(CH 2) m-C (O) NH-(CH 2) m-R b,-S-(CH 2) m-C (O) NH-(CH 2) m-R b,-O-(CHR a) m-C (O) NH-(CHR a) m-R b,-S-(CHR a) m-C (O) NH-(CHR a) m-R b,-NH-(CH 2) m-R b,-NH-(CHR a) m-R b,-NH[(CH 2) mR b] ,-N[(CH 2) mR b] 2,-NH-C (O)-NH-(CH 2) m-R b,-NH-C (O)-(CH 2) m-CHR bR bWith-NH-(CH 2) m-C (O)-NH-(CH 2) m-R b
Each R aAll respectively be selected from following group, that is: the assorted alkyl of hydrogen, (C1-C6) alkyl, (C3-C8) cycloalkyl, cyclohexyl, (C4-C11) cycloalkylalkyl, (C5-C10) aryl, phenyl, (C6-C16) aralkyl, phenmethyl, 2-6 person, 3-8 person encircle assorted alkyl, morpholinyl (morpholinyl), piperazinyl (piperazinyl), high piperazinyl, piperidyl, 4-11 person and encircle assorted alkyl-alkyl, 5-10 person's ring heteroaryl and 6-16 person and encircle heteroaralkyl;
Each R bAll respectively be selected from one of following suitable group, that is :=O ,-OR d, (C1-C3) halogen alkoxyl group ,-OCF 3,=S ,-SR d,=NR d,=NOR d,-NR cR c, halogen ,-CF 3,-CN ,-NC ,-OCN ,-SCN ,-NO ,-NO 2,=N 2,-N 3,-S (O) R d,-S (O) 2R d,-S (O) 2OR d,-S (O) NR cR c,-S (O) 2NR cR c,-OS (O) R d,-OS (O) 2R d,-OS (O) 2OR d,-OS (O) 2NR cR c,-C (O) R d,-C (O) OR d,-C (O) NR cR c,-C (NH) NR cR c,-C (NR a) NR cR c,-C (NOH) R a,-C (NOH) NR cR c,-OC (O) R d,-OC (O) OR d,-OC (O) NR cR c,-OC (NH) NR cR c,-OC (NR a) NR cR cNHC ,-[(O)] nR d,-[NR aC (O)] nR dNHC ,-[(O)] nOR d,-[NR aC (O)] nOR dNHC ,-[(O)] nNR cR c,-[NR aC (O)] nNR cR cNHC ,-[(NH)] nNR cR cWith-[NR aC (NR a)] nNR cR c
Each R cCan respectively be a kind of protecting group, perhaps by R a, perhaps as the another kind of R that selects c, form 5 to 8 Yuans assorted alkyl of ring or heteroaryl with the nitrogen-atoms that is connected separately, and also can comprise one or more identical or different extra heteroatomss, and can be by one or more identical or different R aOr suitable R bBase replaces;
Each R dCan respectively be a kind of protecting group or R a
Each m can respectively be 1 to 3 integer; And
Each n respectively is one 0 to 3 a integer.
On the other hand, the invention provides 2, the prodrug of 4-pyrimidinediamine compounds.This type of prodrug just can possess activity when it exists as prodrug, can keep it not possess active condition before perhaps being converted into the active medicine form under physiological condition or under other working conditions.In prodrug of the present invention, 2, one or more senses of 4-pyrimidinediamine compounds be contained in round and round precursor partly among, this part (in typical case by hydrolysis, Enzyme cracking or some other cracking mechanism) under working conditions makes the molecule cracking and the generation official can be round and round.For example, one-level or secondary amine can be contained among the amide precursor, and under working conditions, this amide precursor cracking produces one-level or secondary amine.Therefore, prodrug of the present invention comprises the blocking group of specific type, is called " blocking group "; this blocking group shields one or more 2; the sense of 4-pyrimidinediamine compounds round and round, and under working conditions cracking produce possess active 2,4-pyrimidinediamine medical compounds.2, can be included in the sense of precursor among partly by blocking group shielding in the 4-pyrimidinediamine compounds and include, but is not limited to round and round: amine (firsts and seconds), hydroxy kind, sulfenyl class (thio-alcohol), carboxyl class, carbonyl class, phenols, pyrocatechol, glycols, alkynes class, phosphoric acid salt etc.Be suitable for shielding this type of sense round and round, generation can the blocking group of cracked prodrug be on record in the world of medicine under institute's requirement condition.These all blocking groups (separate base or its combination) can be included among the prodrug of the present invention.Can be included among the prodrug of the present invention, the specific examples that produces the precursor part of one-level or secondary amine includes, but is not limited to amides, amido formate class, imines class, urea class, phosphniline base class, phosphinylidyne base class and sulfenyl class.Can be included in the prodrug of the present invention, the specific examples that produces the precursor part of sulfenyl includes, but is not limited to the thioether class, for example S-methyl-derivatives (single sulfenyl, disulfide group, sulfenyl, azylthio acetal), silylation thioether class, thioesters class, thiocarbonic ester class, thioamino-formates, asymmetric disulphide etc.Can be included in the prodrug of the present invention, the particular example that the energy cracking produces the precursor part of hydroxyl includes, but is not limited to sulfonic acid esters, ester class and carbonates.Can be included in the prodrug of the present invention, the particular example that can produce the precursor part of carboxyl includes, but is not limited to ester class (comprising silylation ester class, careless amine esters of gallic acid and thioesters class), amides and hydrazides (hydrazides) class.
In a specific embodiment, prodrug of the present invention is the compound shown in the structural formula (I), wherein R cAnd R dProtecting group be blocking group.
With shown in the substituting group displacement structural formula (I) 2, the hydrogen of N2 and N4 position in the 4-pyrimidinediamine will have a negative impact to the activity of compound.Yet the business of this trade person that is familiar with should know, and these nitrogen-atoms can be included among the precursor part, and its cracking under working conditions is produced according to 2 shown in the structural formula (I), 4-pyrimidinediamine.Therefore, in another specific embodiment, prodrug of the present invention is the compound shown in the structural formula (II):
Figure A20091000677100101
Comprise its salt, hydrate, solvate and N-oxide compound, wherein:
R 2, R 4, R 5, R 6, L 1And L 2Such as front structure formula (I) definition; And
R 2bAnd R 4bRespectively be blocking group respectively.
On the other hand, the invention provides a kind of medicinal components and a kind of appropriate carriers, vehicle or thinner that comprises one or more compounds of the present invention and prodrug.The definite character of carrier, vehicle or thinner will depend on the set purposes of this composition, and its scope can contain from being suitable for veterinary purpose or veterinary purpose and can receive and be suitable for human purposes or the acceptable scope of human purposes.
Aspect also having one, the invention provides and can be used for synthetic the present invention's 2, mesosome among 4-pyrimidinediamine compounds and the prodrug.In a specific embodiment, intermediate is the 4-aminopyrimidine shown in the structural formula (III):
Figure A20091000677100111
Comprise its salt, hydrate, solvate and N-oxide compound, wherein R 4, R 5, R 6And L 2Such as previous structural formula (I) definition; LG is picture such as-S (O) 2Me ,-leaving group of SMe or halogen (for example, F, Cl, Br, I) etc.; And R 4cBe hydrogen or blocking group.
In another specific embodiment, intermediate is the 2-aminopyrimidine shown in the structural formula (IV):
Figure A20091000677100112
Comprise its salt, hydrate, solvate and N-oxide compound, wherein R 2, R 5, R 6And L 1Such as previous structural formula (I) definition; LG is picture such as-S (O) 2Me ,-SMe or halogen () leaving group for example: F, Cl, Br, I, and R 2cBe hydrogen or blocking group.
In also having a specific embodiment, intermediate is the 4-amido shown in the following structural formula (V)-or 4-hydroxyl-2-aminopyrimidine:
Figure A20091000677100113
Comprise its salt, hydrate, solvate and N-oxide compound, wherein R 2, R 5, R 6And L 11 such as previous structural formula (I) definition, R 7Be amido or hydroxyl, and R 2cBe hydrogen or blocking group.
In another specific embodiment, intermediate is the cytosine(Cyt) for the N4-replacement shown in the following structural formula (VI):
Figure A20091000677100121
Comprise its salt, hydrate, solvate and N-oxide compound, wherein R 4, R 5, R 6And L 2Such as previous structural formula (I) definition, and R 4cBe hydrogen or blocking group.
Aspect also having one, the invention provides a kind of synthetic the present invention's 2, the method for 4-pyrimidinediamine compounds and prodrug.In a specific embodiment, this method comprises that making a kind of 4-PYRIMITHAMINE shown in the structural formula (III) and a kind of molecular formula of making is HR 2cN-L 1-R 2Amine reaction (L in the formula 1, R 2And R 2cSuch as previous structural formula (IV) definition) with produce a kind of shown in structural formula (I) 2,4-pyrimidinediamine or a kind of prodrug shown in structural formula (II).
In another specific embodiment, this method comprises that making a kind of 2-PYRIMITHAMINE and a kind of molecular formula shown in structural formula (IV) is R 4-L 2-NHR 4cAmine reaction (L in the formula 4, R 4And R 4cSuch as previous structural formula (III) definition) with produce a kind of shown in structural formula (I) 2,4-pyrimidinediamine, or a kind of prodrug shown in structural formula (II).
In also having a specific embodiment, this method comprises makes a kind of 4-amido-2-PYRIMITHAMINE (R wherein shown in the structure formula V 7Be amido) with a kind of molecular formula be R 4-L 2-NHR 4cAmine reaction (L in the formula 2, R 4And R 4cSuch as structural formula (III) definition) with produce a kind of shown in structural formula (I) 2,4-pyrimidinediamine, or a kind of prodrug shown in structural formula (II).Can make also that 4-amido-2-PYRIMITHAMINE and molecular formula are R 4-L 2The compound reaction of-LG, R in the formula 4And L 2Such as previous structural formula (I) definition, LG is a leaving group.
In also having another specific embodiment, this method comprises the 4-hydroxyl shown in a kind of structure formula V of halogenation-2-PYRIMITHAMINE (R 7Be hydroxyl) producing a kind of 2-PYRIMITHAMINE shown in structural formula (IV), and by as mentioned above, make this PYRIMITHAMINE and suitable amine react.
In also having a specific embodiment, this method comprises a kind of cytosine(Cyt) for the N4-replacement as structural formula (VI) shown in of halogenation, producing a kind of 4-PYRIMITHAMINE shown in structural formula (III), and presses the above, makes this PYRIMITHAMINE and a kind of suitable amine react.
Of the present invention 2, the 4-pyrimidinediamine compounds is effective inhibitor of immunocyte (for example loose, have a liking for alkali, neutrophils and/or have a liking for Yihong blood cell cell) degranulation.Therefore, on the other hand, the invention provides the method for such cell degranulation of adjusting (especially suppressing).This method generally includes the present invention's of making degranulation cell and quantity reach effectively regulating or suppress the cell degranulation 2,4-pyrimidinediamine compounds or prodrug, or its acceptable salt, hydrate, solvate, N-oxide compound with/or its composition contact.This method can be used as that a kind of methods of treatment is implemented in vitro or in vivo, is its feature, is caused or relative disease by it with the cell degranulation with treatment or prevention.
Though be not intended to be subjected to the constraint of any theory of operation, yet biochemical data confirms 2, and the 4-pyrimidinediamine compounds is to bring into play its degranulation inhibition effect by (be at least partly by) blocking-up or the crosslinked institute of the high-affinity Fc acceptor activated signal transduction cascade that suppresses IgE (" Fc ε RI ") and IgG (" Fc γ RI ").2, the 4-pyrimidinediamine compounds is by Fc ε RI mediation with by effective inhibitor of the degranulation of Fc γ RI mediation really.Therefore, 2, the 4-pyrimidine compound can be used for suppressing the Fc receptor signal cascade in the cell type (include but not limited to scavenger cell, hypertrophy, have a liking for alkali, neutrophils and/or have a liking for Yihong blood cell cell) of any this kind of demonstration Fc ε RI and Fc γ RI acceptor.
These class methods also allow to regulate (especially suppressing) because of activating the downstream process that this type of Fc receptor signal cascade produces.This downstream process includes, but is not limited to the manufacturing and the release of degranulation, cytokine manufacturing and the lipid medium (for example leukotriene and prostaglandin(PG)) of Fc ε RI mediation and Fc γ RI mediation.This method normally allow the cells contacting some amount that shows Fc acceptor (one of cell type as discussed above) signal cascade can effectively regulate or suppress the cascade of Fc receptor signal and thus signal cascade downstream process that activation causes of the present invention 2,4-pyrimidinediamine compounds or prodrug, or acceptable salt, hydrate, solvate, N-oxide compound and composition.This method may be implemented in vitro or in vivo; be cascaded as feature, caused or relative disease with the Fc receptor signal in order to treatment or prevention as a kind of methods of treatment by it, for example have during degranulation particle specificity chemical mediator release, cytokine release with synthesize, and the release of lipid medium (for example leukotriene and prostaglandin(PG)) and synthesizing and the disease that causes.
Aspect also having one, the present invention is for being released to feature, being discharged that institute is caused or provided the method for the treatment of and preventing with disease that this kind chemical mediator discharges associated by this kind chemical mediator activating chemical mediator that Fc receptor signal cascade (for example Fc ε RI and Fc γ RI signal cascade) caused.These class methods can be implemented on one's body the animal by the animal doctor, perhaps are used on one's body the mankind.These class methods generally include and give doses of the present invention 2 to experimentation on animals object or human experimenter, 4-pyrimidinediamine compounds or prodrug, or its acceptable salt, hydrate, solvate, N-oxide compound and its composition, with treatment or preventing disease.As described in above-mentioned discussion, the activation of Fc ε RI in some immunocyte or the cascade of Fc γ RI receptor signal cause the release of number of chemical material and synthetic, and these chemical substances is pharmacologic mediators of numerous disease.Any this type of disease all can be by method treatment of the present invention or prevention.
For example, in mastocyte and basophilic leukocyte, the activation of Fc ε RI or Fc γ RI signal cascade causes atopy and I type to cross (promptly in receptor activation 1-3 minute it) immediately that sensitive response forms medium (for example, albumen Enzyme such as histamine, class Trypsin Enzyme class etc.) in advance via the degranulation process discharging.This atopy and the anaphylaxis of I type to environment and other anaphylactogen (for example include, but is not limited to, the venom of pollen, insect and/or animal, food, medicine, contrasting colour dyestuff etc.) anaphylaxis, anaphylactoid reaction, spring fever, anaphylaxis conjunctivitis, allergic rhinitis, allergic asthma, atopical dermatitis, eczema, urticaria, mucous membrane pathology, lesion tissue and some gastrointestinal illness.
After the release immediately of the pre-formation medium that degranulation causes, a series of other chemical mediators discharge immediately and are synthetic, comprising platelet activating factor (PAF), Prostaglandins and Leukotrienes element (for example, and cytokines such as TNF α, IL-4, IL-5, IL-6, IL-13 synthetic again and discharging LTC4).First process in these two processes approximately took place after receptor activation in 3-30 minute; Second process generation in about 30 minutes to 7 hours after receptor activation.These " latter stage " media are considered to cause the part reason of chronic sympton in above listed atopy and the type i allergic reaction, they still cause inflammation and inflammatory diseases (osteoarthritis for example in addition, the inflammatory bowel syndrome, ulcerative colitis, clone disease, spontaneous inflammatory bowel disease, the irritable bowel syndromes, spastic colon etc.), (for example, scleroderma slightly scabs, fiber propagation, the scar knurl, operation back scar, pulmonary fibrosis, vasospasm, migraine, perfusion (reperfusion) damage and back myocardial infarction again) and the chemical mediator of dry multiple disease or dry syndrome.These all diseases can be treated or prevent by method of the present invention.
Can comprise and basophilic leukocyte and mastocyte pathology diseases associated by other disease of method treatment of the present invention or prevention.The example of this type of disease includes, but is not limited to tetter such as scleroderma, and heart trouble is as the back myocardial infarction, and lungs are sick as the variation of lung flesh or transformation and chronic obstructive pulmonary disease (COPD), and intestinal tract disease such as inflammatory bowel syndrome (spastic colon).
Of the present invention 2, the 4-pyrimidinediamine compounds also is effective inhibitor that tyrosine-kinase Enzyme Syk swashs Enzyme.Therefore, aspect also having one, the invention provides adjusting (especially suppressing) Syk and swash the active method of Enzyme.This method generally includes to make Syk swash Enzyme or comprise Syk and swashs the cell of Enzyme and the present invention 2 of doses, 4-pyrimidinediamine compounds or prodrug or its acceptable salt, hydrate, solvate, N-oxide compound with/or its composition contact, with effective adjusting or suppress the activity that Syk swashs Enzyme.In a specific embodiment, it is that a kind of isolation or reorganization Syk swash Enzyme that Syk swashs Enzyme.In another specific embodiment, Syk swashs Enzyme and swashs Enzyme by the endogenous of cell (for example mastocyte or basophilic leukocyte) performance or reorganization Syk.This method may be implemented in vitro or in vivo, swashs the Enzyme activity by feature, caused or relative disease by it as methods of treatment treatment or prevention with Syk.
Though be not intended to be subjected to the constraint of any concrete operations theory, but salty letter of the present invention 2, the 4-pyrimidinediamine compounds suppresses the degranulation of cell and the release of other chemical mediator, mainly be to suppress to realize by Syk is swashed Enzyme, Syk swash Enzyme then by the γ chain homodimer of Fc ε RI activate (referring to, for example, Fig. 2).This γ chain homodimer is shared for other Fc acceptor (comprising Fc γ RI, Fc γ RIII and Fc α RI).For all these acceptors, the transduction of signal is by the γ chain homodimer mediation of sharing in the cell.The combination of this receptoroid causes raising and activating of tyrosine-kinase Enzyme (for example Syk swash Enzyme) with gathering.Because these common signal activities, described herein 2, the 4-pyrimidinediamine compounds can be used to regulate (especially suppressing) and has this
Figure A20091000677100151
The signal cascade of the Fc acceptor of chain homodimer (for example Fc ε RI, Fc γ RI, Fc γ RIII and Fc α RI), and this receptoroid institute inductive cell response.
Present known Syk swashs Enzyme and plays an important role in other signal cascade.For example, Syk swashs the effector that Enzyme is B-cell receptor (BCR) signal (people such as Turner, 2000, " immunology today (Immunology Today) " magazine, 21:148-154) and be integral protein (integrin) β (1) in the neutral leukocyte, the crucial composition of β (2) and β (3) generation signal (people such as Mocsai, 2002 years, " immunity " magazine, 16:547-558).Because as herein described various 2, the 4-pyrimidinediamine compounds is the strong inhibitor that Syk swashs Enzyme, therefore can be used for regulating (especially suppressing) any signal cascade by the Syk figure, for example such as the Fc acceptor, BCR and integral protein signal cascade, and this type of signal cascade institute inductive cell response.As well-known in the industry, which kind of specific cells reaction can be conditioned or be suppressed, and partly depends on concrete cell type and receptor signal cascade.Can be by various 2, the 4-pyrimidinediamine compounds is regulated or the non-limitation example of the cell response that suppresses comprises: the breaking of respiratory organs, cell adhesion, cell degranulation, cell are spread, (for example, loose, have a liking for alkali, neutrophils, have a liking for Yihong blood cell and B cell), platelet aggregation and cell maturation (for example, in the B cell) are flowed out in cell migration, phagolysis (for example, in scavenger cell), calcium ion.
Therefore, on the other hand, the present invention provides control method to the signal transduction cascade that Syk plays the part of certain role, especially suppresses method.This kind method generally includes cell and a certain amount of the present invention 2 who makes Syk rely on acceptor or show Syk dependence acceptor, 4-pyrimidinediamine compounds or prodrug, perhaps its acceptable salt, hydrate, solvate, N-oxide compound with/or its composition contact, the signal transduction cascade should be able to effectively be regulated or suppress to used dosage.These class methods also can be used to regulate (especially suppressing) downstream process, perhaps the inductive cell response by the activation of specific Syk dependence signal transduction cascade.This method can be regulated any signal transduction cascade, and whether sharp Enzyme plays a role therein even if we do not know Syk as yet, perhaps just finds its role therein afterwards.This method may be implemented in vitro or in vivo, as a kind of methods of treatment, treatment or prevention rely on the signal transduction cascade with Syk and activate to feature, caused or relative disease by it.The example that the unrestricted example of this type of disease comprises before this being discussed.
Cytology and animal experiment data also confirm, and are of the present invention 2, and the 4-pyrimidinediamine compounds also can be used for treating or preventing the symptom of autoimmune disease and this type of disease.The method that is adopted is generally authorizes the patient who suffers from autoimmune disease or patient's doses of the present invention 2 that the autoimmune disease risk takes place is arranged, 4-pyrimidinediamine compounds or prodrug or its acceptable salt, hydrate, solvate, N-oxide compound and its composition, autoimmune disease and its related symptoms should be able to effectively be treated or prevent to its dosage.Can be with 2, the autoimmune disease of 4-pyrimidinediamine compounds treatment or prevention comprises: activate the disease of (being that part mediates at least) of being mediated by the relevant various diseases (II, III type and the allergy of IV type) of anallergic allergy and those by the Fc γ R signal cascade in the monocyte usually.This type of autoimmune disease includes, but is not limited to those diseases that often is referred to as single organ or unicellular type autoimmune disease, and those are referred to as the disease of general autoimmune disorders usually.Often be referred to as single organ or the non-limiting of unicellular type autoimmune disorders comprises for example: struma lymphomatosa, autoimmune hemolytic anemia, the autoimmune atrophic gastritis of pernicious anemia, the autoimmune encephalomyelitis, Zi body Mian Yi testis inflammation, Goodpasture Cotard (being glomerulonephritis spitting of blood syndrome), the autoimmune thrombopenia, sympathetic ophthalmia, myasthenia gravis, GravesShi disease (being Exophthalmus goiter), primary biliary cirrhosis, chronic active hepatitis, ulcerative colitis and membranous glomerulopathy become.Often being referred to as the non-limiting of general autoimmune disorders comprises for example: whole body property lupus erythematosus, rheumatoid arthritis, Sjogren Cotard (being Sjogren's disease, mouthful xerophthalmia scheorma syndrome), Reiter Cotard (the still undefined triad of a kind of cause of disease comprises urethritis, conjunctivitis and sacroiliitis), polymyositis-dermatomyositis, systemic scleroderma, polyarteritis nodosa, multiple sclerosis and bullous pemphigoid.
The concise and to the point description of illustration
Figure 1 shows that how the explanation anaphylactogen induces IgE to form, and the cartoon synoptic diagram that how discharges preformation medium and other chemical mediator subsequently from mastocyte;
Figure 2 shows that how the cascade of explanation Fc ε R1 signal transduction causes the cartoon synoptic diagram of mastocyte and basophilic leukocyte degranulation;
Figure 3 shows that explanation select to suppress upstream Fc ε RI mediation degranulation compound and suppress Fc ε RI simultaneously to mediate the imaginary point of application cartoon synoptic diagram that degranulation and Ai Nuo mycin (ionomycin) are induced the degranulation compound;
Fig. 4 for explanation some 2,4-pyrimidinediamine compounds, methyl-sulphoxide (DMSO is as control group) and Ai Nuo mycin (ionomycin) are to the Ca in the CHMC cell 2+The chart of flux influence;
The pictorialization compound R 921218 of Fig. 5 and the inhibiting real-time of R926495;
The compound R 921218 of the pictorialization flushing of Fig. 6 and R921302 suppress the active influence;
The compound R 921218 (A) of the data presentation different concns that Fig. 7 provides and R921219 (B) swash the inhibition situation of the range protein phosphorylation in Enzyme downstream to Syk in the IgE receptor signal transductory cascade in activating the BMMC cell;
The endogenous Enzyme of the data presentation that Fig. 8 provides separates thing (LAT) and peptide Enzyme separates thing at compound R 921212 (X), and under R921219 (Y) and the constantly rising situation of R921304 (Z) concentration, Syk swashs the reaction of Enzyme phosphorylation inhibition to dosage;
The data that Fig. 9 provides show that compound R 921219 (A) can compete with Triphosaden the inhibition that Syk swashs Enzyme;
The compound of the data presentation different concns that Figure 10 provides and R9218218 (B) swash the restraining effect of the phosphorylation in Enzyme (and being not that LYN swashs Enzyme) downstream to Syk in the Fc ε RI signal transduction cascade that activates the CHMC cell; Be also shown in known LYN and swash Enzyme inhibitor (PP2) existence down, LYN is swashed the restraining effect of the protein phosphorylation in Enzyme (and being not that Syk swashs Enzyme) downstream;
Figure 11 A-D provide data, is presented in the Fc ε RI signal transduction cascade of BMMC cell, Syk swashed the restraining effect of Enzyme downstream protein phosphorylation;
The pictorialization compound R 921302 of Figure 12 is induced effectiveness in sacroiliitis (" the CAIA ") model at the small white mouse collagen antibodies;
The effectiveness of the pictorialization compound R of in the CAIA model, comparing with the contrast medicament 921302 of Figure 13 with other medicament;
Figure 14's is pictorialization in the collagen-induced sacroiliitis of rat (" CIA ") model, the effectiveness of compound R 921302;
The caption compound R 921302 of Figure 15 suppresses the effectiveness of small white mouse experiment autoimmune encephalomyelitis (" EAE ") and the clinical model of multiple sclerosis;
921302 pairs of the caption of Figure 16 explanation compound Rs are in the effectiveness of the SJL small white mouse for the treatment of with 150 μ g PLP 139-151/200 μ gMTB (CFA) day of beginning.
The detailed description of preferred embodiment
6.1 definition
When being used in herein, following term draw up has following implication:
" Alkyl (Alkyl)" itself or as another substituent some; meaning saturated or undersaturated side chain, straight chain or the ring univalence hydrocarbyl of the carbon atom (being that C1-C6 represents 1 to 6 carbon atom) with described number, is that a carbon atom from female alkane, alkene or alkynes removes a hydrogen atom and gets.Typical alkyl includes, but is not limited to methyl; The ethyl class is ethyl, vinyl, ethynyl for example; The propyl group class is third-1-base, third-2-base, ring third-1-base, third-1-alkene-1-base, third-1-alkene-2-base, third-2-alkene-1-base, ring third-1-alkene-1-base for example; Ring third-2-alkene-1-base, third-1-alkynes-1-base, third-2-alkynes-1-base etc.; Butyl-like is fourth-1-base, fourth-2-base, 2-methyl-third-1-base, 2-methyl-third-2-base, ring fourth-1-base, but-1-ene-1-base, but-1-ene-2-base, 2-methyl-third-1-alkene-1-base, but-2-ene-1-base, but-2-ene-2-base, fourth-1 for example, 3-diene-1-base, fourth-1,3-diene-2-base, ring but-1-ene-1-base, ring but-1-ene-3-base, ring fourth-1,3-diene-1-base, fourth-1-alkynes-1-base, fourth-1-alkynes-3-base, fourth-3-alkynes-1-base etc.; And analogue.Be intended to express in the situation of specific saturation ratio, using as giving a definition it " alkyl group " " thiazolinyl " and " alkynyl " term.In a preferred embodiment, alkyl is (C1-C6) alkyl.
" Alkyl group (Alkanyl)" itself or as another substituent some, mean saturated side chain, straight chain or cycloalkyl, be that a carbon atom from female alkane removes a hydrogen atom and gets.Typical alkyl includes, but is not limited to methyl; Ethyl; The propyl group class is third-1-base, third-2-base (sec.-propyl), ring third-1-base etc. for example; Butyl-like is fourth-1-base, fourth-2-base (secondary-butyl), 2-methyl-third-1-base (isobutyl-), 2-methyl-third-2-base (three grades-butyl), ring fourth-1-base etc. for example; And resemblance.In a preferred embodiment, alkyl group is (C1-C6) alkyl group.
" Thiazolinyl (Alkenyl)" itself or as another substituent some, mean unsaturated side chain, straight chain or cycloalkyl with at least one carbon-to-carbon double bond, be that a carbon atom from female alkene removes a hydrogen atom and gets.One or several of this type of group pair key can be by cis or trans configuration.Typical thiazolinyl includes, but is not limited to vinyl; The propylene base class is third-1-alkene-1-base, third-1-alkene-2-base, third-2-alkene-1-base, third-2-alkene-2-base, ring third-1-alkene-1-base for example; Ring third-2-alkene-1-base; The butylene base class is but-1-ene-1-base, but-1-ene-2-base, 2-methyl-third-1-alkene-1-base, but-2-ene-1-base, but-2-ene-1-base, but-2-ene-2-base, fourth-1 for example, 3-diene-1-base, fourth-1,3-diene-2-base, ring but-1-ene-1-base, ring but-1-ene-3-base, ring fourth-1,3-diene-1-base etc.; And analogue.In a preferred embodiment, thiazolinyl is (C2-C6) thiazolinyl.
" Alkynyl (Alkynyl)" itself or as another substituent some, mean unsaturated side chain, straight chain or cycloalkyl with at least one carbon-to-carbon triple bond, be that a carbon atom from female alkynes removes a hydrogen atom and gets.Typical alkynyl includes, but is not limited to ethynyl; The propine base class is third-1-alkynes-1-base, third-2-alkynes-1-base etc. for example; The butine base class is fourth-1-alkynes-1-base, fourth-1-alkynes-3-base, fourth-3-alkynes-1-base etc. for example; And resemblance.In a preferred embodiment, alkynyl is (C2-C6) alkynyl.
" Alkane two bases (Alkyldiyl)" itself or as another substituent some; mean saturated or unsaturated side chain, straight chain or the ring bivalent hydrocarbon radical of carbon atom (being that C1-C6 represents 1 to 6 carbon atom); be respectively to remove a hydrogen atom, or remove two hydrogen atoms and get from a carbon atom of female alkane, female alkene or female alkynes from two of female alkane, alkene or alkynes different carbon atoms with described number.Each valency at two unit price base centers or divalent radical center can form key with identical or different atom.Typical alkane two bases include, but is not limited to for example second-1 of first two bases, second two base class, 1-two bases, second-1,2-two bases, ethene-1,1-two bases, ethene-1,2-two bases; Glyceryl class for example the third-1,1-two bases, the third-1,2-two bases, the third-2,2-two bases, the third-1,3-two bases, ring the third-1,1-two bases, ring the third-1,2-two bases, third-1-alkene-1,1-two bases, third-1-alkene-1,2-two bases, third-2-alkene-1,2-two bases, third-1-alkene-1,3-two bases, ring third-1-alkene-1,2-two bases, ring third-2-alkene-1,2-two bases, ring third-2-alkene-1,1-two bases, third-1-alkynes-1,3-two bases, or the like; Fourth two base class are fourth-1 for example, 1-two bases, fourth-1,2-two bases, fourth-1,3-two bases, fourth-1,4-two bases, fourth-2,2-two bases, 2-methyl-the third-1,1-two bases, 2-methyl-the third-1,2-two bases, ring fourth-1,1-two bases; Ring fourth-1,2-two bases, ring fourth-1,3-two bases, but-1-ene-1,1-two bases, but-1-ene-1,2-two bases, but-1-ene-1,3-two bases, but-1-ene-1,4-two bases, 2-methyl-third-1-alkene-1,1-two bases, 2-methylene radical (methanylidene)-the third-1,1-two bases, fourth-1,3-diene-1,1-two bases, fourth-1,3-diene-1,2-two bases, fourth-1,3-diene-1,3-two bases, fourth-1,3-diene-1,4-two bases, ring but-1-ene-1,2-two bases, ring but-1-ene-1,3-two bases, ring but-2-ene-1,2-two bases, ring fourth-1,3-diene-1,2-two bases, ring fourth-1,3-diene-1,3-two bases, fourth-1-alkynes-1,3-two bases, fourth-1-alkynes-1,4-two bases, fourth-1,3-diine-1,4-two bases etc.; And analogue.In the situation of hoping specific saturation ratio, use term " alkane two bases (alkanyidiyl) ", " alkene two bases " and " alkynes two bases ".Wherein when hoping two valencys especially on same carbon atom, use term " alkylidene group ".In preferred embodiment, alkane two bases are (C1-C6) alkane two bases.Equally preferably saturated non-cycloalkanes two bases, its group center are at terminal carbon, for example, and first two bases (stretching methyl); Second-1,2-two bases (stretching ethyl); The third-1,3-two bases (stretching propyl group); Fourth-1,4-two bases (stretching butyl); And resemblance (be also referred to as and stretch alkyls, definition as follows).
" Stretch alkyl (Alkyleno)" itself or as another substituent some, mean saturated or unsaturated alkane two bases of the straight chain with two terminal unit price base centers, be that two terminal carbon atoms from the female alkane of straight chain, female alkene or female alkynes respectively remove a hydrogen atom and get.Stretch in the alkyl specific, if having two keys or triple bond, then its position shows in square brackets.Typically stretch alkyl and include, but is not limited to stretch methyl; Stretch the ethyl class as stretching ethyl, stretch vinyl, stretch ethynyl; Stretch the propyl group class as stretching propyl group, stretch third (1) base, stretch third (1,2) dialkylene, stretch third [(1) alkynyl or the like; Stretch butyl-like as stretching butyl, (1) thiazolinyl of stretching fourth, stretching fourth (2) thiazolinyl, stretch fourth (1,3) dialkylene, stretch fourth (1) alkynyl, stretch fourth (2) alkynyl, stretch fourth (1,3) diynyl etc. and analogue.Hoping under the situation of specific saturation ratio, adopting term to stretch alkyl (alkano), stretch thiazolinyl (alkeno) and stretch alkynyl (alkyno).In preferred embodiment, stretch alkyl and stretch alkyl for (C1-C6) or (C1-C3).Same preferable example also for the saturated alkyl of stretching of straight chain, for example, is stretched methyl, stretches ethyl, stretches propyl group, is stretched butyl and resemblance.
" Assorted alkyl (Heteroalkyl)", " Assorted alkyl (Heteroalkanyl)", " Assorted thiazolinyl (Heteroalkenyl)", " Assorted alkynyl (Heteroalkynyl)", " Assorted alkane two bases (Heteroalkyldiyl)" and " The assorted alkyl (Heteroalkyleno) of stretching" itself or as another substituent some, mean alkyl, alkyl group, thiazolinyl, alkynyl, alkane two bases and stretch that one or several carbon atoms in the alkyl are independent separately to be replaced by identical or different heteroatoms or heteroatoms base.The typical heteroatoms and the heteroatoms base of substitutable carbon atom include, but is not limited to-O-,-S-,-S-O-,-NR '-,-PH-,-S (O)-,-S (O) 2-,-S (O) NR ' ,-S (O) 2NR '-and resemblance comprises its combination, and wherein R ' is respectively hydrogen or (C1-C6) alkyl.
" Cycloalkyl (Cycloalkyl)" reach " The assorted alkyl (Heterocycloalkyl) of ring" itself or as another substituent some, mean the ring-type variant of " alkyl " or " assorted alkyl " respectively.With regard to assorted alkyl, a heteroatoms can occupy the position that is connected to the molecule rest part.Typical cycloalkyl includes, but is not limited to cyclopropyl; Cyclobutyl class such as cyclobutyl and cyclobutene base; Cyclopentyl class such as cyclopentyl and cyclopentenyl; Hexamethylene base class such as cyclohexyl and cyclohexenyl; And resemblance.The assorted alkyl of typical ring includes, but is not limited to tetrahydrofuran base (as tetrahydrofuran (THF)-2-base, tetrahydrofuran (THF)-3-base or the like), piperidyl (as piperidines-1-base, piperidines-2-base or the like), morpholinyl (as morpholine-3-base, morpholine-4-yl), piperazinyl (as piperazine-1-base, piperazine-2-base or the like) and analogue.
" Acyclic heteroatoms bridging (Acyclic Heteroatomic Bridge)" mean the divalence bridging, wherein backbone atoms only is heteroatoms and heteroatoms base.Typical acyclic heteroatoms bridging includes, but is not limited to-O-,-S-,-S-O-,-NR '-,-PH-,-S (O)-,-S (O) 2-,-S (O) NR '-,-S (O) 2NR '-and resemblance comprises its combination, and wherein R ' respectively is hydrogen or (C1-C6) alkyl.
" Female aromatic ring system (Parent Aromatic Ring System)" mean the unsaturated ring with conjugated pi electron system or encircle loop systems more.Know clearly it, be included in " female aromatic ring system " definition is that one or several rings are aromatic ring, and one or several rings are for the fused rings system of saturated or unsaturated ring, for example such as fluorenes (fluorene), hydrogen indenes (indane), indenes (indene), Fu (phenalene), naphthane (tetrahydro-naphthalene) or the like.Typical female aromatic ring system includes, but is not limited to aceanthrylene (aceanthrylene), acenaphthene (acenaphthylene), acetyl inferior luxuriant and rich with fragrance (acephenanthrylene), anthracene (anthracene), Azulene (azulene), benzene, (Lv bend) (chrysene), cool (coronene), fluoranthene (fluoranthene), fluorenes (fluorene), oneself is moan (hexacene), hexaphene (hexaphene) Ji Shallot (hexalene), indenes moan (indacene), the s-indenes is moaned, hydrogen indenes (indane), indenes (indene), naphthalene, suffering moan (octacene), Xin Fen (octaphene); hot Shallot (octalene), ovalene (ovalene), penta-2, the 4-diene, penta (pentacene) Wu Shallot (pentalene) that moans, pentaphene (pentaphene) perylene (perylene) Fu (phenalene), luxuriant and rich with fragrance (phenanthrene) Pi (picene), pleiadene, pyrene (pyrene), pyranthrene (pyranthrene), rubine moan (rubicene), naphthane, terphenyl, trinaphthylene and analogue, with and various hydrogen isomer.
" Aryl (Aryl)" itself or as another substituent some, mean the unit price aromatic hydrocarbon radical of carbon atom (being that C5-C15 represents 5 to 15 carbon atoms) with described number, be that a carbon atom from female aromatic ring system removes a hydrogen atom and gets.Typical aryl includes, but is not limited to the group derived from following compound, that is: aceanthrylene (aceanthrylene), acenaphthene (acenaphthylene), acetyl inferior luxuriant and rich with fragrance (acephenanthrylene), anthracene (anthracene), Azulene (azulene), benzene, (Lv bend) (chrysene), cool (coronene), fluoranthene (fluoranthene), fluorenes (fluorene), oneself is moan (hexacene), hexaphene (hexaphene) Liu Shallot (hexalene), as-indenes moan (as-indacene), s-indenes moan (s-indacene), hydrogen indenes (indane), indenes (indene), naphthalene, suffering moan (octacene), Xin Fen (octaphene); hot Shallot (octalene), ovalene (ovalene), penta-2, the 4-diene, penta (pentacene) Wu Shallot (pentalene) that moans, pentaphene (pentaphene) perylene (perylene) Fu (phenalene), luxuriant and rich with fragrance (phenanthrene) Pi (picene), pleiadene, pyrene (pyrene), pyranthrene (pyranthrene), rubine moan (rubicene), terphenyl, trinaphthylene and analogue, with and various hydrogen isomer.In a preferred embodiment, aryl is (C5-C15) aryl, and better is (C5-C10).Special ideal aryl class is cyclopentadienyl, phenyl and naphthyl.
" Aryl aryl (Arylaryl)" itself or as another substituent some; mean from a carbon atom of loop systems remove a hydrogen atom and the unit price aromatic hydrocarbyl; wherein two or more identical or different female aromatic ring systems directly connect together with singly-bound, and the number of this direct shack is than one of the reduced number of the ring of original female aromatic ring system.Typical aryl aryl includes, but is not limited to xenyl, terphenylyl, phenyl-naphthyl, binaphthylyl, xenyl-naphthyl, and resemblance.Carbon atom number part in narration aryl aryl, this type of number means the carbon atom of original each female aromatic ring.For example, (C5-C15) the aryl aryl comprises the aryl aryl of from 5 to 15 carbon for each aromatic ring wherein, for example, and xenyl, terphenylyl, binaphthylyl, phenyl napthyl etc.The female aromatic ring system of each aryl aryl respectively is (C5-C15) aromatics preferably, if be that (C5-C10) aromatics is better.The aryl aryl that wherein all female aromatic ring systems are also preferably identical, for example, xenyl, terphenylyl, binaphthylyl, trinaphthylene base etc.
" Dibenzyl (Biaryl)" itself or as another substituent some, mean to have two aryl aryl that form with the direct-connected identical female aromatic systems of singly-bound.Typical dibenzyl includes, but is not limited to xenyl, binaphthylyl, dianthranide base and resemblance.This type of aromatic ring system is (C5-C15) aromatic ring preferably, is that (C5-C10) aromatic ring is better.Special ideal dibenzyl is an xenyl.
" Aralkyl (Arylalkyl)" itself or as another substituent y some, mean in the non-cycloalkyl that one is keyed to carbon atom (typical situation is terminal or sp 3Carbon atom) hydrogen atom on is that aryl replaces.Typical aralkyl includes, but is not limited to phenmethyl, 2-benzene second-1-base, 2-vinylbenzene-1-base, menaphthyl, 2-naphthalene second-1-base, 2-naphthalene ethene-1-base, naphtho-phenmethyl, 2-naphtho-styroyl-1-base and analogue.Hoping under the situation of expressing concrete alkyl part, use term aralkyl, arylalkenyl and sweet-smelling alkynyl.In preferred embodiment, aralkyl is (C6-C21) aralkyl, and for example the alkyl of aralkyl, alkenyl or alkynyl are (C1-C6) partly, and aryl moiety is (C5-C15).In ideal specific embodiment especially, aralkyl is (C6-C13), and for example the alkyl of aralkyl, alkenyl or alkynyl are (C1-C3) partly, and aryl moiety is (C5-C10).
" Female heteroaromatic ring system (Parent Heteroaromatic Ring System)" mean that one of them or several carbon atoms are separately by female aromatic ring system of identical or different heteroatoms or heteroatom group replacement.The typical heteroatoms or the heteroatom group of alternate c atoms include, but is not limited to: N, NH, P, O, S, S (O), S (O) 2, Si, etc.Particularly including in " female hetero-aromatic ring system " definition for wherein there being one or several rings to be aromatic ring and one or several rings condensed ring system for saturated or unsaturated ring, for example such as benzdioxan, cumarone, chroman (chromane, be coumaran), chromene (chromene, be chromene), indoles (indole), dihydroindole (indoline, i.e. indoline), xanthene (xanthene) or the like.Be also included within " female heteroaromatic ring system " definition the person for comprising common substituent this type of known ring, for example such as benzopyrone (benzopyrone) and 1-methyl isophthalic acid, 2,3,4-tetrazolium.From " female heteroaromatic ring system " definition, need excludedly especially to be and ring polyalkylene glycol (for example encircling macrogol) condensed phenyl.Typical female heteroaromatic ring system includes, but is not limited to acridine (acridine), benzoglyoxaline (benzimidazole), benzisoxa oxazole (benzisoxazole), benzdioxan (benzodioxan), benzo two is disliked luxuriant (benzodioxole), cumarone, benzopyrone (benzopyrone), diazosulfide (benzothiadiazole), benzothiazole (benzothiazole), benzotriazole (benzotriazole), benzoxazine (benzoxaxine), benzoxazoles (benzoxazole), benzoxazoles quinoline (benzoxazoline), carbazole (carbazole), β-Ka Lin (β-carboline), chroman (chromane), chromene (chromene), E quinoline (cinnoline), furans (furan), imidazoles (imidazole), indazole (indazole), indoles (indole), indoline (indoline), indolizine (indolizine), isobenzofuran (isobenzofuran), heterochromatic alkene (isochromene), isoindole (isoindole), isoindoline (isoindoline), isoquinoline 99.9 (isoquinoline), isothiazole (isothiazole), isoxzzole (isoxazole), naphthyridines (naphthyridine), oxadiazoles (oxadiazole), oxazole (oxazole), uncle's pyridine (perimidine), coffee pyridine (phenanthridine), coffee is coughed up quinoline (phenanthroline), coffee piperazine (phenazine), dai piperazine (phthalazine), the pyridine (pteridine) of talking endlessly, purine (purine), pyrans (pyran), pyrazine (pyrazine), pyrazoles (pyrazole), pyridazine (pyridazine), pyridine (pyridine), pyrimidine (pyrimidine), pyrroles (pyrrole), pyrroles's piperazine (pyrrolizine), quinazoline (quinazoline), quinoline (quinoline), quinolizine (quinolizine), quinoxaline (quinoxaline), tetrazolium, thiadiazoles (thiadiazole), thiazole (thiazole), thiophene (thiophene), triazole (triazole), xanthene (xznthene), and analogue.
" Heteroaryl (Heteroaryl)" itself or as another substituent some, mean unit price heteroaryl with described number annular atoms (for example from 5 to 14 annular atomses of " 5-14 member " expression), this group is that an atom from female heteroaryl ring system removes a hydrogen atom and gets.Typical heteroaryl includes, but is not limited to the group derived from following material: acridine (acridine), benzoglyoxaline (benzimidazole), benzisoxa oxazole (benzisoxazole), benzdioxan (benzodioxan), benzo two is disliked luxuriant (benzodioxole), cumarone, benzopyrone (benzopyrone), diazosulfide (benzothiadiazole), benzothiazole (benzothiazole), benzotriazole (benzotriazole), benzoxazine (benzoxaxine), benzoxazoles (benzoxazole), benzoxazoles quinoline (benzoxazoline), carbazole (carbazole), β-Ka Lin (?-carboline), chroman (chromane), chromene (chromene), E quinoline (cinnoline), furans (furan), imidazoles (imidazole), indazole (indazole), indoles (indole), indoline (indoline), indolizine (indolizine), isobenzofuran (isobenzofuran), heterochromatic alkene (isochromene), isoindole (isoindole), isoindoline (isoindoline), isoquinoline 99.9 (isoquinoline), isothiazole (isothiazole), isoxzzole (isoxazole), naphthyridines (naphthyridine), oxadiazoles (oxadiazole), oxazole (oxazole), uncle's pyridine (perimidine), coffee pyridine (phenanthridine), coffee is coughed up quinoline (phenanthroline), coffee piperazine (phenazine), dai piperazine (phthalazine), the pyridine (pteridine) of talking endlessly, purine (purine), pyrans (pyran), pyrazine (pyrazine), pyrazoles (pyrazole), pyridazine (pyridazine), pyridine (pyridine), pyrimidine (pyrimidine), pyrroles (pyrrole), pyrroles's piperazine (pyrrolizine), quinazoline (quinazoline), quinoline (quinoline), quinolizine (quinolizine), quinoxaline (quinoxaline), tetrazolium, thiadiazoles (thiadiazole), thiazole (thiazole), thiophene (thiophene), triazole (triazole), xanthene (xznthene), and analogue, with and various hydrogen base (hydro) isomer.In preferred embodiment, heteroaryl is 5-14 person's ring heteroaryl, and ideal with 5-10 person's heteroaryl.
" Heteroaryl-heteroaryl (Heteroaryl-Heteroaryl)" itself or as another substituent some; mean from a carbon atom of loop systems remove a hydrogen atom and the unit price heteroaryl; wherein two several same or different female heteroaromatic ring systems directly link together with a single key, this directly encircle the connection number of rings lack one than the number of rings of original female heteroaromatic ring system.Typical heteroaryl-heteroaryl includes, but is not limited to: bipyridyl, three pyridyl, pyridyl purine radicals, connection purine radicals or the like.When the narration carbon atom number, this number means the atomicity of original each female heteroaromatic ring system.For example, 5-15 person's ring heteroaryl-heteroaryl refers to that each female heteroaromatic ring system wherein all contains the heteroaryl-heteroaryl of 5 to 15 atoms, bipyridyl for example, three purine radicals or the like.Preferably each female heteroaromatic ring system respectively is 5-15 person's heteroaromatic all, as is that 5-10 person's heteroaromatic is then better.Wherein to be identical heteroaryl-heteroaryl also fine certainly for all female heteroaromatic ring systems.
" Connection heteroaryl (Biheteroaryl)" itself or as another substituent some, mean to have two identical female heteroaromatic ring systems with the direct-connected heteroaryl-heteroaryl of singly-bound.Typical connection heteroaryl includes, but is not limited to bipyridyl, connection purine radicals, diquinolyl base (biquinolinyl) or the like.This type of heteroaromatic ring system is 5-15 person's heteroaromatic rings preferably, is that 5-10 person's heteroaromatic rings is better.
" Heteroaralkyl (Heteroarylalkyl)" itself or as another substituent some, mean that in the non-cycloalkyl one (is typically terminal carbon atom or sp with carbon atom 3Carbon atom) hydrogen atom of Lian Jieing is replaced by heteroaryl.Under the situation of hoping the concrete moieties of expression, use heteroaralkyl, the term of impure aromatic ene base and hetaryne base.In specific embodiment preferably, heteroaralkyl is 6-21 person's heteroaralkyl, and for example the alkyl of heteroaralkyl, alkenyl or alkynyl partly are (C1-C6) alkyl, and its heteroaryl moieties is 5-15-person's heteroaryl.In better specific embodiment, heteroaralkyl is 6-13 person's heteroaralkyl, and for example its alkyl, alkenyl or alkynyl partly are (C1-C3) alkyl, and its heteroaryl moieties is 5-10 person's a heteroaryl.
" Halogen (Halogen or Halo)" itself or as other substituent some, except as otherwise noted, otherwise mean fluorine-based, chloro, bromo and iodo.
" Alkylhalide group (Haloalkyl)" itself or as another substituent some, mean that one or several hydrogen atoms are halogen institute metathetical alkyl.Therefore, term " alkylhalide group " should comprise that single alkyl halide base class, dihalo-alkyls, three alkyl halide base class etc. are up to the perhaloalkyl radical class.For example " (C1-C2) alkylhalide group " this phrase should comprise methyl fluoride, difluoromethyl, trifluoromethyl, 1-fluoro ethyl, 1,1-two fluoro ethyls, 1, and 2-two fluoro ethyls, 1,1,1-trifluoroethyl, perfluor ethyl, or the like.
Group defined above can comprise produce other people recognize substituent general in the industry prefix and suffix altogether.For example " alkyl oxy (alkyloxy) " or " alkoxyl group (alkoxy) " means that a kind of molecular formula formula is-OR " group; " alkylamine (alkylamine) " means that a kind of molecular formula is-NHR " group, and " dialkylamine (dialkylamine) " means that a kind of molecular formula is-NR " R " group, the R in the formula " be alkyl.Again for example " halogen alkoxyl group (haloalkoxy) " or " alkylhalide group oxygen base (haloalkylxy) " means that a kind of molecular formula is-OR " ' base, R in the formula " ' be alkylhalide group.
" blocking group " means when the reactive functional group that is connected in the molecule round and round can shield, weaken or prevent the reactive round and round atomic group of sense.Under typical situation, blocking group can optionally be removed in building-up process if needed.The example of blocking group can be referring to Greene and Wuts, Protective Groups in Organic Chemistry, 3 RdEd., 1999, John Wiley ﹠amp; Sons, NY and Harrison et al., Compendium of Synthetic Organic Methods, Vols.1-8,1971-1996, John Wiley ﹠amp; Sons, NY.Representative amido blocking group includes, but is not limited to formyl radical, ethanoyl, trifluoroacetyl group, phenmethyl, benzene methoxycarbonyl (" CBZ "), special butoxy carbonyl (" Boc "), trimethyl silyl (" TMS "), 2-trimethyl silyl-ethane alkylsulfonyl (" TES "), trityl and the trityl that is substituted, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (" FMOC "), Xiao Ji Quinoa reed oxygen carbonyl (" NVOC ") and resemblance.Representative hydroxy-protective group includes, but is not limited to hydroxyl by acidylate or alkylating group, for example phenmethyl and trityl ether and alkyl oxide, tetrahydropyranyl ethers, three alkane silyl ethers (for example TMS or TIPPS yl) and allyl ether.
" Prodrug (Prodrug)" mean and need under behaviour in service, (for example in vivo) conversion can discharge activity 2, the activity 2 of 4-pyrimidinediamine medicine, 4-pyrimidinediamine compounds (medicine).Prodrug before changing into active medicine often (but might not) do not possess pharmacologically active.In typical case; prodrug is by shielding 2; a functional group in the 4-pyrimidinediamine medicine and obtaining; this functional group it is believed that be under specified behaviour in service with blocking group (progroup; its definition sees below) effect; facilitating relevant partly transform (for example cracking) and generating and possess actively 2,4-pyrimidinediamine medicine institute part is essential.(for example by hydrolysis) can be spontaneously carried out in precursor cracking partly, perhaps by (for example Enzyme, light, acid or the alkali) catalysis of other medium or induce, perhaps change certain physics or environmental parameter (for example changing temperature), perhaps be placed under this unable or environmental parameter.Above-mentioned medium can be endogenous under working conditions, for example throws existing Enzyme or the interior acidic conditions of stomach in the cell that gives prodrug, also can provide from external source.
Be suitable for shielding and possess actively 2, the functional group in the 4-pyrimidinediamine compounds is well-known in the industry to produce the very wide blocking group of prodrug scope.For example hydroxy functional group can be shielded to sulphonate, ester or carbonate precursor partly, and this precursor partly hydrolysis in vivo provides hydroxyl.The amine functional group maskable is acid amides, amido formate, imines, urea, phosphenyl (phosphenyl), phosphono (phosphoryl) or sulfenyl (sulfenyl) precursor part, and this precursor partly hydrolysis in vivo provides amido.The carboxyl maskable be ester (comprising silylation ester and thioesters class), acid amides or acylhydrazine precursor partly, and can be hydrolyzed at live body carboxyl is provided.Other example of due care group and concrete precursor thereof are conspicuous to this gate technique person that is familiar with partly.
" Blocking group (Progroup)" meaning that being used for shielding possesses actively 2,4-pyrimidinediamine medicine is to form the blocking group that precursor can be converted into medicine prodrug partly the time.In typical case, blocking group is by can being connected on the functional group of medicine by the cracked key under the specified working conditions.Therefore, blocking group for can be under specific working conditions cracking discharge precursor that part partly of functional group.As specific examples, molecular formula is-NH-C (O) CH 3Amide precursor partly comprise blocking group-C (O) CH 3
" Fc acceptor (Fc Receptor)" mean a member with the Fc of immunoglobulin (Ig) part (comprising special constant region) bonded cell surface molecule family.The immunoglobulin (Ig) of a specific type of each Fc receptors bind.For example Fc α acceptor (" Fc α R ") is in conjunction with IgA, and Fc ε R is in conjunction with IgE, and Fc γ R is in conjunction with IgG.
Fc α R family be included in polymerization Ig acceptor that the IgA/IgM epithelium transmits, the special acceptor Rc of marrow α RI (being also referred to as CD89), Fc α/μ R and at least two alternately the IgA acceptor (summary is referring to Monteiro ﹠amp recently; Van de Winkel, 2003, Annu.Rev.Immunol. sees the electronic edition of delivering in advance).Fc α RI finds expression in neutral leukocyte, has a liking for Yihong blood cell, on monocyte/macrophage, dentritic cell and starlike (kupfer) cell.Fc α RI comprises a α-chain and has the FcR γ homodimer of the sharp Enzyme of activation theme (ITAM) and phosphorylation Syk in the tenuigenin district.
Fc ε R family comprises two types, is called Fc ε RI and Fc ε RII (being also referred to as CD23).Fc ε RI is anchored into the high-affinity receptor of cell surface (with about 10 for being present in hypertrophy, having a liking for alkali and have a liking among the blood cell cell of Yihong with monomer I gE 10M -1Avidity in conjunction with IgE).Fc ε RI has a α-chain, a beta chain and an above-mentioned γ-chain homodimer.Fc ε RII finds expression in mononuclear phagocyte, B lymph corpuscle, has a liking for the low-affinity receptor on Yihong blood cell and the thrombocyte.Fc ε RII comprises a single polypeptide chain, and does not comprise γ-chain homodimer.
Fc γ R family comprises three types, is called Fc γ RI (being also referred to as CD64), Fc γ RII (being also referred to as CD32) and Fc γ RIII (being also referred to as CD16).Fc γ RI a kind ofly is present in hypertrophy, has a liking for alkali, monokaryon, neutrophils, has a liking among Yihong blood cell, dentritic cell and the phagocytic cell and with monomer I gG and be anchored into the high-affinity receptor of cell surface (with about 10 8M -1Avidity in conjunction with IgG1).Fc γ R comprises a α-chain and γ-chain dipolymer of being shared by Fc α RI and Fc ε RI.
Fc γ RII be find expression in neutral leukocyte, monocyte, have a liking for Yihong blood cell, the low-affinity receptor on thrombocyte and the B lymph corpuscle.Fc γ RII comprises a α-chain, and does not comprise above-mentioned γ-chain homodimer.
Fc γ RIII finds expression in NK, has a liking for low-affinity (acceptor) on Yihong blood cell, scavenger cell, neutrophils and the mastocyte (with 5 * 10 5M -1Avidity in conjunction with IgG1).It comprises a α-chain and γ-chain homodimer of being shared by Fc α RI, Fc ε RI and Fc γ RI.
The one's own profession skill person that is familiar with should be able to find out the sub-cell structure of above various Fc acceptors and in conjunction with character and to show their cell type not qualitative fully.Above-mentioned discussion only reflects that the present skill state of relevant these acceptors is (referring to for example Immunobiology:The Immune System in Health ﹠amp; Disease, 5 ThEdition, Janeway et al., Eds, 2001, ISBN 0-8153-3642-x, Figure 9.30 at pp.371), and be not intended to limit the countless receptor signal cascades that compound described herein can be regulated.
" The degranulation (Fc Receptor-Mediated Degranulation) that Fc is receptor-mediated" or " The Fc acceptor lures The degranulation of leading (Fc Receptor-Induced Degranulation)" mean the degranulation that the Fc receptor signal transductory cascade by the crosslinked initiation of Fc acceptor is caused.
" The degranulation that IgE induces (IgE-Induced Degranulation)" or " The degranulation of Fc ε RI mediation Effect (Fc ε RI-Mediated Degranulation)" mean the degranulation that is caused by with the crosslinked IgE receptor signal transductory cascade that is caused of Fc ε R1 bonded IgE.Crosslinked can inducing by IgE specific allergen or other multivalence binding medium (for example anti-IgE antibodies).Referring to Fig. 2, in loose and basophilic leukocyte, cause the Fc ε RI signal cascade of degranulation can be divided into two stages: upstream and downstream.Upstream phase comprises the process (" Ca among Fig. 2 before all calcium ions mobilization 2+"; Also referring to Fig. 3).Downstream stages comprises calcium ion mobilization and its all downstream processes.Inhibition can act on any point on the cascade of Fc ε RI mediation signal transduction by the compound of Fc ε RI mediation degranulation.The energy selectivity suppresses the compound effects of upstream Fc ε RI mediation degranulation and mobilizes the Fc ε RI signal cascade part of inducing a upstream in calcium ion, and this is partly played restraining effect.In chemical examination based on cell, the compound that selectivity suppresses upstream Fc ε RI mediation degranulation can suppress the mastocyte or the isocellular degranulation of basophilic leukocyte that are activated or excited by IgE specificity allergy unit or binding medium (for example anti-IgE antibodies), but (for example Calcium ionophore Ai Nuo mycin (ionomycin) and the cell degranulation that A23187) activates or excite there is no obvious restraining effect to the degranulation medium of walking around Fc ε RI signal path.
" The degranulation that IgG induces (IgG-Induced Degranulation)" or " The degranulation of Fc γ RI mediation Effect (Fc ε RI-Mediated Degranulation)" mean the degranulation that is caused by with the crosslinked Fc γ RI signal transduction cascade that is caused of Fc γ RI bonded IgG.Crosslinked can inducing by IgE specific allergen or another multivalence binding medium (for example anti-IgE antibodies or fragment antibody).As Fc ε RI signal cascade, in loose and basophilic leukocyte, the cascade of Fc γ RI signal transduction also causes degranulation, and can be divided into two same stages: upstream and downstream.Similar to the degranulation of Fc ε RI mediation, selectivity suppresses the compound effects of upstream Fc γ RI mediation degranulation and mobilizes the upstream end of inducing a little in calcium ion.In chemical examination based on cell, the compound that selectivity suppresses upstream Fc γ RI mediation degranulation can suppress the mastocyte or the isocellular degranulation of basophilic leukocyte that are activated or excited by IgG specificity allergy unit or binding medium (for example anti-IgG antibody or fragment), but (for example Calcium ionophore Ai Nuo mycin (ionomycin) and the cell degranulation that A23187) activates or excite there is no obvious restraining effect to the degranulation medium of walking around Fc γ RI signal path.
" The degranulation that ionophore is induced (Ionophore-Induced Degranulation)" or " Ionophore The degranulation of mediation (Ionophore-Mediated Degranulation)" mean cell (for example mastocyte or the basophilic leukocyte) degranulation that takes place when (as such as Ai Nuo mycin or A23187 etc.) of contact Calcium ionophore.
" Syk swashs Enzyme (Syk Kinsase)" mean the non-acceptor of well-known 72kDa (tenuigenin) the spleen protein tyrosine kinase Enzyme that finds expression in B-cell and other hematopoietic cell.Syk swashs consistent Src-homology 2 (SH2) district, " linker " district and catalytic domain that Enzyme comprises that the vertical row's in two front and back bond is bonded to phosphorylation immunity receptor tyrosine-based and activates theme (" ITAMs "), and (summary of the 26S Proteasome Structure and Function of the sharp Enzyme of Syk is please referring to Sada et al., 2001, J.Biochem. (Tokyo) 130:177-186; Also please referring to Turner et al., 2000, Immunology Today 21:148-154).Syk swash Enzyme broad research as the effector of B-cell receptor (BCR) signal (Turner et al., 2000, the same).Syk swashs Enzyme and plays keying action for adjusting from the tyrosine phosphorylation of the multiple proteins of the important path of immunity receptor derivation, for example Ca 2+Mobilization and mitogen activated protein swash Enzyme (MARK) cascade (referring to for example Fig. 2) and degranulation.Syk swashs also play an important role in the integral protein signal transmission of Enzyme in neutrophils (referring to for example Mocsai et al.2002, Immunity 16:547-558).
With regard to being used in this paper scope, Syk swash Enzyme comprise confirm to belong to Syk family, from the sharp Enzyme of any animal species, include, but is not limited to the sharp Enzyme of the mankind, man like ape, ox, pig and rodents etc.Specifically included isoforming body (isoform) arranged, engage variant, allele variant, mutant, comprise that nature person of existence and people are the producer.The amino acid ordering that this Syk swashs Enzyme is on record, can have access to from gene pool (GENBANK).MRNA can have access to from gene pool with following listed login call number the specific examples that people Syk swashs the different isomerization reformation body coding of Enzyme, that is: gi|21361552|ref|NM__003177.2|, gi|496899|emb|Z29630.1|HSSYKPTK[496899] and gi|15030258|gb|BC011399.1|BC011399[15030258], more than the login call number is taken passages in this only for reference.
The tyrosine-kinase Enzyme of other family can have reactive site similar to the Syk three-dimensional structure or combining site, and this skill person that is familiar with should be able to know from experience the value of this point.Because this structural similarity, can expect that sharp Enzyme that this paper is called " Syk emulation material " is to being played katalysis by the phosphorylation of Syk phosphorylation matrix.Therefore, people will be familiar with the signal transduction cascade of this type of Syk simulacrumy, Syk simulacrumy figure gradually, by the biological respinse of Syk simulacrumy generation, rely on the signal cascade of Syk emulation etc., all can be as herein described 2, the 4-pyrimidinediamine compounds is regulated, and especially suppresses.
" Syk relies on signal cascade (Syk-Dependent Signaling Cascade)" mean that Syk swashs the signal transduction cascade that Enzyme plays a role therein.The unrestricted example that this Syk-relies on signal cascade comprises: Fc α RI, Fc ε RI, Fc γ RI, Fc γ RIII, BCR and integral protein signal cascade.
" Autoimmune disease (Autoimmune Disease)" referring to disease common and that nonallergic allergy (II type, III type and the allergy of IV type) interrelates, this type of allergy is often caused a kind of or some kinds of body fluid and cell mediated immune responses endogenous and foreign immunologic source material by the patient.This autoimmune disease is far different with the disease relevant with anaphylactic hypersensitivity (I type or IgE mediation).
6.2 2, the 4-pyrimidinediamine compounds
Compound of the present invention is generally 2 shown in the structural formula (I), the 4-pyrimidinediamine compounds:
Comprise its salt, hydrate, solvate and N-oxide compound, wherein:
L 1And L 2Independently be selected from separately by a direct key and be connected the group that base constitutes with one;
R 2System is selected from also can be by one or several identical or different R 8(C1-C6) alkyl that replaces, also can be by one or several identical or different R 8The base replacement (C3-C8) cycloalkyl, also can be by one or several identical or different R 8The base replacement cyclohexyl, also can be by one or several identical or different R 8The base replacement 3-8 person encircle assorted alkyl, also can be by one or several identical or different R 8The base replacement (C5-C15) aryl, also can be by one or several identical or different R 8The base replacement phenyl and also can be by one or several identical or different R 85-15 person's ring heteroaryl of base replacement;
R 4The system be selected from hydrogen, also can be by one or several identical or different R 8The base replacement (C1-C6) alkyl, also can be by one or several identical or different R 8The base replacement (C3-C8) cycloalkyl, also can be by one or several identical or different R 8The base replacement cyclohexyl, also can be by one or several identical or different R 8The base replacement 3-8 person encircle assorted alkyl, also can be by one or several identical or different R 8The base replacement (C5-C15) aryl, also can be by one or several identical or different R 8The base replacement phenyl and also can be by one or several identical or different R 85-15 person's ring heteroaryl of base replacement;
R 5System is selected from R 6, also can be by one or several identical or different R 8Replace it (C1-C6) alkyl, also can be by one or several identical or different R 8The base replacement (C1-C4) alkyl group (alkanyl), also can be by one or several identical or different R 8The base replacement (C2-C4) thiazolinyl and also can be by one or several identical or different R 8(C2-C4) alkynyl of base replacement;
R 6Independently be selected from separately hydrogen, electronegativity base ,-OR d,-SR d, (C1-C3) halogen alkoxyl group, (C1-C3) perhalogeno alkoxyl group ,-NR cR c, halogen, (C1-C3) alkylhalide group, (C1-C3) perhaloalkyl radical ,-CF 3,-CH 2CF 3,-CF 2CF 3,-CN ,-NC ,-OCN ,-SCN ,-NO ,-NO 2,-N 3,-S (O) R d,-S (O) 2R d,-S (O) 2OR d,-S (O) NR cR c,-S (O) 2NR cR c,-OS (O) R d,-OS (O) 2R d,-OS (O) 2OR d,-OS (O) NR cR c,-OS (O) 2NR cR c,-C (O) R d,-C (O) OR d,-C (O) NR cR c,-C (NH) NR cR c,-OC (O) R d,-SC (O) R d,-OC (O) OR d,-SC (O) OR d,-OC (O) NR cR c,-SC (O) NR cR c,-OC (NH) NR cR c,-SC (NH) NR cR cNHC ,-[(O)] nR dNHC ,-[(O)] nOR dNHC ,-[(O)] nNR cR cWith-[NHC (NH)] nNR cR c,, also can be by one or several identical or different R 8The base replacement (C5-C10) aryl, also can be by one or several identical or different R 8The base replacement phenyl, also can be by one or several identical or different R 8The base replacement (C6-C16) aralkyl, also can be by one or several identical or different R 8The base replacement 5-10 person's heteroaryl and also can be by one or several identical or different R 86-16 person's heteroaralkyl of base replacement;
R 8System is selected from R a, R b, by one or several identical or different R aOr R bThe R that replaces a, by one or several identical or different R aOr R bReplace-OR a,-B (OR a) 2,-B (NR cR c) 2-(CH 2) m-R b,-(CHR a) m-R b,-O-(CH 2) m-R b,-S-(CH 2) m-R b,-O-CHR aR b,-O-CR a(R b) 2,-O-(CHR a) m-R b,-O-(CH 2) m-CH[(CH 2) mR b] R b,-S-(CHR a) m-R b,-C (O) NH-(CH 2) m-R b,-C (O) NH-(CHR a) m-R b,-O-(CH 2) m-C (O) NH-(CH 2) m-R b,-S-(CH 2) m-C (O) NH-(CH 2) m-R b,-O-(CHR a) m-C (O) NH-(CHR a) m-R b,-S-(CHR a) m-C (O) NH-(CHR a) m-R b,-NH-(CH 2) m-R b,-NH-(CHR a) m-R b,-NH[(CH 2) mR b] ,-N[(CH 2) mR b] 2,-NH-C (O)-NH-(CH 2) m-R b,-NH-C (O)-(CH 2) m-CHR bR bWith-NH-(CH 2) m-C (O)-NH-(CH 2) m-R b
R aEach independently is selected from the assorted alkyl of hydrogen, (C1-C6) alkyl, (C3-C8) cycloalkyl, cyclohexyl, (C4-C11) cycloalkylalkyl, (C5-C10) aryl, phenyl, (C6-C16) aralkyl, phenmethyl, 2-6 person, 3-8 person and encircles assorted alkyl, morpholinyl, piperazinyl, high piperazinyl, piperidyl, 4-11 person and encircle assorted alkyl-alkyl, 5-10 person's heteroaryl and 6-16 person and encircle heteroaralkyl;
R bEach independently be selected from following suitably base :=O ,-OR d, (C1-C3) halogen alkoxyl group ,-OCF 3,=S ,-SR d,=NR d,=NOR d,-NR cR c, halogen ,-CF 3,-CN ,-NC ,-OCN ,-SCN ,-NO ,-NO 2,=N 2,-N 3,-S (O) R d,-S (O) 2R d,-S (O) 2OR d,-S (O) NR cR c,-S (O) 2NR cR c,-OS (O) R d,-OS (O) 2R d,-OS (O) 2OR d,-OS (O) 2NR cR c,-C (O) R d,-C (O) OR d,-C (O) NR cR c,-C (NH) NR cR c,-C (NR a) NR cR c,-C (NOH) R a,-C (NOH) NR cR c,-OC (O) R d,-OC (O) OR d,-OC (O) NR cR c,-OC (NH) NR cR c,-OC (NR a) NR cR cNHC ,-[(O)] nR d,-[NR aC (O)] nR dNHC ,-[(O)] nOR d,-[NR aC (O)] nOR dNHC ,-[(O)] nNR cR c,-[NR aC (O)] nNR cR cNHC ,-[(NH)] nNR cR cWith-[NR aC (NR a)] nNR cR c
R cEach independently is R a, or also can be R cForm 5-8 person with the nitrogen-atoms that it connected separately and encircle assorted alkyl or heteroaryl, assorted alkyl of this ring or heteroaryl also can comprise that one or several identical or different extra heteroatomss also also can be by one or several identical or different R aOr suitable R bBase replaces;
R dEach independently is R a
Each independently is 1 to 3 integer for m; And
Each independently is 0 to 3 integer for n.
In the compound of structural formula (I), L 1And L 2Direct key of independent separately expression or connection base.Therefore, be familiar with this operator and should be appreciated that substituent R 2With/or R 4Can be directly linked to its separately nitrogen-atoms or also can separate with its nitrogen-atoms separately by connecting base.Adopting which kind of connection base is not key point, and typically suitably the connection base includes, but is not limited to (C1-C6) alkane two bases, (C1-C6) stretches the alkyl and alkane two bases of (C1-C6) mixing, and they also can be respectively by one or several identical or different R 8Base replaces, wherein R 8Such as previous structural formula (I) definition.In one embodiment, L 1And L 2Independently be selected from separately a direct key, also can be by one or several identical or different R a, suitable R bOr R 9The base replacement (C1-C3) alkane two bases and also can be by one or several identical or different R a, suitable R bOr R 9The 1-3 person of base replacement assorted alkane two bases, wherein R 9Be selected from (C1-C3) alkyl ,-OR a,-C (O) OR a, also can by one or several identical or different halogens replace it (C5-C10) aryl, its also can by the phenyl of one or several identical or different halogen replacements, also can be by 5-10 person's heteroaryl of one or several identical or different halogen replacements and also can be by 6 Yuans ring heteroaryls of one or several identical or different halogen replacements; R aAnd R bSuch as previous structural formula (I) definition.Can be in order to replace L 1And L 2Concrete R9 base comprise-OR a,-C (O) OR a, phenyl, halobenzene base and 4-halobenzene base, wherein R aSuch as previous structural formula (I) definition.
In another specific embodiment, L 1And L 2Independently be selected from separately and stretch methyl, stretch ethyl and stretch propyl group, they also can be by R 9Base is single to be replaced, wherein R 9Described as defined above.
In all above-mentioned specific embodiments, can be included in R 9Concrete R in the base aBase is selected from hydrogen, (C1-C6) alkyl, phenyl and phenmethyl.
In also having a specific embodiment, L 1And L 2Respectively be direct key, so that of the present invention 2, the 4-pyrimidinediamine compounds is the compound shown in the structural formula (Ia):
Figure A20091000677100341
Comprise its salt, hydrate, solvate and N-oxide compound, wherein R 2, R 4, R 5And R 6Such as previous structural formula (I) definition.Of the present invention 2, the description of other specific embodiment of 4-pyrimidinediamine compounds sees for details hereinafter.
In first specific embodiment of structural formula (I) and compound (Ia), R 2, R 4, R 5, R 6, L 1And L 2Respectively such as previous structural formula (I) and (Ia) definition, its prerequisite is R 2Be not 3,4,5-trimethoxyphenyl, 3,4,5-three (C1-C6) alkoxyl phenyl or
Figure A20091000677100342
R wherein 21, R 22And R 23Respectively as United States Patent (USP) the 6th, 235, the R of No. 746 (its content is included this in the quoted passage mode) 1, R 2And R 3Define.In the specific embodiment of first embodiment, R 21For hydrogen, halogen, also can be by one or several identical or different R 25Straight or branched (C1-C6) alkyl, hydroxyl of base replacement, also can be by one or several identical or different phenyl or R 25(C1-C6) alkoxyl group, the mercaptan of base replacement (SH), also can be by one or several identical or different phenyl or R 25(C1-C6) alkylthio, amido of base replacement (NH2) ,-NHR 26Or-NR 26R 26R 22And R 23Independent separately is also can be by one or several identical or different R 25(C1-C6) straight or branched alkyl of base replacement; R 25System is selected from halogen, hydroxyl, (C1-C6) alkoxyl group, mercaptan, (C1-C6) alkylthio, (C1-C6) alkyl amine group and (C1-C6) dialkyl amino; R 26Independent separately is also can be by one or several identical or different phenyl or R 25The base replacement (C1-C6) alkyl or-C (O) R 27, R wherein 27For also can be by one or several identical or different phenyl or R 25(C1-C6) alkyl of base replacement.
In another specific embodiment of first embodiment, R 21For also can be by the methoxyl group and the R of one or several identical or different halogen replacements 22And R 23Independent separately is also can be by the methyl or the ethyl of one or several identical or different halogen replacements.
At structural formula (I) with (Ia) among second embodiment of compound, R 2, R 4, R 5And L 2Structural formula (I) and (Ia) define L as previously described respectively 1Be direct key, R 6Be hydrogen, its prerequisite is R 2Be not 3,4,5-trimethoxyphenyl, 3,4,5-three (C1-C6) alkoxyl phenyl or
Figure A20091000677100351
R wherein 21, R 22And R 23Definition as above-mentioned first embodiment is similar.
In the 3rd embodiment, structural formula (I) and (Ia) 2, the 4-pyrimidinediamine compounds is got rid of one or several following compounds:
N2, two (4-the ethoxyl phenenyl)-5-of N4-are fluorine-based-2,4-pyrimidinediamine (R070790);
N2, two (2-the p-methoxy-phenyl)-5-of N4-are fluorine-based-2,4-pyrimidinediamine (R081166);
N2, two (4-the p-methoxy-phenyl)-5-of N4-are fluorine-based-2,4-pyrimidinediamine (R088814);
N2, two (2-the chloro-phenyl-)-5-of N4-are fluorine-based-2,4-pyrimidinediamine (R088815);
N2, the two phenyl-5-of N4-are fluorine-based-2,4-pyrimidinediamine (R091880);
N2, two (3-the aminomethyl phenyl)-5-of N4-are fluorine-based-2,4-pyrimidinediamine (R092788);
N2, two (3-the chloro-phenyl-)-5-of N4-are fluorine-based-2,4-pyrimidinediamine (R067962);
N2, two (2, the 5-the 3,5-dimethylphenyl)-5-of N4-are fluorine-based-2,4-pyrimidinediamine (R067963);
N2, two (3, the 4-the 3,5-dimethylphenyl)-5-of N4-are fluorine-based-2,4-pyrimidinediamine (R067964);
N2, two (4-the chloro-phenyl-)-5-of N4-are fluorine-based-2,4-pyrimidinediamine (R0707153);
N2, two (2, the 4-the 3,5-dimethylphenyl)-5-of N4-are fluorine-based-2,4-pyrimidinediamine (R070791);
N2, two (3-the bromophenyl)-5-of N4-are fluorine-based-2,4-pyrimidinediamine (R008958);
N2, two (the phenyl)-5-of N4-are fluorine-based-2, the 4-pyrimidinediamine;
N2, two (the morpholinyl)-5-of N4-are fluorine-based-2, the 4-pyrimidinediamine; With
N2, two ((3-chloro-4-p-methoxy-phenyl))-5-of N4-are fluorine-based-2, the 4-pyrimidinediamine.
In the 4th embodiment, structural formula (I) and compound (Ia) are got rid of the compound of following structural formula (Ib):
Figure A20091000677100361
R wherein 24Be (C1-C6) alkyl; R 21, R 22With R 23The definition of first specific embodiment is similar as described above.
In the 5th embodiment, structural formula (I) and compound (Ia) are got rid of the compound described in the 6th, 235, No. 746 (its content is included in herein in the quoted passage mode) example 1-141 of United States Patent (USP).
In the 6th embodiment, structural formula (I) and compound (Ia) are got rid of this United States Patent (USP) the 6th, 235, molecular formula in No. 746 (1) or molecular formula (Ia) (referring to for example the 1st hurdle the 48th walk to the 7th hurdle the 49th row and the 8th hurdle 9-36 capable disclosed within appearance, this content is included in herein in the quoted passage mode) compound that defined.
In the 7th embodiment, structural formula (I) and compound (Ia) are got rid of wherein R 5For cyano group or-C (O) NHR, work as R 2During for the phenyl that is substituted, wherein R is hydrogen or (C1-C6) alkyl; R 4Encircle assorted alkyl or 5-15 person's heteroaryl for being substituted or not being substituted it (C1-C6) alkyl, (C3-C8) cycloalkyl, 3-8 person; R 6Compound for hydrogen.
In the 8th embodiment, structural formula (I) and compound (Ia) get rid of by the molecular formula (I) of WO 02/04429 and (X) the compound among the compound that defines or any WO of being disclosed in 02/04429 (its disclosure content is included in herein in the quoted passage mode).
At the 9th structural formula (I) with (Ia) among the embodiment of compound, as work as R 5For cyano group or-C (O) NHR, wherein R is hydrogen or (C1-C6) alkyl; R 6Be hydrogen, then R 2Be the group beyond the phenyl that is substituted.
In the tenth embodiment, structural formula (I) and compound (Ia) are got rid of R 2With R 4Be respectively the compound of the pyrroles that is substituted or is not substituted or indole ring (such ring is connected to the rest part of molecule by its theheterocyclic nitrogen atom) separately.
In the 11 embodiment, structural formula (I) and compound (Ia) are got rid of by United States Patent (USP) the 4th, 983, No. 608 formula (I) and (IV) defined compound, or any United States Patent (USP) the 4th, 983 that is disclosed in, the compound in No. 608 (its disclosure content is included in herein in the quoted passage mode).
This operator that is familiar with should be able to know from experience in structural formula (I) and the compound (Ia) R 2And R 4Can be identical or different, and can extensively change the importance of this point.Work as R 2With/or R 4(for example cycloalkyl that replaces arbitrarily, the assorted alkyl of ring, aryl and heteroaryl) this ring can be connected to the rest part of molecule by any available carbon or heteroatoms during for the ring that replaces arbitrarily.Substituting group can be connected to any available carbon and heteroatoms arbitrarily.
At the 12 structural formula (I) with (Ia) among the embodiment of compound, R 2With/or R 4For any substituted phenyl or be substituted its (C5-C15) aryl arbitrarily, but its prerequisite is: (1) works as R 6During for hydrogen, R 2Not 3,4,5-trimethoxyphenyl or 3,4,5-three (C1-C6) alkoxyl phenyl; (2) work as R 2Be 3,4, during the 5-tri-substituted phenyl, then the substituting group of 3-position and 4-position is not methoxyl group or (C1-C6) alkoxyl group simultaneously; Or (3) work as R 6Be hydrogen and R 4Be (C1-C6) alkyl, (C3-C8) cycloalkyl, 3-8 person when encircling assorted alkyl or 5-15 person's heteroaryl, R 5It is the group except that cyano group.Perhaps R 2Prerequisite also can be by first and second described prerequisite of embodiment.The aryl of any replacement or the rest part that phenyl can be connected to molecule by any carbon atom that utilizes.The specific examples of the phenyl of replacement comprises by identical or different R arbitrarily 8The single arbitrarily replacement of base, two replacement or trisubstd phenyl, wherein R 8Structural formula (I) defines as described above, and depends on foregoing prerequisite.When phenyl is single substituting group, R 8Substituting group can be positioned at ortho position, a position or contraposition position.When being positioned at ortho position, a position or contraposition position, R 8Preferably be selected from (C1-C10) alkyl, (C1-C10) branched-chain alkyl, also can be by one or several identical or different R bThe base replace it-O-C (O) OR a, ,-O-(CH 2) m-C (O) OR a,-C (O) OR a,-O-(CH 2) m-NR cR c,-O-C (O) NR cR c,-O-(CH 2) m-C (O) NR cR c,-O-C (NH) NR cR c,-O-(CH 2) m-C (NH) NR cR cWith-NH-(CH 2) m-NR cR c, wherein m, R aAnd R cSuch as previous structural formula (I) definition.In the volunteer embodiment of these compounds ,-NR cR cBe also to comprise one or several identical or different extra heteroatomic 5-6 person's heteroaryls.The specific examples of this type of 5-6 person's heteroaryl includes, but is not limited to oxadiazoles base, triazolyl, thiazolyl, oxazolyl, tetrazyl and isoxazolyl.
In another embodiment of these compounds ,-NR cR cBe the assorted alkyl ring of 5-6 person's saturated rings, this ring also can comprise the heteroatoms that one or several are identical or different.The specific examples of the assorted alkyl of this ring includes, but is not limited to Pyrrolizidine base, pyrazoles pyridine base, imidazolidine base, piperidyl, piperazinyl and morpholinyl.
Also has among the embodiment R at these compounds aIndependent separately be (C1-C6) alkyl and-NR cR cRespectively do for oneself-NHR a, R wherein aBe (C1-C6) alkyl.In a specific embodiment, R 8For-O-CH2-C (O) NHCH3.In another specific embodiment, R 8For-OH.
When phenyl is disubstituted phenyl or tri-substituted phenyl, R 8Substituting group can be positioned at the combination of any position.For example, R 8Substituting group can be positioned at 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, 3,5-, 2,3,4-, 2,3,5-, 2,3,6-, 2,4,5-, 2,4,6-, 2,5,6-or 3,4,5-position.In an embodiment who comprises the disubstituted benzene based compound, substituting group is positioned at the position outside 3,4.In another embodiment, they are positioned at 3,4.In an embodiment who comprises the trisubstituted benzene based compound, substituting group is positioned at 3,4, and the position beyond 5 perhaps also can not allow two substituting groups be positioned at simultaneously on 3,4.In another embodiment, substituting group is positioned at 3,4, on 5.
In this kind disubstituted phenyl and the tri-substituted phenyl, R 8Substituent specific examples comprises above-mentioned various ortho position, the R that a position is relevant with the para-orientation phenyl 8Substituting group.
In another specific examples, can be used for replacing this R of this kind disubstituted phenyl and tri-substituted phenyl 8Substituting group comprise (C1-C6) alkyl, (C1-C6) alkoxyl group, methoxyl group, halogen, chloro, (C1-C6) perhaloalkyl radical ,-CF3, (C1-C6) perhalogeno alkoxyl group and-OCF 3In embodiment preferably, this R 8Substituting group is positioned at 3,4 or 3,5.The specific examples of disubstituted benzene basic ring comprises 3-chloro-4-methoxyl group-phenyl, 3-methoxyl group-4-chloro-phenyl-, 3-chloro-4-Trifluoromethoxyphen-l, 3-trifluoromethoxy 4-chloro-phenyl, 3 preferably, 4-dichloride base-phenyl, 3,4-Dimethoxyphenyl and 3, the 5-Dimethoxyphenyl, its prerequisite is: (1) is as R 4Be one of phenyl of above-mentioned definition, R 5And R 6When respectively being hydrogen, R then 2Be not 3,4,5-three (C1-C6) alkoxyl phenyl or 3,4,5-trimethoxyphenyl; (2) as R 2Be 3,4-Dimethoxyphenyl R 5And R 6When respectively being hydrogen, R then 4Be not 3-(C1-C6) alkoxyl phenyl, 3-p-methoxy-phenyl, 3,4-two-(C1-C6) alkoxyl phenyls or 3,4-Dimethoxyphenyl; (3) work as R 4Be 3-chloro-4-p-methoxy-phenyl, R 5Be halogen or fluorine-based, and R 6When also can be hydrogen, R then 2Be not 3-chloro-4-(C1-C6) alkoxyl phenyl or 3-chloro-4-p-methoxy-phenyl; Or (4) are as R 4Be 3,4-dichlorophenyl, R 5Be hydrogen, (C1-C6) alkyl, methyl, halogen or chloro, and R 6When also can be hydrogen, R then 2Be not to be the mono-substituted phenyl of (C1-C6) alkoxyl group in the contraposition position, above-mentioned (C1-C6) alkoxyl group also can be by one or several identical or different R b,-OH or-NR cR cBase replaces, wherein R bAnd R cSimilar with the description of front structure formula (I); With/or (5) R 2With/or R 4Be not 3,4,5-three (C1-C6) alkoxyl phenyl or 3,4, the 5-trimethoxyphenyl is particularly at R 5And R 6When respectively being hydrogen.
In comprising another embodiment of trisubstituted benzene based compound, tri-substituted phenyl has following structural formula:
Wherein: R 31Be methyl or (C1-C6) alkyl; R 32Be hydrogen, methyl or (C1-C6) alkyl; R 33Be halogen.
At the 13 structural formula (I) with (Ia) among the embodiment of compound, R 2With/or R 4For also can substituted heteroaryl.The typical heteroaryl that conforms to of the 13 embodiment comprises 5 to 15 (more typical situation is 5 to 11) annular atomses therewith, and comprises that one, two, three or four is selected from N, NH, O, S, S (O) and S (O) 2Identical or different heteroatoms or heteroatoms base.Also can substituted heteroaryl can connect (but under typical situation, connecting) by any available carbon atom or heteroatoms respectively to its each C2 or C4 nitrogen-atoms or connect basic L by carbon atom 1Or L 2On.This type of any substituting group can be identical or different, and can be connected to any available carbon atom or heteroatoms.In an embodiment of these compounds, R 5For except that bromo, nitrogen base, trifluoro ylmethyl, cyano group or-base C (O) NHR, wherein R is hydrogen or (C1-C6) alkyl.In another embodiment of these compounds, work as R 2And R 4Respectively for being substituted or when unsubstituted pyrroles or indoles, then this ring is connected to the rest part of molecule by ring carbon atom.also comprise can another embodiment of substituted heteroaryl compound in, this heteroaryl is not to be substituted or by one to four identical or different R 8The heteroaryl that base replaces, wherein R 8Such as previous structural formula (I) definition.This type of also can substituted heteroaryl specific examples include, but is not limited to following heteroaryl:
Figure A20091000677100421
Wherein:
P is one to three integer;
Each
Figure A20091000677100432
Represent a singly-bound or two key separately;
R 35Be hydrogen or R 8, R wherein 8Such as above-mentioned structural formula (I) definition;
X is selected from the group that comprises CH, N and N-O;
Each Y is selected from the group that comprises O, S and NH separately;
Each Y 1Be selected from separately and comprise O, S, SO, SO 2, SONR 36, NH and NR 37Group;
Each Y 2Be selected from separately and comprise CH, CH 2, O, S, N, NH and NR 37Group;
R 36It is hydrogen or alkyl;
R 37Be selected from the group that comprises a hydrogen and a protecting group, preferably hydrogen and or one be selected from following each basic group, that is: R a, R b-CR aR b-O-C (O) R 8,-CR aR b-O-PO (OR 8) 2,-CH 2-O-PO (OR 8) 2,-CH 2-PO (OR 8) 2,-C (O)-CR aR b-N (CH 3) 2,-CR aR b-O-C (O)-CR aR b-N (CH 3) 2,-C (O) R 8,-C (O) CF 3With-C (O)-NR 8-C (O) R 8
A is selected from and comprises O, NH and NR 38Group;
R 38Be selected from the group that comprises alkyl and aryl;
R 9, R 10, R 11And R 12Separate separately, be selected from the group that comprises alkyl, alkoxyl group, halogen, halogen alkoxyl group, amine alkyl and hydroxyalkyl respectively, perhaps R 9And R 10With/or R 11And R 12Form a kind of ketal together;
Each Z respectively is selected from hydroxyl, alkoxyl group, aryloxy, ester, amido formate and alkylsulfonyl;
Q is selected from and comprises-OH, OR 8,-NR cR c,-NHR 39-C (O) R 8,-NHR 39-C (O) OR 8,-NR 39-CHR 40-R b,-NR 39-(CH 2) m-R bWith-NR 39-C (O)-CHR 40-NR cR cGroup;
R 39And R 40Separate separately, be selected from and comprise hydrogen, alkyl, aryl, alkylaryl, arylalkyl and NHR 8Group; And
R a, R bAnd R cSuch as previous structural formula (I) definition.The preferable R of Q bSubstituting group is selected from certainly-C (O) OR 8,-O-C (O) R 8,-O-P (O) (OR 8) 2With-P (O) (OR 8) 2Group.
In 5-15 person's heteroaryl of an embodiment of above-mentioned heteroaryl and other this embodiment according to the present invention, R 8Each independently is selected from and comprises R d,-NR cR c,-(CH 2) m-NR cR c,-C (O) NR cR c,-(CH 2) m-C (O) NR cR c,-C (O) OR d,-(CH 2) m-C (O) OR dAnd-(CH 2) m-OR dGroup, wherein m, R cAnd R dSuch as previous structural formula (I) definition.
In a specific embodiment, R dWith/or R cBe selected from and comprise R aAlso can be by the group of (C3-C8) cycloalkyl of one or several identical or different hydroxyls, amido or carboxyl substituted.
In the another specific embodiment of above-mentioned heteroaryl, each R 35Be a hydrogen atom, (C1-C6) carbochain (comprising methyl, ethyl, sec.-propyl), a cycloalkyl (comprising cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, suberyl, ring octyl group), one
Figure A20091000677100451
Wherein x=1-8 ,-CH 2CONHMe ,-CH 2CH 2NHMe ,-CH 2CH 2CONHMe ,-CH 2CH 2CH 2NHMe or-CH 2CH 2CH 2OCH 3
In another specific embodiment of above-mentioned heteroaryl, the link position of aromatic ring is at 5 or 6.Be understood that R 2Or R 4All can utilize at this and describe the heteroaryl that reaches described in the whole process.
At the 14 structural formula (I) with (Ia) among the embodiment of compound, R 2And R 4Also being respectively separately can substituted phenyl, aryl or heteroaryl, and its prerequisite is: (1) is as L 1Be direct key and R 6(also can comprise R 5) when being hydrogen, R then 2For removing 3,4, the group beyond the 5-trimethoxyphenyl or 3,4,5-three (C1-C6) alkoxyl phenyl; (2) as L 1And L 2Respectively be direct key, R 6Be hydrogen, R 5During for halogen, R then 2And R 4Must not be 3,4 separately simultaneously, 5-trimethoxyphenyl or 3,4,5-three (C1-C6) alkoxyl phenyl; (3) as R 4Be 3-p-methoxy-phenyl or 3-(C1-C6) alkoxyl phenyl, R 2Be 3,4, during the 5-tri-substituted phenyl, then be positioned at 3 and 4 s' substituting group and must not be simultaneously be methoxyl group or (C1-C6) alkoxyl group; (4) as R 2For being substituted phenyl, R 6During for hydrogen, R then 5For except that cyano group or-group C (O) NHR, wherein R is hydrogen or (C1-C6) alkyl; With/or (5) as R 2And R 4Independent of separately being substituted or when unsubstituted pyrroles or indoles, then pyrroles or indoles are connected to all the other parts of molecule by ring carbon atom.Perhaps R 2Also can adopt first or second described prerequisite of embodiment.
In this 14 embodiment of the present invention, R 2And R 4Substituting group can be identical or different.Concrete phenyl, aryl and/or the heteroaryl of also can being substituted comprises this type of cited group among above the 12 and the 13 embodiment.
At the 15 structural formula (I) with (Ia) among the embodiment of compound (comprising above-mentioned the first to the 14 embodiment), R 6Be hydrogen, R 5Be the electronegativity base.This skill person's white silk of being familiar with should be able to find out that the electronegativity base is that electronics is had big magnetism, it can be attracted to atom or atomic radical on itself.The specific examples that meets the electronegativity base of the 14 embodiment requirement includes, but is not limited to-CN ,-NC ,-NO 2, halogen, bromo, chloro, fluorine-based, (C1-C3) alkylhalide group, (C1-C3) perhaloalkyl radical, (C1-C3) fluoroalkyl, (C1-C3) perfluoroalkyl ,-CF 3, (C1-C3) halogen alkoxyl group, (C1-C3) perhalogeno alkoxyl group, (C1-C3) Fluoroalkyloxy, (C1-C3) perfluoro alkoxy ,-OCF 3,-C (O) R a,-C (O) OR a,-C (O) CF 3With-C (O) OCF 3In a specific embodiment, the electronegativity base is halogen-containing electronegativity base, for example-and OCF 3,-CF 3, bromo, chloro or fluorine-based.In another specific embodiment, R5 is fluorine-based, and its prerequisite is that its compound is not the described any compound of the 3rd embodiment.
In the 16 embodiment, structural formula (I) and compound (Ia) are the compound of following structural formula (Ib):
Figure A20091000677100471
With and salt, hydrate, solvate and N-oxide compound, wherein R 11, R 12, R 13And R 14Independently be selected from separately comprise hydrogen, hydroxyl, (C1-C6) alkoxyl group and-NR cR cGroup; R 5, R 6And R cSuch as previous structural formula (I) definition, its prerequisite is as R 13, R 5And R 6Respectively be hydrogen, then R 11And R 12Must not be methoxyl group, (C1-C6) alkoxyl group or (C1-C6) halogen alkoxyl group simultaneously.
In the 17 embodiment, structural formula (I) and compound (Ia) are the compound of following structural formula (Ic):
With and salt, hydrate, solvate and N-oxide compound, wherein:
R 4Be selected from the group that comprises 5-10 person's heteroaryl and 3-hydroxyphenyl;
R 5For F or-CF 3
R 8For-O (CH 2) m-R b, wherein m and R bSuch as previous structural formula (I) description.In a specific embodiment, R 8Be-O-CH 2-C (O) NH-CH 3With/or R 4The 13 the described heteroaryl of embodiment.
In the 18 embodiment, structural formula (I) and compound (Ia) comprise any compound that is selected from table one, this compounds can suppress Fc receptor signal transductory cascade, Syk swashs Enzyme activity, the sharp Enzyme dependence receptor signal transductory cascade of Syk or in vitro examines and determine viewed cell degranulation, also can stipulate that its prerequisite must not be above-mentioned the 3rd compound that embodiment and other embodiment got rid of for this compound.In a specific embodiment, this compounds is through the calibrating of degranulation in vitro (for example example partly one of described degranulation calibration method), has about 20 μ M or less than the IC of 20 μ M 50
In nineteen embodiment, structural formula (I) and compound (Ia) comprise any compound that is selected from table one, measure according in vitro calibrating (for example in one of described calibration method of example part), this compounds can suppress Fc γ R1 or the cascade of Fc ε R1 acceptor, has about 20 μ M or less than the IC of 20 μ M 50, also can stipulate that its prerequisite is that this compounds must not be above-mentioned the 3rd compound that embodiment and other embodiment got rid of.
In the 20 embodiment, the compound of structural formula (Ia) is R wherein 2Be selected from and comprise:
Figure A20091000677100481
Figure A20091000677100482
The compound of group;
R wherein 4, R 8, R a, R b, R c, R dAs described above, R 5Be fluorine atom; R 6Be hydrogen atom, each R 21Respectively be a halogen atom or the alkyl that also can be one or several identical or different halogen groups replacements, R 22And R 23Independently of one another, be respectively a hydrogen atom, also can be the methyl that one or several identical or different halogen groups replace, each m is respectively 1 to 3 integer, and each n is respectively 0 to 3 integer.
In the 21 embodiment, the compound of structural formula (Ia) is R wherein 4Be
Figure A20091000677100483
Compound, the R in the formula 9And R 10Each independently comprises a hydrogen atom, R as defined above, and further 2Be to be one or several identical R 8The base or
Figure A20091000677100484
The phenyl that base is replaced, the R among the latter 35As defined above.A special aspects, work as R 2During for phenyl, one or several R 8Be selected from upward alkoxyl group of a halogen and ring.In one aspect, phenyl is with one or several identical R 8The base metathetical is two to be replaced or tri-substituted phenyl.
In the 22 embodiment, the compound of structural formula (Ia) wherein is R wherein 4Be
Figure A20091000677100491
And R 2Be to be one or several R 8The compound of the phenyl that group replaced.At a particular aspects, one or several R 8Be selected from a halogen and an alkoxyl group.In one aspect, phenyl is to be one or several identical R 8Two replacements of base institute or trisubstd phenyl.
In the 23 embodiment, the compound of structural formula (Ia) is R wherein 4Be to be one or several identical R 8The phenyl that base is replaced, R wherein 2Be
Figure A20091000677100492
R wherein 35Compound as defined above.In special embodiment, R 4Phenyl is by two replacements of same or different halogen atom institute or trisubstd phenyl.In another embodiment, R 4Be by a mono-substituted phenyl of halogen atom.In one aspect, R 35It is hydroxyalkyl.In some aspects, hydroxyalkyl further sense turn to ester group, carbamate (carbamate) etc.
In the 24 embodiment, the compound of structural formula (Ia) is R wherein 4Be
Figure A20091000677100493
Group, the R in the formula 35As defined above, R 2Be by one or several R 8The compound of the phenyl that base replaces.At a particular aspects, R 35Be a hydrogen atom or an alkyl.In yet another aspect, R 2Be by identical or different R 8Two replacements of base (particularly halogen atom) institute metathetical or tri-substituted phenyl.
In the 25 embodiment, the compound of structural formula (Ia) is R wherein 4Be
Figure A20091000677100494
Group, the R in the formula 35As defined above, R 2Be
Figure A20091000677100495
Group, the R in the formula 9And R 10As defined above, and further respectively comprise the compound of a hydrogen atom.In one aspect, R 35Be a hydrogen atom or an alkyl (for example methyl), R 9And R 10Be alkyl group (for example methyl group).
In the 26 embodiment, the compound of structural formula (Ia) is R wherein 4By identical or different R 8The disubstituted phenyl of group displacement, R 2Be
Figure A20091000677100501
Group, the R in the formula 35Compound as defined above.In some aspects, phenyl is replaced by a halogen atom or an alkoxyl group (for example methyl).In certain embodiments, R 35Be a hydrogen atom, an alkyl (for example methyl) or a hydroxyalkyl.In some aspects, hydroxyalkyl further sense turn to ester group, first urethano (carbamate) etc.
In the 27 embodiment, the compound of structural formula (Ia) is R wherein 4Be
Figure A20091000677100503
R in the formula 8And R cAs defined above, R 2Be by one or several identical R 8The two replacements of group displacement or the compound of tri-substituted phenyl.At a particular aspects, R cBe a hydrogen atom or an alkyl.In yet another aspect, R 2Phenyl is by same or different R 8Group (especially halogen atom or Group) two replacements or three replace.
In the 28 embodiment, the compound of structural formula (Ia) is R wherein 4Be Y in the formula 1, Y 2With each R 35Respectively as defined above, R 2Be
Figure A20091000677100506
Group, R in the formula 35Compound as defined above.In the 28 embodiment with regard to R 4, Y 1Be oxygen, Y 2Be NH, one or several R 35Perhaps R 4Partly be alkyl (especially methyl).Aspect some of the 28 embodiment, R 4Two R partly 35Form one partly together with dialkyl group, especially adjacent, be expressed as with NH
Figure A20091000677100511
Gem-dimethyl partly.In some part of the 28 embodiment, with regard to R 2, R 35Be a hydrogen atom or an alkyl (especially methyl).
In the second nineteen embodiment, the compound of structural formula (Ia) is R wherein 4Be
Figure A20091000677100512
R in the formula 9And R 10As defined above, or the compound of a phenyl.In one aspect, phenyl is by one or several identical R 8Base institute is two to be replaced or three replacements.Phenyl especially can be one or several two replacements of halogen atom institute or three replacements, and this halogen atom can be identical, also can be different.R 2In the second nineteen embodiment be
Figure A20091000677100513
Group, the R in the formula 35As defined above.Aspect of the second nineteen embodiment, R 2R 35It is not methyl.The second nineteen embodiment also have one aspect, R 2Be
Figure A20091000677100514
Perhaps
Figure A20091000677100515
Group.
Be applicable to first in the 30 embodiment of the second nineteen embodiment, R 5Be halogen atom, fluorine for example, R 6It is hydrogen atom.
Equally also specifically described is above-mentioned first combination up to the 30 embodiment.
This operator that is familiar with should be able to know from experience described hereinly 2, and the 4-pyrimidinediamine compounds can comprise the important value that is produced this point of functional group of prodrug by the blocking group shielding.This type of prodrug before changing into the active medicine form usually (but might not) do not possess pharmacologically active.Really, many activity of in following table one, describing 2, the 4-pyrimidinediamine compounds is included under the working conditions hydrolyzable or otherwise the cracked precursor is partly.For example, ester group generally produces female carboxylic acid because of acid-catalyzed hydrolysis when the following time of acidic conditions that is exposed to stomach; Or in the following time of alkaline condition that is exposed to enteron aisle or blood, alkali catalyzed hydrolysis takes place.Therefore, when to patient's oral administration medicine supplying, comprise 2 of ester moiety, it is the prodrug of corresponding carboxylic acid that the 4-pyrimidinediamine can be considered, no matter whether its ester-formin has pharmacologically active.In Table 1, many esters 2 that contain of the present invention, 4-pyrimidinediamine when their ester (" prodrug ") form be have active.
In prodrug of the present invention, all available blocking group shielding of any available functional moiety draws prodrug.2, can shield with blocking group in the 4-pyrimidinediamine compounds, it is included in one's duty amine (firsts and seconds), hydroxy kind, sulfenyl class (mercaptan), the carboxyl class etc. of including, but is not limited to of presoma.It is countless that to be suitable for shielding this type of functional group well-known already in the industry to draw under desired working conditions the cracked blocking group.These all blocking groups (separate base or its combination) all can be included in the prodrug of the present invention.
In an illustrative embodiment, prodrug of the present invention is the R in the compound of structural formula (I) cAnd R dExcept the selection of original definition, can also be blocking group.
2 of structural formula (I) is if the hydrogen that is connected to N2 and N4 in the 4-pyrimidinediamine will have a negative impact to compound activity with the substituting group displacement.Yet known to this skill person that is familiar with, these nitrogen can be included in the precursor part, and cracking produces 2 of structural formula (I), 4-pyrimidinediamine under working conditions.Therefore, in another embodiment, prodrug of the present invention is the compound shown in the structural formula (II):
Figure A20091000677100521
Comprise its salt, hydrate, solvate and N-oxide compound, wherein:
R 2, R 4, R 5, R 6, L 1And L 2Such as previous structural formula (I) definition; And
R 2bAnd R 4bIndependent separately is blocking group.The specific examples of the blocking group of this embodiment of the present invention include, but is not limited to (C1-C6) alkyl ,-C (O) CH 3,-C (O) NHR 36With-S (O) 2R 36, R wherein 36Be (C1-C6) alkyl, (C5-C15) aryl and (C3-C8) cycloalkyl.
In the prodrug of structural formula (II), various substituting groups can as above-mentioned first to the 20 described formula of embodiment (I) and compound (Ia), or the combination of this type of embodiment.
This operator that is familiar with should understand chemical compound lot of the present invention and prodrug, and this paper specifically describes and illustrational all cpds kind can present the significance of tautomerism, configuration isomerism, geometric isomerism and optical isomerism.For example, The compounds of this invention and prodrug can comprise one or several palm property center and two keys, therefore can there be steric isomer, for example double bond isomer (being geometrical isomer), enantiomer and diastereomer and its mixture (for example racemic mixture).As another example, can there be some kinds of tautomeric forms in The compounds of this invention and prodrug, comprise enol form, keto-acid and both mixtures.Because all cpds title, formula and compound icon in description and the patent claim can only show one of them possible tautomer, configuration isomer, optical isomer or geometrical isomer, so should be appreciated that the combination that the present invention includes any tautomeric form, configuration isomeric form, optical siomerism form and rotamerism form and above-mentioned various isomeric form with compound that one or more functions as herein described and various isomeric form thereof make up or prodrug.2, in the limited rotation of 4-pyrimidinediamine core texture, it also is possible atropisomer (atrop isomers) occurring, and also specifically is included within the scope of The compounds of this invention.
And, this skill person that is familiar with also should understand when comprise in the listed possible substituting group that can not be used for replacing the substituting group of special groups owing to valency or other reason the time, the purpose of this tabulation should be interpreted as and include only the substituting group that is suitable for replacing this special groups in context.For example, though this skill person that is familiar with should understand R bThe column selection of whole institute all can be used to replace alkyl, but some selection (for example=O) can not be used for replacing phenyl.It should be interpreted as that herein usefulness means includes only spendable substituting group to combination.
The compounds of this invention and prodrug can be by its chemical structure or chemical name identifications.When chemical structure was conflicted with chemical name, the identification of specific compound was as the criterion with chemical structure.
Depend on various substituent character, of the present invention 2,4-pyrimidinediamine compounds and prodrug can be prepared as the form of salt.This type of salt comprises the salt (" pharmaceutically acceptable salt ") that is suitable for medicinal use, is suitable for salt of veterinary purpose or the like.This type of salt can be derived from acid or alkali, as the industry is on record.
In one embodiment, salt is pharmaceutically acceptable salt.Usually, pharmaceutically acceptable salt is hoped pharmacologically active for one or several that keep parent compound substantially and is suitable for offeing medicine in the mankind's salt.Pharmaceutically acceptable salt comprises and mineral acid or the formed acid salt of organic acid.The mineral acid that is suitable for preparing pharmaceutically acceptable acid salt comprises (only unrestricted for illustrating) haloid acid (for example hydrochloric acid, Hydrogen bromide, hydroiodic acid HI etc.), sulfuric acid, nitric acid, phosphoric acid etc.The organic acid that is suitable for preparing pharmaceutically acceptable acid salt comprises (only unrestricted for illustrating) acetate; trifluoroacetic acid; propionic acid; caproic acid; the pentamethylene propionic acid; oxyacetic acid; oxalic acid; pyruvic acid; lactic acid; propanedioic acid; succsinic acid; oxysuccinic acid; suitable-butene dioic acid; instead-butene dioic acid; tartrate; citric acid; palmitinic acid; phenylformic acid; 3-(4-oxybenzene formyl radical) phenylformic acid; styracin; amygdalic acid; alkyl sulfonic acid (methanesulfonic for example; ethane sulfonic acid; 1; 2-ethane-disulfonic acid; 2-hydroxyl ethane sulfonic acid etc.); aromatic sulfonic acid (for example, Phenylsulfonic acid; the 4-chlorobenzenesulfonic acid; the 2-naphthene sulfonic acid; the 4-toluenesulphonic acids); cyclophane sulfonic acid (for example camphorsulfonic acid etc.); 4-methyl bicycle (2.2.2)-oct-2-ene-1-carboxylic acid; glucoheptose; the 3-phenylpropionic acid; trimethylacetic acid; tert.-butylacetic acid; lauryl sulfate; gluconic acid Grains propylhomoserin; the hydroxyl naphthoic acid; Whitfield's ointment; stearic acid; muconic acid etc.
Pharmaceutically acceptable salt is also included within acid proton when being present in parent compound, with metal ion (for example, alkalimetal ion, alkaline-earth metal ions or aluminum ion), ammonium ion replaces formed salt, the perhaps coordination thing that forms with organic bases (for example thanomin, diethanolamine, trolamine, N-methyl glucamine, morpholine, piperidines, dimethylamine, diethylamine etc.).
Of the present invention 2, the 4-pyrimidinediamine compounds with and salt also can be prepared as hydrate, solvate and N-oxide form, as on record in the industry.
6.3 synthetic method
The starting raw material that compound of the present invention and prodrug can adopt market supply with/or synthetic by multiple different synthesis paths with the starting raw material of conventional synthetic method preparation.Be suitable for synthetic of the present inventionly 2, the appropriate method of 4-pyrimidinediamine compounds and prodrug has seen United States Patent (USP) the 5th, 958 for example, and No. 935, disclosed content will be included in herein in the quoted passage mode.Chemical compound lot of the present invention and prodrug are described and its intermediate synthetic specific examples will partly provide at example.All structural formulas (I), (Ia) and compound (II) all can be prepared by these methods being done conventional change.
Can be in order to synthetic the present invention's 2, the various synthesis paths of 4-pyrimidinediamine compounds will be described in a flow process (I)-(XI) for example.In flow process (I)-(XI), similarity number purpose compound has similar structure.Can change in routine these methods, to be suitable for the prodrug of composite structure formula (II).
In an embodiment as an example, compound can be as the illustrational method of following flow process (I) institute from being substituted or unsubstituted uridylic or thiouracil synthesize:
Flow process (I)
Figure A20091000677100561
In flow process (I), R 2, R 4, R 5, R 6, L 1And L 2Such as previous structural formula (I) definition, X is halogen (for example, F, Cl, Br or I), Y and Y ' are independent of separately being selected from the group that comprises O and S.In flow process (I), under standard conditions, use standard halogenating agent POX 3(or other standard halogenating agent) makes uridylic or thiouracil 2 be produced 2, the two halogen pyrimidines 4 of 4-2 and 4 by dihalide.Depend on the R on the pyrimidine 4 5Substituting group, the halogenide of C4 position is more prone to nucleophilic reagent than the halid reactivity of C2 position.This reactive difference can be used to 2 of composite structure formula (I), and the 4-pyrimidinediamine at first makes 2, and two halogen pyrimidines 4 of 4-and 1 normal amine 10 reactions produce 4N-replacement-2-halogen-4-PYRIMITHAMINE 8, then produce 2 of structural formula (I), 4-pyrimidinediamine with amine 6 reactions.2N, two (replacement)-2 of 4N-, 4- pyrimidinediamine 12 and 14 can be respectively by making 2, and the two halogen pyrimidines 4 of 4-obtain with 6 or 10 excessive reactions.
In most of the cases, the C4 halide reaction tends to nucleophilic reagent, as shown in flow process.Yet this skill person that is familiar with should be able to find out R 5Substituting group is which substituting group can change this reactive character.For example, work as R 5During for trifluoromethyl, obtain 50: 50 mixtures of 4N-replacement-4-PYRIMITHAMINE 8 and corresponding 2N-replacement-2-PYRIMITHAMINE.No matter R 5Which kind of substituting group substituting group is, the selectivity of reactive site can be controlled by regulating solvent and other synthetic silk ribbon spare (for example temperature), as well-known in the industry.
Reaction mixture is during with microwave heating, and the speed of reaction can be accelerated described in the flow process (I).When heating by this way, can use following condition: (under 20 bar pressures) are heated to 175 ℃ in ethanol in sealed tube in smith's reaction device (Personal Chemistry), keep 5-20 minute.
The initial substance of uridylic or thiouracil 2 can be available from preparing from commercial source or with the organic chemistry standard technique.Can be used as commercially available uridylic and the thiouracil that initial substance is used in flow process (I) and comprise (only illustrate and be not limited to): uridylic (Aldrich #13,078-8; CAS Registry 66-22-8); 2-sulfenyl-uridylic (Aldrich #11,558-4; CAS Registry 141-90-2), 2,4-two thiouracils (Aldrich #15,846-1; CAS Registry 2001-93-6), 5-acetyl uridylic (Chem.Sources Int ' l 2000; CAS Registry 6214-65-9), 5-azido-uridylic, 5-amido uridylic (Aldrich #85,528-6; CAS Registry 932-52-5), 5-bromouracil (Aldrich #85,247-3; CAS Registry 51-20-7), 5-(trans-the 2-bromo vinyl)-uridylic (Aldrich #45,744-2; CAS Registry69304-49-0), 5-(trans-the 2-chlorovinyl)-uridylic (CAS Registry 81751-48-2), 5-(trans-the 2-carboxyl vinyl)-uridylic, uridylic-5-carboxylic acid (2,4-dihydroxy-pyrimidine-5-carboxylic acid hydrate; Aldrich #27,770-3; CAS Registry 23945-44-0), 5-chlorouracil (Aldrich #22,458-8; CAS Registry 1820-81-1), 5-cyanouracil (Chem.Sources Int ' l 2000; CAS Registry 4425-56-3), 5-ethyl uracil (Aldrich#23,044-8; CAS Registry 4212-49-1), 5-vinyl uridylic (CAS Registry 37107-81-6), 5 FU 5 fluorouracil (Aldrich #85,847-1; CAS Registry 51-21-8), 5-iodouracil (Aldrich #85,785-8; CAS Registry 696-07-1), methyl uracil (thymus pyrimidine; Aldrich #13,199-7; CAS Registry65-71-4), 5-nitrouracil (Aldrich #85,276-7; CAS Registry 611-08-5), uridylic-5-amidosulfonic acid (Chem.Sources Int ' l 2000; CAS Registry 5435-16-5), 5-(trifluoromethyl)-uridylic (Aldrich#22,327-1; CAS Registry 54-20-6), 5-(2,2, the 2-trifluoroethyl)-uridylic (CAS Registry155143-31-6), 5-(pentafluoroethyl group)-uridylic (CAS Registry 60007-38-3), 6-amido uridylic (Aldrich#A5060-6; CAS Registry 873-83-6), uridylic-6-carboxylic acid (vitamin B13; Aldrich #0-840-2; CASRegistry 50887-69-9), 6-6-Methyl Uracil (Aldrich #D 11,520-7; CAS Registry 626-48-2), uridylic-5-amido-6-carboxylic acid (5-amido vitamin B13; Aldrich #19,121-3; CAS Registry #7164-43-4), 6-amido-5-nitroso-group uridylic (6-amido-2,4-dihydroxyl-5-nitroso-group pyrimidine; Aldrich #27,689-8; CAS Registry5442-24-0), uridylic-5-fluorine-based-6-carboxylic acid (5-fluororotic acid; Aldrich #42,513-3; CAS Registry00000-00-0) and uridylic-5-nitro-6-carboxylic acid (5-nitroorotic acid; Aldrich #18,528-0; CAS Registry600779-49-9).Other 5-, 6-and 5, uridylic and thiouracil that 6-replaces can be available from the General Intermediates of Canada of Alberta, Canada Edmonton, Inc., Edmonton, Alberta, CA (network address is: Www.generalintermediates.com) and France Interchim (network address is: Www.interchim.com), perhaps prepare with standard techniques.Countless textbook bibliographies are mentioned suitable synthetic method, please narrate vide infra.
Amine 6 and 10 can be available from commercial source, and also the available standards technology is synthetic.For example, suitable amine can use standard chemical process synthetic from the nitro precursor.Concrete reaction example sees example for details partly.Also please referring to Vogel, 1989, PracticalOrganic Chemistry, Addison Wesley Long-man, Ltd.and John Wiley ﹠amp; Sons, Inc.
This skill person that is familiar with should be able to recognize under some occasion, amine 6 and 10 and/or substituent R on uridylic or the thiouracil 2 5With/or R 6May comprise the functional group that need in synthetic, be protected.The definite classification of the blocking group that should use depend on the classification of the functional group that protects, for be familiar with this skill person this be conspicuous.The principle of selection due care group and connection thereof and the synthesis strategy that removes can be referring to for example Greene ﹠amp; Wuts, Protective Groups inOrganic Synthesis (blocking group in the organic synthesis), 3d Edition, John Wiley ﹠amp; Sons, Inc., New York (1999) and and the bibliography wherein quoted (below " Greene ﹠amp slightly when quoting; Wuts ").
Following flow process (Ia) will illustrate one with 5 FU 5 fluorouracil (Aldrich #32,937-1) as the specific embodiment of the flow process (I) of initial substance:
Flow process (Ia)
Figure A20091000677100591
In flow process (Ia), R 2, R 4, L 1And L 2Such as previous flow process (I) definition.According to flow process (Ia), 5 FU 5 fluorouracil 3 and POCl 3Halogenation draws 2, and 4-dichloride base-5-fluorine pyrimidine 5 produces N2 respectively with excess amine 6 or 10 reactions then, and the disubstituted 5-of N4-is fluorine-based-2,4-pyrimidinediamine 11 or 13.Asymmetric 2N, two replacement-the 5-fluorine-based-2 of 4N-, 4-pyrimidinediamine 9 can be by making 2,4-dichloride base-5-fluorine pyrimidine 5 and normal amine 10 reaction (with draw 2-chloro-N4-replacements-5-fluorine-based-4-PYRIMITHAMINE 7) react with one or several normal amine 6 then and obtain.
Another embodiment for example in, of the present invention 2, the 4-pyrimidinediamine compounds can be by following flow process (IIa) and (IIb) is illustrated, from being substituted or unsubstituted cytosine(Cyt) synthesizes:
Flow process (IIa)
Figure A20091000677100601
Flow process (IIb)
Figure A20091000677100611
In flow process (IIa) with (IIb), R 2, R 4, R 5, R 6, L 1, L 2With X such as previous flow process (I) definition, PG represents blocking group.Referring to flow process (IIa), the outer amine of the C4 of cytosine(Cyt) 20 ring is earlier with suitable blocking group PG protection, to draw the cytosine(Cyt) 22 of N4-protection.About the concrete guide of the blocking group that can be used in this occasion, referring to Vorbr ü ggen and Ruh-Pohlenz, 2001, Handbook of Nucleoside Synthesis (Nucleotide synthesizes handbook), John Wiley ﹠amp; Sons, NY, pp.1-631 (" Vorbr ü ggen ").Under standard conditions, use the standard halogenating agent in the halogenation of C2 position through the cytosine(Cyt) 22 of protection, draw 2-chloro-4N-protection-4-PYRIMITHAMINE 24.After amine 6 reaction, then the outer amine of C4 ring is gone to protect, and with the reaction of amine 10, draw 2 of structural formula (I), the 4-pyrimidinediamine.
Perhaps, make cytosine(Cyt) 20 and amine 10 or 21 reactions of the amine through protecting, draw the cytosine(Cyt) 23 or 27 that N4-replaces respectively by the scheme of flow process (IIb).These substituted cytosine(Cyt) halogenations as described above, go protection (being substituted at N4-under the occasion of cytosine(Cyt) 27), and with amine 6 reaction, draw 2 of structural formula (I), the 4-pyrimidinediamine.
Can be used as that initial substance is used in flow process (IIa) and ready-made commercially available cytosine(Cyt) (IIb) includes, but is not limited to cytosine(Cyt) (Aldrich #14,201-8; CAS Registry 71-30-7), N 4-acetylcytosine (Aldrich #37,791-0; CASRegistry 14631-20-0), 5-flurocytosine (Aldrich #27,159-4; CAS Registry 2022-85-7) and 5-(trifluoromethyl)-cytosine(Cyt).The suitable cytosine(Cyt) that other initial substance that can be used as flow process (IIa) uses can be available from the General Intermediates of Canada of Alberta, Canada Edmonton, Inc., and Edmonton, Alberta, CA ( Www.generalintermediates.com) and French Interchim, France ( Www.interchim.com), maybe can use the standard technique preparation.Countless textbook bibliographies are mentioned suitable synthetic method, please narrate vide infra.
Also have an embodiment for example in, of the present invention 2, the 4-pyrimidinediamine compounds can be by illustrating in the flow process (III), from be substituted or unsubstituted 2-amido-4-ancymidol synthetic, as follows:
Flow process (III)
Figure A20091000677100631
In flow process (III), R 2, R 4, R 5, R 6, L 1, L 2With X such as previous flow process (I) definition, Z in following flow process IV with the leaving group that goes through.In flow process (III), 2-amido-4-ancymidol 30 and amine 6 (or also can with the amine 21 through protection) reaction draws N2-replacement-4-ancymidol 32, and halogenation as discussed previously then draws N2-replacement-4-halogen-2-PYRIMITHAMINE 34.Also can go protection (if for example use in a first step through protection amine 21), then the reaction with amine 10 provides 2 of structural formula (I), 4-pyrimidinediamine.Perhaps ancymidol 30 also can react with acylating agent 31.
The suitable commercially available 2-amido-4-ancymidol 30 that can be used as the initial substance use of flow process (III) includes, but is not limited to 2-amido-6-chloro-4-ancymidol hydrate (Aldrich #A4702-8; CAS Registry 00000-00-0) and 2-amido-6-hydroxyl-4-ancymidol (Aldrich #A5040-1; CAS Registry 56-09-7).2-amido-4-ancymidol 30 that other initial substance that can be used as flow process (III) uses can be available from the General Intermediates ofCanada of Alberta, Canada Edmonton, Inc., and Edmonton, Alberta, CA ( Www.generalintermediates.com) and French Interchim, France ( Www.interchim.com), maybe can use the standard technique preparation.Countless textbook bibliographies are mentioned the synthetic method of living and working as, and please narrate vide infra.
Perhaps of the present invention 2, the 4-pyrimidinediamine compounds also can be by the illustrating of flow process (IV), and is from being substituted or unsubstituted 4-amido-2-ancymidol preparation, as follows:
Flow process (IV)
Figure A20091000677100641
In flow process (IV), R 2, R 4, R 5, R 6, L 1And L 2Such as previous flow process (I) definition, Z represents leaving group.In flow process (IV), the degree that the C2-hydroxyl of 4-amido-2-ancymidol 40 tends to nucleophilic reagent is higher than the C4-amido, causes and amine 6 reactions, draws N2-and replaces-2,4-pyrimidinediamine 42.With compound 44 (this compound comprises a kind of good leaving group Z) or amine 10 reactions, draw 2 of structural formula (I), the 4-pyrimidinediamine subsequently.In fact compound 44 can comprise anyly can be replaced-2 by N2-, the C4-amine metathetical leaving group of 4-pyrimidinediamine 42.The leaving group Z that is fit to includes, but is not limited to halogen, methanesulfonyloxy group (mesyloxy; " OMs "), trifluoromethane sulfonyloxy (" OTf ") and tolysulfonyl oxygen base (tosyloxy; " OTs "), phenylsulfonyloxy (" benzene sulfonate (besylate) ") and m-nitro sulfonyloxy (" nosylate ").Other leaving group that is fit to is conspicuous for this operator that is familiar with.
Substituted 4-amido-2-ancymidol initial substance can obtain on market or be synthetic with standard technique.The textbook of the suitable synthetic method of countless teachings is with reference to being provided in hereinafter.
Also have an embodiment for example in, of the present invention 2, the 4-pyrimidinediamine compounds can illustrate by flow process (V), from 2-chloro-4-amine pyrimidine or 2-amido-4-chloro pyrimidine preparation, shown in following flow process (V):
Flow process (V)
Figure A20091000677100651
In flow process (V), R 2, R 4, R 5, R 6, L 1, L 2With X such as flow process (I) definition, Z such as flow process (IV) definition.In flow process (V), 2-amido-4-chloropyrimide 50 and amido 10 reactions draw 4N-replacement-2-PYRIMITHAMINE 52, then with compound 31 or amine 6 reactions, draw 2 of structural formula (I), the 4-pyrimidinediamine.Perhaps 2-chloro-4-amido-pyrimidine 54 also can react with compound 44, follows and amine 6 reactions, draws the compound of structural formula (I).
Shake-up is suitable as the pyrimidine 50 and 54 that flow process (V) initial substance uses and is the commercial goods, comprises (being not limited to as an example) 2-amido-4,6-dichloro pyrimidine (Aldrich #A4860-1; CAS Registry 56-05-3), 2-amido-4-chloro-6-methoxyl group-pyrimidine (Aldrich #51,864-6; CAS Registry 5734-64-5), 2-amido-4-chloro-6-methylpyrimidine (Aldrich #12,288-2; CAS Registry 5600-21-5) and 2-amido-4-chloro-6-methylthiopyrimidine (Aldrich #A4600-5; CAS Registry 1005-38-5).Other pyrimidine initial substance can be available from the General Intermediates of Canada of Alberta, Canada Edmonton, Inc., and Edmonton, Alberta, CA ( Www.generalintermediates.com) and Interchim, France ( Www.interchim.com), maybe can use the standard technique preparation.Countless textbook bibliographies are mentioned suitable synthetic method, please narrate vide infra.
Perhaps 4-chloro-2-aminopyrimidine 50 also can be prepared shown in flow process (Va):
Flow process (Va)
In flow process (Va), R 5And R 6Such as previous structural formula (I) definition.In flow process (Va), dicarbapentaborane 53 and guanidine reaction draw 2-PYRIMITHAMINE 51.With peracid (as m-chlorine peroxybenzoic acid, trifluoroperacetic acid or urea hydrogen peroxide misfit thing) reaction, draw N-oxide compound 55, halogenation then is to draw 4-chloro-2-PYRIMITHAMINE 50.Corresponding 4-halogen-2-aminopyrimidine can obtain by using suitable halogenating agent.
Also have in addition an embodiment for example in, of the present invention 2, the 4-pyrimidinediamine compounds can be by the illustrating of flow process (V), from 2-chloro-4-amine pyrimidine or 2-amido-4-chloro pyrimidine preparation, shown in following flow process (V):
Flow process (VI)
Figure A20091000677100671
In flow process (VI), R 2, R 4, R 5, R 6, L 1, L 2With X such as previous flow process (I) definition, subscript PG represents the blocking group discussed in the flow process (IIb).In flow process (VI), uridine 60 has the C4 reactive center, can react with amine 10 or the amine through protecting 21, draws the cytidine 62 or 64 that N4-replaces respectively.62 or 64 the acid catalysis that N4-replaces goes provide protection (when the sour unstable protection group of " PG " expression) to draw the cytosine(Cyt) 28 that N4-replaces, then can be in the C2 position by halogenation, and with amine 6 reactions, draw 2 of structural formula (I), the 4-pyrimidinediamine.
Cytidine also can be used as initial substance with similarity method, shown in following flow process (VII) illustrates:
Flow process (VII)
Figure A20091000677100681
In flow process (VI), R 2, R 4, R 5, R 6, L 1, L 2With X such as previous flow process (I) definition, subscript PG represents the blocking group discussed in the flow process (IIb).In flow process (VI),, as uridine 60, cytidine 70 has a C4 reactive center, can react with amine 10 or the amine through protecting 21, draws the cytidine 62 or 64 that N4-replaces respectively.These cytidines 62 and 64 are handled by the described mode of previous flow process (VI) then, draw 2 of structural formula (I), the 4-pyrimidinediamine.
Though flow process (VI) and (VII) with the ribosyl nucleosides for example, the present technique of being familiar with personage should understand corresponding 2 '-deoxyribose nucleosides and 2 ', 3 '-two deoxyribose nucleosides, and comprises that the nucleosides of sugar or the sugar analogue except that ribose can use too.
The many uridine and cytidines of can be in flow process (VI) and being used as initial substance (VII) have been on record in this technology, comprise (only give an example, be not limited to) 5-trifluoromethyl-2 '-cytosine deoxyriboside (Chem.Sources #ABCRF07669; CAS Registry 66,384-66-5), 5-broxuridine (Chem.Sources Int ' l 2000; CAS Registry957-75-5), 5-iodo-2 '-uracil deoxyriboside (Aldrich #1-775-6; CAS Registry 54-42-2); 5-floxuridine (Aldrich #32,937-1; CAS Registry316-46-1), 5-ioduria glycosides (Aldrich #85,259-7; CAS Registry1024-99-3); 5-(trifluoromethyl) uridine (Chem.Sources Int ' l 2000; CAS Registry 70-00-8); 5-trifluoromethyl-2 '-uracil deoxyriboside (Chem.Sources Int ' l 2000; CAS Registry 70-00-8).In addition can be in flow process (VI) and be used as the uridine and the cytidine of initial substance (VII) can be available from the GeneralIntermediates of Canada of Alberta, Canada Edmonton, Inc., Edmonton, Alberta, CA ( Www.generalintermediates.com) and Interchim, France ( Www.interchim.com), maybe can use the standard technique preparation.Countless textbook bibliographies are mentioned the synthetic method of living and working as, and please narrate vide infra.
Of the present invention 2, the 4-pyrimidinediamine compounds also can be by flow process (VIII) and (IX) illustrated, and is synthetic from substituted pyrimidine (for example pyrimidine of chloro-replacement), as follows:
Flow process (VIII)
Flow process (IX)
In flow process (VIII) with (IX), R 2, R 4, L 1, L 2And R aSuch as previous structural formula (I) definition, " Ar " represents aryl.In flow process (VIII), 2,4,6-trichloropyrimidine 80 (Aldrich #T5,620-0; CAS#3764-01-0) with amine 6 reaction, draw the mixture of three kinds of compounds, that is: substituted pyrimidine monoamine, diamines and triamine 81,82 and 83, this mixture usable highly effective liquid chromatography (HPLC) or its routine techniques separate and single from.Monoamine and diamines 81 and 82 can further react with amine 6 and 10, draw N2 respectively, N4, and N6-three replaces-2,4,6-pyrimidine triamine 84 and 85.
N2, N4-pair-replace-2, the 4-pyrimidinediamine can pass through in the method that is similar to flow process (VIII) 2,4-dichloride base-5-methylpyrimidine or 2,4-dichloride base-pyrimidine prepares as initial substance.In this example, do not obtain single substituted pyrimidines amine, but this reaction proceeds corresponding to compound 81, directly draw N2, N4-is two-replace-2, the 4-pyrimidinediamine.
In flow process (IX), 2,4,5,6-tetrachloro-pyrimidine 90 (Aldrich #24,671-9; CAS#1780-40-1) be excess amine 6 reaction, draw the mixture of three kinds of compounds, that is: 91,92 and 93, this mixture usable highly effective liquid chromatography (HPLC) or other routine techniques separate and list from.As described in for example, N2, N4-pair-replace-5,6 ,-dichloride base-2,4-pyrimidinediamine 92 can further react with for example nucleophilic reagent 94 at C6 halogenide place, draws compound 95.Perhaps compound 92 also can change into N2 by suzuki reaction, and N4-pair-replacement-5-chloro-6-aryl-2,4-pyrimidinediamine 97.2,4-pyrimidinediamine 95 by with Bn 3The SnH reaction is converted into 2,4-pyrimidinediamine 99.
This technology personage that is familiar with should be able to find out, by above-mentioned institute's described method for example or of the present invention 2 by other currently known methods synthetic, the 4-pyrimidinediamine, can be used as among synthetic the present invention other 2, the initial substance of 4-pyrimidinediamine compounds and intermediate.Be a specific examples shown in the following flow process (X):
Flow process (X)
Figure A20091000677100711
In flow process (X), R 4, R 5, R 6, L 2And R aSuch as previous structural formula (I) definition.Each R a' each independently is a R a, and and illustrational R aCan be identical or different.With regard to flow process (X), carboxylic acid or ester 100 can be converted into acid amides 104 by reacting with amine 102.In amine 102, R a' with the R of acid or ester 100 aIdentical or different.Carbonic ether 106 can change into amido formate 108 equally.
Second specific examples is shown in following flow process (XI):
Flow process (XI)
In flow process (XI), R 4, R 5, R 6, L 2And R cSuch as previous structural formula (I) definition.With regard to flow process (XI), acid amides 110 or 116 can be separately converted to amine 114 or 118 by the borane reduction reaction with borine first sulphur misfit thing 112.Other is from 2,4-pyrimidinediamine initial substance Synthetic 2, and the appropriate reaction of 4-pyrimidinediamine compounds is conspicuous for this gate technique personage that is familiar with.
Though above-mentioned synthesis flow is many and the use of undeclared blocking group, this skill person that is familiar with should be able to find out in some cases substituent R 2, R 4, R 5, R 6, L 1With/or L 2May comprise the functional group that needs protection.The definite classification (especially) of employed blocking group depend on the classification of the functional group that protects and in specific synthesis flow used reaction conditions, concerning this skill person that is familiar with, this is conspicuous.The principle of selection due care group and connection thereof and the principles of chemistry that remove can be referring to for example above-mentioned Greene ﹠amp; Wuts.
The prodrug of structural formula (II) can prepare it by method described above being carried out necessary conventional the modification.Perhaps 2 of the structural formula (I) that this type of prodrug also can be by the existing due care of order, 4-pyrimidinediamine and suitable blocking group prepared in reaction.Carrying out such reaction and product is gone protection, is well-known with the condition that draws structural formula (II) prodrug.
The bibliography of the process useful of the synthetic PYRIMITHAMINE of countless general introductions and the synthetic method of the described initial substance of flow process (I)-(IX) is on record in the industry.With regard to concrete guide, we recommend following data: Brown to the reader, D.J., " The Pyrimidines (pyrimidine) ", in The Chemistry of Heterocyclic Compounds (chemistry of heterocyclic compound), Volume 16 (Weissberger, A., Ed.), 1962, Interscience Publishers, (ADivision of John Wiley ﹠amp; Sons), New York (" Brown I "), Brown, D.J., " The Pyrimidines ", in The Chemistry of Heterocyclic Compounds, Volume 16, Supplement I (Weissberger, A.andTaylor, E.C., Ed.), 1970, Wiley-Interscience, (A Division of John Wiley ﹠amp; Sons), New York (" Brown II "), Brown, D.J., " The Pyrimidines ", in The Chemistry of HeterocyclicCompounds, Volume 16, Supplement II (Weissberger, A.and Taylor, E.C., Ed.), 1985, AnInterscience Publication (John Wiley ﹠amp; Sons), New York (" Brown III "), Brown, D.J., " ThePyrimidines " in The Chemistry of Heterocyclic Compounds, Volume 52 (Weissberger, A.andTaylor, E.C., Ed.), 1994, John Wiley ﹠amp; Sons, Inc., New York, pp.1-1509 (" Brown IV "), Kenner, G.W.and Todd, A., in Heterocyclic Compounds (heterogeneous ring compound), Volume 6, (Elderfield, R.C., Ed.), 1957, John Wiley, New York, Chapter 7 (pyrimidines (pyrimidine)), Paquette, L.A., Principles of Modern Heterocyclic Chemistry (contemporary heterocyclic chemistry principle), 1968, W.A.Benjamin, Inc., New York, pp.1-401 (uracil synthesis (uridylic is synthetic) pp.313,315; Pyrimidine synthesis (pyrimidine is synthetic) pp.313-316; Amino pyrimidine synthesis (amine pyrimidine synthetic) pp.315), Joule, J.A., Mills, K.and Smith, G.F., Heterocyclic Chemistry (heterocyclic chemistry), 3 RdEdition, 1995, Chapmanand Hall, London, UK, pp.1-516, Vorbr ü ggen, H.and Ruh-Pohlenz, C., Handbook ofNucleoside Synthesis (nucleosides synthesizes handbook), John Wiley ﹠amp; Sons, New York, 2001, pp.1-631 (protection of pyrimidines by acylation (the acidylate protection of pyrimidine) pp.90-91; The silylation ofpyrimidines silanization of the pyrimidine (protection) pp.91-93), Joule, J.A., Mills, K.and Smith, G.F., Heterocyclic Chemistry (heterocyclic chemistry), 4 ThEdition, 2000, Blackwell Science, Ltd, Oxford, UK, pp.1-589 and Comprehensive Organic Synthesis (organic synthesis complete works), Volumes 1-9 (Trost, B.M.and Fleming, I., Ed.), 1991, Pergamon Press, Oxford, UK.
6.4Fc the restraining effect of receptor signal cascade
Of the present invention active 2, the 4-pyrimidinediamine compounds is inhibited to the Fc receptor signal cascade that causes (especially) cell degranulation.As specific examples, this compound suppresses to cause the Fc ε RI and the Fc γ RI signal cascade of immunocyte (as neutrophils, have a liking for Yihong blood cell, mastocyte and basophilic leukocyte) degranulation.Loose and basophilic leukocyte is all played a leading role in the disease (comprising as allergic rhinitis and asthma) of allergen-induced.In figure one, in case in the contact allergy former (especially pollen or parasite) time, allergenic specific IgE antibody system is synthetic by IL-4 (or IL-13) and other courier's activated B cell, transfers IgE class specific antibody to and synthesizes.These allergenic specific IgEs are attached on the high-affinity Fc ε RI.After antigen one combines, produce crosslinked with Fc ε R1 bonded IgE, and activation IgE receptor signal transduction path, thereby cause cell degranulation and the release of a series of chemical mediators subsequently and synthetic, this type of chemical mediator comprises: histamine, albumen Enzyme class (as class Trypsin Enzyme and the short pancreas Enzyme of stomach), leukotriene lipid medium, platelet activating factor (PAF) and prostanoid (as PGD2) and a series of cytokines (comprising TNF-α, IL-4, IL-13, IL-5, IL-6, IL-8, GMCSF, VEGF and TGF-β) such as (as LTC4).These from the release of the medium of mastocyte and basophilic leukocyte and synthetic be the early stage root with late phase response of anaphylactogen institute inductive, and directly related with the downstream events that causes lasting inflammatory status.
It is well-known causing forming the release of medium and the release of other chemical mediator and the molecular events in the synthetic Fc ε RI signal transduction path in advance by degranulation, its description references figure two.With regard to figure two, Fc ε RI is the assorted tetramer acceptor that is made of IgE-bonded α-subunit, β subunit and two γ subunits (γ homodimer).Fc ε RI by multivalence binding medium (comprising as IgE specific allergen or anti-IgE antibodies or fragment) is in conjunction with relevant rapid combination and the activation that swashs Enzyme Lyn of the crosslinked Src of inducing of IgE.Lyn activation theme (ITAMS) of phosphorylation immunity receptor tyrosine-based on β and the γ subunit in cell swashs Enzyme to the γ homodimer thereby cause raising more Lyn to β subunit and more Syk.These sharp Enzymes relevant with acceptor activate by intramolecularly and intermolecular phosphorylation, and other composition on the phosphorylation path (for example Btk swashs Enzyme, LAT and phosphatide Enzyme C-γ PLC-γ).Activated PLC-γ initiation causes protein to swash Enzyme C activation and Ca 2+The path of motion (both be degranulation institute must).The crosslinked mitogen activated protein matter (MAP) that also activates three kinds of main types of Fc ε RI swashs Enzyme, that is: ERK1/2, JNK1/2 and p38.The activation in these paths is very important in the transcriptional regulatory of pro-inflammatory mediator (for example TNF-α and IL-6) and lipid medium leukotriene CA (LTC4).
Though also undeclared, it is believed that shared some common element of Fc γ RI signal cascade and Fc ε RI signal connection cascade mutually.Importantly, comprise phosphorylation and attract the γ homodimer of Syk that as Fc ε RI, Fc γ RI also as Fc ε RI, the activation of Fc γ RI signal cascade causes (especially) degranulation.Sharing the γ homodimer also can be by activity 2, and other Fc acceptor that the 4-pyrimidinediamine compounds is regulated includes, but is not limited to Fc α RI and Fc γ RIII.
The present invention 2, simple mensuration or affirmation during the ability of 4-pyrimidinediamine compounds inhibition Fc receptor signal cascade can in vitro be examined and determine.Affirmation mediates the inhibiting suitable calibrating of degranulation to Fc ε RI and partly introduces at example.In typical case's calibrating, cell (for example hypertrophy or basophilic leukocyte) with the degranulation ability of carrying out Fc ε RI mediation is grown in the presence of IL-4, STEM CELL FACTOR (SCF), IL-6 and IgE earlier to increase Fc ε RI performance, and be exposed to of the present invention 2, in the 4-pyrimidinediamine test compound, and stimulate with anti-IgE antibodies (or IgE specific allergen also can).After the cultivation, by Fc ε RI signal cascade activate and discharge with/or the quantity available standards technology of synthetic chemical mediator or other chemical media quantitatively and with cellular control unit (promptly stimulate but be not exposed to cell in the test compound) medium of release or the quantity of media compare.All measured media or media quantity are compared with control group and are reduced by 50% test compounds concentration, are called the IC of test compounds 50The desired use of compound is partly depended in the mastocyte that is used to examine and determine or the source of basophilic leukocyte, for be familiar with this operator this be conspicuous.For example, be people or other animal that can be used as the acceptable or known clinical model of specified disease if compound is used for the treatment of or prevents human specified disease, the convenient source of mastocyte or basophilic leukocyte.Therefore, depend on its specific end use, hypertrophy or basophilic leukocyte can take from animal-origin widely (scope from for example low wait Mammals as small white mouse and rat to dog, sheep and be used for other Mammals of clinical trial, for example monkey, chimpanzee and ape arrive the people up to higher mammal).The object lesson of the cell that is suitable in vitro examining and determine includes, but is not limited to rodent or human basophilic leukocyte, rat is had a liking for the alkali leukemia cell line, the former mouse mastocyte mouse mastocyte " BMMC " of marrow (for example derived from) and single from organizing for example former human mastocyte of lung from vertebra blood (" CHMC ") or other.Single from and the method for cultivating these cell types be on record or be provided at this paper example partly (referring to for example: Demo et al., 1999, Cytometry 36 (4): 340-348 and the sequence number that awaits the reply simultaneously are 10/053,355, date of application is the patent application case in November 8 calendar year 2001, and its disclosure content is incorporated in herein in the quoted passage mode).Certainly, other other type immunocyte that plays degranulation when Fc ε RI signal cascade activates also can use, and comprises and for example has a liking for Yihong blood cell.
As this skill person that is familiar with should be able to find out, quantized medium or media and non-key.Unique requirement be it must be the cascade of Fc receptor signal cause or activate discharge and synthetic medium or media.For example with regard to Fig. 1, the activation of Fc ε RI signal cascade in loose and basophilic leukocyte causes a lot of downstream events.For example, the activation of Fc ε RI signal cascade causes the release at once (promptly after receptor activation within 1-3 minute) of a series of pre-formation chemical mediators and media by degranulation.Therefore, in one embodiment, medium that is quantized or media can have specificity (promptly be present in the particle, but be not present in the tenuigenin of cell usually) to particle.Can be quantized to judge and to confirm of the present inventionly 2 that the example of 4-pyrimidinediamine compounds active particle specificity medium or media includes, but is not limited to particle specificity Enzyme (for example hexosamine Enzyme and class Trypsin Enzyme) and particle specificity composition (for example histamine and thrombotonin).The calibrating that quantizes this type of factor is well-known, and often at the market public offering.For example, class Trypsin Enzyme and hexosamine Enzyme discharge the cell that cleavable matrix (in case fluorescent is sent in this matrix cracking) can be arranged by cultivation, adopt conventional art to record gained fluorescent amount then and it is quantized.This kind cleavable fluorogenic substrate is that sell the market.For example fluorescent swashs energy matrix Z-Gly-Pro-Arg-AMC (Z=benzene methoxycarbonyl; The smoked grass of AMC=7-amido-4-methyl; BIOMOLResearch Laboratories, Inc., Plymouth Meeting, PA 19462, catalog number (Cat.No.) P-142) and Z-Ala-Lys-Arg-AMC (Enzyme Systems Products, a division of ICN Biomedicals, Inc., Livermore, CA 94550,, catalog number (Cat.No.) AMC-246) can be used to quantize the quantity of the class Trypsin Enzyme that discharged.Fluorescent swash can matrix 4-methyl cymenyl-N-ethanoyl-β-D-amido glucoside (Sigma, St.Louis, MO, catalog number (Cat.No.) 69585) can be used to quantize discharge the quantity of hexosamine Enzyme.Histamine discharges available commercially available Enzyme connection immunosorption calibrating (ELISA), and for example (Beckman-Coulter Inc.) quantizes Immunotech histamine Enzyme connection immunosorption calibrating #IM2015.The concrete grammar that quantizes class Trypsin Enzyme, hexosamine Enzyme and histamine release will partly be introduced at example.These calibratings any all can be used to judge or confirm of the present invention 2, the activity of 4-pyrimidinediamine compounds.
Still with regard to figure one, degranulation only be among several reactions that caused of Fc ε RI signal cascade one.In addition, the activation of this signal path causes cytokine and chemical hormone (chemokines) (IL-4 for example, IL-5, IL-6, TNF-α, IL-13 and MIP1-α) synthetic again and discharge and the release of leukotriene lipid medium, platelet activating factor (PAF) and prostaglandin(PG)s such as (as LTC4).Therefore, of the present invention 2, the 4-pyrimidinediamine compounds also can one or several activating cellss discharge and institute's synthetic medium comes its activity is assessed by quantizing.
Different with particle specificity composition discussed above is that these " late period " media do not discharge after the activation of Fc ε RI signal cascade at once.Therefore,, should guarantee carefully that the activating cells substratum must cultivate the sufficiently long time, be planned synthetic (if needs are arranged) and the required time of release of quantized medium to guarantee to finish institute when when these late periods, medium quantized.Usually, PAF and lipid medium (for example leukotriene C4) release in 3-30 minute behind Fc ε RI activated.Cytokine and other medium in late period release in about 4-8 hour behind Fc ε RI activated.The incubation time that is suitable for concrete medium is conspicuous for this operator that is familiar with.Direct concretely and examine and determine and partly to introduce at example.
Discharged specific late period medium the available any standard technique of amount quantize.In one embodiment, this measures available Enzyme connection immunosorption calibrating (ELISA) mensuration.The Enzyme connection immunosorption calibrating (ELISA) of TNF α, the IL-4 that is suitable for quantizing being discharged, IL-5, IL-6 and/or IL-13 amount can be available from for example Biosource International with detecting box, Inc., Camarillo, CA 93012 (referring to following catalog number (Cat.No.): KHC3011, KHC0042, KHC0052, KHC0061 and KHC0132).The ELISA that is suitable for leukotriene C4 (LTC4) burst size that quantization cell discharges detects box can be available from Cayman Chemical Co., Ann Arbor, MI 48108 (referring to for example catalog number (Cat.No.) 520211).
In typical case, measure as chemical examination in vitro, with regard to medium release, of the present invention active 2, the 4-pyrimidinediamine compounds will manifest about 20 μ M or be lower than the IC of this value with regard to Fc ε RI mediation degranulation 50, as described above or example partly in vitro shown in one of calibrating.Certainly, this skill person that is familiar with should understand and shows low IC 50The compound of (for example 10 μ M, 1 μ M, 100nM, 10nM, 1nM or even the lower order of magnitude) is useful especially.
This skill person that is familiar with should understand that also various medium discussed above can induce different side effects, perhaps identical side effect is shown different potency ratingses.For example, lipid medium LTC4 is strong vasoconstrictor, and institute's inductive vasoconstriction effect is higher approximately more than 1000 times than histamine.As another example, except that mediation atopy or the allergy of I type, cytokine also can cause tissue engineered and cell proliferation.Therefore, though it is useful suppressing previous release and the synthetic compound that any chemical mediator is discussed, but this skill person that is familiar with will understand and suppress above-mentioned some kinds and even all release of media and the special purposes of synthetic compound, because this compounds can improve or avoid multiple fully and even all are by this class particular medium medium institute inductive side effect.For example, suppress all three types of media (particle specificity medium, lipid medium and cytokine medium) to treatment or the i.e. property sent out I type allergy of prevention and relative chronic sympton.
The compounds of this invention suppress more than one type media (as particle specificity medium or late period medium) ability that discharges can be with above-mentioned various in vitro calibratings (or other suitable in vitro calibrating) by measuring the corresponding IC that represent every type of medium 50Define.Allly can suppress the The compounds of this invention that more than one type media discharge, with typically show every kind of institute's examination medium type less than the IC about 20 μ M 50For example, one discharges (IC with regard to histamine 50 Histamine) show the IC of 1 μ M 50, and put out the synthetic and release (IC of plain LTC4 with regard to white three 50 LTC4) show the IC of 1nM 50Compound suppress i.e. property sent out (particle specificity) and medium release in late period.As another specific examples, show the IC of 10 μ M 50 Class Trypsin Enzyme, 1 μ M IC 50 LTC4IC with 1 μ M 50 IL-4Compound suppress the i.e. release of the property sent out (particle specificity) medium, lipid medium and cytokine medium.Though above-mentioned specific examples has only been used the IC of a representative medium in each type 50, but this skill person that is familiar with will understand and might obtain majority and even whole IC that comprises one or several type media 50With regard to certain specific compound and some specific end use, need to determine IC 50The media quantity of data and classification should be conspicuous to this skill person that is familiar with.
As long as do some conventional changes, just can utilize similar calibrating to confirm restraining effect to the signal transduction cascade of other Fc acceptors (for example Fc α RI, Fc γ RI and Fc γ RIII) initiation.For example, compound suppresses the ability of Fc γ RI signal transduction can use the calibrating affirmation similar with calibrating described above, but will be, rather than with IgE and IgE specific allergen or antibody with IgG and IgG specific allergen or antibody culturing cell activating Fc γ RI signal cascade.Required suitable cell type, activation media and the quantized media of needs of restraining effect of confirming other Fc acceptor (the Fc acceptor that for example comprises the γ homodimer) should be conspicuous to this skill person that is familiar with.
The useful especially compound of one class is can be with similar substantially IC 50Release various 2 that suppress type particle specificity medium and late period medium at once, the 4-pyrimidinediamine compounds.The IC of similar each media type of finger of cardinal principle 50Gap each other is within 10 times of scopes.Another kind of useful especially compound is can be with similar substantially IC 50Suppress 2 of type particle specificity medium, lipid medium and the release of cytokine medium at once, the 4-pyrimidinediamine compounds.In a specific embodiment, this compounds is with similar substantially IC 50Suppress the release of some media, that is: histamine, class Trypsin Enzyme, hexosamine Enzyme, IL-4, IL-5, IL-6, IL-13, TNF α and LTC4.This compounds improves (especially) or prevents that fully early stage the and late phase response relevant with atopy or property I type allergy at once is particularly useful.
If same compound can possess the ability that whole required type media discharge that suppresses, that yes desirable more only.Yet we also can find the mixture of the compound that comes to the same thing.For example, suppress first compound that particle specificity medium discharges can with the release that suppresses the cytokine medium with/or second compound of synthetic be used in combination.
Except that above-mentioned Fc ε RI or Fc γ RI degranulation path, the degranulation of mastocyte and basophilic leukocyte also can be induced by other media.For example Ai Nuo mycin (a kind of Calcium ionophore of walking around cell early stage Fc ε RI or Fc γ RI signal transduction mechanism) is directly induced the calcium running that triggers degranulation.With regard to Fig. 2, activated PLC γ causes the path that causes transfer of (especially) calcium ion and degranulation subsequently again.As described, this Ca 2+Mobilization triggers in Fc ε RI signal transduction path very late.As mentioned above and as shown in Figure 3, the Ai Nuo mycin is directly induced the Ca that causes degranulation 2+Mobilize and Ca 2+Running.Other induces the ionophore of degranulation to comprise A23187 in this way.The ability of inducing the ionophore (for example Ai Nuo mycin) of particle effect to walk around Fc ε RI and Fc γ RI signal cascade commitment can be used as by blocking-up or suppress aforesaid early stage Fc ε RI or Fc γ RI signal cascade is brought into play its degranulation specially and suppressed active active compound of the present invention and recognize with the counting screen of judging and use.The special compound that suppresses Fc ε RI and Fc γ RI mediation degranulation not only suppresses degranulation and the rapid release of histamine, class Trypsin Enzyme and other granular contents subsequently, but also suppresses to cause the scorching activated path of urging of TNF α, IL-4, IL-13 and lipid medium (as LTC4) release.Therefore, the compound of degranulation that can suppress this kind early stage Fc ε RI and Fc γ RI mediation is specially not only blocked or is suppressed acute atopy or the allergy of I type, and blocking-up or suppress to relate to the late phase response of multiple inflammatory mediator.
The The compounds of this invention of special early stage Fc ε RI of inhibition and Fc γ RI mediation degranulation (for example with regard to the release of particle specificity medium, has less than the IC about 20 μ M for suppressing Fc ε RI and Fc γ RI mediation degranulation 50, and with regard to the cell that IgE or IgG excite in conjunction with media, be the composition of measuring in the in vitro calibrating) compound, but significantly do not suppress ionophore inductive degranulation.In one embodiment, if the IC of shown ionophore inductive degranulation in the calibrating in vitro 50Greater than about 20 μ M, then this compounds promptly is considered to and not obvious inhibition ionophore inductive degranulation.Certainly, show that more the macroion carrier is induced the IC of degranulation 50, or not suppressing ionophore, to induce the degranulation active compound be useful especially.In another embodiment, if according in vitro calibrating mensuration, the Fc ε RI that it is shown and the degranulation of Fc γ RI mediation and the IC of ionophore inductive degranulation 50Difference is greater than 10 times, and this compound promptly is considered to significantly not suppress ionophore inductive degranulation.Be suitable for measuring the IC of ionophore inductive degranulation 50Calibration method comprise the calibration method of any aforementioned degranulation, but need to do following the modification, that is: be with Ai Nuo mycin or A23187 (A.G.Scientific, SanDiego, CA produces) etc. degranulation induce Calcium ionophore to excite or active cells, rather than anti-IgE antibodies or a kind of IgE specific allergen.Assess of the present invention specificly 2, the 4-pyrimidinediamine compounds suppresses ionophore and induces the specific calibration method of the ability of degranulation partly to provide at example.
This skill person that is familiar with should be able to find out that the compound that Fc ε RI mediation degranulation is presented high selectivity has special purpose, because this compounds is selected aiming Fc ε RI cascade and do not disturbed other degranulation mechanism.The compound that Fc γ RI mediation degranulation is presented high selectivity equally also has special purpose, because this compounds is selected aiming Fc γ RI cascade, and does not disturb other degranulation mechanism.The compound that presents high selectivity induces degranulation (for example the Ai Nuo mycin is induced degranulation) high more than 10 times to the selectivity ratios ionophore of Fc ε RI or Fc γ RI mediation degranulation.
Biological chemistry and other data validation are as herein described 2, and the 4-pyrimidinediamine compounds is that Syk swashs the active strong inhibitor of Enzyme.For example, use single from Syk swash in the Enzyme experiment, 24 tested 2, in the 4-pyrimidinediamine compounds, the sharp Enzyme catalysis phosphorylation of the Syk of whole inhibiting peptide matrix except that two, its IC 50Occupy the submicrogram molecular range.All the other compounds suppress phosphorylation in the submicrogram molecular range.In addition, in the in vitro calibrating of adopting mastocyte, 16 compounds all suppress Syk and (for example swash Enzyme matrix, PLC-γ 1, LAT) and Syk swash protein (for example JNK, p38, Erk1/2 and the PKB in Enzyme downstream, phosphorylation at the trial), rather than in cascade, occupy the protein (for example Lyn) that Syk swashs the Enzyme upstream.The phosphorylation of Lyn matrix be not tested 2, the 4-pyrimidinediamine compounds suppresses.And with regard to following compound, we observe them and in biochemical assays Syk are swashed the active restraining effect (IC of Enzyme 503 within the 1650nM scope) and they in mastocyte, Fc ε R1 is mediated the restraining effect (IC of degranulation 50Within 30 to 1850nM scopes) exist height correlation to concern between the two: R950373, R950368, R921302, R945371, R945370, R945369, R945365, R921304, R945144, R945140, R945071, R940358, R940353, R940352, R940351, R940350, R940347, R940343, R940338, R940323, R940290, R940277, R940276, R940275, R940269, R940255, R935393, R935372, R935366, R935310, R935309, R935307, R935304, R935302, R935293, R935237, R935198, R935196, R935194, R935193, R935191, R935190, R935138, R927050, R926968, R926956, R926931, R926891, R926839, R926834, R926816, R926813, R926791, R926782, R926780, R926757, R926753, R926745, R926715, R926508, R926505, R926502, R926501, R926500, R921218, R921147, R920410, R909268, R921219, R908712, R908702.
Therefore, the present invention's 2, the activity of 4-pyrimidinediamine compounds also can be swashed in active biochemistry of Enzyme or the cell calibrating by Syk and confirm.Let us is seen following Fig. 2 again, and in the Fc of mastocyte and basophilic leukocyte ε RI signal cascade, Syk swashs Enzyme to LAT and PLC-γ 1 phosphorylation, causes (especially) degranulation.These activity all can be in order to confirm the present invention's 2, the activity of 4-pyrimidinediamine compounds.In one embodiment, by make single from Syk swash Enzyme or its active fragments and swash in the presence of the Enzyme matrix (for example known in signal cascade, be the synthetic peptide or the protein of Syk phosphorylation) and 2 at Syk, the contact of 4-pyrimidinediamine compounds, whether assessment Syk swashs Enzyme and the matrix phosphorylation is confirmed activity.Perhaps calibrating also can be undertaken by the cell of the sharp Enzyme of performance Syk.This type of cell can endogenous mode show the sharp Enzyme of Syk, also can make the sharp Enzyme of its performance reorganization Syk by processing.These cells also can show Syk and swash Enzyme matrix.Be suitable for carrying out the cell of this affirmation calibrating, and the method for the suitable cell of processing is conspicuous for this operator that is familiar with.Be suitable for confirming 2, the object lesson of active biochemistry of 4-pyrimidinediamine compounds and cell calibrating will partly be introduced at example.
Usually in fact, the compound as the sharp Enzyme inhibitor of Syk will present and the active relevant IC of the sharp Enzyme of Syk 50, for example Syk swashs Enzyme synthetic substrate or endogenous matrix is carried out the ability of phosphorylation, occupy about 20 μ M or the following scope of 20 μ M in calibrating or the cell calibrating in vitro.This skill person that is familiar with should understand and shows low IC 50The compound of value (for example in 10 μ M, 1 μ M, 100nM, 10nM, 1nM and even lower scope) has useful especially value.
6.5 purposes and composition
As discussed above described, active compound of the present invention plays restraining effect to the signal cascade (for example Fc ε RI and Fc γ RI signal cascade) of Fc acceptor (the Fc acceptor that especially comprises the γ homodimer), the release that causes the chemical mediator in (especially) cell by degranulation or other process with synthesize.Also as discussed above described, active compound also is that strong Syk swashs the Enzyme inhibitor.Because these activity, active compound of the present invention can be used for various in vitro, in vivo and under (ex vivo) situation that exsomatizes, regulate or suppress Syk and swash Enzyme, Syk and swash Enzyme signal cascade, cascade of Fc receptor signal of figure therein and the biological respinse that is caused by this class signal cascade.For example, in one embodiment, compound can be used in vitro or in vivo and suppress Syk and swash Enzyme in any Syk of presenting almost swashs the cell type of Enzyme.They also can be used to regulate Syk and swash the Enzyme signal transduction cascade of figure therein.This kind Syk relies on the signal transduction cascade and includes, but is not limited to Fc ε RI, Fc γ RI, Fc γ RIII, BCR and the cascade of integral protein signal transduction.Compound also can be used in vitro or in vivo, suppresses cell or the biological respinse that the cascade of this kind Syk dependence signal transduction causes to regulate with (especially).This type of cell or biological respinse include, but is not limited to that respiratory organs breaks, cell adhesion, cell degranulation, cell are spread, cell migration, cell are gathered, phagolysis, cytokine is synthetic and release, cell maturation and Ca 2+Running.Importantly, compound suppresses Syk in vivo and swashs Enzyme as a kind of treatment means, swashs the disease that Enzyme is fully movable or part mediates to treat or to prevent by Syk.Can swash the disease mediated non-limitative example of Enzyme with the Syk of this compounds for treating or prevention will more go through hereinafter.
In another embodiment, active compound can be used as a kind of treatment means, regulate or suppress Fc receptor signal cascade and the degranulation of Fc ε RI with Fc γ RI mediation, with treat or prevent with the release of this type of Fc receptor signal cascade or degranulation chemical mediator or synthesize feature, caused and relative disease.This type of treatment can be offerd medicine under animal doctor's occasion on one's body the animal, and the mankind also can offer medicine.To discharge by this class chemical mediator, synthetic or degranulation is a feature, caused or relevant by it with it, the disease of therefore available this active compound treatment or prevention comprises (only as an example, and also unrestricted) atopy or the super quick or anaphylaxis of supersensitivity, allergy is (as anaphylaxis conjunctivitis, allergic rhinitis, atopic asthma, atopic dermatitis and food anaphylaxis), low scar is (as scleroderma, the proliferative fibrosis, keloid, operation back scar, pulmonary fibrosis, vasospasm, migraine, reperfusion injury and back myocardial infarction), with the disorganization diseases associated (as chronic obstructive pulmonary disease (COPD), cardiac branch trachitis (cardiobronchitis) and back myocardial infarction), with the tissue inflammation diseases associated (as the irritable bowel syndrome, spastic colon and inflammatory bowel disease), inflammation and scabbing.
Except treating the above countless disease, cytology and animal experiment data validation are described herein 2, and the 4-pyrimidinediamine compounds also can be used for treatment or prevention autoimmune disease, and the various symptoms that interrelate with this type of disease.Can be with 2, the autoimmune disease of 4-pyrimidinediamine compounds treatment or prevention generally includes because the disease that endogenous and foreign immunologic source or antigenic body fluid and tissue injury that cell-mediated reaction causes are brought.This type of disease often is called the disease that nonallergic (being II type, III type and IV type) allergy causes.
As mentioned above, the allergy of I type normally after the specific exogenous antigen of contact the release of mastocyte and basophilic leukocyte have pharmacological active substance (as histamine) and cause.As mentioned above, this type of I type is reflected at figure in the numerous disease, comprises allergic asthma, allergic rhinitis etc.
II type allergy (claiming cell-cytotoxic reaction, cell complement-dependent solubilizing reaction, cell induction allergy again) be immunoglobulin (Ig) and cell or tissue antigen partly or with the antigen of having combined closely with cell or tissue or the result of haptens reaction.Disease common and that the allergy of II type interrelates includes, but is not limited to autoimmune hemolytic anemia, erythroblastic fetalis (erythroblastosis fetalis) and GoodpastureShi disease (glomerulonephritis spitting of blood syndrome).
III type allergy (claiming the toxicity compound substance super quick, the super quick or immunocomplex matter allergy of soluble complex matter again) be solubility circulating antigen-immunoglobulin complex matter in blood vessel or in the tissue deposition cause, and be attended by acute inflammatory reaction in immunocomplex matter deposition site.The non-limitative example of standard type III type reaction disease comprises: glomerulonephritis, multiple sclerosis and the bullous pemphigoid of ArthusShi reaction (arthus' response), rheumatoid arthritis, serum sickness, whole body property lupus erythematosus, some type.
IV type allergy (often being called cellularity, cell-mediated property, tardy property or the allergy of tuberculin type) is that the T-lymphocyte of sensitization causes owing to contact a specific antigen.The indefiniteness that is listed as IV type reaction disease comprises for example: contact dermatitis and allograft rejection.
The autoimmune disease that interrelates with above any nonallergic allergy is all available of the present invention 2, treatment of 4-pyrimidinediamine or prevention.It is the autoimmune disease of feature that these methods are suitable for being used for treating or preventing with one organ or single cell type by it, include, but is not limited to struma lymphomatosa, autoimmune hemolytic anemia, the autoimmune atrophic gastritis of pernicious anemia, the autoimmune encephalomyelitis, Zi body Mian Yi testis inflammation, Goodpasture Cotard (being glomerulonephritis spitting of blood syndrome), the autoimmune thrombopenia, sympathetic ophthalmia, myasthenia gravis, GravesShi disease (being Exophthalmus goiter), primary biliary cirrhosis, chronic active hepatitis, ulcerative colitis and membranous glomerulopathy become, and often be referred to as the general autoimmune disorders, include, but is not limited to whole body property lupus erythematosus, rheumatoid arthritis, the Sjogren Cotard (is a Sjogren's disease, mouth xerophthalmia scheorma syndrome), (the still undefined triad of a kind of cause of disease comprises urethritis to the Reiter Cotard, conjunctivitis and sacroiliitis), polymyositis-dermatomyositis, systemic scleroderma, polyarteritis nodosa, multiple sclerosis and bullous pemphigoid.
This skill person that is familiar with will understand that above listed autoimmune disease has many serious symptoms that are attended by, if can alleviate these symptoms, even if can not effect a radical cure the autoimmune disease that causes these symptoms, also still has great therapeutic value.These symptoms and potential disease condition thereof are many to be monocyte Fc γ R signal cascade activated consequences.Because of the present invention 2, the 4-pyrimidinediamine is the strong inhibitor of this kind Fc γ R signal in monocyte and other cell, these methods will and/or be applied aspect the countless ill symptomses of listed autoimmune disease more than preventing in treatment.
As a specific examples, rheumatoid arthritis (RA) is causing swelling, pain and joint stiffness that whole body involves and tenderness in typical case.The feature of rheumatoid arthritis is the synovial membrane chronic inflammation, has filled up intensive lymphocyte in interior.Synovial membrane under normal circumstances is one deck cell thickness, nowadays becomes and fills up cell, forms the lymphoid tissue form, comprising dendritic cells, T-cell, B-cell, NK cell, phagocytic cell and plasma cell bunch.The destruction that this process and countless immunopathogenesis mechanism (comprising the formation of antigen-immunoglobulin (Ig) complex body) finally cause the joint integrity is caused deformity, permanent afunction and joint and bone erosion on every side thereof.The above method can be used for treating or improving a kind of, some kinds and even all these symptoms of rheumatoid arthritis.Therefore, with regard to rheumatoid arthritis, alleviate or improve usually and during any symptom that rheumatoid arthritis interrelates when obtaining, can think that aforesaid method has result of treatment (it generally defines and will be discussed further below), no matter treat the internal circulating load that whether has reached treatment potential rheumatoid arthritis itself simultaneously and reduced Rheumatoid factors, polyclonal (" RF ").
As another specific examples, the classical symptom of whole body property lupus erythematosus (" SLE ") is: fever, arthralgia (arthrodynia), sacroiliitis, serositis (pleuritis or pericarditis).With regard to whole body property lupus erythematosus, when obtaining when alleviating or improving usually any symptom that interrelates with whole body property lupus erythematosus, can think that aforesaid method has result of treatment, no matter whether treatment has reached the purpose of treatment potential whole body property lupus erythematosus itself simultaneously.
As another specific examples, every symptom of multiple sclerosis (" MS ") disables the patient, comprising: disturb its visual acuity, cause ghost image, disturb and walk with trick motor functions such as hands, cause gatism, spasm to twitch and insensitive (sense of touch, pain, to temperature sensitivity).With regard to multiple sclerosis,, can think that aforesaid method has result of treatment, no matter whether treatment has reached the purpose of treatment potential multiple sclerosis itself simultaneously when improving or slow down above-mentioned any one or multinomial disabling during the symptom process.
When being used for the treatment of or prevent this type of disease, active compound is with single form, a kind of or some kinds of active compound mixed forms or can be used for treating this type of disease with other and the medicament mixed of relevant symptom or the form dispensing of combination with disease.Active compound also can with can be used for treating for example steroid of other disease or sick medicament, membrane stabilizer, the 5LO inhibitor, synthetic and the acceptor inhibitor of leukotriene, the IgE similar shape changes or the IgE synthetic inhibitor, the IgG similar shape changes or IgG synthesizes, β-activator, class Trypsin Enzyme inhibitor, Asprin, the COX inhibitor, methotrexate (methotrexate), anti-tumor necrosis factor medicine (anti-TNF drugs), retuxin, the PD4 inhibitor, the p38 inhibitor, PDE4 inhibitor and antihistaminic agent (above listed only be some examples) mix or the combination dispensing.Active compound can prodrug itself form or comprise active compound or the dispensing of the compound medicine form of prodrug.
The medicinal components that comprises active compound of the present invention (or its prodrug) can be made grinding, emulsification, encapsulate, carry secretly or the manufacturing of lyophilization method by conventional mixing, dissolving, granulation, coated tablet (dragee).This type of composition can use one or more physiologically acceptable carriers, thinner, vehicle or auxiliary agent allotment in the usual way, to help that the effective ingredient processing and preparing is become the preparation that can be used as drug use.
As previously mentioned, active compound or prodrug can itself, perhaps allocate as pharmaceutical compositions with the form of its hydrate, solvate, N-oxide compound or pharmaceutically acceptable salt.In typical case, free bronsted lowry acids and bases bronsted lowry is easier is dissolved in the aqueous solution than corresponding for this type of salt, but also can prepare the salt that solubleness is lower than corresponding free bronsted lowry acids and bases bronsted lowry.
Pharmaceutical compositions of the present invention is applicable to any types of administration almost, comprise as local, eye drips, in oral, the buccal, whole body, nose, injection, through skin, internal rectum, intravaginal etc., or be suitable for sucking or the form of insufflation dispensing.
With regard to topical administration, (respectively) active compound or prodrug can be formulated into solution, gel, ointment, creme, suspension etc., as well-known in the industry.
Whole body dispensing prescription comprises the prescription that is designed to drug administration by injection, in for example subcutaneous, intravenously, intramuscular, the sheath or peritoneal injection administration person, and is designed to through skin, and per mucous membrane is sucked or through lung dispensing person.
The preparation that is fit to injection comprises sterile suspensions, solution or the emulsion of (respectively) active compound in water or oily matchmaker's liquid.Also can comprise blender in the composition, for example suspend, stablize and/or dispersion agent.The packaged form of injection can be single dose form (a for example ampoule), also can adopt the multi-dose container form, and can comprise sanitas.
Perhaps injection can also provide by powder type, and dissolves it again with suitable carrier (including but not limited to aseptic apirogen water, damping fluid, glucose solution etc.) before use.For this purpose, (respectively) active compound can be with any on record in the industry technology drying (for example low-pressure refrigeration drying), and dissolving more before use.
For per mucous membrane dispensing preparation, can adopt in the prescription to be fit to penetrate the suitable permeate agent of desiring permeability barrier.This type of permeate agent is on record in the industry.
With regard to oral administration, pharmaceutical compositions can conventional method be prepared as such as lozenge, tablet or capsule form, can adopt acceptable vehicle in the preparation, for example tamanori (as: pregelatinization W-Gum, polyethylene Pyrrolizidine ketone or Vltra tears), weighting agent (as: lactose, Microcrystalline Cellulose or secondary calcium phosphate), lubricant ((as: Magnesium Stearate, talcum or silica), collapse powder (as: yam starch or sodium starch glycollate) or wetting agent (as: sodium lauryl sulfate).Sugar-coat, film or enteric coating are wrapped by on record method in the industry in this type of tablet outside.The compound that is particularly suitable for oral administration medicine supplying comprises compound R 940350, R935372, R935193, R927050 and R935391.
The liquid formulation of oral administration can be made into the form such as elixir, solution, syrup or suspension, also can be made into drying products, make the patient before taking medicine to take after the suitable solvent allotment of water or other.This type of liquid formulation can prepare by ordinary method, and can adopt pharmaceutically acceptable additive, for example suspension agent (as sorbitol syrups, derivatived cellulose or hydrogenation edible-fat), emulsifying agent (as Yelkin TTS or kordofan gum), non-aqueous solvent (as, Prunus amygdalus oil, oily ester, ethanol, cremophore (the solubilizing agent series that BASF AG produces) or fractionation rape oil etc.) and sanitas (as methyl or propyl para-hydroxybenzoate or Sorbic Acid).This type of preparation also can comprise buffering salt, sanitas, correctives, tinting material and the sweeting agent of appropriate amount.
The preparation of oral administration can suitably be allocated, and makes its controlledly release of active compounds or prodrug, as well-known.
With regard to the oral cavity included the clothes administration, this type of composition can be prepared the form that becomes tablet or lozenge according to a conventional method.
With regard to per rectum and transvaginal administration path, (respectively) active compound can be formulated into solution (being used for retention enema) and contains cocoa butter or the suppository or the ointment of conventional suppository bases such as other glyceryl ester.
With regard to intranasal administration or with regard to suction or insufflation administration, (respectively) active compound or prodrug can be sent with the aerosol spray form in pressurized package or atomizer easily with suitable sprays (for example Refrigerant 12, trichlorofluoromethane, dichloro tetrafluoro ethane, fluorocarbon, carbonic acid gas or other suitable gas).With regard to the pressurized aerosol spraying, dose unit can calculate by the metering valve that specified amount is provided.Be used for the capsule of sucker or insufflator and the powdered mixture that cartridge case (as capsule and the cartridge case of being made by gelatin) can be formulated into inclusion compound and suitable powder matrix such as lactose or starch.
An object lesson that is suitable for carrying out with commercially available nose inner sprayer unit the aqueous suspension liquid formula of interanasal administration comprises following composition: active compound or prodrug (0.5-20 mg/ml), benzalkonium chloride (benzalkonium chloride, 0.1-0.2 mg/ml), Polysorbate 80 ( 80; 0.5-5 mg/ml), the carboxymethyl dimension plain sodium of dimension or Microcrystalline Cellulose (1-15 mg/ml), phenylethyl alcohol (1-4 mg/ml) and glucose (20-50 mg/ml).The pH value of last suspension is adjustable to the scope from about pH5 to pH7, and typical pH value is about 5.5.
Be suitable for suction to the specific examples of the waterborne suspension of The compounds of this invention (especially compound R 921218) dispensing comprise 1-20 mg/ml compound or prodrug, 0.1-1% (volume/volume) Polysorbate 80 (
Figure A20091000677100882
80), 50mM Citrate trianion and 0.9% sodium-chlor.
When being used for eye drip, (respectively) active compound or prodrug can be modulated into the solution that is suitable for eye drip, emulsion, suspension etc.It is various that to be suitable for carrying out the medium of intraocular dispensing in the field of business to compound be well-known.Some are concrete, non-limitative example can be referring to United States Patent (USP) the 6th, 261, No. 547, United States Patent (USP) the 6th, 197, No. 934, No. the 6th, 056,950, United States Patent (USP), United States Patent (USP) the 5th, 800, No. 807, No. the 5th, 776,445, United States Patent (USP), United States Patent (USP) the 5th, 698, No. 219, No. the 5th, 521,222, United States Patent (USP), United States Patent (USP) the 5th, 403, No. 841, No. the 5th, 077,033, United States Patent (USP), United States Patent (USP) the 4th, 882, No. the 4th, 738,851, No. 150 and United States Patent (USP).
With regard to the prolongation of effect dispensing, (respectively) active compound or prodrug can be modulated for slowly-releasing (depot) preparation of implanting or intramuscularly is offerd medicine.Active ingredient can be with suitable polymeric material or hydrophobic substance (as: as acceptable finish or emulsion) or ion exchange resin or microsolubility derivative (as slightly soluble salt) preparation.Perhaps also can adopt make become paster or circular adhesive plaster through the slow release of active compounds of skin administration system, absorb for transdermal penetration.For this purpose, penetration enhancers can be in order to help the transdermal penetration of (respectively) active compound.Suitably can be referring to as United States Patent (USP) the 5th, 407 through the description of skin absorption adhesive patch, No. 713, United States Patent (USP) the 5th, 352, No. 456, United States Patent (USP) the 5th, 332, No. 213, No. the 5th, 336,168, United States Patent (USP), United States Patent (USP) the 5th, 290, No. 561, United States Patent (USP) the 5th, 254, No. 346, No. the 5th, 164,189, United States Patent (USP), United States Patent (USP) the 5th, 163, No. 899, No. the 5th, 088,977, United States Patent (USP), United States Patent (USP) the 5th, 087, No. the 5th, 008,110, No. 240, United States Patent (USP) and United States Patent (USP) the 4th, 921, No. 475.
Also can use other drug delivery system in addition.Liposome and emulsion are the well-known examples that can be used for sending (respectively) active compound or prodrug.Also can use some organic solvent, for example methyl-sulphoxide (DMSO) has bigger toxic cost but often will pay.
This type of pharmaceutical compositions (when needed) can be packaged as the packing that comprises one or many dosage effective ingredient or the box-like formula of adjusting.Packing can comprise such as tinsel or plastics film, for example Blister Package.Can have the instructions of taking explanation on packing or the adjustment box.
6.6 effective dose
(respectively) of the present invention active compound or prodrug, or its composition, be used in usually reach be intended to reach result's significant quantity, the effective required dosage of the disease specific that will treat of treatment or prevention for example.(respectively) compound can be used for the therapeutic administration reaching the expection result of treatment, or preventive administration is to reach preventive effect.Result of treatment refer to effect a radical cure or improve the potential disease of intending treatment with/or radical cure or improvement a kind of or some kind symptoms relevant with potential disease, sensuously or on the state of an illness making moderate progress thereby patient report is said, though patient still suffers from this potential disease.For example, a compound is offerd medicine to suffering from patient hypersensitive, be not only in potential anaphylaxis and obtain radical cure or when improving, and be that the severity of symptom relevant with allergy the conscious contact allergy of patient is former after alleviates or the time length shortening, all should be considered as obtaining result of treatment.As another example, with regard to treatment asthma, if breath state makes moderate progress after asthma attack, perhaps asthma attack frequency reduces, and severity alleviates, and all should be considered as obtaining result of treatment.Result of treatment also comprises the progress of preventing or slowing down disease, no matter whether symptom makes moderate progress.
With regard to preventive administration, can give this compound to there being the patient who suffers from one of above-mentioned disease to throw.For example,, before medicine is given in throwing, can throw earlier and give this compound, to avoid or to improve the anaphylaxis that may occur this medicine if whether the patient is irritated in confused situation to a certain concrete medicine.Perhaps also can be used as preventive dosage, to avoid diagnosing the patient's who suffers from potential disease paresthesia epilepsy.For example can before the expection contact allergy is former, give this compound to allergy patient's throwing.Compound also can carry out preventive dosage to the healthy individual who contacts the anaphylactogen that can cause one of above-mentioned disease repeatedly, with prophylactic outbreak.For example can throw and give this compound, in order to avoid allergic symptom appears in this this individual to the healthy people who contacts the known anaphylactogen that can cause allergic reaction (as: latex) repeatedly.Compound also can be thrown to the asthma patient and be given this compound before participate in triggering the activity of asthma attack in advance, to avoid the outbreak of asthma fully, perhaps alleviated its severity.
The dosage of compound administration depends on multiple factor, comprise mode, be used for preventive or treatment, intend bioavailability of severity, patient's age of treatment disease and body weight, particular active compounds or the like as the disease specific of intending treatment, administration.Which kind of effective dose decision adopts fully within this operator's the limit of power of being familiar with.
Effective dose can be assessed from vitro examining and determine at first.For example, the preliminary dosage that is used in animal can be formulated into the IC that the blood circulation that makes this active compound or serum-concentration reach this compound 50Or more than, as in vitro calibrating (for example in vitro CHMC calibrating or BMMC calibrating, and other in the partly described in vitro calibrating of example) shown in.Calculate with reference to the bioavailability of specific compound and to reach the required dosage of this kind blood circulation or serum-concentration fully within this operator's the limit of power of being familiar with.As guide, the reader can be referring to Fingl ﹠amp in Goodman and Gilman ' s ThePharmaceuticalBasis of Therapeutics (pharmacological basis of the treatment) book; Woodbury, " General Principles (general rule) " chapter (Chapter 1, pp.1-46, latest edition, Pagamonon Press) and wherein listed reference.
Preliminary dosage also can be assessed according to data (for example animal model) in vivo.Can be used for test compound treatment or prevent the animal model of drug effect of above-mentioned various diseases in the field of business on record.Can be about the description of super quick or anaphylactoid suitable animal model referring to Foster, 1995, Allergy 50 (21Suppl): 6-9, discussion 34-38, Tumaset al, 2001, J.Allergy Clin.Immunol.107 (6): 1025-1033.The suitable animal model of allergic rhinitis is described can be referring to Szelenyi et al, and 2000, Arzneimittelforschung 50 (11): 1037-42; Kawaguchi (Kawaguchi) et al, 1994, Clin.Exp.Allergy 24 (3): 238-244 and Sugimoto (China fir is originally) et al, 2000, Immunopharmacology 48 (1): 1-7.Describing when animal model that anaphylaxis conjunctivitis is suitable can be referring to Carreras etal, and 1993, Br.J.Ophthalmol.77 (8): 509-514; Saiga (congratulate in the west) et al, 1992, Ophthalmic Res.24 (1): 45-50; With Kunert et al, 2001, Invest.Ophthalmol.Vis.Sci.42 (11): 2483-2489.The suitable animal model of systemic mastocytosis is described can be referring to O ' Keefe et al, 1987, J.Vet.Intern.Med.1 (2): 75-80 and Bean-Knudsen et al, 1989, Vet.Pathol.26 (1): 90-92.The suitable animal model of high IgE syndrome is described can be referring to Claman et al, 1990, Clin.Immunol.Immunopathol.56 (1): 46-53.The suitable animal model of B cell woods crust knurl is described can be referring to Hough et al, 1998, Proc.Natl.Acad.Sci.USA95:13853-13858 and Hakim et al, 1996, J.Immunol.157 (12): 5503-5511.(describing as the suitable animal model of atopical dermatitis, atopic eczema and atopic asthma can be referring to Chan et al for atopic disorder, 2001, J.Invest.Dermatol.117 (4): 977-983 and Suto (palpus rattan) et al, 1999, Int.Arch.Allergy Immunol.120 (Suppl1): 70-75.Usually this skill person that is familiar with can utilize this type of information to do conventional change, obtains to be suitable for human dosage.Other appropriate animal model will partly be described at example.
The scope of the exemplary value scope of dosage from about 0.0001 or 0.001 or 0.01 milligram/kg/day to about 100 milligrams/kg/day, but can be higher or lower than this scope, depend on multiple factor, comprise compound activity, its bioavailability, administering mode and above-mentioned other factors.The numerical value of dosage and the timed interval of medication can vary with each individual, to keep the plasma concentration that treatment or the required compound of preventive effect are provided.For example, but the medication of compound per week is once, per week medication several times (for example, every other day), once a day or every day several times, depend on multiple factor, comprise administration mode, intend the disease specific of treatment and prescriber's judgement.In the situation of local application or selection picked-up, for example when region of interest local application, effective partial concn of (respectively) active compound may be irrelevant with plasma concentration.This skill person that is familiar with can draw best local application's dosage fully, and need not pass through unnecessary experimentation.
(respectively) compound if can provide treatment or preventive effect, and there is no great toxicity, and is ideal naturally.The toxicity available standards pharmacology rules of (respectively) compound are measured.The dosage of toxicity and treatment (or prevention) effect is than being therapeutic index.The compound that therapeutic index is high is comparatively desirable.
This invention was described as above already.Following example only illustrates, and unrestricted meaning.
7. example
7.1 it is synthetic to pressing flow process (I)-(V) Synthetic 2, parent material that the 4-pyrimidinediamine compounds is useful and intermediate
Prepared by the following method a series of to by flow process (I)-(V) Synthetic 2, the single replacements-PYRIMITHAMINE of parent material that the 4-pyrimidinediamine compounds is useful and N4-and the single replacements-4-pyrimidinediamine of N2-(list replacement nucleophilicity aromatics reacts (SNAR) product).Be suitable for the synthetic single condition that replaces nucleophilic aromatic reaction (SNAR) with 2-chloro-N4-(3,4-stretches the ethylenedioxy phenyl)-5-fluorine-based-4-PYRIMITHAMINE (R926087) is for exemplifying routine explanation.
Figure A20091000677100941
Figure A20091000677100951
Figure A20091000677100961
Figure A20091000677100971
Figure A20091000677100981
Figure A20091000677100991
Figure A20091000677101001
Figure A20091000677101011
Figure A20091000677101031
Figure A20091000677101041
Figure A20091000677101051
Figure A20091000677101061
Figure A20091000677101071
Figure A20091000677101081
Figure A20091000677101091
Figure A20091000677101111
Figure A20091000677101121
Figure A20091000677101131
Figure A20091000677101141
Figure A20091000677101151
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Figure A20091000677101171
Figure A20091000677101181
Figure A20091000677101191
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Figure A20091000677101211
Figure A20091000677101221
Figure A20091000677101231
Figure A20091000677101241
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7.5 of the present invention 2, the 4-pyrimidinediamine suppresses the receptor-mediated degranulation of Fc ε RI
A series ofly all show of the present inventionly 2 to cultivate cells calibrating that human mast cell (CHMC) and mouse bone marrow cells derived cell (BMMC) carry out, the 4-pyrimidinediamine compounds has the ability that inhibition IgE induces degranulation.The inhibition of degranulation is weighed by the burst size of measuring particle specific factor class Trypsin Enzyme, histamine and hexosamine Enzyme under low cell density and high-cell density.The release of lipid medium and synthetic restraining effect system are by measuring the release assessment of leukotriene LTC4, and the release of cytokine and synthetic suppress system by the Quantitative Monitoring to TNF-α, IL-6 and IL-13.Class Trypsin Enzyme and hexosamine Enzyme system are described quantitative with fluorogenic substrate by each example respectively.Histamine, TNF α, IL-6, IL-13 and LTC4 system use following commercially available ELISA to detect box (calibrating of Enzyme connection immunosorption is with detecting box) quantitatively: histamine (Immunotech #2015, Beckman Coulter), TNF α (Biosource#KHC3011), IL-6 (Biosource #KMC006), IL-13 (Biosource #KHC0132), LTC4 (CaymanChemical #520211).The experimental program of various calibratings is provided in down.
7.5.1 the cultivation of human mast cell and basophilic leukocyte
Mankind's hypertrophy and basophilic leukocyte are cultivated (also please referring to related U.S. patent application case sequence number 10/053, the 355 described method of applying for January 8 calendar year 2001, its content is disclosed in herein in the quoted passage mode) by the following stated from the negative progenitor of CD34-.
7.5.5.1STEMPRO-34 the preparation of perfect medium
For preparation STEMPRO-34 perfect medium (" CM "), with 250 milliliters of STEMPRO-34 TMSerum free medium (" SFM "; GibcoBRL, catalog number (Cat.No.) 10640) add in the filter flask.To wherein adding 13 milliliters of STEMPRO-34 nutritional supplements (" NS "; GibcoBRL, catalog number (Cat.No.) 10641) (preparation method's description is as follows in detail).The NS container washes with about 10 milliliters of serum free mediums (SFM), and this scavenging solution is added in the filter flask.Adding 5 milliliters of L-Grains amine amide (200mM; Mediatech, catalog number (Cat.No.) MT 25-005-CI and 5 milliliters of 100X penicillin/streptomycin (" pen-strep "; HyClone, catalog number (Cat.No.) SV30010) afterwards, add SFM and make volume reach 500 milliliters, and filtering solution.
The maximum variable factor of preparation perfect medium when (CM) is the serum free medium (SFM) that adds melting and blending means of nutritional supplement (NS) before.Nutritional supplement (NS) should melt in 37 ℃ water-bath, and carries out eddy flow and stir, rather than whirling motion is stirred or concussion, till it becomes solution fully.When eddy flow stirs, note observing and have or not still undissolved lipid floating on it.If there is lipid floating, nutritional supplement (NS) outward appearance is inhomogeneous, then should put back in the water-bath, repeats the eddy flow whipping procedure, when its outward appearance is even till.Sometimes this composition is dissolved among the solution immediately, will then can not dissolve sometimes through several eddy flow stirring cycle sometimes at all.If through several hrs, nutritional supplement (NS) still fails to dissolve, and it is abandoned, and melts a new nutritional supplement in addition.Quality looks uneven that nutritional supplement (NS) should not use after melting.
7.5.1.2CD34+ the propagation of cell
Use following substratum and the method less CD34-positive (CD34+) population of cells (1-5 * 10 of quantity when initial 6Individual cell) breeds to the negative progenitor of the bigger CD34-of quantity (about 2-4x10 9Individual cell).CD34+ cell (from same contributor) is to derive from Allcells company (California Berkeley).Because the common CD34+ cell that provides of Allcells company is in quality with quantitatively be the fluctuation that has to a certain degree, should be with in cell transfer to the 15 milliliter tapered tube of newly sending, and in substratum (CM), breed to 10 milliliters before use.
At the 0th day, viable cell (phase-bright (life is flourishing) phase cell) is carried out cell counting, and with the rotation of 1200 rev/mins of speed, cell is rolled be spherolite.Make cell be suspended in again and contain 20 millimicro grams per milliliter recombinant human STEM CELL FACTOR (" SCF "; Peprotech, Catalog No.300-07) and in the substratum (" CM/SCF/flt-3 substratum ") of the human flt-3 ligands of 20 millimicro grams per milliliters (Peprotech, Catalog No.300-19), density is 275,000 cells/ml.About the 4th day or the 5th day, carry out cell counting, check the density of culture, and add fresh " CM/SCF/flt-3 " substratum, be 275,000 cells/ml with the density dilution.About the 7th day, culture is transferred in the sterile glass tube, carry out a cell counting again.Pair cell centrifugation under 1200 rev/mins of rotating speeds, and to suspend again in fresh " CM/SCF/flt-3 " substratum be the density of 275,000 cells/ml.
This circulation repeated since the 0th day, repeated 3-5 time altogether in the phase in propagation.
Bigger in culture quantity, in a plurality of culturing bottles, cultivate, and when needing resuspending, should before carrying out cell counting, be integrated into the content in all culturing bottles in the single container.Can draw a cell counting accurately like this, and guarantee processing the relative uniformity of whole population of cells.Before merger, not contamination phenomenon be arranged at each culturing bottle of microscopically individual inspiration, in order to avoid cause the pollution of whole population of cells.
Between 17-24 days, culture can begin to enter the paracme (that is to say that sum is the necrocytosis of 5-10% approximately), can not breed rapidly as before again.During this period should every day pair cell monitor because cultivate fall flat and might within short 24 hours, take place.As falling into a decline, the pair cell counting, rotation separated 15 minutes under 850 rev/mins of rotating speeds, with the density of 350,000 cells/ml resuspending again in " CM/SCF/flt-3 " substratum, so that inducing culture thing cell carries out once to twice division again.Every day, pair cell monitored, to prevent to cultivate failure.
15% above necrocytosis is arranged in the progenitor substratum, and when in substratum, beginning some chips to occur, can begin the negative progenitor of CD34-is broken up.
7.5.1.3 the negative progenitor of CD34-is divided into the mucous membrane mastocyte
Carry out the operation of subordinate phase, the negative progenitor of the CD34-of propagation is converted into the mucous membrane mastocyte of differentiation.The cultivation human mast cell of these mucous membranes (" CHMC ") is available from by isolating CD34+ cell in the Cord blood, and handles as stated above, makes its propagation become the negative progenitor of CD34-group.For obtaining the negative progenitor of CD43-, the resuspending circulation of substratum is identical with the above, and just culture is implanted with the density of 425,000 cells/ml, and adds 15% substratum in addition at the 4th or the 5th day, and does not carry out cell counting.In addition, the cytokine composition of substratum is also revised, and makes it comprise SCF (human STEM CELL FACTOR) (200 millimicro grams per milliliter) and recombinant human interleukin-6 (recombinant human interleukin-6 (IL-6)) (200 millimicro grams per milliliters; Peprotech, Catalog No.200-06 is formulated as the substratum of 100 mcg/ml again in aseptic 10mM acetate) (" CM/SCF/IL-6 substratum ").
I and II stage continue about 5 week.Some necrocytosiss were obviously arranged in the culture during 1-3 week, and some chips occur; During 2-5 week, account for the not very big a part of culture of per-cent for some time and no longer be in suspended state, but be attached to the surface of culture vessel.
As at I in the stage, when each round-robin the 7th day suspends again to culture, should be before carrying out cell counting being integrated in the single container of content in all culturing bottles, to guarantee to the relative uniformity of the processing of whole population of cells.Before merger, not contamination phenomenon be arranged at each culturing bottle of microscopically individual inspiration, in order to avoid cause the pollution of whole population of cells.
During each culturing bottle of merger, about 75% of volume is transferred in the common container, in culturing bottle, stayed about 10 milliliters nutrient solution.Kowtow with the angle of acute angle in the culturing bottle side that remaining culture liq is housed and to hit, strike down attached to the cell on the bottle wall.Kowtowing once more and hit, come off fully then so that make attached to the cell on the bottle wall with kowtowing for the first time rectangular position, the place of hitting.
Culturing bottle is the miter angle several minutes of swaying, and then remaining culture liq is poured among the counting container.Cell is implanted in the bottle then earlier with 950 rev/mins of rotating speed rotations 15 minutes, implants 35-50 milliliter (density is 425,000 cells/ml) for every bottle.
7.5.1.4CD34-negative progenitor is divided into connective tissue mast cells
Propagation group by the above method prepares the negative progenitor of CD34-handles then, makes it form the short pancreas Enzyme positive (reticular tissue) phenotype of class Trypsin Enzyme/stomach.Institute's employing method is identical with above-mentioned mucous membrane mastocyte, but replaces IL-6 (human interleukin-6) with IL-4 (human interleukin-4) in substratum.The cell that is obtained has the feature of typical connective tissue mast cell.
7.5.1.5CD34-negative progenitor is divided into basophilic cell
By the propagation group of the negative progenitor of the described preparation of 7.5.1.3 CD34-, in order to form the propagation group of basophilic cell.The treatment process of CD34-negative cells is identical with the described treatment process of mucous membrane mastocyte, but replaces IL-6 (human interleukin-6) with IL-3 (Ro 24-7472/000-3) in substratum.
The activation of (7.5.2CHMC cultivation human mast cell) low cell density IgE (immunoglobulin E): the calibrating of class Trypsin Enzyme and LTC4 (leukotriene C)
For duplicating 96 hole U-shape base plate Enzyme targets (Costar 3799), the tubulin (MT) (137mM NaCl, 2.7mM KCl, the 1.8mM CaCl that comprise 2%MeOH and 1%DMSO (dimethyl sulfoxide (DMSO)) that are preparing 2, 1.0mM MgCl 2, 5.6mM glucose, 20mM Hepes damping fluid (pH value 7.4), 0.1% bovine serum albumin (A4503 of Sigma company)) in add the diluted chemical compound liquid or the control sample of 65 microlitres.Human mast cell (CHMC) be will cultivate and spherolite (980 rev/mins of rotating speeds, 10 minutes) and resuspending will be prepared in the tubulin (MT) of preheating.The cell that on each 96 hole Enzyme target, adds 65 microlitres.According to each specific cultivation human mast cell (CHMC) contributor's degranulation activity, loading capacity is the 1000-1500 cells/well.Mix four times, and cultivated 1 hour down at 37 ℃.In 1 hour nurturing period, preparation 6X anti-immunoglobulin (IgE) solution (the anti-human immunoglobulin of rabbit (IgE, 1 mg/ml, Bethyl Laboratories A80-109A), dilution is 1: 167 concentration in the MT damping fluid).6X anti-immunoglobulin E (IgE) solution that on each suitable Enzyme target, adds 25 microlitres, irritation cell.In unprovoked contrast Enzyme target hole, inject tubulin (MT) solution of 25 microlitres.Adding anti-immune protein E (IgE) back mixes twice.30 minutes nurturing period, examine and determine damping fluid (0.1MHepes damping fluid (pH value 7.5), 10% weight/volume glycerine, 10 μ M heparin (Sigma H-4898), 0.01%NaN with class Trypsin Enzyme in 1: 2000 ratio 3) dilution 20mM class Trypsin Enzyme matrix mother liquor ((Z-Ala-Lys-Arg-AMC 2TFA; Enzyme SystemsProducts, #AMC-246)).Centrifugal rotation Enzyme target is 10 minutes under 1000 rev/mins of rotating speeds, cell is rolled be spherulitic.25 microlitre supernatant liquids are transferred on the 96 hole black matrix Enzyme targets, and in each hole, added the class Trypsin Enzyme matrix solution of the new dilution of 100 microlitres.Enzyme target was at room temperature cultivated 30 minutes.Learn the optical density(OD) that reads 355 millimicrons/460 millimicrons of precision at spectrophotometer target readout instrument.
Also according to supplier's explanation, detect box (calibrating of Enzyme connection immunosorption is with detecting box) with suitable ELISA the supernatant liquid (extent of dilution is by each contributor population of cells experience is measured, so that the sample determination value remains within the scope of typical curve) through suitably dilution is carried out the quantitative assay of leukotriene C4 (LTC4).
(7.5.3CHMC cultivation human mast cell) high-cell density IgE (immunoglobulin E) activation: the calibrating of degranulation (class Trypsin Enzyme, histamine), leukotriene (LTC4) and cytokine (TNF α, IL-13)
Cultivate human mast cell (CHMC) in CM (perfect medium) with IL-4 (interleukin 4) (20 millimicro grams per milliliter), SCF (STEM CELL FACTOR) (200 millimicro grams per milliliter), IL-6 (interleukin-6) (200 millimicro grams per milliliter) and human IgE (immunoglobulin E) (CP 1035K, Cortx Biochem company, 100-500 millimicro grams per milliliter is because of from generation to generation different) sensitization 5 days.Carry out cell counting after the sensitization, roll and be spherulitic (1000 rev/mins of rotating speeds, 5-10 minute), and with 1-2 * 10 6The density of cells/ml is suspended in MT (tubulin) damping fluid again.In each hole, inject 100 microlitre cell suspending liquids and 100 microlitre diluted chemical compound liquid.Final medium liquid concentration is 0.5%DMSO (dimethyl sulfoxide (DMSO)).(5%CO under 37 ℃ 2) cultivated one hour.After with compound treatment 1 hour, resist-the IgE irritation cell with 6X.Mix the hole that is marked with cell, and allow Enzyme target (5%CO under 37 ℃ 2) cultivated one hour.Cultivate after 1 hour, cell is rolled be spherulitic (1000 rev/mins of rotating speeds, 10 minutes), in each hole, collect 200 microlitre supernatant liquids, note the not cell of dispellet shape.The supernatant liquid plate is placed on ice.For the time 7 hours step (vide infra) be that 1: 500 supernatant liquid carries out class Trypsin Enzyme calibrating to diluting.Cell granulations is suspended in 240 microlitres again to be contained in the perfect medium (CM) of 0.5%DMSO (dimethyl sulfoxide (DMSO)) and corresponding concentration compound.Cultivate human mast cell (CHMC) at 37 ℃ of (5%CO 2) cultivated 7 hours down.After the cultivation, cell rolled be particulate state (1000 rev/mins of rotating speeds, 10 minutes), from each hole, collect 225 microlitres, place under-80 ℃ of temperature, when ELISA calibrating (calibrating of Enzyme connection immunosorption) is ready till.According to supplier's explanation, carry out the ELISA calibrating with sample (extent of dilution is by each contributor population of cells experience is measured, so that the sample determination value remains within the scope of typical curve) through suitably dilution.
7.5.4 BMMC (bone marrow mast cell) high-cell density IgE (immunoglobulin E) activation: the calibrating of degranulation (hexosamine Enzyme, histamine), leukotriene (LTC4) and cytokine (TNF α, IL-6)
7.5.4.1WEHI-the preparation of conditioned medium
The WEHI-conditioned medium is 37 ℃ in humidification, temperature, contain 5%CO 2In the incubator of/95% air, (source: Mediatech, Hernandon VA) are aided with 10% heat inactivation foetal calf serum (source: FBS with Iscove ' s Modified Eagles Media substratum; JRH Biosciences, Kansas City, MO), the 2 mercapto ethanol of 50 μ M (source: Sigma, St.Louis, MO) and 100 international unit/ml penicillin-Streptomycin sulphate (source: Mediatech) cultivate mouse marrow monokaryon blood cell WEHI-3B cell (source: American Type Culture Collection, Rockville MD) obtains.Initial cell suspending liquid is implanted with 200,000 cells/ml density, then during two weeks every 3-4 days with 1: 4 ratio centrifugation.Skim acellular supernatant liquid, be divided into sample aliquot, place under-80 ℃ of temperature preserve standby.
The preparation of (7.5.4.2BMMC bone marrow mast cell) substratum
The composition of BMMC substratum comprises: 20% WEHI-conditioned medium, 10% heat inactivation FBS (foetal calf serum) (source: JHR Biosciences), 25mM HEPES, pH value 7.4 (source: Sigma), 2mM L-glu famine (source: Mediatech), 0.1mM non-essential amino acid (source: Mediatech), 1mM Sodium.alpha.-ketopropionate (source: Mediatech), 50 μ M 2 mercapto ethanols (source: Sigma) (originate: Mediatech) be dissolved in RPMI 1640 substratum (source: Mediatech) with the penicillin-Streptomycin sulphate of 100 international unit/milliliters.Be preparation BMMC substratum, all compositions are joined among the aseptic IL filtration unit, before using, filter by 0.2 μ m strainer.
7.5.4.3 embodiment
Bone marrow mast cell (BMMC) in the BMMC substratum with 666x10 3The cell density of cells/ml the murine stem cell factor (murine SCF) (20 millimicro grams per milliliter) and the anti-dinitrophenol antibody of individual plant (monoclonal anti-DNP) (10 millimicro grams per milliliters, source: Clone SPE-7, Sigma #D-8406) sensitization is spent the night.After the sensitization, pair cell is counted, cell is rolled be spherulitic (1000 rev/mins of rotating speeds, 5-10 minute), and with 1-3 * 10 6The density of cells/ml is suspended in MT (tubulin) damping fluid again.Inject the cell suspending liquid of 100 microlitres and the diluted chemical compound liquid of 100 microlitres to each hole.Final matchmaker's liquid concentration is 0.5%DMSO (dimethyl sulfoxide (DMSO)).At 37 ℃ of (5%CO 2) cultivated 1 hour down.After 1 hour compound treatment, with 6X stimulator (60 millimicro grams per milliliter DNP-BSA (dinitrophenol-bovine serum albumin)) irritation cell.Mix the hole that is marked with cell, allow target at 37 ℃ of (5%CO 2) cultivated 1 hour down.Cultivate after 1 hour, cell is rolled be spherulitic (1000 rev/mins of rotating speeds, 10 minutes), in each hole, collect 200 microlitre supernatant liquids, note the not cell of dispellet shape, and with in the pipe of the cleaning of its transfer or on the 96 hole Enzyme targets.The supernatant liquid target is placed on ice.When being, in 4-5 hour the step (vide infra), carry out hexosamine Enzyme calibrating.Cell granulations is suspended in 240 microlitres again to be contained in the WEI-conditioned medium of 0.5%DMSO (dimethyl sulfoxide (DMSO)) and corresponding concentration compound.Bone marrow mast cell (BMMC) is at 37 ℃ of (5%CO 2) cultivated 4-5 hour down.After the cultivation, cell rolled be particulate state (1000 rev/mins of rotating speeds, 10 minutes), from each hole, collect 225 microlitres, place under-80 ℃ of temperature, when ELISA calibrating (calibrating of Enzyme connection immunosorption) is ready till.According to supplier's explanation, carry out the ELISA calibrating with sample (extent of dilution is by each contributor population of cells experience is measured, so that the sample determination value remains within the scope of typical curve) through suitably dilution.
Hexosamine Enzyme calibrating: inject 50 microlitre hexosamine Enzyme matrix (4-methyl cymenyls-N-ethanoyl-β-D-glucosaminide in each hole on solid black 96 hole assaying tables; 2mM).On hexosamine Enzyme matrix, add 50 microlitre bone marrow mast cell (BMMC) supernatant liquids, place 30 minute down, and in the time of 5,10,15 and 30 minutes, on spectrophotometer, read reading at 37 ℃.
7.5.5 basophilic leukocyte IgE or dirt mite activation: histamine discharges calibrating
The calibrating of basophilic leukocyte activation is with picking up from the dust mite allergy contributor and removing most erythrocytic human peripheral vessel blood with the dextran settlement action and carry out.Human peripheral vessel blood mixes with 3% Dextran T 500 in 1: 1 ratio, and makes RBC (red corpuscle) sedimentation 20-25 minute.Upper half part is with the D-PBS (Du Shi sterile phosphate buffer saline liquid) of 3 times of volumes dilution, and under room temperature (RT) with 1500 rev/mins of rotating speed centrifugal settling cells.The sucking-off supernatant liquid is with isopyknic MT (tubulin) damping fluid washed cell.At last cell is suspended among the MT-damping fluid that contains 0.5%DMSO (dimethyl sulfoxide (DMSO)) again, makes its volume be equivalent to original volume of blood.On 96 hole tissue cultivating plates at the bottom of the V-shape, in the presence of 0.5%DMSO (dimethyl sulfoxide (DMSO)), make three group of 80 microlitre cell and 20 microlitre compound.Detect 8 compound concentrations altogether, draw 10 dose point response curves that comprise maximum value (stimulation) and minimum value (non-stimulated).Cell is at 37 ℃, 5%CO 2Cultivated under the condition 1 hour, add subsequently 20 microlitre 6X stimulator (1 mcg/ml is anti--IgE (source: 667 antibody units/milliliter house dust mite (originating: Antigen Laboratories)) Bethyl Laboratories).Cell is at 37 ℃, 5%CO 2Under stimulated 30 minutes.Culture plate at room temperature with 1500 rev/mins of centrifugal rotations of rotating speed 10 minutes, is collected 80 microlitre supernatant liquids, and the histamine ELISA detection box of producing with Immunotech company carries out the histamine content analysis.The ELISA calibrating is undertaken by supplier's explanation.
7.5.6 result
Low density CHMC (cultivation human mast cell) verification result (7.5.2 joint), high-density BMMC (bone marrow mast cell) verification result (7.5.4 joint) and basophilic leukocyte verification result (7.5.5 joint) are as shown in table 1.The verification result of high-density CHMC (cultivation human mast cell) (7.5.3 joint) is shown among the table 2.In table 1 and table 2, all report values are IC 50Value (unit is μ M)." 9999 " value representation IC 50＞10 μ M, thereby the activity that under 10 μ M concentration, can not measure.Most compound of being measured has the concentration less than 10 μ M, wherein many IC 50On sub-micro mole rank.
7.6 2, the 4-pyrimidinediamine suppresses the receptor-mediated degranulation of Fc γ RI
Compound R 921218, R921302, R921303, R940347, R920410, R927050, R940350, R935372, R920323, R926971 has shown of the present invention 2 with R940352 in the calibrating similar to the description of the 7.5th joint, the 4-pyrimidinediamine compounds is to the inhibition ability of the receptor-mediated degranulation of Fc γ RI, unique exception be cells contacting be not IgE (immunoglobulin E), but activate by the anti-human immunoglobulin G Fab of rabbit (rabbitanti-human IgG Fab fragment) (source: Bethyl Laboratories, Catalog No.A80-105).
All determined compounds all show other IC of sub-micro volumetric molar concentration level 50Value.
Figure A20091000677106111
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Figure A20091000677106901
7.7 of the present invention 2, the 4-pyrimidinediamine compounds suppresses upstream IgE acceptor cascade selectively
For confirming of the present invention 2, the 4-pyrimidinediamine compounds has many energy by blocking or suppressing early stage IgE (immunoglobulin E) receptor signal transductory cascade and bring into play it to active restraining effect, induce the influence of degranulation to detect to the Ai Nuo mycin of some kinds of compounds in the cell calibrating, its result as described below.
7.7.1CHMC (cultivation human mast cell) low cell density Ai Nuo mycin activates: class Trypsin Enzyme calibrating
Ai Nuo mycin inductive mastocyte degranulation calibrating is undertaken by the described method of CHMC low density IgE activation calibrating (saving referring to 7.5.2 above), different is: in 1 hour cultivation, the mould cellulose solution of Ai Nuo of preparation 6X (5mM Ai Nuo mycin (source: Signma I-0634) be dissolved among the MeOH (mother liquor), dilution is 1: 416.7 (2 a μ M ultimate density) in the MT damping fluid), add the 6X Ai Nuo mycin of 25 microlitres on suitable target, pair cell stimulates.
7.7.2 basophilic leukocyte Ai Nuo mycin activation: histamine discharges calibrating
Ai Nuo mycin inductive basophilic leukocyte degranulation calibrating activates the described method of calibrating (saving referring to 7.5.5 above) by basophilic leukocyte IgE calibrating or dirt mite and carries out, different is that cell stimulates with the Ai Nuo mycin of 20 microlitres, 2 μ M after cultivating with compound.
7.7.3 result
With IC 50The Ai Nuo mycin of value (the μ M of unit) report induces the degranulation verification result to list among the above-mentioned table 1.In tested active compound (promptly IgE being induced the inhibited compound of degranulation), the overwhelming majority induces degranulation to there is no restraining effect to the Ai Nuo mycin, thereby has confirmed that these active compounds optionally suppress (or upstream) IgE receptor signal transductory cascade in early days.
Anti-IgE in the CHMC cell (cultivation human mast cell) induces or the Ai Nuo mycin induces calcium current to go out by measuring, and we have confirmed this kind result of some active compounds.At these Ca 2+Flow out in the check, the R902420 antagonism-IgE-of the R921218 of 10 μ M and 10 μ M induces Ca 2+Outflow has restraining effect, but to Ai Nuo mycin inductive Ca 2+Flow out (referring to Fig. 4) like water off a duck's back.
7.8 of the present invention 2, the restraining effect of 4-pyrimidinediamine compounds is at once
For verifying the property at once of its effect, of the present invention some 2, the 4-pyrimidinediamine adds in the cell calibrating described above simultaneously with anti--IgE antibody activator.The IgE that all tested compounds all play blocking-up CHMC cell induces degranulation, and its blocking-up degree is equivalent to compound the CHMC cell be cultivated 10 minutes or 30 minutes viewed blocking-up degree in advance before acceptor is crosslinked.
7.9 the kinetics of pharmacological activity in vitro
With the flushing experiment compound R 921218, R921302, R921219, R926240, R940277, R926742, R926495, R909243 and R926782 are detected.In these experiments, CHMC cell or promptly be engraved in 1.25 μ M compounds and have down (zero-time) or after compound is removed in flushing, activated with anti--IgE antibody again every 30 minutes, 60 minutes or 120 minutes.After these compounds were removed 30 minutes, its inhibition ability significantly went down, and showed to expect that maximum degranulation suppresses effect, and mastocyte must keep in touch with these compounds all the time.The identical result of other compound exhibits who is tested.
7.10 toxicity: T cell and B cell
The cell calibrating of carrying out with B cell and T cell shows that compound of the present invention can play them and suppress active or effect, and can not bring toxicity to immunocyte.The embodiment of toxicological detection as shown below.
(7.10.1Jurkat T-cell) toxicity
The Jurkat cell is diluted to 2 * 10 in full RPMI (10% heat inactivation foetal calf serum) nutrient solution 5The concentration of cells/ml is at 37 ℃ of at, 5%CO 2Environment was cultivated 18 hours down.(final medium liquid concentration is 0.5%DMSO (dimethyl sulfoxide (DMSO)), and (handle the source through tissue culture medium: Costar) go up injection 65 microlitre density is 7.7x10 to culture plate at the bottom of 96 hole V-shapes 1.5%MeOH) to containing 65 microlitre 2X compounds 5The cell culture fluid of cells/ml.With its mixing, and at 37 ℃, 5%CO 2Environment cultivated 18-24 hour down.Wandering cells analysis of accounts by the cell scattering of light is assessed toxicity.
(7.10.2BJAB B-cell) toxicity
BJAB places RPMI1640 substratum+10% heat-inactivated fetal bovine serum, 1xL-glutamine, 1x penicillin, 1x streptavidin and 1x beta-mercaptoethanol at 37 ℃ and 5%CO with logarithmic phase (log phase) B-cell strain 2Environment cultivate down.At first collect BJAB, rotation separates, and it is suspended in the substratum again, makes its density reach 7.7 * 10 5Cells/ml.At the mixture that in tissue culturing plate at the bottom of the 96 hole V-shapes, mixes two part of 65 microlitre cell and 65 microlitre compounds in the presence of the 0.1%DMSO (dimethyl sulfoxide (DMSO)).Wandering cells analysis of accounts by the cell scattering of light is assessed toxicity.
7.10.3 toxicity: cell fluorescence is active to be detected
Be implanted into 50 milliliters cell (density 1 * 10 to each hole of containing 50 microlitre compounds 6/ milliliter).Final medium liquid concentration is 0.5%DMSO (dimethyl sulfoxide (DMSO)), 1.5%MeOH.Concussion culture plate 1 minute is with cell and compound.At 37 ℃, 5%CO 2Environment cultivated 18 hours down.Next day, take out 50 microlitre cells from each hole, add 50 microlitre cell fluorescences calibrating reagent (Invitrogen).With plate concussion 1 minute.On photofluorometer, read reading.
7.10.4 result
With IC 50The T-cell and the B-cytotoxicity verification result of value (the μ M of unit) report are shown among the above-mentioned table 2.Except that several exceptions (seeing Table 1), all tested compounds under effective inhibition concentration to B-cell and the equal nontoxicity of T-cell.The verification result that former B-cell is carried out is identical therewith.
7.11 animal is to 2, the 4-pyrimidinediamine has tolerance
Shown that with compound R 921218, R921219 and R921302 compound of the present invention can show restraining effect to animal under the toxigenous dosage being lower than.
7.11.1R921218
R921218 is a secular non-clinical safety research project, and its conclusion shows that rodents and non-rodent have good tolerance to this medicament.The result that toxicology/non-clinical safety is tested to R921218 may be summarized as follows: during 14 days by a definite date repeated doses toxicity research, with the dosage of comparing high times of the human dosage that produces the expection drug effect non-rodent (rabbit and primate) is carried out the nasal cavity dispensing, and rodent (small white mouse and rat) carry out offeing medicine in the oral cavity in and all do not show the toxicity of limit drug threshold dose.In the core pharmacology proof test combination that cardiovascular systems, respiratory system and/or central nervous system function are carried out, do not find any adverse consequences.Do not show in the genetoxic test and may cause sudden change or bring out distored evidence, be exposed on the skin and do not find adverse side effect in the eyes yet.It below is the short discussion of crucial toxicity research.
With 2.1,4.5 or 6.3 milligrams/kilogram/days dosage baboon (Cynomolgus monkeys) has been carried out 14 days by a definite date repeated doses interanasal administration toxicity research.Its life parameters comprises: clinical observation, body weight, the situation of ingesting, eye examination, blood pressure, Electrocardioscopy, hematological examination, clinical chemistry, urine assay, immunotoxicology assessment, overall postmortem, organ weight, the assessment of poisonous substance kinetics and histopathological examination (comprising nasal cavity).All do not find the bad result that caused by R621218 in parameter in any one research, NOAEL (=no observed adverse effectlevel (not having observed the adverse consequences)) dosage that research draws is 6.3 milligrams/kg/day.
With 1.7,3.4 or 5.0 milligrams/kilogram/days dosage New Zealand's white rabbit has been carried out 14 days by a definite date repeated doses interanasal administration toxicity research.Its life parameters comprises: clinical observation, body weight, the situation of ingesting, eye examination, hematological examination, clinical chemistry, overall postmortem, organ weight, the assessment of poisonous substance kinetics and histopathological examination (comprising nasal cavity).All do not find the bad result that caused by R621218 in parameter in any one research, NOAEL (=noobserved adverse effect level (not having observed the adverse consequences)) dosage that research draws is 5.0 milligrams/kg/day.
7.11.2R921219
Inquire in the research at preliminary dosage, 600 milligrams/kilogram disposable oral dosage is considered to NOEL (=no observed effect level (nothing is observed effect)) dosage, and 200 milligrams/kg/day or higher repetition (7 days) dosage are not then tolerated.
In vitro Salmonellas-intestinal bacteria/Mammals-microsome finds that against sudden change calibrating (Ames coomb's test Coomb) test strain TA1537 is the test positive to R921219, and metabolic activation can exist or not exist, and has confirmed an original result of study.To other 4 test strain, R921219 does not show any adverse consequences.In the calibrating of chromosome aberration in vitro, do not find that R921219 has any clastogenic potential possibility.
7.11.3R921302
Rodent was once carried out several non-GLP (the good drug safety inspection specification of GLP=Good Laboratory Practice) toxicity exploratory development, found that small white mouse can tolerate 7 days to 1000 milligrams/kilogram oral dosage.With 100,300 and 1000 milligrams/kilogram dosage small white mouse has been carried out 14 days oral toxicity research.1000 milligrams/kilogram dosage can't tolerate, and 300 milligrams/kilogram dosage evidence suggests that then the histopathology that can cause external genital changes.100 milligrams/kilogram dosage quilt identification in the research is the dosage of NOAEL (nothing is observed effect).With 100 milligrams/kilogram once a day, twice of 100 milligrams of/kilogram every day, 300 milligrams/kilogram once a day with 300 milligrams of/kilogram every days twice dosage small white mouse has been carried out 28 days by a definite date oral toxicity test.Fact proved R921302 300 milligrams/kilogram once a day or every day twice dosage all belong to and can not tolerate.Lower dosage (100 milligrams/kilogram once a day or every day twice) be it seems well-tolerated (clinical and the histopathology result is still unknown).Concerning rat, it seems equal well-tolerated (clinical and histopathology result still unknown) in 32 days with 50,150 and 300 milligrams/kilogram dosed administrations.
In vitro Salmonellas-intestinal bacteria/Mammals-microsome finds that against sudden change calibrating (Ames coomb's test Coomb) the test strain TA98 with S9 and TA1537 is the test positive to R921302.To other 3 test strain, R921302 does not show any detrimentally affect.In the calibrating of chromosome aberration in vitro, do not find that R921302 has any clastogenic potential possibility.
7.12 2, the 4-pyrimidinediamine can pass through oral bioavailability
To of the present invention 2 more than 50, the 4-pyrimidinediamine compounds has carried out the check of oral bioavailability rate.In order to carry out this research, compound is melted at various medium liquids (for example PEG 400 solution and CMC suspension), and rat is carried out intravenously and oral administration.After the administration, blood sampling extraction plasma sample is with the plasma concentration of high-performance liquid chromatography (LC)/tandem mass spectrometry (LC/MS/MS) mensuration compound.The pharmacokinetics analysis is finished according to plasma concentration data.Interested pharmacokinetic parameter comprises: Cl (CL), steady-state distribution volume (Vss), final transformation period (t 1/2) and oral bioavailability rate (%F).
It is many 2 that these pharmacokinetics studies show that, the 4-pyrimidinediamine compounds can oral utilization, and its bioavailability (%F) is the highest can to reach 50% (scope is within 0-50).Its transformation period is 0.5 to 3 hour.Especially compound R 940350, R935372, R935193, R927050 and R935391 have presented good oral bioavailability rate and transformation period in rat.Therefore, these researchs confirmed these 2, the 4-pyrimidinediamine compounds is suitable for oral administration.
7.13 compound is effectively irritated to treating
Compound R 926109, R921218, R921219, R921302, R926495, R926508, R926742, R926745 and R945150 once carried out in vivo assessment to the effect of treatment allergic symptom in small white mouse passivity allergic (PCA) model.This model provides induces directly measuring of degranulation to tissue mast cell IgE-.In this model, order has contacted the antigenic animal of IgE and has been exposed under anaphylactogen excites, and measures because the perviousness of the skin heart system that the release of mastocyte histamine causes changes according to the variation that leaks into the dye quantity in the surrounding tissue.Compound can be measured from the dyestuff that extraction is infiltrated the tissue at an easy rate to the inhibition of the mediation material of direct regulation and control mastocyte degranulation.
7.13.1 the description of research approach and result of study
In PCA (models of passive skin irritability) calibrating, give the anti-dinitrophenol(DNP) of small white mouse intradermal injection (DNP) IgE antibody (the-1 day), make its passive sensitization.Give animal injection treatment medicament (the 0th day) at predetermined reagent.At intravenous injection and human serum albumin's conjugated dinitrophenol(DNP) (HSA-DNP) and the blue dyestuff (Evans bluedye) of Yi Wensishi and measure the mudulation effect of the loose fine and closely woven degranulation of skin of medicament.The enhancement of the crosslinked and mastocyte degranulation inductive vascular permeability subsequently of the IgE acceptor that is produced is measured by measuring the dyestuff quantity that enters tissue of exosmosing.Dyestuff is with methane amide extraction from tissue, then 620 millimicrons of optical densitys that read extract.The inhibition effect of chemicals treatment suppresses expression to handle the per-cent of comparing with medium liquid, promptly with A 620Decline per-cent represent.
Two kinds of compounds are arranged after tested as the positive control agent: histamine antagonist diphenhydramine and 5-hydroxytryptamine antagonist cyproheptadine.Two kinds of mediation materials (histamine and serotonin) all discharge when small white mouse mastocyte IgE-mediation degranulation takes place.Two kinds are all suppressed PCA (models of passive skin irritability) with reference to compound and react; Cyproheptadine is used in the later test as routine.Cyproheptadine can repeat to suppress the PCA reaction of 61%+/-4% (8 milligrams/kilogram, i.p., 30 minutes pretreat time, n=23 test).
7.13.1.1 result
Observed in the test with R921218, R926109, R921219 and RR921302 dosage increase and enhanced to the dose-dependent inhibition effect of Fc ε R-mediation vascular leakage amount.These compounds or with solution formula form (67%PEG/33% citrate buffer agent) or with aqeous suspension form (1.5%Avicel (Avicel)) administration.These results show in compound plasma concentration, effect and in vitro exist very strong correlationship between the usefulness three in vivo.The compound R 921219 that usefulness is the strongest promptly has activity (dosage level reaches 68% inhibition when being 100 milligrams/kilogram) under about 10 mcg/ml cycle exposure, and more weak molecule R921302 only can reduce by 42% plasma extravasation under 100 milligrams of/kilogram dosage levels.In addition, the time length that is exposed in the blood internal recycle compound directly shows in suppressing active duration length.Be determined as the most stable compound R of metabolism 921302 and can suppress vascular exosmosis 1-2 hour before the antigen induction receptor signal occurs in pharmacokinetic study, its effect begins to weaken subsequently.Table 3 and table 4 brief summary the above data.
Figure A20091000677106981
Figure A20091000677106982
Compound R 926495, R926508, R926742, R926745 and R926150 also can suppress passivity allergic (PCA) reaction (data are not in this demonstration) with PEG type prescription oral administration.
7.14 compounds for treating asthma is effective
The effect of compound R 921218, R921302, R926495, R926508, R926742 and R921219 treatment asthma is showed among the sheep model allergic asthma.Sheep bronchoconstriction occurs in being exposed to suction antigen (ascaris suum Ascaris suum) back several minutes, (EAR, the early allergic response) stage of anaphylaxis in early days maximum airflow occurs and blocks.The release of preformed mast cell mediated material may be the reason of the early stage airflow obstruction of this kind.Except that EAR (early stage anaphylaxis), the sheep model also allows compound that we further assess us to late asthmatic response (LAR, lateasthmatic reaction) the local or result of treatment of the non-specific air flue allergy (AHR, airway hyperresponsiveness) that causes of anaphylactogen excitation among a small circle with owing to air flue.On chimera, after several hours, begin to occur AHR (air flue allergy) at contact antigen, and the time in the longest sustainable two weeks.The result of the following stated shows that the compound of being tested has inhibition owing to mastocyte discharges a succession of chain reaction incident that cytokine causes.
7.14.1 research embodiment
In the sheep model of allergic asthma, by endotracheal intubation subjects is given to sheep with the aerosol form throwing, throw the antigen that gives from ascaris suum (Ascaris suum) extraction with aerosol form to sheep subsequently, sheep has natural supersensitivity to this kind antigen.Exciting of anaphylactogen causes direct tracheae to shrink (comprising early stage anaphylaxis and tardy anaphylaxis) and lasting non-specific air flue allergy (AHR).The symptom that three features of kind and human allergic asthma patient are seen on one's body is quite similar.The activity of test medicine is by measuring lung resistibility (R L) change measure, this data based transpulmonary pressure power, flow and respiratory capacity numerical value calculate.Pick up from same bellwether behind the historical control data after the physiological saline treatment and anaphylactogen excitation, adopt data comparison shows that at EAR (anaphylaxis in early days) stage R LValue sharply rises, and continues about 2-3 hour after the anaphylactogen excitation always.LAR (tardy anaphylaxis) shows as more unconspicuous R LValue rises, and it started from the anaphylactogen excitation about 5-6 hour afterwards, was eased after 8 hours after the anaphylactogen excitation.After excitation 24 hours, carried out mensuration to carbachol (carbachol) dose response, to judge whether having AHR (air flue allergy), the latter's definition is to make R LFrom the baseline 400% needed carbachol dosage that rises.(this numerical value is called excitation R LFrom baseline 400% required carbachol excitation concentration (provocative concentration at400%, the PC that rise 400).This data one same individuality gives physiological saline contrast aerosol and throws the historical control data after giving and compare with gained data behind the ascaris suum excitation.
7.14.2 result
All tested compounds have all shown the restraining effect to LAR (tardy anaphylaxis) and AHR (non-specific air flue allergy), and wherein some medicine one has shown the restraining effect to EAR (early stage anaphylaxis).Be shown in the table 5 in the active test of a series of assessments, in the different pretreat time, with the optimum response of some kinds of different solutions and the resulting every kind of compound of suspension formulation.As if R921218 depends on prescription to the effectiveness of EAR (early stage anaphylaxis), and dosage is 30 milligrams of every bellwethers, it seems that with 10% ethanolic soln aerosol form administration effect is best.R926495, R926742, R926508 and R921219 are with the dosage of 45 milligrams of every bellwethers, in the administration in preceding 60 minutes of anaphylactogen excitation, the result shows and has blocked LAR (tardy anaphylaxis) and AHR (non-specific air flue allergy) fully with the form of aqeous suspension.Except these back period parameters, also alleviated the symptom of EAR (early stage anaphylaxis) greatly with R921219, R926508 or R926495 treatment.With 45%PEG400/55% citrate buffer medium liquid the effect of RR921302 is studied.Under these conditions, R921302 influences to matchmaker's liquid as 60 minutes dosage with 30 milligrams of every bellwethers before excitation.
Clear this compounds that shows of these data can be blocked the asthma reaction of supersensitivity sheep.Compare with the historical control data, all compounds have all suppressed AHR (air flue allergy) and LAR (tardy anaphylaxis) significantly.(early stage anaphylaxis) has significant inhibitory effect (being respectively 54%, 21% and 33%) to EAR for R921219, R926508 and R926495.In contrast to this, when R921218, R921302 and R926742 offer medicine with the aqeous suspension form, to EAR (early stage anaphylaxis) unrestraint effect.
Table 5
Compound is to the effect of sheep model allergic asthma for example
Figure A20091000677107011
7.15 compound is effective to treatment asthma
The effect of R921304 and R921219 treatment asthma has also obtained demonstration in the small white mouse model of allergic asthma.
7.15.1 research embodiment
Small white mouse carries out sensitization with ovalbumin (egg albumen) via the intraperitoneal route of administration the 0th day and the 7th day in the presence of adjuvant (alum).After one week, small white mouse is at the 14th day, the 15th day and the 16th day (strict model) or give mode at the 14th day (more undemanding model) in nose and carry out excitation with ovalbumin.This sensitization and excitation process cause air flue allergy and lung inflammation, and this is two prevailing symptoms of human allergic asthma.In the small white mouse model, in vivo the air flue reaction is measured with whole body plethysmography, to obtain PENH value (enhancedPause, Buxco Electronics).PENH is one and comprises peak inspiratory capacity (PIF), peak amount venting one's pent-up feelings (PEF), inspiratory duration, the dimensionless quantity of the time and the time of releiving of feeling elated and exultant, and is considered to weigh an actual parameter of air flue reaction.To the reaction (OVA) of anaphylactogen excitation with only compared by the animal of physiological saline excitation.After the excitation 24 hours, small white mouse is exposed under the methacholine (a kind of muscarinic receptor agonist) of escalated dose, causes smooth muscle contraction.With only compare with the small white mouse of physiological saline excitation, the small white mouse that is subjected to the ovalbumin excitation shows significant air flue allergy to methacholine.In addition, compared with the small white mouse that only is subjected to the physiological saline excitation by the small white mouse of ovalbumin excitation, on air flue, observe tangible cellular infiltration.This kind cellular infiltration mainly is feature with the eosinophil, but a spot of neutrophil(e) cell and the monocytic tissue that enters also occur.
The credibility of the micromolecular inhibitor of mastocyte degranulation being assessed with this model has obtained checking aspect some.At first, adopt the small white mouse kind (W/W that lacks mastocyte v) test carried out confirms that the reaction of ovalbumin inductive depends on the existence of mastocyte.In the small white mouse kind that lacks mastocyte, ovalbumin sensitization and excitation do not cause the allergy of air flue and the infiltration of eosinophil.Secondly, mast cell stabilizers Cromoglycic Acid (Cromolyn) can be blocked air flue allergy of ovalbumin inductive and inflammation (data not shown).In addition, steroid class such as dexamethasone (dexamethasone) and budesonide (budesonide) have supported to assess with this model the credibility of the compound (especially not being the asthma reaction by the mediation of mastocyte stabiliser) of treatment asthma reaction to the retarding effect of methacholine inductive bronchoconstriction.
7.15.2 result
The effect of R921304 from the 7th day to the 16th day giving the mode successive administration in the nose 10 days, last three administrations all before physiological saline or ovalbumin excitation 30 minutes at every turn.Compare with the small white mouse for the treatment of with medium liquid, R921304 can suppress by the air flue allergy of ovalbumin inductive to methacholine.
In very not strict testing program, small white mouse only at the 14th day with the ovalbumin excitation once, throw the R921219 in the 67%PEG400/33% citrate buffer of being dissolved in that gives 70 milligrams of/kilogram dosage in the subcutaneous injection mode then, and after 30 minutes with physiological saline or ovalbumin excitation, this test shows that R921219 has blocked air flue allergy and cellular infiltration phenomenon fully.
In more not urgent experimental program, wherein this type of mouse only at the 14th day with zygote protein excitation once, preceding 30 minutes R921219 subcutaneous administration of salt solution or zygote protein excitation in 70 milligrams/kilogram in the 67%PEG400/33% citrate buffer, prove that R921219 blocks ovum protein-inductive air flue fully and crosses sensitivity and cellular infiltration.
These results are clear, and R921219 and the R921304 of showing can effectively suppress the air flue reaction in the small white mouse model of allergic asthma.
7.162 the 4-pyrimidinediamine compounds suppresses the protein phosphorylation effect that Syk swashs the Enzyme downstream in activating mastocyte
2,4-pyrimidinediamine compounds is tested in the BMMC cell (bone marrow mast cell) by the IgE receptor activation with compound R 921218, R218219 and R921304 the inhibition effect that Syk swashs the protein phosphorylation effect in Enzyme downstream.
In calibrating, BMMC cell (bone marrow mast cell) was being cultivated 1 hour in the presence of the test compound of different concns (0.08 μ M, 0.4 μ M, 2 μ M and 10 μ M) under 37 ℃.This type of cell is subsequently with anti--IgE antibody excitation, as previously mentioned.After 10 minutes, dissolved cell is with electrophoretic method (SDS PAGE) isolated cell protein.
After the electrophoresis, with the protein phosphorylation effect shown in immunoblotting (immunoblot) evaluation graph 7,10 and the 11A-D.Antibody is available from following source: Cell Signaling Technology, Beverley, MA.
Referring to Fig. 7,10 and 11A-D, shown test compound suppresses the protein phosphorylation of Syk upstream in the cascade of IgE receptor signal, rather than the protein phosphorylation in Syk downstream, thereby the IgE that had both confirmed these compounds inhibition upstreams induces degranulation, and has confirmed that these compounds swash Enzyme by inhibition Syk and realize that it suppresses active effect.
7.172 4-pyrimidinediamine compounds suppresses Syk and swashs Enzyme in biochemical assays
To some kinds 2, the 4-pyrimidinediamine compounds is tested, and is carrying out in the biochemical fluorescent polarization calibrating from the sharp Enzyme of Syk with single to understand, and this compounds swashs the inhibition ability of Enzyme catalysis phosphorylation to the Syk of peptide matrix.In this test, compound is at sharp Enzyme damping fluid (20mM HEPES, pH value 7.4,5mM MgCl 2, 2mM MnCl 2, 1mM DTT, 0.1 mg/ml acetylize bovine) be diluted to 1%DMSO (dimethyl sulfoxide (DMSO)).The compound that is dissolved among the 1%DMSO (dimethyl sulfoxide (DMSO)) (ultimate density 0.2%DMSO) at room temperature mixes with ATP (Triphosaden)/matrix solution.Add Syk and swash Enzyme (source: Upstate, Lake Placid NY), make it reach end reaction volume 20 microlitres, at room temperature cultivate reaction 30 minutes.Final ferment reaction conditions is: 20mM HEPES, pH 7.4,5mM MgCl 2, 2mM MnCl 2, 1mM DTT, 0.1 mg/ml acetylize bovine, 0.125 nanogram(ng) Syk, 4uM ATP (Triphosaden), 2.5uM peptide matrix (source: biotin-EQEDEPEGDYEEVLE-CONH2, SynPepCorporation).According to manufacturer's (manufacturer's name: explanation PanVera Corporation), with EDTA (ethylenediamine tetraacetic acid (EDTA)) (ultimate density: 10mM)/anti-phosphorus base tyrosine antibody (1X ultimate density)/fluorescent phosphorus base tracer peptide agent (0.5X ultimate density) adds in the FP dilution buffer liquid with stopped reaction, obtains the cumulative volume of 40 microlitres.Culture plate was cultivated under room temperature 30 minutes in the dark.Culture plate reads on Polarion fluorescent polarising sheet readout instrument (Tecan).Use by manufacturers (PanVera Corporation) and data-switching is become existing phosphorus base peptide content for competing the calibration curve that in tyrosine-kinase Enzyme assay cartridges, provides with phosphorus base peptide rival.
The calibrating the result as shown in the following Table 6:
Figure A20091000677107041
Figure A20091000677107051
Figure A20091000677107061
These data presentation are worked as IC 50In the time of within the sub-micro molar range, all tested compounds except that R945142 and R909236, all swash the Enzyme phosphorylation to Syk and play restraining effect.Work as IC 50In the time of within micro-molar range, all tested compounds all have the effect that Syk swashs the Enzyme phosphorylation that suppresses.
7.18 compound is effective to the treatment autoimmunity
At reverse passive Arthus reaction (reverse passive Arthus reaction), a kind of acute Ag-Ab mediates tissue injury's model, and in some kinds of autoimmunities and inflammatory disease model, we carried out some 2, the 4-pyrimidinediamine compounds is to the autoimmune disorders assessment of effect in vivo.The similarity of these models is all to have (IC-triggered) diseases associated with inflammation that a special antigen mediation immunocomplex brings out and disorganization subsequently.Immunocomplex (IC) can cause the activation of the cell of surperficial Fc γ R of performance and Fc ε R in the deposit of anatomical site (for example be deposited on the experimental encephalomyelitis (EAE) that central nervous system (CNS) causes and be deposited on the collagen-induced property sacroiliitis (CIA) that synovial membrane causes), particularly mastocyte, scavenger cell and neutrophil(e) cell, thus the release of cytokine and neutrophil's chemotaxis caused.The activation of inflammatory reaction has caused the reaction of downstream effect device, comprises that oedema, hemorrhage, neutrophil(e) cell are soaked into and the release of proinflammatory medium.The incident that these immunocomplexs (IC) are brought out is difficult with identification in the autoimmune illness; But many investigators had shown the Fc γ R signal path in these animal models already and have suppressed the sickness rate of disease and severity are alleviated greatly.
7.18.1 compound is effective to the small white mouse Arthus reaction
In the reverse passive Arthus reaction of small white mouse (RPA reaction), shown that compound R 921302, R926891, R940323, R940347 and R921303 suppress the effect that IC (immunocomplex) brings out the inflammation cascade in vivo.
7.18.1.1 model
The series of human autoimmune disease all can involve the infringement of immunocomplex (IC) mediated acute sex organization, comprises vasculitis syndrome, bad serum syndrome, whole body property lupus erythematosus (SLE), rheumatoid arthritis, Gourde(G) Paasche Che Shi disease (glomerulonephritis spitting of blood syndrome) and glomerulonephritis.The classical trial model of immunocomplex (IC) mediation tissue injury is reverse passive Arthus reaction (RPA reaction).The RPA reaction model is to inquire into the model in vivo very easily of the local inflammation reaction that there is no whole body reaction that IC brought out.The antibody (Abs) (rabbit resists-OVA (ovalbumin) IgG) that subcutaneous injection is special to chicken egg white, intravenous injection subsequently (IV) antigen (Ags), specifically, injection egg ovalbumin (ovalbumin, OVA), cause the blood vessel deposit of IC (immunocomplex) on every side and the inflammatory reaction of rapid spread, it is characterized in that oedema, neutrophil's infiltration and hemorrhage of injection site.Many aspects of small white mouse RPA reaction (reverse passive Arthus reaction) model have rheumatoid arthritis, SLE (whole body property lupus erythematosus) similar with glomerulonephritis patient's inflammatory reaction.
7.18.1.2 research embodiment
In this model system, test compounds will give several time point administrations before at Abs (antibody) and Ags (antigen).Rabbit is anti--and OVA IgG (rabbit antiovalbumin immunoglobulin G) solution (50 micrograms are dissolved in 25 microlitres/small white mouse) injects in the subcutaneous injection mode, and intravenous injection immediately is dissolved in the egg white powder solution (20 milligrams/per kilogram of body weight) that contains the blue dyestuff of 1% Yi Wensishi.Oedema and hemorrhage degree are measured from C57BL/6 small white mouse skin of back, with the pointer of the blue dyestuff of Yi Wensishi as the local organization infringement.As the contrast material is the polyclone man rabbit igg of purifying.
The pretreat time that gave test compounds before Ab/Ag (antibody/antigen) excitation is depended on pharmacokinetics (PK) character of every individual compound.Inducing Arthus reaction and four hours,, obtain tissue so that oedema is assessed with the peaceful and comfortable execution of small white mouse.This model system allows our the in vivo behavior of the many inhibitor of screening rapidly.
7.18.1.3 result
All tested compounds are all with oral administration.
R921302 demonstrates the dosage interdependence that oedema is formed and suppresses (being respectively 49.9%, 93.2% and 99.1%) with 50 milligrams/kilogram, 100mg/kg milligram/kilogram and 200 milligrams of/kilogram dosage before Ab/Ag (antibody/antigen) excitation 60 minutes during to the administration of C57Bl6 small white mouse.In addition, R921302 not only demonstrates the preventative restraining effect to oedema, but also has shown the therapeutic effect, when giving compound with 100 milligrams of/kilogram dosage in 30 minutes after excitation, can suppress 7735% to oedema.
R940323 and R926891 were respectively 32.4% and 54.9% to the inhibition effect of oedema during with 200 milligrams of/kilogram dosed administrations in 60 minutes before excitation.During these compound oral administrations, its bioavailability is much smaller, and its whole body exposure level is than R921302 little 50 times (data not shown).During with 100 milligrams of/kilogram dosed administrations, it was 89% that its oedema suppresses effect to R940347 preceding 2 hours of excitation.
With the pretreat time administration of 200 milligrams of/kilogram dosage and 30 minutes, 60 minutes and 120 minutes the time, the inhibition degree of 921303 pairs of oedema of compound R is respectively 100%, 100% and 93.6%.When dosage was respectively 50 milligrams/kilogram, 100 milligrams/kilogram and 200 milligrams/kilogram, this compound had shown that also the dosage interdependence suppresses, and its inhibiting rate is respectively 65.4%, 81.2% and 100%.Table 7 has been summed up the result of each compound test in generalized mode.
Figure A20091000677107091
Figure A20091000677107101
Figure A20091000677107111
* nd=undetermined.N/A=is inapplicable
7.18.2 compound is induced in the arthritis model effective at the small white mouse collagen antibodies
The in vivo effect of the autoimmune disease of compound R 921302 is shown in small white mouse model collagen antibodies and induces among the sacroiliitis (CAIA).
7.18.2.1 model
The collagen-induced sacroiliitis (CIA) of rodent often is used as one of experimental model of IC (immunocomplex) induced tissue infringement.Throw to small white mouse or rat and to give the II Collagen Type VI and can cause that be the immune response of feature with the remote key cartilage with struvite destruction of bone and surrounding tissue while swelling.CIA (collagen-induced sacroiliitis) is useful on as the potential possible compound for the treatment of rheumatoid arthritis and other chronic inflammatory condition medicine in order to assessment usually.
In recent years, a new technology occurred in CIA (collagen-induced sacroiliitis) model trial, this technology is used the antibody-mediated CIA of anti-II Collagen Type VI antibody induction.The advantage of this method is: disease inductive time compactness (state of an illness occurring in 24-48 hour after intravenous injection (IV) antibody); All can cause sacroiliitis to the small white mouse kind of CIA sensitivity with to CIA in the resistive small white mouse kind; It is the desired procedure of screening the medicine of anti-inflammatory rapidly.
At the 0th day, with Arthrogen- Monoclonal antibody induces sacroiliitis with cocktail agent (source: Chemicon International Inc.) Balb/c strain small white mouse is carried out intravenous injection (every mouse 2 milligrams).After 48 hours, at peritoneal injection 100 microlitre LPS (lipopolysaccharides).At the 4th day, toe looks may edema.By the 5th day, one or two claws (especially back leg) began rubescent swelling.From the 6th day backward, Hong Zhong claw guarantees 1-2 week to the major general.Under study for action, write down the clinical symptom of inflammation, so that the severity of claw oedema is assessed.Arthritic severity is recorded as the score summation (the highest possible score is 8) of two rear solid ends of each animal.The inflammation degree of relevant claw is calculated by the diameter of claw.Simultaneously body weight change is monitored.
Animal is treated in bringing out since the 0th day in sacroiliitis.Test chemical combination and control compound are offerd medicine by the mode of original definite PK (pharmacokinetics) analytical results (q.d.) or every day twice (b.i.d) with once a day.
When research finishes (after bringing out sacroiliitis 1-2 week),, from the long-range claw that blocks of shin bone, and weigh its weight with endless knife with the peaceful and comfortable execution of small white mouse.Obtain the mean value ± mean value standard error (SEM) of every treated animal from the clinography of each animal every day, and calculate the weight of each test group animal rear solid end when off-test, and it is noted.And should obtain the histopathological evaluation of claw.
7.18.2.2 result
The remarkable arthritic development that suppressed is given in the throwing of R921302, has alleviated severity of disease (statistical probability p＜0.005), as (Figure 12) shown in average sacroiliitis clinography variation every day.(Figure 13) compares with the medium liquid control group, and the treatment group is average sacroiliitis record minimizing every day 71-92% from the 4th day to the 14th day.Compare with control group in animal according to the degree of the claw inflammation of claw weight determination and to alleviate (Figure 13) to some extent with the R921302 treatment.Finish in research be, the degree of swelling is by measured average claw weight assessment, this assessment shows that with the R921302 treatment one group alleviates 99.9% (p＜0.002) than control group.
Histopathological evaluation to the claw that excises shows and the consistent tangible synovitis of CIA (collagen-induced sacroiliitis).The animal of handling with physiological saline or medium liquid shows tangible sick the damage; And only find the slightest disease damage with a treated animal of R921302 treatment.The propagation of synovial membrane causes the joint to thicken.Fibroblast increases, and is attended by the infiltration of intensive neutrophil(e) cell, lymphocyte, monocyte, scavenger cell and plasma cell.The hyperemia, the hemorrhage and swelling that also have vascular proliferation simultaneously and follow with it.The joint space exists pannus formation and cartilage infringement.Approaching normal through one group joint of treatment, perhaps only show limited inflammation, but cartilage is involved.
Table 8. is respectively organized histopathology assessment average (0-15)
Figure A20091000677107131
(matchmaker's liquid p=0.1) is compared, and through the R050 treatment, dosage is that 100 milligrams of/kilogram every days clinical score of sacroiliitis and claw swelling of twice animal on average alleviates 20% with untreated control group.Press rear solid end thickness (p=0.1) and measure, claw swelling has been subjected to the unanimity of the moon 20% than untreated control group (matchmaker's liquid).The dosage level of R050 does not show the sacroiliitis sign when reaching 30 milligrams/kilogram.
R070 is a kind of salt of R050, compare with untreated control group (matchmaker's liquid) during with dosage level twice administration every day of 50 or 100 milligrams/kilogram, can distinguish and on average suppress clinical disease symptom 39.75% (p＜0.0002) or 35.28% (p＜0.0004).Claw thickness reduces about 50%.
R429 is a kind of salt of R363, with twice of every day, compare the average respectively inhibition that shows the clinical score of sacroiliitis of 23.81% (p＜0.05) or 20.82% (p=0.05) during each 50 or 100 milligrams/kilogram dosed administration with the control group of untreated (matchmaker's liquid).Claw thickness reduces too.
R347 does not influence the sacroiliitis score at the dosage level (every day twice, each 30 and 100 milligrams/kilogram) of tested person.
7.18.3 compound is effective to the collagen-induced sacroiliitis of rat
The in vivo effect of compound R 921302 treatment autoimmune disorderss shows with the collagen-induced sacroiliitis of rat (CIA) model.
7.18.3.1 the description of model
The feature of rheumatoid arthritis (RA) is chronic crucial inflammation, causes irreversible cartilage infringement gradually.Suffer from patient's the synovial tissue of RA (rheumatoid arthritis) the abundant IC that contains IgG (immunocomplex) is arranged.Though these complex bodys are in that to play which kind of effect aspect the pathogeny of disease and the pathology still disputable actually, IC (immunocomplex) links up by Fc γ R and protometrocyte.
CIA (collagen-induced sacroiliitis) is the animal model for the RA (rheumatoid arthritis) that people accept extensively, and it can cause with pannus formation and degenerative joint is the chronic inflammation synovitis of feature.In this model, carry out subcutaneous immunization with incomplete Freund's adjuvant emulsive original I type i collagen and in 10 days or 11 days, cause struvite multiple sacroiliitis, and joint damage occurring in 3 to 4 weeks subsequently.
7.18.3.2 research embodiment
Syngeneic LOU rat the 0th day with original I type i collagen immunity sensitization, in prevention embodiment and treatment embodiment, assess the effect of R921302 then respectively.In the prevention embodiment, give the R921302 of various dose from the immune sensitization in the oral administration mode.In the treatment embodiment, (300 milligrams/kilogram, oral, once a day), that continues kills the 28th day of rat always with the R921302 treatment in the beginning in the 10th day that begins to occur in the sacroiliitis clinical symptom.In two embodiments, obtain clinical score every day, weekly twice body weight of weighing.At the 28th day, obtain the radiograph data, and detect the serum level of II Collagen Type VI antibody with ELISA (calibrating of Enzyme connection immunosorption).
7.18.3.3 result
Immunity sensitization or the 10th day front and back, clinical CIA (collagen-induced sacroiliitis) symptom appears in rat, and the sacroiliitis score that shows as them increases (Figure 14).After the 10th day, only the average sacroiliitis score with the rat of matchmaker's liquid treatment rises gradually, reaches 6.75 ± 0.57 to the 28th day average clinical score.Compare remarkable attenuating (p＜0.01) 10-28 days average clinical score than matchmaker's liquid control group with the animal of high dosage (300 milligrams/kg/day) R921302 treatment from day (the 0th day) beginning of immune sensitization.Significantly lower since the 16th day sacroiliitis score with the rat of 300 milligrams of/kilogram dosage treatment from the morbidity beginning, this difference continues to observe the 28th day always and studies till the final day.Blindness radiograph score (scale 0-6) the matchmaker liquid control group that CIA (collagen-induced sacroiliitis) obtained on the 28th day is 4.8 ± 0.056, then be respectively 2.5 ± 0.0.16,2.4 ± 0.006 and 0.13 ± 0.000001 with the score of the prevention embodiment animal of 75,150 and 300 milligrams/kg/day dosage treatment respectively once a day, and begin once a day must to be divided into 0.45 ± .031 with the animal of 300 milligrams/kg/day treatment from disease incidence.Carry out in the R921302 treatment with 300 milligrams/kg/day dosage, no matter be preventative (from immune sensitization time) and when disease incidence begin treatment, can both prevent erosion, and reduce soft tissue swelling.Similarly, the R921302 treatment also makes the anti-II Collagen Type VI of serum antibody significantly reduce (not display data).
7.18.4 compound is effective to mouse experiment autoimmune encephalomyelitis
The in vivo effect of 921302 pairs of autoimmune disorderss of compound R shows with experimental autoimmunity encephalomyelitis (EAE) mouse model.
7.18.4.1 model description
EAE (experimental autoimmune encephalomyelitis) is a useful model of multiple sclerosis (MS), and multiple sclerosis is that CNS (central nervous system) white matter immunocyte soaks into CNS (central nervous system) autoimmune disorders that is caused.The inflammation of spinal cord and being damaged have subsequently been caused gradual paralysis.Anthropoid disease is the same, and EAE (experimental autoimmune encephalomyelitis) activates relevant with the autoreaction tip of T cell and myelin protein (for example myelin basic protein (MBP), proteolipid albumen (PLP) or myelin oligodendroglia albumen (MOG)).Activated neural antigen specific T-cells passes hemato encephalic barrier, causes monocyte infiltration and the demyelination phenomenon concentrated.EAE (experimental autoimmune encephalomyelitis) can induce responsive mouse species immunity sensitization by cooperate adjuvant with the special albumen of myelin.In the mouse model in these researchs, the paralysis of hind leg and tail just occurred on the 10th day after the immune sensitization, during the 10th day to the 14th day, severity of disease reaches peak value, and a part natural remission and recurrence also can occur by the cycle up to the 35th day.Result described below shows that test medicament (R921302) has the potentiality that suppress the severity of disease and the symptomatic recurrence that wards off disease, and causes symptomatic recurrence to cause owing to the immunocyte Fc γ R mediated cell factor discharges.
7.18.4.2 research embodiment
In EAE (experimental autoimmune encephalomyelitis) SJL mouse model, every mouse is all with PLP/CFA (150 microgram PLP139-151 are dissolved in company with 200 microgram CFA in the isogamete at four positions of 0.05 milliliter of trailing flank and make 0.2 milliliter emulsion altogether, are used to induce EAE) sensitization.In the embodiment that suppresses, from day (the 0th day) of immune sensitization just the R921302 with matchmaker's liquid or various dose pass through oral administration.In the treatment embodiment, from state of an illness appearance, just animal is separated, so that obtain each treated animal that has similar average clinical score from the beginning, give the experiment medicine of matchmaker's liquid or various dose frequency then by oral administration.In two groups of embodiments, clinical score is monitored every day, and body weight is measured weekly twice.
7.18.4.3 result
After the PLP immunity sensitization 10 days, clinical EAE (experiment autoimmune encephalomyelitis) symptom appearred in the SJL mouse, showed as average clinical score raise (Figure 15).Only benumb score with the animal of matchmaker's liquid treatment from the day (the 0th day) of immune sensitization and rise gradually, average score reached the peak value of 5.1+0.3 by the 14th day.When the disease peak (the 14th day), with once a day, 100 milligrams/kilogram of each dosage or every day twice, the average clinical score of the animal of each 100 milligrams of/kilogram treatments of dosage significantly reduces (being respectively p＜0.05,4.3+1.3 and 4.3+1.4).By the 16th day, all animals all showed the part alleviation of average clinical severity of symptom, and this is the typical phenomenon of SJL model.Up to all animals all sacrificed the 30th day, remain its tangible statistical significance (p＜0.05) with the remarkable lower clinical score of the animal of twice administration R921302 every day of 100 milligrams of/kilogram dosage.These lower scores during whole treatment are reflected on the accumulative total weight index (CWI, cumulative weight index) of significantly lower accumulative total disease index (CDI, cumulative disease index) and rising, and are as described in Table 9.In a treated animal of only treating with matchmaker's liquid, symptomatic recurrence appears in 2/5 mouse.In animal with dosage treatment 100 milligrams/kilogram/every day, 3/8 mouse recurrence.With every day twice, in the mouse of each 100 milligrams of/kilogram dosage treatment, there is not a mouse recurrence to occur.
Table 9
From the immune sensitization with 150 microgram PLP 139-151/200 microgram MTB's (CFA)
The SJL female mice that Rigel compound R 921302 is treated
Figure A20091000677107161
CDI=to the+26 day accumulative total disease index
CWI=to the+23 day accumulative total weight index
A=is by non-EAE factor, with the relevant damage of ingesting, and the perhaps animal of putting to death because of hydrocephalus
Exist difference (p＜0.05) between two tail t calibrating demonstration control groups that b=student carries out and the experimental group with statistical significance.
From day (the 11st day) beginning twice of one every day of disease symptoms appearance, the CDI of the SJL mouse of the R921302 treatment of each 200 milligrams of/kilogram dosage (accumulative total disease index) has shown the decline (p=0.003) (mouse with the R921302 treatment is 53.5 ± 16.9, and the mouse of only treating with matchmaker's liquid is 72.9 ± 8.9) with statistical significance.In addition, compare, with outstanding descend (2/12) of recurrence quantity of the mouse of R921302 treatment with recurrence quantity (7/11) only with the mouse of matchmaker's liquid treatment.Experimental result is shown in table 10 and Figure 16.
Table 10
From the morbidity beginning with the SJL female mice of Rigel compound R 921302 treatments
Figure A20091000677107171
CDI=was up to the+27 days accumulative total disease index
7.18.5 of the present invention 2, the 4-pyrimidinediamine compounds can suppress the T-cell-stimulating
7.18.5.1 describe
Of the present invention 2, the ability that the 4-pyrimidinediamine suppresses the T-cell-stimulating is shown by carrying out a series of the calibrating with Jurkat T-cell strain and former generation T-cell culture medium.As the response that T-cell receptors (TCR) is stimulated, the inhibition of Jurkat T-cell-stimulating is by quantizing measuring to adjusted of cell surface marker CD69.The inhibition throughput of former generation T-cell-stimulating turns into to helping the release of cytokines of stimuli responsive to measure to the TCR/CD28 spoke, and above-mentioned cytokine comprises tumour necrosis factor (TNF) α, interleukin II (IL-2), interleukin-4 (IL-4), interferon-gamma (IFN γ) and granulocyte macrophage-colony stimulating factor (GMSCF).
7.18.5.2Jurkat the screening that the T-cell-stimulating suppresses
Human Jurkat T-cell (clone N) is routinely in RPMI 1640 substratum (source: Mediatech) be aided with 10% foetal calf serum (FBS) (source: Hyclone), cultivate among penicillin and the Streptomycin sulphate.Screening process was carried out within three days.
At first day of screening, the cell of cultivation rotated separation (1000 rev/mins, 5 minutes) on whizzer, and is suspended in 3.0 * 10 again 5Among the RPMI+5%FBS of cells/ml density.Second day of screening is with cell centrifugation 5 minutes under 1000 rev/mins of rotating speeds, 1.3 * 10 5Again be suspended among the RPMI+5%FBS under the cells/ml density.With (source: Corning) among each hole of culture plate, 96 holes at the bottom of this cell suspending liquid adding U-shaped of 85 microlitres.With the compound of 85 microlitres or only be that the RPMI+5%FBS (in contrast) of dilution adds in each hole, cultivated 1 hour down at 37 ℃.Then by adding 25 microlitre 8X solution in the cell onboard, under 500 millimicro grams per milliliters with anti--TCR (C305) irritation cell.Cell was cultivated 20 hours down at 37 ℃ then.
At the 3rd day of screening, culture plate with 2500 rev/mins of rotating speed rotations 1 minute, was removed substratum then on Beckman GS-6R whizzer.(anti--CD69-APC antibody of dilution in 1: 100 (source: BectonDickenson) place among the PBS+2%FBS) is cultivated plate 20 minutes under 4 degree among each hole subsequently in the dark to add 50 microlitre staining fluids.In each hole, add 150 microlitre dcq buffer liquid (PBS+2%FBS) then, culture plate is descended centrifugal rotations 1 minute at 3000 rev/mins.The skim supernatant liquid suspends spherical particle under whirling motion is stirred gently again once more.Add 75 microlitre PBS+2%FBS+Cytofix (dilution in 1: 4) subsequently, the plate of whirling motion stir culture gently wraps with Aluminium Foil Package.Cell measures with the flow cytometer of band automatic fluid treatment system on the plate.
The compound and the solvent of different concns compare separately, to judge the inhibition degree IC of every kind of compound to the T-cell-stimulating 50It is of the present invention 2 that table 11 is depicted as, the representative IC of 4-pyrimidinediamine compounds 50Value.
7.18.5.3 the separation of former generation T-cell
In rIL-2, grow, be suspended in available from the human donor's of health E8-4E8 PBMC or proliferative T cell and carry out (1500 rev/mins of centrifugations among the PBS, 8-10 minute), and be suspended in again in 100 milliliters of RPMI perfect mediums (1% green grass or young crops-Streptomycin sulphate, 1%L-glutamine, 10mM HEPES).Cell coat in the T175 flask (37 ℃, 5%CO 2), allow monocyte to adhere to 2-3 hour.After monocyte adheres to, gather the cell of no tack, count with hematimeter, with PBS flushing several times, density with the 1.54E6 cells/ml is suspended in Yssels perfect medium (modification IMDM substratum contains 1% people AB serum, 1% green grass or young crops-Streptomycin sulphate, 1%L-glutamine, 10mM HEPES) again then.Then 90 microlitre cell diluents are joined in Yssel ' s substratum and be diluted in the compound of 2X concentration, at 37 ℃ of (5%CO 2) cultivated 30 minutes down.After this pre-incubation step is finished, compound/cell mixture is transferred on the stimulation plate by the following stated.
7.18.5.4 generate the screening of carrying out for suppressing to be excited the cytokine of former generation T cell
On 96 orifice plates with 5 mcg/ml α CD3 (source: BD PharMingen, Catalog#555336)+be dissolved in PBS (no Ca 2+/ Mg 2+) in 10 mcg/ml α CD28 (source: Beckman Coulter, Catalog#IM1376) at 37 ℃ of (5%CO 2) be coated with 3-5 hour down, stimulate plate with preparation.After stimulating the antibody cultivation to finish, remove above-mentioned cocktail agent, before adding former generation T cell/compound liquid, wash this plate three times with PBS.
Compound/cell mixture is shifted on the stimulation plate, at 37 ℃ of (5%CO 2) cultivated 18 hours down.Cytositimulation or, with the moons 1 50 the microlitre supernatant liquid from each hole, transfer to 96 hole screen plates (source: Corning PVDF FilterPlates), they (2000 rev/mins of centrifugations, 2-3 minute), perhaps being used for ELISA or LUMINEX immediately measures, perhaps that it is freezing under-80 ℃, for using in the future.
By manufacturers's explanation, detect box (source: R﹠amp with Quantikine Human IL-2 ELISA; D Systems, Catalog# D2050) carry out interleukin II ELISA calibrating, and under 450 millimicrons of wavelength with the spectrophotometric determination absorption spectrum.Deduct blank value, specific absorption is scaled pg/mL unit according to typical curve.
The operation of the multiple immunoassays of Luminex that TNF, IL-2, GMSCF, IL-4 and IFN γ are carried out is undertaken by the explanation of manufacturers (Upstate Biotechnology) basically.Basically be 50 microlitre samples to be diluted in 50 microlitres calibratings thinner and 50 microlitres cultivate among the damping fluid, at room temperature cultivated in the dark 1 hour with the detection antibody of 100 microlitres dilution then.With lavation buffer solution washing and filtering plate twice, the spoke with the dilution of 100 microlitres helps reagent (SAV-RPE) at room temperature to cultivate in the dark 30 minutes then.With plate washing three times, carry out bead (magnetic bead) identification and RPE fluorometric assay at last with the Luminex instrument.
The compound and the solvent of different concns compare separately, to judge the inhibition degree IC of every kind of compound to the T-cell-stimulating 50It is of the present invention 2 that table 11 is depicted as, the representative IC of 4-pyrimidinediamine compounds 50Value.
7.18.6 of the present invention 2, the 4-pyrimidinediamine compounds suppresses the activation of B-cell
7.18.3.1 describe
Of the present invention 2, the activation capability that the 4-pyrimidinediamine compounds suppresses the B-cell is examined and determine former generation B-cell with fluorescence-activated cell sorter (FACS) and is shown in the cell surface marker calibrating.As the inhibition of former generation B-cell-stimulating of the response that stimulates of B-cell receptor (BCR) by quantizing measuring of cell surface marker CD69 to adjusted.
7.18.6.2 the separation of former generation B-cell
The former generation B-of people cellular segregation is from buffycoat, the leukocytic cream that this forms between red corpuscle and thrombocyte when being the centrifugation of anti-freezing human blood, also available CD19-
Figure A20091000677107201
Bead (magnetic bead) s separates from fresh human blood with FACS.Buffycoat is available from medical college of Stanford University Blood Center, and this buffycoat is to be prepared by blood bank on the same day, and (with ice cube) refrigerated storage is transported.Buffycoat (about 35 milliliters) is placed 500 milliliters of sterile centrifuge conically shapeds, in cooled on ice, then to contain 0.2%BSA (source: Sigma:A7638) and Trisodium Citrate (0.1%, source: Sigma:S-5570) the cold PBS dilution of (P-B-C), make its total amount reach 200 milliliters, and mix gently.Fresh human blood picks up from the blood donor, and is placed in the vacuum vessel that contains heparin (a pipe vacuum vessel can be gathered about 8.5 milliliters of blood).Blood is in cooled on ice, transfers in 50 milliliters the Falcon test tube in (20 milliliters/pipe) or the 500 milliliters of sterile centrifuge conically shapeds, with the P-B-C dilution of equivalent.
25 milliliters of dilute bloods or buffycoat are poured on the cold ficoll (ficoll), make its layering, put back on ice then., carry out density gradient centrifugation under 4 ℃ and separated (Beckman GS-6R type whizzer) 45 minutes, at 2000 rev/mins with the stratified blood of ficoll so that peripheral blood monocyte (PBMC) is separated from red blood cell (RBC) and granulocyte.Sucking-off top water layer is up to 1 inch place above the PBMC layer.From per two ficoll pipes, the PBMC layer is transferred to (about 10 milliliters/pipe) in 50 milliliters of falcon pipes of a cleaning.The PBMC that shifts is with ice-cold PBS and 0.2%BSA (P-B) dilution 5x, and 1400 rev/mins and 4 ℃ of following centrifugations 20 minutes.Sucking-off supernatant liquid (some muddiness of possibility sometimes) is suspended in PBMC among 25 milliliters of P-B again, pair cell counting (with dilution in 1: 5), and place on ice.
By the explanation of manufacturers use with bead (magnetic bead) (
Figure A20091000677107211
) bonded is anti--CD19 antibody pair cell carries out forward and selects.Press the quantity of 5% estimation B-cell of the PBMS cell of being counted, add that each cell needs about 10 magnetic beads to attract, and the density (4 * 10 of bead stock (magnetic bead liquid) 8Magnetic bead/milliliter) calculates CD19-
Figure A20091000677107212
Beads (magnetic bead) (source: CD19-coated dyna beads M-450 (pabB),
Figure A20091000677107213
) requirement roughly.In 5 milliliters of pipes, use Magnet with 5 milliliters in P-B to CD19-
Figure A20091000677107215
The magnetic bead washed twice adds among the PBMC that suspends then.Then mixture is passed through
Figure A20091000677107216
Magnet, the washing several times are to separate and magnetic bead bonded cell.
7.18.6.3 screening suppresses the compound of B-cell-stimulating
After the separation, use CD19-
Figure A20091000677107218
Take off the operation under 30 ℃ of magnetic bead liquid and removed magnetic bead and antibody in 45 minutes.Typical yield is every part of buffycoat 2 * 10 7The B-cell.Washing B-cell, and be suspended in again among RPM I1640+10%FBS+ penicillin/streptavidin+1 millimicro grams per milliliter IFN α 8 with 1E6 cells/ml density.Make cell at 37 ℃ and 5%CO 2Under standing over night.
Second day, with cell washing and be suspended in again among the RPMI+2.5%FBS, make it reach 1 * 10 6The density of cells/ml.Then cell is assigned to 96 orifice plates at the bottom of the V-shape (source: Corning) in each hole, every hole 65 microlitre cells.Add the 2x compound of 65 microlitres with robot to each hole, make final DMSO concentration reach 0.2%, and cultivated 1 hour down at 37 ℃.Use 20 microlitre 7.5x α-IgM (final 5 mcg/ml) pair cell to stimulate then 24 hours available from Jackson laboratories.At the 3rd day centrifugal separating cell,, analyze (using scattering of light) with FACS gating viable cell with CD69 dyeing.
The compound and the solvent of different concns compare separately, to judge the inhibition degree IC of every kind of compound to the B-cell-stimulating 50It is of the present invention 2 that table 11 is depicted as, the representative IC of 4-pyrimidinediamine compounds 50Value.
7.18.7 of the present invention 2, the 4-pyrimidinediamine compounds suppresses macrophage activation
7.18.7.1 describe
Of the present invention 2, the 4-pyrimidinediamine compounds suppresses differentiated macrophages activated ability can be excited the cytokine that scavenger cell discharged by mensuration and show.We quantize the tumor necrosis factor alpha (TNF α) of the response of IgG or LPS stimulation and the release of interleukin 6 (IL-6) conduct.
7.18.7.2 the purification of human macrophage and cultivation
By the explanation of manufacturers, with monocyte separate, dedicated equipment (source: Miltenyi biotec#130-045-501) with PBMC (peripheral blood monocyte) (source: Allcells # PB002) be starting materials purification CD14+ monocyte.With the per-cent of flow cytometry (flow cytometry) detection CD14+ cell, its purity is assessed.Can obtain in typical case＞90% purity.Then the CD14+ cell of purifying is placed culture dish (6 * 10 6Cell/150 centimetre TC culture dish, in adorn 15 milliliters of substratum) with 100 millimicro grams per milliliter M-CSF (source: Macrophage-SFM Pepro Tech #300-25) (scavenger cell serum free medium) (source: Gibco#12065-074), make its differentiation 5 days.After 5 days finished, cellular form and cell surface marker (CD14, HLA-DR, B7.1, B7.2, CD64, CD32 and CD16) showed the existence of sophisticated differentiated macrophages.
7.18.7.3IgG (immunoglobulin G) stimulates
(source: VWR #62402-959) coating is to mix immunoglobulin G while (IgG) (source: Jackson Immunoresearch lab#009-000-003) for Immulon 4HBX 96 well culture plates, coating amount 10 micrograms/hole, under 4 ℃, place and spend the night, or placed 1 hour down at 37 ℃.The a oppositely contrast of preparation in addition, 2 fragments apply with F (ab '), so that the assessment background stimulates.With 200 microlitre PBS washed twice, rinse out unconjugated antibody.The compound that adds 20 microlitre 5X in each hole adds the scavenger cell of the 15k that places 80 microlitre liquid (15000) differentiation that scrapes from culture plate subsequently.Cell was cultivated 16 hours in one 37 ℃ cultivating container, collected supernatant liquid and carried out the Luminex of IL-6 and TNF α is analyzed, and method is similar to the description of the former generation T cell that rises.
7.18.7.4LPS (lipopolysaccharides) stimulates
Carry out LPS (lipopolysaccharides) when stimulating, 10 microlitre 10X storing solutions are added to cultivate in advance in ready cell-compound liquid, make its ultimate density reach 10 millimicro grams per milliliters.Cultivated 16 hours at 37 ℃ of following pair cells then, supernatant liquid is analyzed by the above method.
The compound and the solvent of different concns compare separately, to judge the IC of every kind of compound to every kind of cytokine 50It is of the present invention 2 that table 11 is depicted as, the representative IC of 4-pyrimidinediamine compounds 50Value.
Figure A20091000677107241
Figure A20091000677107251
Figure A20091000677107261
Figure A20091000677107271
Figure A20091000677107281
Figure A20091000677107291
Figure A20091000677107301
Figure A20091000677107321
Figure A20091000677107331
Figure A20091000677107351
Figure A20091000677107361
Figure A20091000677107381
Figure A20091000677107391
Figure A20091000677107401
Figure A20091000677107411
Figure A20091000677107421
Figure A20091000677107431
Figure A20091000677107451
Figure A20091000677107461
Figure A20091000677107471
Figure A20091000677107481
Figure A20091000677107491
Figure A20091000677107511
Figure A20091000677107541
Figure A20091000677107551
Figure A20091000677107561
Figure A20091000677107571
Figure A20091000677107581
Figure A20091000677107591
Figure A20091000677107611
Figure A20091000677107621
Figure A20091000677107631
Figure A20091000677107641
Figure A20091000677107661
Figure A20091000677107671
Figure A20091000677107681
Figure A20091000677107691
Figure A20091000677107711
Figure A20091000677107721
Figure A20091000677107741
Figure A20091000677107751
Figure A20091000677107761
Figure A20091000677107771
Figure A20091000677107781
Figure A20091000677107791
Though above invention has compared detailed description,, obviously still can carry out some change and modification, and not exceed within the scope of the described right application in back so that understand.Therefore, described particular embodiment should be regarded as illustrating, and should not regard the scope restriction as, and the present invention is not limited within the details scope described above, but can make amendment in the appended suitable scope of right application clause.
No matter document that all are quoted in this application and patent are all quoted at this as just bibliography under which kind of situation.

Claims (3)

1。A kind of compound, it is selected from following compound or its salt:
Segment number is 7.4.30,7.4.31,7.4.32,7.4.33,7.4.34,7.4.35,7.4.36,7.4.37,7.4.38,7.4.39,7.4.40,7.4.41,7.4.42,7.4.43,7.4.44,7.4.45,7.4.46,7.4.47,7.4.48,7.4.49,7.4.50,7.4.51,7.4.52,7.4.53,7.4.54,7.4.55,7.4.56,7.4.57,7.4.58,7.4.59,7.4.60,7.4.61,7.4.62,7.4.63,7.4.64,7.4.65,7.4.66,7.4.67,7.4.68,7.4.69,7.4.70,7.4.71,7.4.72,7.4.73,7.4.74,7.4.75,7.4.76,7.4.77,7.4.78,7.4.79,7.4.80,7.4.81,7.4.82,7.4.83,7.4.84,7.4.85,7.4.86,7.4.87,7.4.88,7.4.89,7.4.90,7.4.101,7.4.102,7.4.103,7.4.104,7.4.105,7.4.106,7.4.107,7.4.108,7.4.109,7.4.110,7.4.111,7.4.112,7.4.113,7.4.114,7.4.115,7.4.116,7.4.117,7.4.118,7.4.119,7.4.120,7.4.121,7.4.122,7.4.123,7.4.124,7.4.125,7.4.126,7.4.127,7.4.128,7.4.129,7.4.130,7.4.131,7.4.132,7.4.133,7.4.134,7.4.135,7.4.136,7.4.137,7.4.138,7.4.147,7.4.148,7.4.149,7.4.150,7.4.152,7.4.153,7.4.154,7.4.155,7.4.156,7.4.157,7.4.158,7.4.159,7.4.160,7.4.161,7.4.162,7.4.163,7.4.164,7.4.165,7.4.166,7.4.167,7.4.168,7.4.169,7.4.170,7.4.171,7.4.172,7.4.173,7.4.174,7.4.175,7.4.176,7.4.177,7.4.178,7.4.179,7.4.180,7.4.183,7.4.186,7.4.187,7.4.194,7.4.195,7.4.196,7.4.197,7.4.198,7.4.199,7.4.200,7.4.201,7.4.202,7.4.203,7.4.204,7.4.205,7.4.206,7.4.207,7.4.208,7.4.209,7.4.210,7.4.211,7.4.213,7.4.245,7.4.247,7.4.248,7.4.249,7.4.253,7.4.254,7.4.255,7.4.259,7.4.260,7.4.261,7.4.270,7.4.271,7.4.277,7.4.281,7.4.282,7.4.290,7.4.291,7.4.292,7.4.293,7.4.294,7.4.295,7.4.297,7.4.303,7.4.306,7.4.307,7.4.314,7.4.316,7.4.319,7.4.320,7.4.321,7.4.322,7.4.323,7.4.324,7.4.325,7.4.326,7.4.327,7.4.328,7.4.329,7.4.342,7.4.343,7.4.344,7.4.345,7.4.346,7.4.347,7.4.348,7.4.349,7.4.350,7.4.351,7.4.352,7.4.353,7.4.354,7.4.355,7.4.356,7.4.357,7.4.358,7.4.359,7.4.360,7.4.361,7.4.362,7.4.372,7.4.373,7.4.374,7.4.375,7.4.376,7.4.377,7.4.378,7.4.379,7.4.380,7.4.381,7.4.382,7.4.384,7.4.385,7.4.386,7.4.387,7.4.388,7.4.389,7.4.390,7.4.391,7.4.392,7.4.393,7.4.394,7.4.395,7.4.396,7.4.397,7.4.398,7.4.399,7.4.400,7.4.401,7.4.402,7.4.403,7.4.404,7.4.405,7.4.406,7.4.407,7.4.408,7.4.410,7.4.411,7.4.412,7.4.413,7.4.414,7.4.415,7.4.416,7.4.417,7.4.418,7.4.419,7.4.420,7.4.421,7.4.422,7.4.423,7.4.424,7.4.425,7.4.426,7.4.427,7.4.428,7.4.429,7.4.430,7.4.431,7.4.432,7.4.433,7.4.434,7.4.435,7.4.436,7.4.437,7.4.438,7.4.439,7.4.440,7.4.441,7.4.442,7.4.443,7.4.444, or the compound of 7.4.445.
2. pharmaceutical composition, it comprises the described compound of claim 1 and pharmaceutically acceptable carrier, vehicle and/or thinner.
3. the described compound of claim 1 is used for the treatment of application in the medicine of autoimmune disorders in preparation.
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CN102746337A (en) * 2012-06-21 2012-10-24 成都苑东药业有限公司 2,4-pyrimidinediamine compound and preparation method thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102746337A (en) * 2012-06-21 2012-10-24 成都苑东药业有限公司 2,4-pyrimidinediamine compound and preparation method thereof
CN102746337B (en) * 2012-06-21 2014-12-17 成都苑东药业有限公司 2,4-pyrimidinediamine compound and preparation method thereof

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