CN110300586A - Anti-inflammatory composition comprising IRAK and JAK inhibitor - Google Patents

Anti-inflammatory composition comprising IRAK and JAK inhibitor Download PDF

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CN110300586A
CN110300586A CN201880012183.5A CN201880012183A CN110300586A CN 110300586 A CN110300586 A CN 110300586A CN 201880012183 A CN201880012183 A CN 201880012183A CN 110300586 A CN110300586 A CN 110300586A
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R·C·X·布莱斯
R·A·加利安
S·I·J·德沃斯
D·阿曼蒂尼
P·克莱门特-拉克鲁瓦
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Galapagos NV
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    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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Abstract

The invention discloses the composition of second of compound comprising compound of formula I and with JAK inhibitory, wherein R1、R2It is as defined herein with Cy.The present invention relates to composition, preparation method, the pharmaceutical composition comprising the composition and the treatment methods for using it, and the method includes passing through the prevention of application the compound of the present invention and/or treatment inflammatory disease, autoimmune disease, proliferative diseases, allergic disease, graft rejection, the disease for being related to cartilage update damage, congenital cartilage deformity and/or the disease relevant to IL6 or interferon hypersecretion.

Description

Anti-inflammatory composition comprising IRAK and JAK inhibitor
Invention field
The present invention relates to for preventing and/or treating inflammatory disease, autoimmune disease, proliferative diseases, allergia disease Disease, graft rejection, be related to cartilage update damage disease, congenital cartilage deformity and/or with IL6 or interferon hypersecretion phase The composition of the disease of pass.In a specific aspect, the composition includes the first chemical combination with IRAK inhibitory activity Object and second of compound with JAK inhibitory.The present invention also provides comprising combined pharmaceutical composition of the invention, with And prevented by applying combination of the invention and/or treatment disease including inflammatory disease, autoimmune disease, proliferative diseases, Allergic disease, graft rejection, the disease for being related to cartilage update damage, congenital cartilage are lopsided and/or divide with IL6 or interferon Secrete the method for excessive relevant disease.
Background of invention
Kinases participates in many basic processes of stechiology, such as protein phosphorylation.Particularly, protein and lipid Activation, growth, differentiation and the survival of kinases participation cell.Protein kinase can be divided into that of preferential phosphorylation tyrosine residue A little and those of preferential phosphorylation serine and/or threonine residues.
For many years, kinases has been developed as the very important target (Cohen, 2009) of exploitation anti-inflammatory drug.Especially Ground, interleukin 1 receptor associated kinase (IRAK), more particularly IRAK-4 have been identified as in inflammation and itself exempt from Work in epidemic disease (Ringwood and Li, 2008；Wang et al., 2009).
IRAK is expressed in many cell types and is mediated from including interleukin-1 (IL-1) and toll sample receptor (TLR) Various kinds of cell receptor signal.In IRAK family, it has been identified that 4 members, i.e. IRAK 1-4 (Wang et al., 2009), and IRAK-4 (the newest member of the family) represents attractive therapeutic targets (Li et al. people, 2002).In fact, IRAK-4 is considered as the key protein matter kinases of the early stage downstream activation in IL-1 receptor and TLR (other than TLR3), It is conducted via the fast activating enabling signal of IRAK-1 and IRAK-2, congenital immunity is caused to respond.Moreover, other interleukins, example Such as IL-18 and IL-33, signal transduction is carried out dependent on IRAK-4.Therefore, these cell factors participate in the disease of pathogenic course (for example, fibre modification (Li et al. people, 2014；McHedlidze et al., 2013；Rankin et al., 2010) and atopic dermatitis (Salimi et al., 2013)) it is potential targeted condition by IRAK-4 inhibitor for treating.
Inactive IRAK-4 mutant is being expressed in the mouse of wild type, is observing and is touched to by several TLR agonists The complete resistance of the septic shock of hair and impaired response to IL-1.In addition, express inactive IRAK-4 mutant rather than The mouse of wild type several autoimmune disease models such as rheumatoid arthritis (Koziczak-Holbro et al., 2009) it and is partly protected in multiple sclerosis (Staschke et al., 2009).Interestingly, rheumatoid joint Scorching and Patients with SLE serum is shown activates plasmacytoid dendritic cells (Chiang in a manner of IRAK-4 dependence Et al., 2011).Finally, observing that recurrent is suppurative thin in the children with the genetic defect for causing IRAK-4 inactive Bacterium infection.Due to not observing these pyogenic infections, IRAK-4 signal in the adult for carrying inactivation IRAK-4 mutation System is seemingly extra for some aspects of adult congenital immunity.
The imbalance of the signal transduction component of innate immune system is also increasingly considered important in cancer starting and progress Factor (Rhyasen and Starczynowski, 2015).In fact, proving that IL-1 is raw in growth of tumour cell, blood vessel on evidence At serve in, invasion, drug resistance and transfer directly (Carmi et al., 2013；Vidal-Vanaclocha et al., 2000).Separately Outside, depending on the background of tumour cell, TLR participates in a large amount of tumours that promote and responds.As the required of IL-1 receptor and TLR signal transduction Medium, IRAK family kinase represent promising cancer drug target.In addition, several cancer types have been displayed dependent on MYD88 Activated form, be the linkers in the downstream TLR and IL-1R, activate IRAK-4.In such as diffusivity large B cell lymph Activation is identified in tumor (DLBCL) (Ngo et al., 2011) and Waldenstrom macroglobulinemia (Treon et al., 2012) MYD88 mutation.In addition report supports IRAK-4 in oncology, especially T- cell acute lymphoblastic leukaemia (T-ALL) effect (Li et al. people, 2015) in.Have shown that the pharmacology of IRAK-4 inhibits enhancing T-ALL to the quick of chemotherapeutics Perception.
Have shown that IL-33 in the development of fibre modification and allergic disease, especially asthma and atopic dermatitis In work (Nabe, 2014).Since the cell factor carries out signal transduction (Kroeger etc. by IRAK-4 dependent pathway People, 2009), these diseases can also represent the target of IRAK-4 inhibitor.
Finally, it is active dependent on IL-1 to have shown that itself several inflammatory disease, and therefore, IL-1 blocks property biology system Agent shows some benefits to these patients.Gout, juvenile idiopathic arthritis, Mu-Wei disease, familial Mediterranean fever, shellfish He Qiete disease, adult onset Still disease are the examples (Dinarello et al., 2012) of itself inflammatory disease.
It can help to mitigate the disease outcome of immune-inflammatory diseases with the conduction of little molecules in inhibiting cytokine signaling (Sundberg et al., 2014).Particularly, cell factor can defend in pathogen and infection to work in organism.However, When develop immune-inflammatory diseases new treatment when, on the one hand it is essential that selection participate in can not damage it is appropriate and/or first Target in the case where its immune response in repressed approach, because inhibiting multiple cytokine response approach may mistake simultaneously Degree weakens immune system.However, it is difficult to realize drug to the selectivity of kinases (Bain et al., 2003；Fabian et al., 2005), but in order to avoid relevant side effect of missing the target, this selectivity is highly desirable to, especially in long-term treatment Under background (Broekman et al., 2011；Dy and Adjei, 2013；Force and Kolaja, 2011).
Particularly, display recently is adjoint uses IL-1 blocking agent (anakinra (Anakinra)) and TNF α blocking agent (Etanercept (Etanercept)) cause neutrophils reduce and infection risk increase (Genovese et al., 2003, EMEA public statement EMEA/31631/02,05Feb 2003).The discovery emphasizes selectivity in developing new drug object When be key element, and therefore, it is intended that exploitation can be selectively adjusted signal transduction path without influencing other approach Compound can especially be selectively adjusted compound of the IL-1 response without influencing TNF α signal transduction path.
Janus kinases (JAK) is the cytoplasm that the cytokine signaling from membrane receptor is conducted to STAT transcription factor Tyrosine kinase.Describe four kinds of JAK family members, JAK1, JAK2, JAK3 and TYK2.When cell factor is in conjunction with its receptor When, JAK family member autophosphorylation and/or turn phosphorylation each other, STAT phosphorylation then occurs, then moves to nucleus To adjust transcription.JAK-STAT intracellular signal transduction act on interferon, most of interleukins and various kinds of cell because Son and endocrine factor, such as EPO, TPO, GH, OSM, LIF, CNTF, GM-CSF and PRL (Vainchenker W. et al. (2008))。
The combination of genetic model and the research of small molecule JAK inhibitor discloses the treatment potentiality of some JAK.Mouse and the mankind Science of heredity confirms that JAK3 is immunosupress target (O ' Shea J. et al. (2004)).JAK3 inhibitor is successfully brought into be faced Bed exploitation, is used primarily for organ-graft refection, but was also used for other immunoinflammatory indications, such as rheumatoid joint later Scorching (RA), psoriasis and Crohn disease (http://clinicaltrials.gov/).
TYK2 is a kind of potential target that the immune-inflammatory diseases that research confirms are knocked out by human genetics and mouse (Levy D. and Loomis C. (2007)).
JAK1 is the new target drone in immune-inflammatory diseases region.JAK1 and other JAK carries out heterodimericization, thin to conduct The proinflammatory disease signal transduction of intracellular cytokine driving.Therefore, it is contemplated that inhibit JAK1 and/or other JAK for a large amount of inflammatory diseases and The Other diseases of the signal transduction driving mediated by JAK have treatment benefit.
Current therapy is simultaneously unsatisfactory, therefore, there is still a need for identify can be used for preventing and/or treating inflammatory disease, Autoimmune disease, proliferative diseases, allergic disease, graft rejection, the disease for being related to cartilage update damage, congenital cartilage Otherization with the correlation side effect of missing the target reduced of deformity and/or disease relevant to IL6 or interferon hypersecretion Close object.
Brief description
Fig. 1 is described compared with excipient (solid diamond), 1mg/kg bid (black triangle), 3mg/kg bid (ten Font), the exemplary compounds 1 of 10mg/kg bid (asterisk) and 30mg/kg bid (solid circles) are in treatment CIA model Clinical score from the 31st day to the 46th day.
Fig. 2 is described compared with excipient (solid diamond), 7.5mg/kg bid (black triangle) and 15mg/kg bid The exemplary compounds XXb and compound 1 (30mg/kg bid) of (Saint Andrew's cross shape)+compound XXb (15mg/kg bid) The clinical score of (solid circles) in treatment CIA model from the 31st day to the 41st day.
Invention summary
The present invention, which has been based on the discovery that, can be used for preventing and/or treating inflammatory disease, autoimmune disease, proliferative disease Disease, allergic disease, graft rejection, be related to cartilage update damage disease, congenital cartilage deformity and/or with IL6 or interference The present composition of the relevant disease of plain hypersecretion.In a particular aspect, the composition includes to have IRAK suppression Make the first active compound and second of compound with JAK inhibitory.
The present invention also provides prevent comprising combined pharmaceutical composition of the invention, and by applying combination of the invention And/or treatment disease method, the disease include inflammatory disease, autoimmune disease, proliferative diseases, allergic disease, Graft rejection, the disease for being related to cartilage update damage, congenital cartilage are lopsided and/or related to IL6 or interferon hypersecretion Disease.
In a particular aspect, JAK inhibitor has JAK1 inhibitory activity.In an embodiment particularly, JAK inhibitor is JAK1 selective depressant.
In another particular aspect, IRAK inhibitor has IRAK4 inhibitory activity.In an embodiment particularly In, IRAK inhibitor is IRAK4 selective depressant.
Therefore, in the first aspect of the invention, composition of the invention is provided, it includes:
A) compound of Formulas I
Wherein
Cy is
Optionally by the R of one or more independent choices3Substituted monocycle C3-7Naphthenic base, or
Optionally by the R of one or more independent choices3What is replaced includes one or two independently selected from N, S and O Heteroatomic 4-7 unit monocycle Heterocyclylalkyl；
R1For
- H,
--SO3H,
-- P (=O) (OH)2,
-C1-4Alkyl,
-- C (=O)-(includes one or two heteroatomic 4-7 unit monocycle Heterocyclylalkyl independently selected from N, S and O), Or
-- C (=O) C1-6Alkyl, wherein C1-6Alkyl is optionally by the R of one or more independent choices4Group replaces；
R2For H or C1-4Alkyl；
Each R3Independently selected from
- OH,
=O,
Halogen, and
-C1-4Alkyl；
Each R4Independently selected from:
--NR5aR5b,
-- C (=O) OH,
Optionally by the C of one or more independent choices1-4Alkyl-substituted includes one or two independently selected from N, S With the heteroatomic 4-7 unit monocycle Heterocyclylalkyl of O, and
-- NHC (=O)-C1-4Alkyl-NH2；And
R5aAnd R5bIt independently is H or C1-4Alkyl；
Or the salt of its officinal salt or solvate or solvate；With
B) with second of compound of JAK inhibitory.
In a particular aspect, composition of the invention is provided for preventing and/or treating inflammatory disease, autoimmunity Disease, proliferative diseases, allergic disease, graft rejection, be related to cartilage update damage disease, congenital cartilage deformity and/ Or disease relevant to IL6 or interferon hypersecretion.
In one aspect, combination of the invention can inhibit IRAK kinase families member, more particularly IRAK-4.
In one aspect, combination of the invention can inhibit jak kinase family member, more particularly JAK1.
On the other hand, combination of the invention can show the selectivity to IRAK-4 and JAK1, and can get and change Kind safety and lower related side effects of missing the target.In a particular aspect, combination of the invention can be the choosing of IL-1 Selecting property inhibitor.
In an additional aspect of the present invention, also unexpectedly confirm, it is of the invention compared with each member of individual application The effect of combination shows increase.In a particular aspect, combination of the invention can show synergistic effect, can permit Reduce the dosage of every kind of component of the present composition.This can advantageously be avoided taking unnecessary medication amount and kept simultaneously Effect, thus reduce the risk of unfavorable medical event.
On the other hand, the present invention provides pharmaceutical compositions, and it includes compositions of the invention and pharmaceutical carrier, tax Shape agent or diluent.In a particular aspect, pharmaceutical composition, which can additionally comprise, to be suitble to combine with the compound of the present invention The other therapeutic activity ingredients used.One more particularly aspect, other therapeutic activity ingredients be for treat inflammatory disease, Autoimmune disease, proliferative diseases, allergic disease, graft rejection, the disease for being related to cartilage update damage, congenital cartilage The reagent of deformity and/or disease relevant to IL6 or interferon hypersecretion.
Moreover, the composition of the invention in pharmaceutical composition disclosed herein and treatment method is making and using When be pharmaceutical.
In another aspect of the invention, the present invention provides a kind of treatments to suffer from selected from those herein set forth illness simultaneously And especially inflammatory disease, autoimmune disease, proliferative diseases, allergic disease, graft rejection, be related to cartilage update damage Disease, congenital cartilage deformity and/or relevant to IL6 or interferon hypersecretion disease mammal, particularly people The method of class, the method includes applying a effective amount of pharmaceutical composition as of the invention described herein or combination.
The present invention also provides the pharmaceutical compositions for medicine, and it includes compositions of the invention and suitable medicinal load Body, excipient or diluent.In a particular aspect, pharmaceutical composition is for preventing and/or treating inflammatory disease, itself exempt from Epidemic disease, proliferative diseases, allergic disease, graft rejection, the disease for being related to cartilage update damage, congenital cartilage deformity And/or disease relevant to IL6 or interferon hypersecretion.
On the other hand, therein the present invention provides the method for synthesizing the compound in the present composition Representative synthetic schemes and approach are published in this article later.
By considering that subsequent detailed description, other objects and advantages are apparent to those skilled in the art.
It should be appreciated that the compound of the present invention can be metabolized generation bioactive metabolites.
Detailed description of the invention
Definition
Following term has the meaning being set forth below, and can be used for understanding description of the invention and desired extent.
When description the present invention (its may include compound, the pharmaceutical composition containing the compound and use the compound With the method for composition) when, unless otherwise stated, following term (if present) has following meanings.Should also Understand, when being described herein, any part that definition is listed herein below can be replaced by a variety of substituent groups, and each definition Be intended to by it is such it is substituted be partly comprised in list as follows they in the range of.Unless otherwise stated, term " replaces " be defined as described below.It will be further understood that as used herein, term " group " and " base " are considered It is interchangeable.
The article that may be used herein "one" and "an" refer to one (kind) or more than one (kind) (i.e. at least one (kind)) the article grammar object.For example, " analog " refers to a kind of analog or more than one analog.
" alkyl " refers to the linear chain or branched chain aliphatic hydrocarbon with specified carbon atom number.Specific alkyl has 1 to 6 carbon atom Or 1 to 4 carbon atom.Branch refers to that one or more alkyl (such as methyl, ethyl or propyl) are connected to linear alkyl chain.Specifically Alkyl be methyl (- CH3), ethyl (- CH2-CH3), n-propyl (- CH2-CH2-CH3), isopropyl (- CH (CH3)2), normal-butyl (-CH2-CH2-CH2-CH3), tert-butyl (- CH2-C(CH3)3), sec-butyl (- CH2-CH(CH3)2), n-pentyl (- CH2-CH2- CH2-CH2-CH3), n-hexyl (- CH2-CH2-CH2-CH2-CH2-CH3) and 1,2- dimethylbutyl (- CHCH3)-C(CH3)H2- CH2-CH3).Specific alkyl has 1 to 4 carbon atom.
" alkenyl " refers to monovalent olefinic (unsaturation) hydrocarbyl group with specified carbon atom number.Specific alkenyl has 2 To 8 carbon atoms, and more particularly there are 2 to 6 carbon atoms, can be linear chain or branched chain, and have at least 1 A and 1 to 2 olefinic unsaturated sites in particular.Specific alkenyl includes vinyl (- CH=CH2), positive acrylic (- CH2CH=CH2), isopropenyl (- C (CH3)=CH2) etc..
" alkylidene " refers to the divalent alkenyl group group with specified carbon atom number, particularly has 1 to 6 carbon atom, more special Not there is 1 to 4 carbon atom, can be straight chain or branch.The exemplary radicals of the term be for example methylene (- CH2), ethylidene (- CH2-CH2) or-CH (CH3)-etc..
" alkynylene " refers to the divalent alkynyl radical group with specified carbon atom number and specified three bond numbers, particularly has 2 to 6 carbon Atom more particularly has 2 to 4 carbon atoms, can be straight chain or branch.The exemplary radicals of the term are such as-C ≡C-、-CH2- C ≡ C- and-C (CH3)H-C≡CH-。
" alkoxy " refers to group O- alkyl, wherein the alkyl has the carbon atom of specified quantity.Specifically, which refers to Group-O-C1-6Alkyl.Specific alkoxy is methoxyl group, ethyoxyl, positive propoxy, isopropoxy, n-butoxy, tertiary fourth oxygen Base, sec-butoxy, n-pentyloxy, positive hexyloxy and 1,2- dimethyl butyrate oxygroup.Specific alkoxy is lower alkoxy, that is, is had There is 1 to 6 carbon atom.More specific alkoxy has 1 to 4 carbon atom.
" amino " refers to group-NH2。
" aryl " refers to through the univalent aromatic hydrocarbon derived from the single carbon atom of parent aromatic ring system one hydrogen atom of removing Group.Specifically, aryl refers to the aromatic ring structure (monocycle or fused polycycle) with specified annular atom number.In particular, the art Language includes the group comprising 6 to 10 ring members.Specific aryl group includes phenyl and naphthalene.
" naphthenic base " refer to specified annular atom number non-aromatic hydrocarbons ring structures (monocycle, fused polycycle, bridging it is polycyclic or Loop coil).Naphthenic base can have 3 to 12 carbon atoms, especially 3 to 10, more particularly 3 to 7 carbon atoms.For example, Such naphthenic base includes single ring structure, such as cyclopropyl, cyclobutyl, cyclopenta, cyclohexyl and suberyl.
" cyano " refers to group-CN.
" halogenated " or " halogen " refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).Special halogen is fluorine or chlorine.
When the group for describing compound or being present on compound, " miscellaneous " refers to one in the compound or group Or multiple carbon atoms are replaced by nitrogen, oxygen or sulfur heteroatom.It is miscellaneous to can be applied to any one of above-mentioned hydrocarbyl group, example Such as there is 1 to 4, particularly there is 1,2 or 3 hetero atom, more generally 1 or for example single hetero atom of 2 hetero atoms Alkyl such as miscellaneous alkyl, naphthenic base such as Heterocyclylalkyl, aryl such as heteroaryl etc..
" heteroaryl " refers to aromatic ring structure (monocycle or fused polycycle), and it includes one or more independently selected from O, N and S Hetero atom and specify number annular atom.Particularly, aromatic ring structure can have 5 to 9 ring members.Heteroaryl can be example Such as five yuan or hexa-atomic of monocycles or by condensed five-membered ring and hexatomic ring or two condensed hexatomic rings or (as another reality Example) the condensed bicyclic ring structures that are formed of two condensed five-membered rings.Each ring may include at most four be generally selected from nitrogen, sulphur and The hetero atom of oxygen.Typically, heteroaryl ring include at most 4 hetero atoms, more typically at most 3 hetero atoms, more typically at most 2 A hetero atom, such as single hetero atom.In one embodiment, heteroaryl ring includes at least one theheterocyclic nitrogen atom.Heteroaryl Ring nitrogen can be alkalinity, such as in the situation of imidazoles or pyridine, or substantially non-alkaline, such as in indoles Or in the situation of pyrroles's nitrogen.In general, basic nitrogen atom present in heteroaryl groups (any amino-substituent including ring) Quantity should be less than five.
The example of five unit monocycle heteroaryls include, but are not limited to pyrrole radicals, furyl, thienyl, imidazole radicals, furazanyl, Oxazolyl, dislikes triazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, triazolyl and tetrazole radical at oxadiazoles base.
The example of single six-membered rings heteroaryl includes but is not limited to pyridyl group, pyrazinyl, pyridazinyl, pyrimidine radicals and triazine radical.
Specific example comprising being fused to another pentacyclic bicyclic heteroaryl includes but is not limited to imidazoles Benzothiazolyl and imidazo imidazole radicals.
The specific example of bicyclic heteroaryl comprising being fused to a pentacyclic hexatomic ring includes but is not limited to benzo furan It mutters base, benzothienyl, benzimidazolyl, benzoxazolyl, different benzoxazolyl, benzo isoxazolyl, benzothiazolyl, benzene And isothiazolyl, isobenzofuran-base, indyl, isoindolyl, indolizine base, purine radicals (such as adenine, guanine), indazole Base, pyrazolopyrimidine base, triazolopyrimidinyl and Pyrazolopyridine base.
The specific example of bicyclic heteroaryl comprising two condensed hexatomic rings includes but is not limited to quinolyl, isoquinolin Base, pyridopyridine base, quinoxalinyl, quinazolyl, cinnoline base, phthalazinyl, naphthyridines base and pteridyl.Specifically heteroaryl is Derived from thienyl, pyrrole radicals, benzothienyl, benzofuranyl, indyl, pyridyl group, quinolyl, imidazole radicals, oxazolyl Those of with pyrazinyl.
The example of representative heteroaryl includes following heteroaryl:
Wherein each Y is selected from > C=O, NH, O and S.
" Heterocyclylalkyl " refers to the non-aromatic monocycle being saturated entirely, fused polycycle, loop coil or the polycyclic ring structure of bridging, and it includes one The annular atom of a or multiple hetero atoms and specified quantity independently selected from O, N and S.Heterocyclylalkyl ring structure can have 4 to 12 ring members, especially 4 to 10 ring members, more particularly 4 to 7 ring members.Each ring may include at most four It is typically chosen from the hetero atom of nitrogen, sulphur and oxygen.Typically, heterocycloalkyl ring include at most 4 hetero atoms, more typically at most 3 A hetero atom, more generally at most 2, for example single hetero atom.The example of heterocycle includes but is not limited to azetidinyl, oxygen Azetidinyl, Thietane base, pyrrolidinyl (for example, 1- pyrrolidinyl, 2- pyrrolidinyl and 3- pyrrolidinyl), tetrahydro Furyl (for example, 1- tetrahydrofuran base, 2- tetrahydrofuran base and 3- tetrahydrofuran base), tetrahydro-thienyl are (for example, 1- tetrahydro thiophene Pheno base, 2- tetrahydro-thienyl and 3- tetrahydro-thienyl), piperidyl is (for example, 1- piperidyl, 2- piperidyl, 3- piperidyl and 4- piperazine Piperidinyl), THP trtrahydropyranyl (for example, 4- THP trtrahydropyranyl), tetrahydro thiopyranyl (for example, 4- tetrahydro thiopyranyl), Quinoline base, thio-morpholinyl, dioxanes base or piperazinyl.
Term " heterocycloalkenyl " as used herein refers to " Heterocyclylalkyl " comprising at least one double bond.The tool of heterocycloalkenyl Body example is shown in the example of following exemplary:
Wherein each W is selected from CH2, NH, O and S；Each Y is selected from NH, O, C (=O), SO2And S；And each Z is selected from N or CH.
The specific example of monocycle is shown in the example of following exemplary:
Wherein each W and Y are independently selected from-CH2,-NH- ,-O- and-S-.
The specific example of fused bicyclic is shown in the example of following exemplary::
Wherein each W and Y are independently selected from-CH2,-NH- ,-O- and-S-.
The specific example of bridged bicyclic is shown in the example of following exemplary:
Wherein each W and Y are independently selected from-CH2,-NH- ,-O- and-S-, and each Z is selected from N or CH.
The specific example of loop coil is shown in the example of following exemplary:
Wherein each Y is selected from-CH2,-NH- ,-O- and-S-.
" hydroxyl " refers to group-OH.
" oxo " refers to group=O.
" substituted " refers to the base that wherein one or more hydrogen atoms are substituted by identical or different substituent group each independently Group.
" sulfo group " or " sulfonic acid " refers to group such as-SO3H。
" mercaptan " refers to group-SH.
Term " being replaced by one or more " as used herein refers to one to four substituent group.In one embodiment, It refers to one to three substituent group.In a further embodiment, refer to one or two substituent group.Still further real It applies in scheme, refers to a substituent group.
" thio alkoxy " refers to group-S-alkyl, wherein the alkyl has the carbon atom of specified quantity.Particularly, should Term refers to group-S-C1-6Alkyl.Specific thio alkoxy is thiornethoxy group, thio ethoxy, just thio propoxyl group, different Thio propoxyl group, just thio butoxy, different thio butoxy, tertiary thio butoxy, secondary thio butoxy, just thio amoxy, Just thio hexyloxy and 1,2- dimethyl thio butoxy.Specific thio alkoxy is lower thioalkoxy, that is, has 1 To 6 carbon atoms.More specific alkoxy has 1 to 4 carbon atom.
Ordinary technicians in the field of organic synthesis it should be appreciated that stable, chemically feasible heterocycle (no matter it is Aromatics or it is non-aromatic) in heteroatomic maximum quantity be to be determined by the size of ring, degree of unsaturation and heteroatomic valence 's.In general, heterocycle can have one to four hetero atom, as long as heteroaromatic rings are chemically feasible and stable.
" pharmaceutically acceptable " refers to that the corresponding mechanism of the country by federal or management organization, state government or in addition to the U.S. is ratified Or it can ratify or be listed in United States Pharmacopeia (US Pharmacopeia) or other generally acknowledged pharmacopeia for animal (and particularly It is the mankind) in.
" officinal salt " refers to the salt of the compounds of this invention of pharmaceutically acceptable and expectation pharmacological activity with parent compound. Particularly, such non-toxic salts can be inorganic or organic acid addition salt and base addition salts.Particularly, such salt includes: (1) acid-addition salts formed with inorganic acid, the inorganic acid are such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid；Or with have The acid-addition salts that machine acid is formed, the organic acid are such as acetic acid, propionic acid, caproic acid, pentamethylene propionic acid, glycolic, pyruvic acid, cream Acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3- (4- hydroxy benzoyl) Benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2- ethane-disulfonic acid, 2- ethylenehydrinsulfonic acid, benzene sulfonic acid, 4- chlorine Benzene sulfonic acid, 2- naphthalene sulfonic acids, 4- toluenesulfonic acid, camphorsulfonic acid, 4- methyl bicyclic [2.2.2]-oct-2-ene -1- formic acid, glucoheptose Acid, 3- phenylpropionic acid, trimethylace tonitric, butylacetic acid, lauryl sulfate, gluconic acid, glutamic acid, hydroxynaphthoic acid, water Poplar acid, stearic acid, muconic acid etc.；Or (2) when the acid proton being present in parent compound by metal ion (for example, alkali is golden Belong to ion, alkaline-earth metal ions or aluminium ion) when replacing or with organic base (such as ethanol amine, diethanol amine, triethanolamine, N- Methylglucosamine etc.) coordination when the salt that is formed.For example only, salt further comprises sodium salt, sylvite, calcium salt, magnesium salts, ammonium Salt, tetraalkylammonium salt etc.；And when compound contains basic functionality, the salt including nontoxic organic or inorganic acid, such as Hydrochloride, hydrobromate, tartrate, mesylate, acetate, maleate, oxalates etc..Term " it is pharmaceutical sun from Son " refers to the acceptable cation counterbalancing ion of acidic functionality.Such cation is such as sodium, potassium, calcium, magnesium, ammonium, four alkane Base ammonium cation etc..
" pharmaceutically acceptable excipient " refers to the diluent applied together with the compounds of this invention, auxiliary agent, excipient or carrier.
" prodrug " refers to cleavable moiety and can become by solvolysis or under physiological conditions having in vivo There is the compound of the compounds of this invention of pharmaceutical active, the derivative including the compounds of this invention.Such example includes but not It is limited to choline ester derivant etc., N- alkyl morpholine ester etc..
" solvate " refers to the compound form usually by solvolysis reaction and solvent association (associated).It should Physical association includes hydrogen bonding.Conventional solvents include water, EtOH, acetic acid etc..The compound of the present invention can be prepared as example Crystal form, and solvation or hydration can be carried out.Suitable solvate includes pharmaceutical acceptable solvates, such as is hydrated Object, and further comprise the solvate and non-stoichiometric solvate of stoichiometry.In some cases, for example, When in the lattice of one or more solvent molecules incorporation crystalline solid, solvate can be separated." solvate " includes Solution phase and separable solvate.Representative solvate includes hydrate, alcoholate and methylate.
" individual " includes the mankind.Term " mankind ", " patient " and " individual " is used interchangeably herein.
" effective quantity " refers to be enough to realize the compounds of this invention of the disease treatment when being administered to individual to treat disease Amount." effective quantity " can change according to compound, disease and its severity and age, the weight of individual to be treated etc..
" prevention " refers to that the risk of disease or obstacle is suffered from or occurred to reduction (that is, making the disease before seizure of disease At least one clinical symptoms do not occur in the individual for being likely to be exposed at pathogenic agent or the easy infection disease).
Term " prevention and treatment " is related with " prevention ", and refers to that purpose is the measure or journey of prevention and non-treatment or healing disease Sequence.The non-limiting example of control measure may include administration of vaccines；There is thrombosis wind to due to cannot for example move Inpatient admission's low molecular weight heparin of danger；And access malaria be endemic disease or the higher geography of the risk being infected with malaria Antimalarial agent such as chloroquine is administered when region in advance.
In one embodiment, " treatment " of any disease or illness refers to that improving the disease or illness (prevents to be somebody's turn to do Disease or performance, degree or the seriousness for mitigating its at least one clinical symptoms).In another embodiment, " treatment " be Referring to improves at least one indistinguishable body parameter of individual.In another embodiment, " treatment " refers in physics aspect It adjusts disease or illness (such as stable symptom experienced) or adjusts disease or illness (such as resistate in terms of physiology Manage parameter) or both.In a further embodiment, " treatment " refers to the process for slowing down disease.
Term " inflammatory disease " as used herein refers to including illness below: rheumatoid arthritis, osteoarthritis, Juvenile idiopathic arthritis, psoriasis, psoriasis arthropathica, ankylosing spondylitis, allergy airway disorders (example Such as, asthma, rhinitis), chronic obstructive pulmonary disease (COPD), inflammatory bowel disease (IBD, for example, Crohn disease, ulcerative colitis), The morbid state that sarcoidosis, endotoxin cause is (for example, bypass surgery infectious-related complication or facilitate such as chronic heart failure Chronic endotoxin state) and it is related to the related disease of cartilage, such as the related disease in joint.Particularly, which refers to class wind Wet arthritis, psoriatic arthritis, ankylosing spondylitis, osteoarthritis, allergy airway disorders (for example, asthma), Sarcoidosis, chronic obstructive pulmonary disease (COPD) and inflammatory bowel disease.More particularly, the term refer to rheumatoid arthritis, sarcoidosis, Psoriatic arthritis, ankylosing spondylitis and inflammatory bowel disease.
Term " autoimmune disease " as used herein refers to including disease below: obstructive airway diseases, including Illness such as COPD, asthma (for example, intrinsic asthma, extrinsic asthma, dust asthma, asthma in children), it is especially chronic Or obstinate asthma (for example, late asthma and airway hyperreactivity), bronchitis, including bronchial asthma, systematicness Lupus erythematosus (SLE), lupus erythematosus,cutaneous, membranous lupus nephritis, dermatomyositis, Sjogren syndrome, multiple sclerosis, silver bits Disease, scheroma, type-1 diabetes mellitus and its associated complication, atopic eczema (atopic dermatitis), thyroiditis (bridge this first shape Adenositis and autoimmune thyroiditis), contact dermatitis and other eczematous dermatitis, inflammatory bowel disease (for example, Crohn disease and Ulcerative colitis), atherosclerosis and amyotrophic lateral sclerosis, alopecia areata, vitiligo.Particularly, the term refer to COPD, Asthma, lupus erythematosus,cutaneous, membranous lupus nephritis, alopecia areata, vitiligo, type-1 diabetes mellitus and inflammatory bowel disease.
Term " proliferative diseases " as used herein refers to illness such as cancer (for example, leiomyosarcoma of uterus or preceding Column gland cancer), myeloproliferative disorders are (for example, polycythemia vera, primary thrombocytosis and myleo become Property), leukaemia (for example, Acute Meyloid sample leukaemia, acute and chronic lymphoblastic leukemia), Huppert's disease, Psoriasis, restenosis, chorionitis or fibre modification.Particularly, which refers to cancer, leukaemia, Huppert's disease and silver bits Disease.
Term " cancer " as used herein refer to skin or organ be such as, but not limited to breast, prostate, lung, The pernicious or benign growths of cell in kidney, pancreas, stomach or intestines.Cancer is easy to infiltrate into adjacent tissue, and spreads (transfer) To remote organ, such as bone, liver, lung or brain.Term cancer as used herein include metastatic cancer cell type (such as But it is not limited to melanoma, lymthoma, leukaemia, fibrosarcoma, rhabdomyosarcoma and mastocytoma) and tissue cancer type (such as, but not limited to colorectal cancer, prostate cancer, Small Cell Lung Cancer and non-small cell lung cancer, breast cancer, cancer of pancreas, bladder Cancer, kidney, gastric cancer, glioblastoma, primary carcinoma of liver, oophoroma, prostate cancer and leiomyosarcoma of uterus).Particularly, Term " cancer " refer to acute lymphoblastic leukemia, Acute Meyloid sample leukaemia, adrenocortical carcinoma, cancer of anus, appendix cancer, Astrocytoma, atypia monster sample/Rhabdoid tumor, basal-cell carcinoma, cholangiocarcinoma, bladder cancer, osteocarcinoma (osteosarcoma and evil Property fibrous histiocytoma), brain stem glioma, brain tumor, brain and spinaloma, breast cancer, bronchophyma, Hugh Burkitt lymph Tumor, cervical carcinoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, craniopharyngioma, skin T Cell lymphoma, embryoma, carcinoma of endometrium, ependymoblastoma, ependymoma, cancer of the esophagus, Ewing's sarcoma family it is swollen Between tumor, cancer eye, retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinal associated cancers tumour, gastrointestinal stromal tumor (GIST), gastrointestinal tract Cell plastid tumor, gonioma, glioma, hairy cell leukemia, head and neck cancer, liver cell (liver) cancer, Hodgkin lymphoma, Hypopharyngeal cancer, intraocular melanoma, islet-cell tumour (endocrine pancreas), Kaposi sarcoma, kidney, Langerhans cell tissue are thin Born of the same parents' increase disease, laryngocarcinoma, leukaemia, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, Chronic myelogenous leukemia, hairy cell leukemia, liver cancer, non-small cell lung cancer, Small Cell Lung Cancer, Burkitt lymphoma, skin T are thin Born of the same parents' lymthoma, Hodgkin lymphoma, non-Hodgkin lymphoma, lymthoma, macroglobulinemia Waldenstron, pith mother cells Tumor, medulloepithelioma, melanoma, celiothelioma, carcinoma of mouth, chronic myelogenous leukemia, myeloid leukemia, Huppert's disease, The pernicious fibre of nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, carcinoma of mouth, oropharyngeal cancer, osteosarcoma, bone Dimension property histocytoma, oophoroma, epithelial ovarian cancer, ovarian germ cell tumor, the low pernicious potentiality tumour of ovary, cancer of pancreas, It is former on papilloma, parathyroid carcinoma, carcinoma of penis, pharynx cancer, the pineal body achiblastoma of intermediate differentiation, pinealoblastoma and curtain Beginning neuroectodermal tumors, hypophysoma, plasmacytoma/Huppert's disease, pleuropulinonary blastoma, Primary Central Nervous system System lymthoma, prostate cancer, the carcinoma of the rectum, nephrocyte (kidney) cancer, retinoblastoma, rhabdomyosarcoma, salivary-gland carcinoma, meat Tumor, the tumour of Ewing's sarcoma family, Kaposi sarcoma, Sezary syndrome, cutaneum carcinoma, Small Cell Lung Cancer, carcinoma of small intestine, soft tissue Sarcoma, squamous cell carcinoma, gastric cancer, Supratentorial primitive neuroectodermal tumour, t cell lymphoma, carcinoma of testis, throat cancer, thymoma With thymic carcinoma, thyroid cancer, carcinoma of urethra, uterine cancer, sarcoma of uterus, carcinoma of vagina, carcinoma of vulva, waldenstrom macroglobulinemia Disease and wilms' tumor.
Term " leukaemia " as used herein refers to that blood and blood form the tumor disease of organ.Such disease can Marrow and immune system dysfunction are caused, causes host that infection and bleeding easily occurs.Particularly, term leukaemia refers to acute Myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) and chronic lymphoblastic leukaemia (CLL)。
Term " allergic disease " as used herein refers to the illness for being characterized in that the super quick obstacle of immune system, Including allergy airway disorders (such as asthma, rhinitis), nasosinusitis, eczema and nettle rash and food allergy or To the allergy of insect venom.
Term " asthma " as used herein refer to be characterized in that with no matter which kind of reason (endogenous, exogenous or both； Allergy or non-allergy) airway contraction relevant pulmonary air flow variation any lung disorder.Term asthma can be with one A or multiple adjectives to indicate reason are used together.
Term " graft rejection " as used herein refers to such as pancreas islet, stem cell, marrow, skin, muscle, cornea group It knits, neuronal tissue, heart, lung, cell, tissue or the entity device for combining the heart-lung, kidney, liver, intestines, pancreas, tracheae or esophagus The allograft of official or the acute or chronic repulsion of xenograft or graft versus host disease.
Term " being related to the disease that cartilage updates damage " as used herein includes following illnesss, such as osteoarthritis, silver Consider sick arthritis, juvenile rheumatoid arthritis, urarthritis, septic or infectional arthritis, reactive joint to be worth doing Inflammation, reflex sympathetic dystrophy, algodystrophy, Tietze syndrome or costal chondritis, fibromyalgia, osteochondritis, Neurogenic or neuropathic arthritis, arthropathy, the arthritis such as Kashin-Beck's disease of region form, Mseleni disease and Handigodu disease；Denaturation caused by fibromyalgia, systemic loupus erythematosus, chorionitis and tatanic ridge Column is scorching.
Term " congenital cartilage deformity " as used herein includes following illnesss, such as heredity chondrolysis, cartilage Depauperation and pseudoachondroplasia are especially but not limited to microtia, earless, metaphysial chondrodysplasia and phase Close illness.
Term " disease relevant to IL6 hypersecretion " as used herein includes following illnesss, such as Castleman Disease, Huppert's disease, psoriasis, Kaposi sarcoma and/or Pathology of Mesangial Proliferative Glomerulonephritis.
Term " disease relevant to interferon hypersecretion " as used herein includes following illnesss, for example, systematicness and Lupus erythematosus,cutaneous, lupus nephritis, dermatomyositis, Sjogren syndrome, psoriasis, rheumatoid arthritis.
" composition of the invention " and equivalents mean include general formulas as described herein one or more chemical combination Object, if context allows, which includes officinal salt and solvate, such as hydrate and officinal salt is molten Agent compound.Similarly, if context allows, intermediate (no matter whether themselves is claimed) Shi Yeyi is referred to Taste including their salt and solvate.
When reference is made to range, such as, but not limited to C1-8Alkyl, the reference of range are contemplated that indicate the model Each member in enclosing.
The acid and acid derivative form of other derivatives of the compound of the present invention are active, but its acid-sensitive form The advantages of dissolubility, histocompatbility or sustained release are provided usually in mammalian organism (Bundgard, H, 1985). Prodrug includes acid derivative well known to those skilled in the art, such as by making parent acid react the ester prepared with suitable alcohol, Or the amide or acid anhydrides or mixed acid anhydride prepared by reacting parent acid compound with substituted or unsubstituted amine.It is derived from The simple aliphatic series or aromatic ester, amide and acid anhydrides for the acidic-group being suspended on the compounds of this invention are particularly useful prodrugs. In some cases, it is desired to prepare diester-type prodrug, such as (acyloxy) Arrcostab or ((alkoxy carbonyl) oxygroup) Arrcostab. Particularly, such prodrug is the C of the compounds of this invention1-8Alkyl, C2-8Alkenyl, C6-10The aryl and (C optionally replaced6-10Virtue Base)-(C1-4Alkyl) ester.
Term " isotopic variations " as used herein refers at the one or more atoms for constituting such compound The compound of isotope comprising unnatural proportions.For example, ' isotopic variations ' of compound may include one or more Non radioactive isotope, such as deuterium (2H or D), carbon -13 (13C), nitrogen (15N) etc..It should be appreciated that substituted carrying out the isotope In compound, following atoms (when it is present) can change so that for example any hydrogen can be2H/D, any carbon can be13C, or Any nitrogen can be15N, and the presence and replacement of such atom can be determined by those skilled in the art.Equally, for example Gained compound can be used in the case of drug and/or substrate tissue distribution research, and the present invention may include that preparation has radiation The isotopic variations of property isotope.Radioactive isotope tritium (that is,3H) and carbon-14 (that is,14C) due to being easily incorporate into and being easy Detection and especially suitable for the purpose.Furthermore, it is possible to prepare by Positron emitting isotopes (such as11C、18F、15O and13N) replace Compound, and the compound can be used for positron emission computerized tomography (PET) research in check substrate receptor occupy feelings Condition.
It is also understood that bond characters or sequence difference or its steric arrangement with identical molecular formula but its atom Different compounds is known as " isomers ".The different isomers of steric arrangement is known as " stereoisomer ".
The stereoisomer being not mirror-images of each other is known as " diastereoisomer ", and those are that cannot be overlapped mirror image each other Be known as " enantiomter ".When compound has asymmetric center, such as it is from 4 different group bondings, it is understood that there may be A pair of of enantiomter.Enantiomter can carry out characterizing by the absolute configuration of its asymmetric center and by Cahn and R- the and S- ordering rule of Prelog describes, or describes by way of molecule rotatory polarization optical plane and be appointed as the right side Rotation is left-handed (that is, being respectively (+) or (-)-isomers).Chipal compounds can be with or mixtures thereof single enantiomter Form exists.The mixture of enantiomter comprising equal proportion is known as " racemic mixture ".
" tautomer " refers to can mutually transition form and set in hydrogen atom and electronics with specific compound structure Change the different compound of aspect.Therefore, two kinds of structures can reach balance by pi-electron and atom (usually H) movement.Example Such as, enol and ketone are tautomers, because they can be by rapidly being mutually converted with acid or alkali process.Tautomerism is existing Another example of elephant is the sour form and nitro versions of phenyl nitromethane, is formed again by with acid or alkali process 's.
Tautomeric form may be related to the optimum chemical reactivity and bioactivity for reaching interested compound.
The compounds of this invention can have one or more asymmetric centers；Therefore, such compound can be with single (R)-(S)-stereoisomer or in the form of its mixture prepare.
Unless otherwise stated, meaning to wrap to the description of specific compound or name in specification and claims Include its individual enantiomter and its mixture, racemate etc..For establish its spatial chemistry and to stereoisomer into The isolated method of row is well-known in the art.
It should be appreciated that the compound of the present invention can be metabolized, bioactive metabolites are generated.
The present invention
The present invention, which has been based on the discovery that, can be used for preventing and/or treating inflammatory disease, autoimmune disease, proliferative disease Disease, allergic disease, graft rejection, be related to cartilage update damage disease, congenital cartilage deformity and/or with IL6 or interference The present composition of the relevant disease of plain hypersecretion.In a particular aspect, the composition includes to have IRAK suppression Make the first active compound and second of compound with JAK inhibitory.
The present invention also provides prevent comprising combined pharmaceutical composition of the invention, and by applying combination of the invention And/or treatment disease method, the disease include inflammatory disease, autoimmune disease, proliferative diseases, allergic disease, Graft rejection, the disease for being related to cartilage update damage, congenital cartilage are lopsided and/or related to IL6 or interferon hypersecretion Disease.
In a particular aspect, JAK inhibitor has JAK1 inhibitory activity.In an embodiment particularly, JAK inhibitor is JAK1 selective depressant.
In another particular aspect, IRAK inhibitor has IRAK4 inhibitory activity.In an embodiment particularly In, IRAK inhibitor is IRAK4 selective depressant.
Therefore, in the first aspect of the invention, composition of the invention is provided, it includes:
A) compound of Formulas I:
Wherein
Cy is
Optionally by the R of one or more independent choices3Substituted monocycle C3-7Naphthenic base, or
Optionally by the R of one or more independent choices3What is replaced includes one or two independently selected from N, S and O Heteroatomic 4-7 unit monocycle Heterocyclylalkyl；
R1For
-H,
--SO3H,
-- P (=O) (OH)2,
-C1-4Alkyl,
-- C (=O)-(includes one or two heteroatomic 4-7 unit monocycle Heterocyclylalkyl independently selected from N, S and O), Or
-- C (=O) C1-6Alkyl, wherein C1-6Alkyl is optionally by the R of one or more independent choices4Group replaces；
R2For H or C1-4Alkyl；
Each R3Independently selected from:
- OH,
=O,
Halogen, and
-C1-4Alkyl；
Each R4Independently selected from:
--NR5aR5b,
-- C (=O) OH,
Optionally by the C of one or more independent choices1-4Alkyl-substituted includes one or two independently selected from N, S With the heteroatomic 4-7 unit monocycle Heterocyclylalkyl of O, and
-- NHC (=O)-C1-4Alkyl-NH2；And
R5aAnd R5bIt independently is H or C1-4Alkyl；
Or the salt of its officinal salt or solvate or solvate；With
B) with second of compound of JAK inhibitory.
In one embodiment, composition of the invention includes the compound of Formulas I, and wherein Cy is monocycle C3-7Naphthenic base. In a specific embodiment, Cy is cyclohexyl.
In one embodiment, composition of the invention include Formulas I compound, wherein Cy be by one, two or The R of three independent choices3Substituted monocycle C3-7Naphthenic base.In a specific embodiment, Cy be by one, two or The R of three independent choices3Substituted cyclohexyl.In another specific embodiment, Cy is by one or two R3Replace Monocycle C3-7Naphthenic base.In an embodiment particularly, Cy is by the R of one or two independent choice3Substituted hexamethylene Base.
In one embodiment, composition of the invention includes the compound of Formulas I, and wherein Cy is to include one or two Heteroatomic 4-7 unit monocycle Heterocyclylalkyl independently selected from N, S and O.In a specific embodiment, Cy is tetrahydro pyrrole It mutters base or tetrahydro thiopyranyl.In an embodiment particularly, Cy is
In one embodiment, composition of the invention include Formulas I compound, wherein Cy be by one, two or The R of three independent choices3What is replaced includes one or two heteroatomic 4-7 unit monocycle heterocycle alkane independently selected from N, S and O Base.In another embodiment, Cy is THP trtrahydropyranyl or tetrahydro thiopyranyl, respectively by one, two or three The R of independent choice3Replace.In a specific embodiment, Cy is by one or two R3What is replaced includes one or two Heteroatomic 4-7 unit monocycle Heterocyclylalkyl independently selected from N, S and O.In another specific embodiment, Cy is tetrahydro Pyranose or tetrahydro thiopyranyl, respectively by the R of one or two independent choice3Replace.In an implementation particularly In scheme, Cy isOrIt is respectively by the R of one or two independent choice3Replace.
In one embodiment, composition of the invention includes the compound of Formulas I, wherein each R3Selected from OH ,=O, halogen Element and C1-4Alkyl.In a specific embodiment, each R3Selected from OH ,=O, F and-CH3.In an embodiment party particularly In case, each R3Selected from OH and CH3.In another embodiment particularly, each R3For F.In another embodiment party particularly In case, each R3For=O.
In one embodiment, composition of the invention includes the chemical combination of Formula II a, IIb, IIc, IId, IIe or IIf Object:
Wherein R1And R2As described above.
In one embodiment, composition of the invention includes the compound of any of Formulas I-IIf, wherein R1For H、-SO3H or-P (=O) (OH)2。
In one embodiment, composition of the invention includes the compound of any of Formulas I-IIf, wherein R1For C1-4Alkyl.In a specific embodiment, R1For-CH3、-CH2CH3Or-CH (CH3)2.In an embodiment party particularly In case, R1For-CH3。
In one embodiment, composition of the invention includes the compound of any of Formulas I-IIf, wherein R1For-C (=O)-(including one or two heteroatomic 4-7 unit monocycle Heterocyclylalkyl independently selected from N, S and O).It is specific at one Embodiment in, R1For-C (=O)-pyrrolidinyl.
In one embodiment, composition of the invention includes the compound of any of Formulas I-IIf, wherein R1For-C (=O) C1-6Alkyl.In a specific embodiment, R1For-C (=O) C1-6Alkyl, wherein C1-6Alkyl is selected from-CH3、- CH2CH3、-CH2CH2CH3Or-CH2(CH(CH3)2)。
In one embodiment, composition of the invention includes the compound of any of Formulas I-IIf, wherein R1-C (=O) C1-6Alkyl, wherein C1-6Alkyl is by the R of one or more independent choices4Replace.In a specific embodiment, R1 For-C (=O) C1-6Alkyl, wherein C1-6Alkyl is selected from-CH3、-CH2CH3、-CH2CH2CH3Or-CH2(CH(CH3)2), respective quilt The R of one or more independent choices4Replace.In another specific embodiment, R1For-C (=O) C1-6Alkyl, wherein C1-6 The R that alkyl is selected independently by one or two4Replace.In an embodiment particularly, R1For-C (=O) C1-6Alkane Base, wherein C1-6Alkyl is selected from-CH3、-CH2CH3、-CH2CH2CH3Or-CH2(CH(CH3)2), respectively by one or two independence The R of selection4Replace.
In one embodiment, the compound of composition of the invention comprising any of Formulas I-IIf, and R4For- NR5aR5b, wherein R5aAnd R5bIt is each independently H or C1-4Alkyl.In a specific embodiment, R5aAnd R5bRespectively solely It is on the spot H ,-CH3Or-CH2CH3.In an embodiment particularly, R5aFor H and R5bFor H ,-CH3Or-CH2CH3.? In one most specific embodiment, R4For-NH2、-NHCH3Or-N (CH3)2。
In one embodiment, composition of the invention includes the compound of any of Formulas I-IIf, wherein R4For-C (=O) OH.
In one embodiment, composition of the invention includes the compound of any of Formulas I-IIf, wherein R4To appoint Selection of land is by the C of one or more independent choices1-4Alkyl-substituted includes one or two hetero atom independently selected from N, S and O 4-7 unit monocycle Heterocyclylalkyl.In a specific embodiment, R4For morpholinyl, piperidyl or piperazinyl, respectively appoint Selection of land is by the C of one or more independent choices1-4Alkyl replaces.In another specific embodiment, R4For comprising one or Two heteroatomic 4-7 unit monocycle Heterocyclylalkyls independently selected from N, S and O, optionally by a C1-4Alkyl replaces.? In one embodiment particularly, R4For the heteroatomic 4-7 unit monocycle comprising one or two independently selected from N, S and O Heterocyclylalkyl, optionally by-a CH3Replace.In another embodiment particularly, R4For morpholinyl, piperidyl or Piperazinyl, respectively optionally by one or more-CH3Replace.In a most specific embodiment, R4For morpholinyl, piperazine Piperidinyl or piperazinyl, respectively optionally by-a CH3Replace.
In one embodiment, composition of the invention includes the compound of any of Formulas I-IIf, wherein R4For- NHC (=O)-C1-4Alkyl-NH2.In a specific embodiment, R4For-NHC (=O)-CH2-NH2。
In one embodiment, composition of the invention includes the compound of any of Formulas I-IIf, wherein R1For-C (=O) CH2NH2,-C (=O) CH2NHCH3,-C (=O) CH2N(CH3)2,-C (=O) CH2CH2N(CH3)2,-C (=O) CH (NH2) CH(CH3)2,-C (=O) CH2CH2C (=O) OH ,-C (=O) CH (NH2)CH2C (=O) OH ,-C (=O) CH (NH2)CH2CH2C (=O) OH ,-C (=O) CH (CH (CH3)2) NHC (=O) CH2NH2、
In one embodiment, composition of the invention includes the compound of any of Formulas I-IIf, wherein R2For H.
In one embodiment, composition of the invention includes the compound of any of Formulas I-IIf, wherein R2For C1-4Alkyl.In a specific embodiment, R2For-CH3。
In one embodiment, composition of the invention includes the compound of Formulas I, wherein the compound is selected from:
6- [6- [2- (2- Hydroxy-ethoxy)-ethyoxyl] -5- (ttetrahydro-pyran -4- base amino)-imidazo [4,5-b] Pyridin-3-yl]-nicotinic acid nitrile,
6- { 5- (1,1- dioxo-tetrahydro -2H- thio-pyrylium -4- base amino) -6- [2- (2- Hydroxy-ethoxy)-ethoxy Base]-imidazo [4,5-b] pyridin-3-yl }-nicotinic acid nitrile,
6- { 6- [2- (2- Hydroxy-ethoxy)-ethyoxyl] -5- [((cis- -1,4) -4- hydroxy-4-methyl-cyclohexyl) - Methyl-amino]-imidazo [4,5-b] pyridin-3-yl }-nicotinic acid nitrile,
6- { 6- [2- (2- Mehtoxy-ethoxy)-ethyoxyl] -5- [methyl-(ttetrahydro-pyran -4- base)-amino]-imidazoles And [4,5-b] pyridin-3-yl-nicotinic acid nitrile,
6- [6- [2- (2- Mehtoxy-ethoxy)-ethyoxyl] -5- (ttetrahydro-pyran -4- base amino)-imidazo [4,5- B] pyridin-3-yl]-nicotinic acid nitrile,
6- { 5- (3- Hydroxy-cyclohexylamino) -6- [2- (2- Hydroxy-ethoxy)-ethyoxyl]-imidazo [4,5-b] pyrrole Pyridine -3- base }-nicotinic acid nitrile,
6- { 5- (4,4- Difluoro-cyclohexyl amino) -6- [2- (2- Hydroxy-ethoxy)-ethyoxyl]-imidazo [4,5-b] Pyridin-3-yl }-nicotinic acid nitrile,
Sulfuric acid it is mono- (2- 2- [3- (5- cyanopyridine -2- base) -5- (ttetrahydro-pyran -4- base amino) -3H- imidazo [4, 5-b] pyridine -6- base oxygroup]-ethyoxyl }-ethyl) ester,
(S) -2- amino -3- metliyl-butyric acid 2- { 2- [3- (5- cyanopyridine -2- base) -5- (ttetrahydro-pyran -4- base ammonia Base) -3H- imidazo [4,5-b] pyridine -6- base oxygroup]-ethyoxyl }-ethyl ester,
(S) -2- amino -3- metliyl-butyric acid 2- { 2- [3- (5- cyanopyridine -2- base) -5- (ttetrahydro-pyran -4- base ammonia Base) -3H- imidazo [4,5-b] pyridine -6- base oxygroup]-ethyoxyl }-ethoxal salt,
6- [6- [2- (2- hydroxyl-oxethyl) ethyoxyl] -5- [[(cis- -3,4) -4- hydroxy tetrahydro pyrans -3- base] ammonia Base] imidazo [4,5-b] pyridin-3-yl] pyridine -3- formonitrile HCN,
6- [6- [2- (2- hydroxyl-oxethyl) ethyoxyl] -5- [((cis- -1,4) -4- hydroxy-4-methyl cyclohexyl) ammonia Base] imidazo [4,5-b] pyridin-3-yl] pyridine -3- formonitrile HCN,
6- [5- [((cis- -1,4) -4- hydroxy-4-methyl-cyclohexyl)-Methyl-amino] -6- [2- (2- methoxyl group ethoxy Base) ethyoxyl] imidazo [4,5-b] pyridin-3-yl] pyridine -3- formonitrile HCN,
6- [5- [((cis- -1,4) -4- hydroxy-4-methyl cyclohexyl) amino] -6- [2- (2- methoxy ethoxy) ethoxy Base] imidazo [4,5-b] pyridin-3-yl] pyridine -3- formonitrile HCN,
2- [2- [3- (5- cyano -2- pyridyl group) -5- (tetrahydropyran -4-base amino) imidazo [4,5-b] pyridine -6- Base] oxygroup ethyoxyl] ethyl 2- (dimethylamino) acetic acid esters,
2- [2- [3- (5- cyano -2- pyridyl group) -5- (tetrahydropyran -4-base amino) imidazo [4,5-b] pyridine -6- Base] oxygroup ethyoxyl] ethyl 2- aminoacetate,
2- [2- [3- (5- cyano -2- pyridyl group) -5- (tetrahydropyran -4-base amino) imidazo [4,5-b] pyridine -6- Base] oxygroup ethyoxyl] ethyl 2- (methylamino) acetic acid esters,
2- [2- [3- (5- cyano -2- pyridyl group) -5- (tetrahydropyran -4-base amino) imidazo [4,5-b] pyridine -6- Base] oxygroup ethyoxyl] ethyl (2S)-pyrrolidines -2- formic acid esters,
2- [2- [3- (5- cyano -2- pyridyl group) -5- (tetrahydropyran -4-base amino) imidazo [4,5-b] pyridine -6- Base] oxygroup ethyoxyl] ethyl (2S) -2- [(2- glycyl) amino] -3- metliyl-butyric acid ester,
2- [2- [3- (5- cyano -2- pyridyl group) -5- (tetrahydropyran -4-base amino) imidazo [4,5-b] pyridine -6- Base] oxygroup ethyoxyl] ethyl 2- (morpholine -4- base) acetic acid esters,
2- [2- [3- (5- cyano -2- pyridyl group) -5- (tetrahydropyran -4-base amino) imidazo [4,5-b] pyridine -6- Base] oxygroup ethyoxyl] ethyl 2- (4- methylpiperazine-1-yl) acetic acid esters,
2- [2- [3- (5- cyano -2- pyridyl group) -5- (tetrahydropyran -4-base amino) imidazo [4,5-b] pyridine -6- Base] oxygroup ethyoxyl] ethyl 3- (dimethylamino) propionic ester,
2- [2- [3- (5- cyano -2- pyridyl group) -5- (tetrahydropyran -4-base amino) imidazo [4,5-b] pyridine -6- Base] oxygroup ethyoxyl] ethyl 2- (dimethylamino) acetic acid ester oxalate,
2- [2- [3- (5- cyano -2- pyridyl group) -5- (tetrahydropyran -4-base amino) imidazo [4,5-b] pyridine -6- Base] oxygroup ethyoxyl] ethyl 2- amion acetic acid ester oxalate,
2- [2- [3- (5- cyano -2- pyridyl group) -5- (tetrahydropyran -4-base amino) imidazo [4,5-b] pyridine -6- Base] oxygroup ethyoxyl] ethyl 2- (methylamino) acetic acid ester oxalate,
2- [2- [3- (5- cyano -2- pyridyl group) -5- (tetrahydropyran -4-base amino) imidazo [4,5-b] pyridine -6- Base] oxygroup ethyoxyl] ethyl (2S)-pyrrolidines -2- formic acid ester oxalate,
(3S) -3- amino -4- [2- [2- [3- (5- cyano -2- pyridyl group) -5- (tetrahydropyran -4-base amino) imidazo [4,5-b] pyridine -6- base] oxygroup ethyoxyl] ethyoxyl] -4- ketobutyric acid hydrochloride,
(4S) -4- amino -5- [2- [2- [3- (5- cyano -2- pyridyl group) -5- (tetrahydropyran -4-base amino) imidazo [4,5-b] pyridine -6- base] oxygroup ethyoxyl] ethyoxyl] -5- oxopentanoic acid hydrochloride,
2- [2- [3- (5- cyano -2- pyridyl group) -5- (tetrahydropyran -4-base amino) imidazo [4,5-b] pyridine -6- Base] oxygroup ethyoxyl] ethyl (2S) -2- [(2- glycyl) amino] -3- metliyl-butyric acid ester oxalate,
2- [2- [3- (5- cyano -2- pyridyl group) -5- (tetrahydropyran -4-base amino) imidazo [4,5-b] pyridine -6- Base] oxygroup ethyoxyl] ethyl 2- (morpholine -4- base) acetic acid ester oxalate,
2- [2- [3- (5- cyano -2- pyridyl group) -5- (tetrahydropyran -4-base amino) imidazo [4,5-b]] pyridine -6- Base] oxygroup ethyoxyl] ethyl 2- (4- methylpiperazine-1-yl) acetic acid ester oxalate,
2- [2- [3- (5- cyano -2- pyridyl group) -5- (tetrahydropyran -4-base amino) imidazo [4,5-b] pyridine -6- Base] oxygroup ethyoxyl] ethyl 3- (dimethylamino) propionic acid ester oxalate, and
4- [2- [2- [3- (5- cyano -2- pyridyl group) -5- (tetrahydropyran -4-base amino) imidazo [4,5-b] pyridine - 6- yl] oxygroup ethyoxyl] ethyoxyl] -4- ketobutyric acid.
In one embodiment, composition of the invention includes the compound of Formulas I, wherein the compound is 6- [6- [2- (2- Hydroxy-ethoxy)-ethyoxyl] -5- (ttetrahydro-pyran -4- base amino)-imidazo [4,5-b] pyridin-3-yl]-cigarette Nitrile.
In one embodiment, composition of the invention includes the compound of Formulas I, wherein the compound is not 6- [6- [2- (2- Hydroxy-ethoxy)-ethyoxyl] -5- (ttetrahydro-pyran -4- base amino)-imidazo [4,5-b] pyridin-3-yl] - Nicotinic acid nitrile.
In one embodiment, composition of the invention includes the compound of Formulas I, wherein the compound is (S) -2- Amino -3- metliyl-butyric acid 2- 2- [3- (5- cyanopyridine -2- base) -5- (ttetrahydro-pyran -4- base amino) -3H- imidazo [4, 5-b] pyridine -6- base oxygroup]-ethyoxyl }-ethyl ester.
In one embodiment, composition of the invention includes the compound of Formulas I, wherein the compound is not (S)- 2- amino -3- metliyl-butyric acid 2- { 2- [3- (5- cyanopyridine -2- base) -5- (ttetrahydro-pyran -4- base amino) -3H- imidazo [4,5-b] pyridine -6- base oxygroup]-ethyoxyl }-ethyl ester.
In another embodiment, composition of the invention includes the compound with JAK inhibitory.In a spy In fixed embodiment, the compound with JAK inhibitory is compound disclosed in WO2010/010190.
In another embodiment, composition of the invention includes the compound with JAK1 inhibitory activity.At one In specific embodiment, the compound with JAK1 inhibitory activity is the compound according to Formula X Xa or XXb:
In a specific embodiment, composition of the invention includes:
A) it is selected from 6- [6- [2- (2- Hydroxy-ethoxy)-ethyoxyl] -5- (ttetrahydro-pyran -4- base amino)-imidazo [4,5-b] pyridin-3-yl]-nicotinic acid nitrile and (S) -2- amino -3- metliyl-butyric acid 2- { 2- [3- (5- cyanopyridine -2- base) -5- (four Hydrogen-pyrans -4- base amino) -3H- imidazo [4,5-b] pyridine -6- base oxygroup]-ethyoxyl-ethyl ester compound；With
B) it is selected from the compound of the compound according to Formula X Xa and XXb.
In a specific embodiment, composition of the invention includes:
A) it is selected from 6- [6- [2- (2- Hydroxy-ethoxy)-ethyoxyl] -5- (ttetrahydro-pyran -4- base amino)-imidazo [4,5-b] pyridin-3-yl]-nicotinic acid nitrile and (S) -2- amino -3- metliyl-butyric acid 2- { 2- [3- (5- cyanopyridine -2- base) -5- (four Hydrogen-pyrans -4- base amino) -3H- imidazo [4,5-b] pyridine -6- base oxygroup]-ethyoxyl-ethyl ester compound；With
B) according to the compound of Formula X Xb.
In an embodiment particularly, composition of the invention includes:
A) 6- [6- [2- (2- Hydroxy-ethoxy)-ethyoxyl] -5- (ttetrahydro-pyran -4- base amino)-imidazo [4,5- B] pyridin-3-yl]-nicotinic acid nitrile；With
B) according to the compound of Formula X Xb.
In one embodiment, the compound in the present composition is not isotopic variations.
In one aspect, according to the compound in the present composition of embodiment any one of described herein with free alkali Form exists.
It in one aspect, is pharmaceutically acceptable according to the compound in the present composition of embodiment any one of described herein Salt.
It in one aspect, is describedization according to the compound in the present composition of embodiment any one of described herein Close the solvate of object.
It in one aspect, is describedization according to the compound in the present composition of embodiment any one of described herein Close the solvate of the officinal salt of object.
Although briefly listing the specified group of each embodiment, the chemical combination of the present composition respectively above Object includes that several or each embodiments wherein in other general formulas existing for above-mentioned general formula and this paper are selected from for each change Measure the compound of the special member or one or more of group that respectively specify that.Therefore, the present invention is intended to include in its range All combinations of interior such embodiment.
Although briefly listing the specified group of each embodiment respectively above, the compound of the present invention can be with It is one or more of variables (such as R group) selected from one or more embodiment party according to any general formula listed above The compound of case.Therefore, the present invention is intended to include all combinations of the variable of any disclosed embodiment within its scope.
Optionally, present invention further contemplates that excluding one or more of specifying variable or its group from group or embodiment It closes.
In some aspects, the present invention provides the prodrug and derivative of the compound of above-mentioned general formula.Prodrug is combination of the present invention The derivative of compound in object has the group of metabolism cleavable and passes through solvolysis or change in physiological conditions For in vivo with the compound of the present invention of pharmaceutical activity.Such example includes but is not limited to choline ester derivant etc., N- Alkyl morpholine ester etc..
The acid and acid derivative form of other derivatives of the compound of composition of the invention are active, but acid-sensitive Sense form provided usually in mammalian organism the advantages of dissolubility, histocompatbility or sustained release (Bundgard, H, 1985).Prodrug includes acid derivative well known to those skilled in the art, such as by making parent acid react preparation with suitable alcohol Ester, or by making parent acid compound react with substituted or unsubstituted amine the amide or acid anhydrides or mixed acid anhydride of preparation.Spread out The simple aliphatic series or aromatic ester, amide and acid anhydrides for being born from the acidic-group being suspended on the compounds of this invention are preferred prodrugs. In some cases, it is desired to prepare diester-type prodrug, such as (acyloxy) Arrcostab or ((alkoxy carbonyl) oxygroup) alkyl Ester.It is especially useful that the C of the compounds of this invention1To C8Alkyl, C2-C8Alkenyl, aryl, C7-C12Substituted aryl and C7- C12Alkyl aryl.
Pharmaceutical composition
When being used as drug, composition of the invention is typically administered with pharmaceutical compositions.Such composition can Reactive compound of the invention to be prepared in a manner of known to drug field and comprising at least one Formulas I.In general, of the invention Composition be administered with pharmacy effective dose.The practical dosage of the compound of the present invention is usually true according to correlation circumstance by doctor Fixed, the situation includes illness to be treated, selected administration route, the compound of the present invention being actually administered, individual patient Age, weight and response, the severity of patient symptom etc..
Pharmaceutical composition of the invention can be administered through a variety of ways, and the approach includes oral, rectum, transdermal, skin Under, it is intra-articular, intravenous, intramuscular and intranasal.Depending on expected route of delivery, the compound of the present invention is preferably formulated as to infuse It penetrates or Orally administered composition or is formulated as paste, lotion or patch for cutaneous penetration.
Compounds for oral administration can take the form of bulk liquids solution or suspension or bulk powder.So And the composition more commonly exists with unit dosage forms in order to accurately be administered.Term " unit dosage forms ", which refers to, to be suitable for making The physical discrete unit of human individual and other mammals are used for for single dose, each unit, which contains, to be computed the phase of can produce Hope the active material and suitable pharmaceutical excipient, solvent or carrier of the predetermined amount of therapeutic effect.Typical unit dosage forms packet Include pre-filled, pre-weighing the ampoule or syringe of liquid composition, or pill, tablet, glue under solid composite situation Wafer etc..In such composition, composition of the invention is usually that (about 0.1 to about 50% weight is preferred for accessory constituent About 1 to about 40% weight), and remainder contributes to be formed the various solvents or carrier and processing aid of desired dosage form.
Liquid form suitable for oral administration may include with buffer, suspending agent and dispersing agent, colorant, corrigent Deng suitable aqueous or non-aqueous vehicles.Solid form may include for example any following compositions or the sheet with similarity The compound of invention: adhesive, such as microcrystalline cellulose, tragacanth or gelatin；Excipient, such as starch or lactose；Disintegration Agent, such as alginic acid, Primogel or cornstarch；Lubricant, such as magnesium stearate；Glidant, such as colloidal silicon dioxide； Sweetener, such as sucrose or saccharin；Or corrigent, such as peppermint or orange taste corrigent.
Injectable composition be normally based on injectable Sterile Saline or phosphate buffered saline (PBS) or it is known in the art its Its injectable carrier.As previously mentioned, the reactive compound of the present composition in such composition is usually secondary group Point, typically about 0.05 to 10% weight, and remainder is injectable carrier etc..
Transdermal composition is usually formulated as topical ointments or cream comprising active constituent, the active constituent Amount is usually following ranges: about 0.01 to about 20% weight, preferably from about 0.1 to about 20% weight, preferably from about 0.1 to about 10% weight Amount, more preferably from about 0.5 to about 15% weight.When being formulated as ointment, active constituent is usually miscible soft with paraffinic base or water Cream substrate combination.Optionally, active constituent is configured to cream by usable such as Oil-in-water emulsifiable paste agent matrix.It is such transdermal Preparation is well known in the art, and generally includes other ingredient to enhance the stabilization of the dermal permeation of active constituent or preparation Property.All such known preparation capable of permeating skin and ingredient are included within the scope of the present invention.
Composition of the invention can also be applied by transdermal device.Therefore, storage cavern or porous can be used in cutaneous penetration Membranous type patch or solid matrix class patch are realized.
For Orally-administrable, injectable or can the said components of composition of local administration be only representative.It is other Material and processing technology etc. are listed in Remington ' s Pharmaceutical Sciences, and the 17th edition, 1985, Mack In the 8th part of Publishing Company, Easton, Pennsylvania, it is incorporated into herein as reference.
Composition of the invention can also be administered with sustained release forms or from slow releasing pharmaceutical delivery system.Representative sustained release material The description of material can be found in Remington ' s Pharmaceutical Sciences.
Following formulation examples have been illustrated can be with representative pharmaceutical composition prepared in accordance with the present invention.However, this Invention is not limited to following pharmaceutical compositions.
Preparation 1- tablet
It can be in the form of dried powder and dry by the composition of the invention of the compound comprising Formulas I and JAK inhibitor Dry gelatin adhesive is mixed with the weight ratio of about 1:2.A small amount of magnesium stearate can be added as lubricant.The mixture can be 240-270mg tablet (reactive compound of the invention of the every Formulas I containing 80-90mg) is formed in tablet press machine.
Preparation 2- capsule
It can be by the composition of the invention of the compound comprising Formulas I and JAK inhibitor in the form of dried powder and shallow lake Powder diluent is mixed with the weight ratio of about 1:1.The mixture can be filled into capsule (every capsule Formulas I containing 125mg of 250mg Reactive compound of the invention).
Preparation 3- liquid
It can be by the composition (125mg) of the invention of the compound comprising Formulas I and JAK inhibitor and sucrose (1.75g) It is mixed with xanthan gum (4mg), and obtained mixture is mixed, make it through No. 10 mesh U.S. sieves, then prepared with prior Microcrystalline cellulose and sodium carboxymethylcellulose (11:89,50mg) aqueous solution mixing.Sodium benzoate can be diluted with water (10mg), corrigent and colorant, and be added under stiring.It is then possible to which the water of sufficient amount is added under stiring.Then, The water of sufficient amount can be added further to reach the total volume of 5mL.
Preparation 4- tablet
It can be in the form of dried powder and dry by the composition of the invention of the compound comprising Formulas I and JAK inhibitor Dry gelatin adhesive is mixed with the weight ratio of about 1:2.A small amount of magnesium stearate can be added as lubricant.The mixture can be 450-900mg tablet (reactive compound of the invention of 150-300mg Formulas I) is formed in tablet press machine.
Preparation 5- injection
It is sterile the composition of the invention of the compound comprising Formulas I and JAK inhibitor can be dissolved or suspended in buffering To the concentration of about 5mg/mL in saline injectable aqueous medium.
Preparation 6- topical formulations
Stearyl alcohol (250g) and albolene (250g) can be melted at about 75 DEG C, then can be added and be dissolved in water (about The composition (50g) of the invention of the compound comprising Formulas I and JAK inhibitor in 370g), methylparaben (0.25g), Buddhist nun The mixture of golden propyl ester (0.15g), NaLS (10g) and propylene glycol (120g) is moored, and can be stirred to get mixed Object is closed until its condensation.
Treatment method
In one embodiment, the present invention is provided to the composition of the invention of medicine or include combination of the invention The pharmaceutical composition of object.In a specific embodiment, the present invention is provided to prevent and/or treat inflammatory disease, from Body immunological diseases, proliferative diseases, allergic disease, graft rejection, the disease for being related to cartilage update damage, congenital cartilage are abnormal The composition of the invention of shape and/or disease relevant to IL6 or interferon hypersecretion includes composition of the invention Pharmaceutical composition.
In another embodiment, the present invention provides composition of the invention or the drug comprising composition of the invention Composition is used to prepare for preventing and/or treating inflammatory disease, autoimmune disease, proliferative diseases, allergia disease Disease, graft rejection, be related to cartilage update damage disease, congenital cartilage deformity and/or with IL6 or interferon hypersecretion phase The drug of the disease of pass.
It is dynamic the present invention is provided to prevent and/or treat the lactation with following diseases in terms of other treatment method The method of object: inflammatory disease, proliferative diseases, allergic disease, graft rejection, is related to cartilage update damage at autoimmune disease The disease of wound, congenital cartilage deformity and/or disease relevant to IL6 or interferon hypersecretion, wherein this method includes applying With a effective amount of composition of the invention or one or more pharmaceutical compositions described herein for being used to treat or prevent the illness Object.
In one embodiment, the present invention provides the pharmaceutical composition comprising composition and another therapeutic agent of the invention Object.In a specific embodiment, other therapeutic agents are for treating inflammatory disease, autoimmune disease, proliferative disease Disease, allergic disease, graft rejection, be related to cartilage update damage disease, congenital cartilage deformity and/or with IL6 or interference The therapeutic reagent of the relevant disease of plain hypersecretion.
In one embodiment, the present invention is provided to prevent and/or treat the composition of the invention of inflammatory disease Or the pharmaceutical composition comprising composition of the invention.In a specific embodiment, the inflammatory disease is selected from class wind It is wet arthritis, psoriatic arthritis, ankylosing spondylitis, osteoarthritis, allergy airway disorders, sarcoidosis, chronic Obstructive lung disease (COPD) and inflammatory bowel disease.In an embodiment particularly, the inflammatory disease is selected from rheumatoid Arthritis, sarcoidosis, psoriatic arthritis, ankylosing spondylitis and inflammatory bowel disease.
In another embodiment, the present invention provides composition of the invention or the drug comprising composition of the invention Composition is used to prepare the drug for preventing and/or treating inflammatory disease.It is described in a specific embodiment Inflammatory disease is selected from rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, osteoarthritis, allergy air flue Disease, sarcoidosis, chronic obstructive pulmonary disease (COPD) and inflammatory bowel disease.In an embodiment particularly, the inflammatory Disease is selected from rheumatoid arthritis, sarcoidosis, psoriatic arthritis, ankylosing spondylitis and inflammatory bowel disease.
In terms of another treatment method, the present invention provides the mammal of prevention and/or treatment with inflammatory disease Method, wherein the method includes apply a effective amount of composition of the invention or it is one or more it is described herein for treating or The pharmaceutical composition for preventing the illness.In a specific embodiment, the inflammatory disease is selected from rheumatoid joint Inflammation, psoriatic arthritis, ankylosing spondylitis, osteoarthritis, allergy airway disorders, sarcoidosis, chronic obstructive pulmonary Sick (COPD) and inflammatory bowel disease.In an embodiment particularly, the inflammatory disease is selected from rheumatoid arthritis, knot Save disease, psoriatic arthritis, ankylosing spondylitis and inflammatory bowel disease.
In one embodiment, the present invention provides the pharmaceutical composition comprising composition and another therapeutic agent of the invention Object.In a specific embodiment, other therapeutic agents are the reagent for treating inflammatory disease.In a specific implementation In scheme, the inflammatory disease is selected from rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, osteoarthritis, metamorphosis Reactive airway disorders, sarcoidosis, chronic obstructive pulmonary disease (COPD) and inflammatory bowel disease.In an embodiment particularly In, the inflammatory disease is selected from rheumatoid arthritis, sarcoidosis, psoriatic arthritis, ankylosing spondylitis and inflammatory bowel disease.
In one embodiment, the present invention is provided to prevent and/or treat the group of the invention of autoimmune disease Close object or the pharmaceutical composition comprising composition of the invention.In a specific embodiment, the autoimmune disease Selected from COPD, asthma, lupus erythematosus,cutaneous, membranous lupus nephritis, alopecia areata, vitiligo, type-1 diabetes mellitus and inflammatory bowel disease.
In another embodiment, the present invention provides composition of the invention or the drug comprising composition of the invention Composition is used to prepare the drug for preventing and/or treating autoimmune disease.In a specific embodiment, The autoimmune disease is selected from COPD, asthma, lupus erythematosus,cutaneous, membranous lupus nephritis, alopecia areata, vitiligo, I type glycosuria Disease and inflammatory bowel disease.
In terms of another treatment method, it is dynamic that the present invention provides the lactation of prevention and/or treatment with autoimmune disease The method of object, wherein the method includes applying a effective amount of composition of the invention or one or more described herein for controlling Treat or prevent the pharmaceutical composition of the illness.In a specific embodiment, the autoimmune disease be selected from COPD, Asthma, lupus erythematosus,cutaneous, membranous lupus nephritis, alopecia areata, vitiligo, type-1 diabetes mellitus and inflammatory bowel disease.
In one embodiment, the present invention provides the pharmaceutical composition comprising composition and another therapeutic agent of the invention Object.In a specific embodiment, other therapeutic agents are the reagent for treating autoimmune disease.It is specific at one In embodiment, the autoimmune disease is selected from COPD, asthma, lupus erythematosus,cutaneous, membranous lupus nephritis, alopecia areata, white Insane wind, type-1 diabetes mellitus and inflammatory bowel disease.
In one embodiment, the present invention is provided to prevent and/or treat the combination of the invention of proliferative diseases Object or pharmaceutical composition comprising composition of the invention.In a specific embodiment, the proliferative diseases are selected from Cancer, leukaemia, Huppert's disease and psoriasis.
In another embodiment, the present invention provides composition of the invention or the drug comprising composition of the invention Composition is used to prepare the drug for preventing and/or treating proliferative diseases.In a specific embodiment, institute It states proliferative diseases and is selected from cancer, leukaemia, Huppert's disease and psoriasis.
In terms of another treatment method, the present invention provides prevention and/or treatment suffers from the mammal of proliferative diseases Method, wherein the method includes applying a effective amount of composition of the invention or one or more described herein for treating Or the pharmaceutical composition of the prevention illness.In a specific embodiment, the proliferative diseases are selected from cancer, white blood Disease, Huppert's disease and psoriasis.
In one embodiment, the present invention provides the pharmaceutical composition comprising composition and another therapeutic agent of the invention Object.In a specific embodiment, other therapeutic agents are the reagent for treating proliferative diseases.It is specific real at one It applies in scheme, the proliferative diseases are selected from cancer, leukaemia, Huppert's disease and psoriasis.
In one embodiment, the present invention is provided to prevent and/or treat the combination of the invention of allergic disease Object or pharmaceutical composition comprising composition of the invention.In a specific embodiment, the allergic disease is wet Rash.
In another embodiment, the present invention provides composition of the invention or the drug comprising composition of the invention Composition is used to prepare the drug for preventing and/or treating allergic disease.In a specific embodiment, institute Stating allergic disease is eczema.
In terms of another treatment method, the present invention provides prevention and/or treatment suffers from the mammal of allergic disease Method, wherein the method includes applying a effective amount of composition of the invention or one or more described herein for treating Or the pharmaceutical composition of the prevention illness.In a specific embodiment, the allergic disease is eczema.
In one embodiment, the present invention provides the pharmaceutical composition comprising composition and another therapeutic agent of the invention Object.In a specific embodiment, other therapeutic agents are the reagent for treating allergic disease.It is specific real at one It applies in scheme, the allergic disease is eczema.
In one embodiment, the present invention is provided to prevent and/or treat the composition of the invention of graft rejection Or the pharmaceutical composition comprising composition of the invention.In a specific embodiment, the graft rejection is graft Anti- host disease.
In another embodiment, the present invention provides composition of the invention or the drug comprising composition of the invention Composition is used to prepare the drug for preventing and/or treating graft rejection.It is described in a specific embodiment Graft rejection is graft versus host disease(GVH disease).
In terms of another treatment method, the present invention provides the mammal of prevention and/or treatment with graft rejection Method, wherein the method includes apply a effective amount of composition of the invention or it is one or more it is described herein for treating or The pharmaceutical composition for preventing the illness.In a specific embodiment, the graft rejection is graft versus host disease(GVH disease).
In one embodiment, the present invention provides the pharmaceutical composition comprising composition and another therapeutic agent of the invention Object.In a specific embodiment, other therapeutic agents are the reagent for treating graft rejection.In a specific implementation In scheme, the graft rejection is graft versus host disease(GVH disease).
In one embodiment, the present invention is provided to prevent and/or treat the disease for being related to cartilage and updating damage Composition of the invention or the pharmaceutical composition comprising composition of the invention.It is described to relate in a specific embodiment And it is ankylosing spondylitis that cartilage, which updates the disease of damage,.
In another embodiment, the present invention provides composition of the invention or the drug comprising composition of the invention Composition is used to prepare and is related to the drug for the disease that cartilage updates damage for preventing and/or treating.It is specific real at one It applies in scheme, it is described to be related to cartilage to update the disease of damage being ankylosing spondylitis.
In terms of another treatment method, the present invention provides prevention and/or treatment suffers from and is related to the disease that cartilage updates damage The method of the mammal of disease, wherein the method includes applying a effective amount of composition of the invention or one or more this paper The pharmaceutical composition for being used to treat or prevent the illness.It is described to be related to cartilage more in a specific embodiment The disease newly damaged is ankylosing spondylitis.
In one embodiment, the present invention provides the pharmaceutical composition comprising composition and another therapeutic agent of the invention Object.In a specific embodiment, other therapeutic agents are for treating the Remedies for diseases for being related to cartilage and updating damage.? It is described to be related to cartilage to update the disease of damage being ankylosing spondylitis in one specific embodiment.
In one embodiment, the present invention is provided to prevent and/or treat the of the invention of congenital cartilage deformity Composition or pharmaceutical composition comprising composition of the invention.
In another embodiment, the present invention provides composition of the invention or the drug comprising composition of the invention Composition is used to prepare the drug for preventing and/or treating congenital cartilage deformity.
In terms of another treatment method, the present invention provides prevention and/or treatment suffers from the lactation of congenital cartilage deformity The method of animal, wherein the method includes applying a effective amount of composition of the invention or one or more described herein be used for Treat or prevent the pharmaceutical composition of the illness.
In one embodiment, the present invention provides the pharmaceutical composition comprising composition and another therapeutic agent of the invention Object.In a specific embodiment, other therapeutic agents are the therapeutic agent for treating congenital cartilage deformity.
In one embodiment, related to IL6 or interferon hypersecretion the present invention is provided to prevent and/or treat Disease composition of the invention or pharmaceutical composition comprising composition of the invention.
In another embodiment, the present invention provides composition of the invention or the drug comprising composition of the invention Composition is used to prepare the drug for preventing and/or treating disease relevant to IL6 or interferon hypersecretion.
In terms of another treatment method, the present invention provides prevention and/or treatment suffers from and IL6 or interferon hypersecretion The method of the mammal of relevant disease, wherein the method includes apply a effective amount of composition or one kind of the invention or It is a variety of described herein for treating or preventing the pharmaceutical composition of the illness.
In one embodiment, the present invention provides the pharmaceutical composition comprising composition and another therapeutic agent of the invention Object.In a specific embodiment, other therapeutic agents are for treating disease relevant to IL6 or interferon hypersecretion Therapeutic agent.
Injection dosage horizontal extent is about 0.1mg/kg/h at least 10mg/kg/h, is injected about 1 hour to about 120 small When, and especially 24 hours to 96 hours.The preload that about 0.1mg/kg can also be administered to about 10mg/kg or higher doses pushes away Note is to obtain enough steady-state levels.For the human patients of 40kg to 80kg, it is contemplated that maximum accumulated dose no more than about 1g/ It.
In order to prevent and/or treat long-term illness (such as neurodegenerative disorder), therapeutic scheme usually extends to several months or number Year, therefore being administered orally is preferred for patient convenience and tolerance.For oral administration, representative scheme be one to Secondary routine dose/the day four (1-4), the secondary routine dose/day especially one to three (1-3) are typically one to two (1-2) secondary routine Dosage/day, and most typical is the secondary routine dose/day in one (1).Alternatively, for depot drug product, the representativeness side of oral administration Case is every other week once, once a week and once a day.Particularly, dosage can be every 1 to 14 day once, particularly Ground is every 1 to 10 day primary, is even more particularly every 1 to 7 day primary, and most particularly every 1 to 3 day primary.
Using these mode of administration, each dosage provides the composition of the invention of about 1mg to about 1000mg, special agent Amount respectively provides about 10mg to about 500mg, especially about 30mg to about 250mg.
Transdermal dosage compositions are generally selected to provide and be similar to or lower than the blood level for using injection dosage to obtain.
It, usually can be according to the suggestion of doctor and under doctor's supervision, with above-mentioned dosage when for preventing illness breaking-out Level, which is given, has the patient that the disease risk occurs to apply composition of the invention.There is the patient that particular condition risk occurs usually to wrap It includes those of family history with the illness or has passed through genetic test or screened that for determining and being especially susceptible to occur the illness A bit.
Composition of the invention can be used as single-activity agent administration, or can be with other therapeutic agent combination medicine-feedings, institute State other therapeutic agents include show same or like therapeutic activity and have determined for the combination medicine-feeding be safety and Effective other compounds of the invention.In a specific embodiment, the co-administered of two kinds of (or a variety of) activating agents Allow to significantly reduce the dosage of every kind of activating agent ready for use, to reduce the side effect observed.
In one embodiment, composition of the invention or the pharmaceutical composition comprising composition of the invention are as medicine Object application.In a specific embodiment, described pharmaceutical composition additionally comprises other active constituents.
In one embodiment, composition of the invention and other therapeutic agent co-administered be used to treat and/or Prevention is related to the disease of inflammation, and specific activating agent includes but is not limited to immunomodulator such as imuran, corticosteroid (for example, prednisolone or dexamethasone), cyclophosphamide, cyclosporin A, tacrolimus, mycophenolate mofetil (mycophenolate mofetil), muromonab-CD3 (muromonab-CD3) (OKT3, such as)、 ATG, aspirin, Paracetamol, brufen, naproxen and piroxicam.
In one embodiment, composition of the invention and other therapeutic agent co-administered be used to treat and/or Prevent arthritis (for example, rheumatoid arthritis), specific activating agent includes but is not limited to antalgesic, non-steroidal anti-inflammatory drugs (NSAID), steroids, synthesis DMARD (such as, but not limited to methotrexate (MTX), leflunomide, sulfasalazine, Anranofin, golden sulphur Fourth disodium, penicillamine (penicillamine), chloroquine, hydroxychloroquine, imuran, tropsch imatinib (tofacitinib), bar West for Buddhist nun (baricitinib), good fortune he replace Buddhist nun (fostamatinib) and cyclosporin) and biology DMARD is (such as, but not limited to Infliximab, Etanercept, adalimumab, Rituximab and Abatace (abatacept)).
In one embodiment, composition of the invention and other therapeutic agent co-administered be used to treat and/or Prevent proliferative disorders, specific activating agent includes but is not limited to: methotrexate (MTX), formyl tetrahydrofolic acid (leukovorin), Ah Mycin (adriamycin), prednisone, bleomycin, cyclophosphamide, 5 FU 5 fluorouracil, taxol, docetaxel, Changchun are new Alkali, vincaleukoblastinum, vinorelbine, Doxorubicin, tamoxifen, Toremifene, megestrol acetate, Anastrozole, Goserelin, Anti-HER 2 monoclonal antibody is (for example, HerceptinTM), capecitabine, RALOXIFENE HCL, EGFR inhibitor (such asTarcevaTM、ErbituxTM), VEGF inhibitor is (for example, AvastinTM), proteasome inhibitor (for example, VelcadeTM)、With hsp90 inhibitor (for example, 17-AAG).Furthermore it is possible to by the compound comprising Formulas I and The composition of the invention and other therapies (including but not limited to radiotherapy or surgical operation) of JAK inhibitor are administered in combination. In a specific embodiment, proliferative disorders are selected from cancer, myeloproliferative disease or leukaemia.
In one embodiment, composition of the invention and other therapeutic agent co-administered be used to treat and/or Preventing autoimmune disease, specific activating agent includes but is not limited to: glucocorticoid, cytostatic agent (such as purine Analog), alkylating agent (for example, mustargen (cyclophosphamide), nitroso ureas, platinum complexes of the invention etc.), antimetabolite (example Such as, methotrexate (MTX), imuran and purinethol), cytotoxic antibiotics (for example, actinomycin D, anthracene nucleus, mitomycin C, Bleomycin and mithramycin), antibody (for example, anti-CD20, anti-CD25 or AntiCD3 McAb (OTK3) monoclonal antibody, With), cyclosporin, tacrolimus, rapamycin (sirolimus), interferon is (for example, IFN- β), TNF binding protein (for example, infliximab, according to that former times is general or adalimumab), mycophenolate, fingomode and more Coccus shell element.
In one embodiment, composition of the invention and other therapeutic agent co-administered are for treating and/or in advance Anti- graft rejection, specific activating agent includes but is not limited to: calcineurin inhibitors are (for example, cyclosporin or tacrolimus (FK506)), mTOR inhibitors (for example, sirolimus, everolimus), antiproliferative (for example, imuran, Mycophenolic Acid), Corticosteroid (for example, prednisolone, hydrocortisone), antibody are (for example, the anti-IL-2R α receptor antibody of monoclonal, Bali former times Monoclonal antibody, daclizumab), Anti-TNF-α-T- cell antibody is (for example, anti-anti-thymocyte globulin (ATG), anti-lymphocyte Globulin (ALG)).
In one embodiment, composition of the invention and other therapeutic agent co-administered be used to treat and/or Prevention of asthma and/or rhinitis and/or COPD, specific activating agent includes but is not limited to: beta-2-adrenoreceptor agonists (example Such as, salbutamol, Levalbuterol, Terbutaline and bitolterol), adrenaline (sucking or tablet), cholilytic drug (for example, Ipratropium Bromide) and glucocorticoid (oral or sucking).Long-acting beta 2- agonist is (for example, salmeterol, good fortune are not Special sieve, bambuterol and release oral salbutamol), sucking steroids and long-acting bronchodilator combination (for example, fluorine For Kathon CG/salmeterol, Budesonide/formoterol), leukotriene antagonist and synthetic inhibitor be (for example, montelukast, bundle Lu Site and Zileuton), medium release inhibitor (for example, cromoglycate and Ketotifen), IgE response biological regulator (for example, Ao Mazuo monoclonal antibody), antihistamine (for example, cetirizine, cinnarizine, fexofenadine) and vasoconstrictor (for example, Oxymetazoline (oxymetazoline), Xylometazoline (xylometazoline), naphazoline (naphazoline) and bent horse Oxazoline).
Furthermore it is possible to composition of the invention is administered in combination with the acute disease therapy for being used for asthma and/or COPD, it is such Therapy include oxygen or helium-oxygen gas mixture (heliox) application, atomization salbutamol or Terbutaline (optionally with cholilytic drug (example Such as, ipratropium (ipratropium)) combination), systemic steroids (it is oral or intravenous, for example, prednisone, prednisone Dragon, methylprednisolone, dexamethasone or hydrocortisone), intravenous salbutamol, non-specific beta-2-agonists (injection or sucking, For example, adrenaline, dilabron (isoetarine), isoprel, orciprenaline), cholilytic drug (IV or Atomization, for example, glycopyrronium bromide (glycopyrrolate), atropine, ipratropium), methyl xanthine (theophylline, aminophylline, benzyl Amine theophylline), inhalation anesthetic (for example, isoflurane, fluothane, enflurane), ketamine and vein with bronchiectasis effect Interior magnesium sulfate.
In one embodiment, composition of the invention and other therapeutic agent co-administered be used to treat and/or Prophylaxis of inflammatory bowel disease (IBD), specific activating agent includes but is not limited to: improving the synthetic glucocorticoid of disease (for example, sprinkling Buddhist nun Pine, budesonide), immunomodulator (for example, methotrexate (MTX), leflunomide, sulfasalazine, mesalazine, imuran, Ismipur and cyclosporin) and improve the biological agent of disease, immunomodulator (infliximab, adalimumab, Rituximab and A Batasai (abatacept)).
In one embodiment, composition of the invention and other therapeutic agent co-administered be used to treat and/or Prevent SLE, specific activating agent includes but is not limited to: human monoclonal antibodies (Baily wood monoclonal antibody (belimumab)) improve disease Antirheumatic drug (DMARD), such as Anti-Malarial (for example, plaquenil, hydroxychloroquine), immunosuppressor are (for example, first ammonia Pterin and imuran), cyclophosphamide and Mycophenolic Acid, immunosuppressive drug and antalgesic, such as non-steroidal anti-inflammatory drugs, opiate (for example, dextropropoxyphene and compound codeine and paracetamol (co-codamol)), opioid are (for example, hydrocodone, hydroxyl can Ketone, MS Contin or methadone) and Duragesic.
In one embodiment, composition of the invention and other therapeutic agent co-administered are for treating and/or in advance Anti- psoriasis, specific activating agent includes but is not limited to: local treatment agent, such as contains coal tar, anthraline (dithranol) (Dithranol), corticosteroid (such as Desoximetasone (TopicortTM), Fluocinonide, vitamine D3 class Like object (for example, Calcipotriol), the bath foam of Ah's glycerol and retinoids (etretinate, vitamin A acid, tazarotene), moisturizing Lotion, medicinal cream agent and ointment；Systemic therapy agent, such as methotrexate (MTX), cyclosporin, retinoids, thioguanine, hydroxyl Base urea, salicylazosulfapyridine, mycophenolate, imuran, tacrolimus, fumarate or biological agent, such as AmeviveTM、 EnbrelTM、HumiraTM、RemicadeTM、RaptivaTMWith excellent spy gram monoclonal antibody (IL-12 and IL-23 blocking agent).Furthermore it is possible to The compound of the present invention and other therapies are administered in combination, the therapy includes but is not limited to light therapy or photochemotherapy (example Such as, psoralen and ultraviolet A phototherapy method (PUVA)).
In one embodiment, composition of the invention and other therapeutic agent co-administered be used to treat and/or Prevent allergy, specific activating agent includes but is not limited to: antihistamine (such as cetirizine, diphenhydramine, Fexofenadine Fixed, left alerlisin), glucocorticoid (for example, prednisone, betamethasone, beclomethasone, dexamethasone), adrenaline, tea Alkali or anti-leukotriene (such as Montelukast or zafirlukast), cholilytic drug and decongestant.
It will be apparent for a person skilled in the art that any mode for delivering two or more therapeutic agents to patient includes The a part of co-administered as same therapeutic scheme.Although can be given simultaneously using single formulation (i.e. as single drug composition) Two or more activating agents of medicine, but this is not required.The activating agent can be administered with different preparations in different time.
Chemical synthesis process
It summarizes
Following conventional methods and step can be used from the starting material preparation being easy to get in the compound of the present invention.It should Understand, when providing typical or preferred preparation condition (that is, reaction temperature, time, the molar ratio of reactant, solvent, pressure etc.) When, unless otherwise stated, other preparation conditions can also be used.Optimum reaction condition can with the specific reactant used or Solvent and change, but such condition can be determined by those skilled in the art by routine optimisation procedures.
In addition, it will be apparent for a person skilled in the art that may need to commonly use protecting group to prevent certain functional groups Undesirable reaction occurs.Suitable condition for the suitable protecting group of particular functional group and for protecting and being deprotected Selection is well known in the art.For example, T.W.Greene and P.G.M.Wuts, Protecting Groups in Organic Synthesis,Wiley-Blackwell；4th Revised edition (2006) and reference cited therein A large amount of protecting groups and its introducing and removal are described in document (Wuts and Greene, 2006).
Method following detailed description of is provided to prepare the compound of the present composition as defined above and compare real Apply example.The compound of the present composition can be as known to organic synthesis field technical staff or the raw materials and reagents that are commercially available To prepare.
Unless otherwise stated, all reagents are commerical grade and are directly employed without upon receipt further Purifying.It is reacted under an inert atmosphere using the anhydrous solvent being commercially available.Unless otherwise stated, in all other feelings SILVER REAGENT solvent is used under shape.Column chromatography is carried out on silica gel standard items (30 μm to 70 μm).Use the silica gel of precoating 60F-254 plate (with a thickness of 0.25mm) carries out thin-layer chromatography.In 400MHz Bruker Avance spectrometer or It is recorded on 300MHzBruker Avance DPX spectrometer1H NMR spectra.1The chemical shift (δ) of H NMR spectra is relative to work It is reported for interior target tetramethylsilane (δ 0.00) or appropriate residual solvent peak with million numbers (ppm).Multiplicity is with unimodal (s), bimodal (d), triplet (t), quartet (q), quintet (quin), multiplet (m) and broad peak (br) are provided.Electron spray MS Spectrum is on Waters platform LC/MS spectrometer or using with Waters Acquity PDA detector and SQD matter The Waters Acquity UPLC of spectrometer is obtained.The column used: UPLC BEH C181.7 μm, 2.1 × 5mm VanGuard are pre- Column and 1.7 μm of C18 of Acquity UPLC BEH, 2.1 × 30mm column or 1.7 μm of C18 of Acquity UPLC BEH, 2.1 × 50mm column.All methods use MeCN/H2O gradient.MeCN and H2O contains 0.1% formic acid or 0.05%NH3.Prepare LCMS: The column used, 5 μm of 30 × 100mm of ODB (preparing column) of Waters XBridge Prep C18 and Waters XBridge 5 μm of C18,4.6mm × 100mm (analytical column).All methods use MeCN/H2O gradient.MeCN and H2O contains 0.1% formic acid Or 0.1% diethylamine.
Abbreviated list used in Table I experimental section
The compounds of this invention is synthetically prepared
The sheet according to formula (XXa) is generally described in WO 2013/189771 (Van ' t Klooster et al., 2013) The compound of inventive composition simultaneously discloses data.
Similarly, the sheet according to formula (XXb) is generally described in WO 2010/149769 (Menet and Smits, 2010) Invention compound simultaneously discloses data.The synthesis of salt and suitable is described in WO2015/117980 and WO2015/117981 Preparation.
The general synthetic method of embodiment 1
1.1 synthetic methods are summarized
1.2 conventional method
1.2.1 conventional method A
Corresponding 6- is added into the anhydrous THF solution for the NaH (2 equivalents, 60% dispersion oil) for being cooled to 0 DEG C Amino-nicotinic acid nitrile (1.1 to 1.2 equivalent).At 0 DEG C after 30 minutes, it is added the chloro- 3- nitropyridine of 2- (1 equivalent), at room temperature It is stirred to react, and is monitored by UPLC-MS.If reaction is not completed, it will react cooling at 0 DEG C again, add NaH is added followed by amine.Reaction mixture is poured into ice water and is stirred 2 hours.Sediment is filtered out, H is used2O is washed simultaneously It air-dries under vacuum, obtains desired compound.
The exemplary synthesis of conventional method A:
6- (the chloro- 3- nitropyridine -2- base amino of 6-)-nicotinic acid nitrile (Int 1)
To cooling NaH (2.07g, 51.81mmol, 2 equivalent, 60% dispersion oil) at 0 DEG C in anhydrous THF 6- amino-nicotinic acid nitrile (3.4g, 28.5mmol, 1.1 equivalent) is added in solution in (50mL).At 0 DEG C after 30 minutes, it is added Simultaneously reaction 16 hours is stirred at room temperature in 2,6- dichloro-3-nitropyridine (5g, 25.91mmol, 1 equivalent).The reaction is cooled to 0 DEG C, NaH (0.5g, 13mmol, 0.5 equivalent) is added and is stirred to react at 0 DEG C 1 hour, is then stirred at room temperature 2 hours. Reaction mixture is poured into ice water, and is stirred 2 hours.Sediment is filtered out, H is used2O is washed and is air-dried under vacuum.It will Obtained solid is collected into MeCN (75mL), is stirred at room temperature 30 minutes 1 hour, is then stirred 1 hour at 0 DEG C.So Afterwards, it is filtered and is washed with MeOH, obtain desired compound.
1H NMR(400MHz,DMSO-d6)δ10.81(1H,br s),8.73(1H,dd),8.61(1H,d),8.31(1H, dd),8.01(1H,dd),7.36(1H,d)。
1.2.2 conventional method B
Corresponding amine (1.1 is added into the chloro- 3- nitro-pyridine -2- base aminoderivative of 6- (1 equivalent) in DMSO Equivalent) and DIPEA (2 equivalent), then by reaction mixture microwave heating at 110-130 DEG C, until reaction is completed.It will mixing Object H2O dilution, filters out sediment and air-dries under vacuum, obtain desired compound.
The exemplary synthesis of conventional method B:
6- [3- nitro -6- (ttetrahydro-pyran -4- base amino)-pyridine -2- base amino]-nicotinic acid nitrile (Int 8)
To in DMSO (20mL) 6- (the chloro- 3- nitropyridine -2- base amino of 6-)-nicotinic acid nitrile (Int 1,4g, 14.51mmol, 1 equivalent) in ttetrahydro-pyran -4- base amine (1.65mL, 15.96mmol, 1.1 equivalent) and DIPEA is added (5.05mL, 29.02mmol, 2 equivalent), then microwave heating reaction mixture 20 minutes at 130 DEG C.The mixture is used H2O and Et2O dilution, filters out sediment and air-dries under vacuum, obtain desired compound.
1H NMR(300MHz,DMSO-d6)δ11.38(1H,s),8.78(1H,d),8.47-8.63(2H,m),8.39(1H, dd),8.17(1H,d),6.27(1H,d),3.98-4.12(1H,m),3.91(2H,d),3.52(2H,t),1.94(2H,d), 1.33-1.67(2H,m)。
1.2.3 conventional method C
NBS (1.1 to 2 equivalent) is added to 3- nitropyridine -2,6- diamino radical derivative (1 equivalent) in anhydrous MeCN Solution in, be stirred at room temperature reaction, and pass through UPLC-MS and monitor.If being not fully complete reaction, it is added in addition NBS is not until having surplus stock.The precipitating to be formed is filtered out, Et is used2O is washed and is air-dried under vacuum, obtains desired chemical combination Object.
The exemplary synthesis of conventional method C:
6- [the bromo- 3- nitro -6- of 5- (ttetrahydro-pyran -4- base amino)-pyridine -2- base amino]-nicotinic acid nitrile (Int11)
NBS (2.04g, 11.46mmol, 1.3 equivalent) is added to 6- [3- nitro -6- (ttetrahydro-pyran -4- base amino) - Pyridine -2- base amino]-nicotinic acid nitrile (Int 8,3g, 8.81mmol, 1 equivalent) is in the solution in anhydrous MeCN (150mL) and in room It is stirred to react under temperature 4 hours.NBS (0.31g, 1.76mmol, 0.2 equivalent) is added and is stirred for reaction 16 hours at room temperature. The sediment to be formed is filtered out, Et is used2O is washed and is air-dried under vacuum, obtains desired compound.
1H NMR(300MHz,DMSO-d6)δ11.10(1H,br s),8.80(1H,m),8.34-8.50(3H,m),7.72 (1H,d),4.05-4.25(1H,m),3.93(2H,m),3.38-3.55(2H,m),1.70-1.87(4H,m)。
1.2.4 conventional method D
Corresponding alcohol (5 equivalent) is added portionwise in anhydrous 1,4- dioxanes or as solvent in LiOtBu (3 equivalent) In solution in correspondent alcohol.Then 2- amino -3- bromopyridine derivative (1 equivalent) is added, is subsequently added into CuI (0.6 equivalent).It will Reaction is heated to 80-120 DEG C, or is heated to 110-150 DEG C under microwave irradiation, until reaction is completed.The mixture is toppled over Into ice water, or 1N HCL aqueous solution is added.Sediment is filtered out, and is air-dried under vacuum.Then, pass through the quick color of silica gel Spectrum purifying residue, obtains desired compound.
The exemplary synthesis of conventional method D:
6- [5- [2- (2- Hydroxy-ethoxy)-ethyoxyl] -3- nitro -6- (ttetrahydro-pyran -4- base amino)-pyridine -2- Base amino]-nicotinic acid nitrile (Int 19)
By LiOtBu (2.87g, 35.8mmol, 3 equivalent) be added portionwise 2- (2- Hydroxy-ethoxy)-ethyl alcohol (5.7mL, 59.7mmol, 5 equivalents) in the solution in anhydrous 1,4- dioxanes (50mL).6- [the bromo- 3- nitro -6- (tetrahydro-pyrrole of 5- is added Mutter -4- base amino)-pyridine -2- base amino]-nicotinic acid nitrile (Int 11,5.0g, 11.9mmol, 1 equivalent), it is subsequently added into CuI (1.36g, 7.2mmol, 0.6 equivalent).Then, reaction is heated to 120 DEG C 4 hours.The mixture is cooled to 0 DEG C, is added The HCL aqueous solution (50mL) of 1N and be stirred at room temperature mixture 20 minutes.It filters out sediment and does under vacuum It is dry.Then, residue is purified by flash chromatography on silica gel, the DCM solution elution of use 0 to 5%MeOH obtains desired chemical combination Object.
MW (calculated value): 444.45；MW (measured value): 445.18ES+.
1H NMR(400MHz,DMSO-d6)δ11.42(1H,s),8.75-8.79(1H,m),8.47-8.50(1H,m), 8.38(1H,dd),7.87(1H,d),7.69(1H,s),4.58-4.72(1H,m),4.21-4.26(2H,m),4.11-4.21 (1H,m),3.94(2H,dd),3.82(2H,dd),3.44-3.57(6H,m),1.81-1.90(2H,m),1.74(2H,qd)。
1.2.5 conventional method E
Orthoformic acid three is added in the solution in anhydrous MeOH to 2,6- diamino -5- nitropyridine derivatives (1 equivalent) Methyl esters (for 2, the 6- diamino -5- nitropyridine derivatives of 0.1mmol, about 0.1mL) and formic acid (for the 2 of 0.1mmol, 6- diamino -5- nitropyridine derivatives, about 0.1mL).Then, NH is added4Cl (4 equivalent) and Zn (4 to 5 equivalent), and will mix It closes object and is heated to 70 DEG C until the reaction is complete.Then, which is cooled to room temperature.
If observing that precipitating is formed while cooling, solid is filtered out, and use DCM/CHCl3With 2% aqueous formic acid Aqueous post-processing is carried out, desired compound is obtained.
If do not precipitated while cooling, solvent is evaporated, residue is then purified by flash chromatography on silica gel, is obtained To desired compound.
The exemplary synthesis of conventional method E:
6- [6- [2- (2- Hydroxy-ethoxy)-ethyoxyl] -5- (ttetrahydro-pyran -4- base amino)-imidazo [4,5-b] Pyridin-3-yl]-nicotinic acid nitrile (compound 1)
To 6- [5- [2- (2- Hydroxy-ethoxy)-ethyoxyl] -3- nitro -6- (ttetrahydro-pyran -4- base amino)-pyridine - 2- base amino] orthoformic acid is added in the solution in anhydrous MeOH (60mL) in-nicotinic acid nitrile (Int 19,2.3g, 5.2mmol, 1 equivalent) Trimethyl (10mL) and formic acid (10mL).Then, NH is added4Cl (1.1g, 20.7mmol, 4 equivalent) and Zn (1.4g, 20.7mmol, 4 equivalents), and the mixture is heated to reflux 2 hours.It is added MeOH (30mL), and reaction mixture is heated back Stream 1 hour.
The mixture is cooled to room temperature, the sediment of formation is filtered, and is dried under vacuum.It is added MeOH (100mL) With formic acid (2mL), and obtained mixture is stirred at reflux 1 hour.The mixture is cooled to room temperature, is poured into ice water, The precipitating formed is filtered, and is dried under vacuum.Solid is suspended in DCM and CHCl3Mixture in, pass through diatomite mistake Filter, and filtrate is washed with 2% aqueous formic acid.By organic phase Na2SO4It is dried, filtered and concentrated to doing, obtains desiredization Close object.
MW (calculated value): 424.46；MW (measured value): 425.40ES+
1H NMR(400MHz,DMSO-d6)δ9.00(1H,dd),8.89-8.95(1H,m),8.75(1H,s),8.62(1H, dd),7.59(1H,s),6.04(1H,d),4.61-4.69(1H,m),4.22(2H,dd),4.05-4.17(1H,m),3.90- 3.98(2H,m),3.83(2H,dd),3.50-3.60(6H,m),1.99(2H,m),1.55-1.70(2H,m)。
1.2.6 conventional method F
At room temperature, by compound 1, corresponding carboxylic acid (1.5 equivalent), DMAP (1.5 equivalent) and EDCI (2.25 equivalent) Mixture stirred in DCM, until reaction complete.By reaction H2O is quenched, and is extracted with DCM, then uses MgSO4Drying is organic Layer is simultaneously evaporated to dryness.Residue is purified by flash chromatography on silica gel, obtains desired compound.
The exemplary synthesis of conventional method F:
(S) -2- tertbutyloxycarbonylamino -3- metliyl-butyric acid 2- { 2- [3- (5- cyanopyridine -2- base) -5- (tetrahydro - Pyrans -4- base amino) -3H- imidazo [4,5-b] pyridine -6- base oxygroup]-ethyoxyl }-ethyl ester (Int 30)
At room temperature, by compound 1 (42mg, 0.1mmol, 1 equivalent), boc- (S)-valine (33mg, 0.15mmol, 1.5 equivalents), the mixture of DMAP (19mg, 0.15mmol, 1.5 equivalent) and EDCI (45mg, 0.225mmol, 2.25 equivalent) exists Stirring 3 hours in DCM (5mL).By reaction H2O is quenched, and is extracted with DCM, then uses MgSO4Dry organic layer is simultaneously evaporated to dryness. Residue is purified by flash chromatography on silica gel, the n-heptane solution elution of use 0 to 100%EtOAc obtains desired compound.
1.2.7 conventional method G: at salinization method
Raw material is dissolved in the hot mixt of hot EtOAc or EtOAc and MeOH (5/1).Oxalic acid is added into the hot solution (the EtOAc solution of 0.2M, 1 equivalent).Sediment is formed, Et is used in filtering2O is rinsed and dried, and obtains the oxalate form of raw material Desired compound.
The exemplary synthesis of conventional method G:
(S) -2- amino -3- metliyl-butyric acid 2- { 2- [3- (5- cyanopyridine -2- base) -5- (ttetrahydro-pyran -4- base ammonia Base) -3H- imidazo [4,5-b] pyridine -6- base oxygroup]-ethyoxyl }-ethoxal salt (compound 10)
Compound 9 (100mg, 0.19mmol, 1 equivalent) is dissolved in hot EtOAc (10mL), and is added into the hot solution Oxalic acid (the EtOAc solution of 0.2M, 0.96mL, 0.19mmol, 1 equivalent).The sediment formed is filtered, Et is used2O is rinsed and is done It is dry, obtain desired compound.
1.2.8 (3,4 is cis-) -3- amino-ttetrahydro-pyran -4- alcohol (Int 28) synthesis
1.2.8.1 step i): 3 ,-two ring of 7- dioxa [4.1.0] heptane to m- chlorine benzylhydroperoxide (23.51g, 136.2mmol, 2 equivalents) 3,6- dihydro -2H- pyrans is added in the solution in DCM (15mL), and (5.73g, 68.1mmol, 1 work as Amount) solution in DCM (10mL).The reaction mixture is stirred at room temperature 6 hours, later, m- chlorine benzylhydroperoxide is added (11.76g, 68.1mmol, 1 equivalent).The reaction mixture is stirred at room temperature 16 hours and is then filtered.Use Na2SO3、 NaHCO3Saturated solution and water washing filtrate.Then, through Na2SO4Dry organic layer, is filtered and concentrated in vacuo to obtain desired Compound is directly used in next step without further purification.
1.2.8.2 step ii): (3,4 is trans-) -3- benzylamino-ttetrahydro-pyran -4- alcohol
By-two ring of 3,7- dioxa [4.1.0] heptane (2.7mmol, 1 equivalent) and benzylamine, (300 μ L, 2.7mmol, 1 work as Amount) mixture in EtOH (10mL) heats 18 hours at a reflux temperature.Then, it evaporates EtOH and passes through silica gel column chromatography Thick material is purified, DCM:MeOH:NH is used4OH10:1:0.1 elution, obtains desired compound.
1.2.8.3 step iii): N- benzyl-N- ((3,4 is trans-) -4- hvdroxv-tetrahydro-pvran -3- base)-benzamide
Chlorobenzoyl chloride (78 μ L, 0.68mmol, 1 equivalent) is added dropwise to (3,4 is trans-) -3- benzyl ammonia from abovementioned steps Base-ttetrahydro-pyran -4- alcohol (140mg, 0.68mmol, 1 equivalent) and TEA (280 μ L, 2.03mmol, 3 equivalent) are at DCM (2mL) In ice cooling solution in.The reaction mixture is stirred at room temperature 1 hour.Then, this is washed with 2N HCL aqueous solution to mix Close object twice.With DCM aqueous layer extracted, then through Na2SO4Dry combined organic layer, is filtered and concentrated in vacuo to obtain desiredization Close object.
1.2.8.4 step iv): (3,4 is cis-) -3- benzylamino-ttetrahydro-pyran -4- alcohol
At 0 DEG C, to N- benzyl-N- ((3,4 is trans-) -4- hvdroxv-tetrahydro-pvran -3- base)-benzamide (220mg, 0.71mmol, 1 equivalent) dropwise addition thionyl chloride (195 μ L, 2.68mmol, 3.8 equivalent) in the solution in DCM (2.5mL).? The reaction mixture is stirred at room temperature 4 hours, be then concentrated in vacuo.The HCL aqueous solution (2mL) of 6N is added into residue and incites somebody to action Obtained mixture heats 18 hours at a reflux temperature.After cooling, sediment is filtered out, be washed with water and is extracted with EtOAc Filtrate.Et is added into water layer2Simultaneously the NaOH aqueous solution of 2N is added so that the mixture is in alkalinity in O.Separate each phase, with DCM and EtOAc aqueous phase extracted.Then, by combined organic layer through Na2SO4It dries, filters and is concentrated in vacuo, obtain desired compound.
1.2.8.5 step v): (3,4 is cis-) -3- amino-ttetrahydro-pyran -4- alcohol
By (3,4 is cis-) -3- benzylamino-ttetrahydro-pyran -4- alcohol (100mg, 0.48mmol, 1 equivalent) in MeOH Solution in (3mL) at room temperature, in the H of 1atm2It is lower to be hydrogenated 1.5 hours with 10% Pd/C (40mg).Pass through diatomite mistake Catalyst is filtered out, filtrate is washed and evaporated with MeOH, obtains desired compound.
1.2.9 the synthesis of (cis- -1,4) -1- methyl -4- methylamino-cyclohexanol (Int 29)
1.2.9.1 step i): (cis- -1,4)-(4- hydroxy-4-methyl-cyclohexyl)-t-butyl carbamate
To suspension of the cis- -4- amino -1 methyl cyclohexanol (1.0g, 7.74mmol, 1 equivalent) in MeCN (15mL) Middle addition di-tert-butyl dicarbonate (1.85g, 8.47mmol, 1.1 equivalent), and the mixture is stirred at room temperature 16 hours.It crosses Sediment is filtered, is washed and is dried with hexane, obtain desired compound.
1.2.9.2 step ii): (cis- -1,4) -1- methyl -4- methylamino-cyclohexanol
At room temperature, to LiAlH4It is added portionwise in 2.0M solution (7mL, 14.0mmol, 4.9 equivalent) in THF (suitable Formula -1,4)-(4- hydroxy-4-methyl-cyclohexyl)-t-butyl carbamate (660mg, 2.9mmol, 1 equivalent).At room temperature, It stirs the reaction mixture 1 hour, then stirs 45 minutes under reflux.Reaction mixture is cooled to room temperature, is then added Water and THF.It filters out sediment and is washed with THF.It concentrates the filtrate to dry, obtains desired compound.
1.2.10. intermediate 34:2- [2- [3- (5- cyano -2- pyridyl group) -5- (tetrahydropyran -4-base amino) imidazo [4,5-b] pyridine -6- base] oxygroup ethyoxyl] ethyl (2S) -2- [[2- (tertbutyloxycarbonylamino) acetyl group] amino] -3- Metliyl-butyric acid ester
By compound 9 (170mg, 0.40mmol, 1 equivalent), 2- (tertbutyloxycarbonylamino) acetic acid (Boc-Gly-OH, 105mg, 0.60mmol, 1.5 equivalent), EDCI (173mg, 0.90mmol, 2.25 equivalent) and DMAP (73mg, 0.6mmol, 1.5 Equivalent) it mixes and is stirred at room temperature overnight in DCM (4mL).With salt water quenching reaction, with DCM extraction and having merging Machine mutually evaporates, and obtains desired compound.
Table II is used to synthesize the intermediate of the compounds of this invention
SM=raw material, Mtd=method, the quality of MS Mes ' d=measurement
The preparation of 2 the compound of the present invention of embodiment
2.1 compound, 3 6- { 6- [2- (2- Hydroxy-ethoxy)-ethyoxyl] -5- [((cis- -1,4) -4- hydroxyl -4- first Base-cyclohexyl)-Methyl-amino]-imidazo [4,5-b] pyridin-3-yl }-nicotinic acid nitrile
Formaldehyde (3.1 μ L, 0.11mmol, 1 equivalent) is added to compound 12 (50mg, 0.11mmol, 1 equivalent) in TFA/ In solution in DCM (2mL, 1/1) mixture.It is stirred at room temperature after 30 minutes, is added NaBH (OAc)3(47mg, 0.22mmol, 2 equivalents) and the reaction 1 hour is stirred at room temperature.Then, reaction mixture is evaporated and passes through preparative HPLC-MS purification of crude product obtains desired compound.
2.2. compound 8: mono- (2- { 2- [3- (5- Cyano-pyridin -2- base) -5- (ttetrahydro-pyran -4- base ammonia of sulfuric acid Base) -3H- imidazo [4,5-b] pyridine -6- base oxygroup]-ethyoxyl }-ethyl) ester
Compound 1 (84mg, 0.2mmol, 1 equivalent) is added to pyridine-SO3(127mg, 0.8mmol, 4 work as compound Amount) it is heated to reflux 16 hours in the solution in pyridine (5mL) and by the reaction.Then, mixture dry doubling is evaporated to pass through Flash chromatography on silica gel purifying, with 100%EtOAc to 100% (DCM/MeOH/AcOH/H2O:90/10/1/1 it) elutes, then uses 100% (DCM/MeOH/AcOH/H2O:85/15/2/2 it) elutes, obtains desired compound.
2.3. compound 9 (S) -2- amino -3- metliyl-butyric acid 2- { 2- [3- (5- Cyano-pyridin -2- base) -5- (tetrahydro - Pyrans -4- base amino) -3H- imidazo [4,5-b] pyridine -6- base oxygroup]-ethyoxyl }-ethyl ester
TFA (0.5mL) is added to Int 30 (20mg, 0.032mmol, 1 equivalent) in the solution in DCM (10mL) simultaneously The mixture is stirred at room temperature 1 hour.With the NaHCO of saturation3Aqueous solution quenching reaction is simultaneously extracted with EtOAc.Through MgSO4It is dry Dry organic layer is simultaneously evaporated to dryness.By residue solvent system DCM/Et2The pentane solution of O recrystallizes, and obtains desired chemical combination Object.
2.4. compound 16:2- [2- [3- (5- cyano -2- pyridyl group) -5- (tetrahydropyran -4-base amino) imidazo [4, 5-b] pyridine -6- base] oxygroup ethyoxyl] ethyl 2- aminoacetate
Int 31 (195mg, 0.34mmol, 1 equivalent) is placed in TFA/DCM mixture (1/5mL) and is stirred at room temperature Mix the reaction 2 hours.Then, it by reaction mixture dilution with toluene and is evaporated to dryness.By residual collection in DCM, with full The NaHCO of sum3Aqueous solution washs and is evaporated to dryness organic phase.Residue is dissolved in minimal amount of DCM, is added a large amount of Et2O simultaneously makes the solid to be formed be deposited in drag.Solvent is carefully removed, stays in solid in flask, pentane and mistake is added Filter solid obtains desired compound.
2.5. compound 17:2- [2- [3- (5- cyano -2- pyridyl group) -5- (tetrahydropyran -4-base amino) imidazo [4, 5-b] pyridine -6- base] oxygroup ethyoxyl] ethyl 2- (methylamino) acetic acid esters
Int 32 (205mg, 0.34mmol, 1 equivalent) is placed in TFA/DCM mixture (1/5mL) and is stirred at room temperature Mix the reaction 2 hours.Then, it by reaction mixture dilution with toluene and is evaporated to dryness.By residual collection in DCM, with full The NaHCO of sum3Aqueous solution washs and is evaporated to dryness organic phase.Residue is dissolved in minimal amount of DCM, a large amount of Et are added2O And the solid to be formed is made to be deposited in drag.Solvent is carefully removed, stays in solid in flask, pentane is added and is filtered solid Body obtains desired compound.
1.1. compound 18:2- [2- [3- (5- cyano -2- pyridyl group) -5- (tetrahydropyran -4-base amino) imidazo [4, 5-b] pyridine -6- base] oxygroup ethyoxyl] ethyl (2S)-pyrrolidines -2- formic acid esters
Int 33 (211mg, 0.34mmol, 1 equivalent) is placed in TFA/DCM mixture (1/5mL) and is stirred at room temperature Mix the reaction 2 hours.Then, it by reaction mixture dilution with toluene and is evaporated to dryness.By residual collection in DCM, with full The NaHCO of sum3Aqueous solution washs and is evaporated to dryness organic phase.Residue is dissolved in minimal amount of DCM, a large amount of Et are added2O And the solid to be formed is made to be deposited in drag.Solvent is carefully removed, stays in solid in flask, pentane is added and is filtered solid Body obtains desired compound.
2.6. compound 19:2- [2- [3- (5- cyano -2- pyridyl group) -5- (tetrahydropyran -4-base amino) imidazo [4, 5-b] pyridine -6- base] oxygroup ethyoxyl] ethyl (2S) -2- [(2- glycyl) amino] -3- metliyl-butyric acid ester
Int 34 (212mg, 0.31mmol, 1 equivalent) is placed in TFA/DCM mixture (1/5mL) and is stirred at room temperature Mix the reaction 2 hours.Then, toluene is added and is evaporated to dryness solvent.Residue is dissolved in DCM, saturation is added NaHCO3After aqueous solution, there is solid to be settled out.Solid is filtered and dried, desired compound is obtained.
2.7 compound 27:(3S) -3- amino -4- [2- [2- [3- (5- cyano -2- pyridyl group) -5- (tetrahydropyran -4-base Amino) imidazo [4,5-b] pyridine -6- base] oxygroup ethyoxyl] ethyoxyl] -4- oxo-butynic acid hydrochloride
By solution (4M, 0.4mL, 1.6mmol, 5 equivalent) of the HCl in 1,4- dioxanes be added to Int 35 (222mg, 0.32mmol, 1 equivalent) in the solution in 1,4- dioxanes (4mL).The mixture is stirred at room temperature 3 hours, then evaporates It is extremely dry.By residual collection HCl in the solution (4M, 6mL) in Isosorbide-5-Nitrae-dioxanes and be stirred at room temperature reaction 2 hours, Then the HCl solution (4M, 2mL) in 1,4- dioxanes is added again.It is stirred at room temperature after 1 hour, solvent is evaporated To doing, desired compound is obtained.
2.8. compound 28:4S) -4- amino -5- [2- [2- [3- (5- cyano -2- pyridyl group) -5- (tetrahydropyran -4-base Amino) imidazo [4,5-b] pyridine -6- base] oxygroup ethyoxyl] ethyoxyl] -5- oxo-pentanoic acid hydrochloride
Solution (4M, 0.41mL, 1.63mmol, 5 equivalent) of the HCl in 1,4- dioxanes is added to Int 36 (232mg, 0.33mmol, 1 equivalent) is in the solution in 1,4- dioxanes (4mL).The mixture is stirred at room temperature 3 hours, Then it is evaporated to dryness.In the solution (4M, 6mL) in 1,4- dioxanes and this is stirred at room temperature in HCl in residual collection Reaction 2 hours.Solvent is evaporated to dryness, desired compound is obtained.
2.9. compound 33:4- [2- [2- [3- (5- cyano -2- pyridyl group) -5- (tetrahydropyran -4-base amino) imidazo [4,5-b] pyridine -6- base] oxygroup ethyoxyl] ethyoxyl] -4- oxo-butynic acid
Int 37 (90mg, 0.16mmol, 1 equivalent) is placed in TFA/DCM mixture (1/5mL) and is stirred at room temperature The reaction 2 hours.Then, toluene is added and is evaporated to dryness solvent.Residue, use 0 to 6% are purified by flash chromatography on silica gel The DCM solution of MeOH elutes, and the product then precipitated from MeOH obtains desired compound after filtration.
The illustrative the compound of the present invention of Table III
SM=raw material, Mtd=method, the quality of MS Mes ' d=measurement
The NMR data of Table IV representative compound of the invention
Biological examples
3 analyzed in vitro of embodiment
The IC of 3.1 mankind IRAK-450Measurement
The IC of IRAK-4 is measured in the measurement of radioactivity filter plate50Value.The principle of the measurement is using [γ-33P]ATP It is incorporated into RIP140 substrate after enzyme IRAK-4 phosphorylation with ATP measurement33P.By (will be used in sample loading to filter plate Collector, PerkinElmer) and 6 subsequent wash steps removals it is unbonded33P.By MicroScintTM-20 (PerkinElmer, 6013621) is measured after being added to filter plate by scintillation counter (Topcount, PerkinElmer) It is mixed in RIP14033P。
The water dilution series of 5 μ L test compounds (since 20 μM or 6.6 μM of maximum concentrations, are laid in by 100%DMSO Solution preparation, 1/5 dilution) (final DMSO concentration is 1% in response analysis) is added into each hole.IRAK-4(Carna Biosciences, 09-145) and RIP140 (SEQ ID1, reference table VII) made respectively with 10ng/mL and 4 μM of ultimate density With.In 25mM Tris pH 7.5,0.025%Triton X-100,5mM MnCl2In 2mM DTT, enzyme and substrate are diluted To the total volume of 11 μ L.By be added in buffer identical with enzyme and substrate diluted 9 μ L, 1 μM of ATP (Sigma, A6419-5G)+0.25μCi[γ-33P] ATP (PerkinElmer, NEG602K001MC) starts to react.By mixture at 30 DEG C Culture 45 minutes.150mM phosphoric acid (VWR, 1.00573.1000) by the way that 25 μ L are added terminates reaction.Sample is transferred to filtering Plate simultaneously uses the radioactivity of scintillation counter measurement incorporation.
Use 10 μM of staurosporines (1%DMSO) as positive control (100% inhibits)；Solvent (water+1%DMSO) conduct Negative control (0% inhibits).
The external mankind IRAK-4IC of Table V the compounds of this invention50
Cpd# hIRAK-4IC50(nM)
1 6.35
2 25.4
3 21.7
4 523
5 13.8
6 4.85
7 14.3
8 51.4
9 13.5
11 14.3
12 0.95
13 39.1
14 3.45
3.2 Kinase Selectivities analyze (wide group)
In REACTION BIOLOGY (Reaction Biology Corp., 1Great Valley Parkway, Suite 2Malvern, PA 19355, USA) radioactive kinase measurement in measure human kinase inhibition.
In order to measure the IC of compound50, (since 10 μM (maximum concentration), it is serially diluted using 3 times) with 10 dosage Test compound.Dose-response curve by being fitted remaining enzymatic activity % (compareing relative to DMSO) derives IC50Value.
3.3 Kinase Selectivities analyze (concentration group)
The purpose of the analysis is to measure the compounds of this invention to the activity and selectivity of the human kinase of selected range, when this A little kinases can cause when being suppressed undesirable side effect (Dy and Adjei, 2013；Force and Kolaja, 2011).
3.3.1 scheme is measured
The IC for the kinases that misses the target is measured in the measurement of radioactivity filter plate50Value.The principle of the measurement is using [γ-33P] ATP and ATP measurement is incorporated into peptide substrates after tyrosine phosphorylation33P.By the way that (collection will be used in sample loading to filter plate Device, PerkinElmer) and 6 subsequent wash steps removals it is unbonded33P.By MicroScint TM-20 (PerkinElmer, 6013621) is added to after filter plate and measures peptide by scintillation counter (Topcount, PerkinElmer) It is mixed in substrate33P。
The water dilution series of 5 μ L test compounds (since 20 μM or 6.6 μM of maximum concentrations, are laid in by 100%DMSO Solution preparation, 1/5 dilution) (final DMSO concentration is 1% in response analysis) is added into each hole.Enzyme and peptide substrates are with best Concentration uses (referring to Table VI).Enzyme and substrate are diluted to the total volume of 11 μ L in analysis buffer.By be added with enzyme Diluted 9 μ L ATP+ [γ-in buffer identical with substrate33P] ATP starts to react.The mixture is cultivated at 30 DEG C.It is logical Cross the 150mM phosphoric acid termination reaction that 25 μ L are added.Sample is transferred to filter plate and putting using scintillation counter measurement incorporation Penetrating property.
The exemplary kinases that the concentration of incubation time, analysis buffer composition and ATP, enzyme and substrate is reported in Table VI is de- In target analysis.
Use 10 μM of staurosporines (1%DMSO) as positive control (100% inhibits)；Solvent (water+1%DMSO) conduct Negative control (0% inhibits).
Table VI human kinase misses the target the condition in inhibition analysis
Table VII human kinase misses the target peptide substrates used in inhibition analysis
The external human kinase of the illustrative the compounds of this invention of Table VIII misses the target IC50
3.3.2 conclusion
For the data of Table VIII compared with the data of Table V, the inhibition effect that display the compound of the present invention misses the target to kinases is low In IRAK-4.These data confirm that the compound of the present invention have selectivity to IRAK-4, thus limit and miss the target suppression with kinases Make the risk of relevant side effect.
3.4 cell analysis: the TNF α that CL097 is activated in PBMC, which discharges, to be inhibited
The compound of the present invention is tested using the human peripheral blood mononuclear cell (PBMC) of primary separation in cell analysis, with The secretion of measurement inflammatory cytokine TNF α when carrying out TLR activation using specific TLR7/8 agonist CL097.Pass through the mankind TNF α Enzyme Linked Immunoadsorbent Assay (ELISA) scheme quantifies the release of TNF α protein in cell culture supernatant.
3.4.1 people's primary PBMC is separated from people's buffy coat
By people's buffy coat (being provided by Croatian Institute for Transfusion Medicine) 4 It DEG C keeps overnight, and being handled at second day to separate PBMC.Use Ficoll-PaqueTM PLUS(GE HealthCare, 17-1440-02) PBMC separated by density gradient centrifugation.It is diluted using sterile PBS (1 ×) 1:4 isometric Buffy coat, and in suitable 50mLMake 35mL at the top of 15mL Ficoll-Paque PLUS in pipe Careful layering.At room temperature, by the pipe with 1500rpm centrifugation 35 minutes.Without acceleration or interruption.After centrifugation, top is removed Plasma layer carefully separates monocyte ring, and transfers them to newPipe.Isolated cell suspending liquid is existed 50mL is diluted in PBS, then at room temperature with 1300rpm centrifugation 10 minutes.2 other washing steps are carried out in PBS Collect lag with cell, by the way that cell precipitate is resuspended in 50mL AKL lysis buffer (150mM NH4Cl, 10mM NaHCO3, 1mM Na2EDTA, pH 7.4) in, it is then gently mixed to dissolve remaining red blood cell.Then, at room temperature, will 50mL suspension then removes supernatant, and cell precipitate is resuspended in culture medium with 1300rpm centrifugation 10 minutes (RPMI 1640 (Gibco, 21875)+heat inactivation 30 minutes at 56 DEG C 10% fetal calf serum (FBS, Biowest, S1810)+ Pen/Strep (Gibco, 15240)) in.
3.4.2 the compound processing and triggering in PBMC analysis
Cell is counted using hematology analyzer (Sysmex XS-500i), and by it with 4.0 × 105It is a thin Born of the same parents/hole density bed board is in the 160 μ L culture mediums in 96 well culture plates.Then, in 37 DEG C and 5%CO2Under, by being added 20 The compound solution of 10 times of concentration of μ L preculture 1 hour together with test compound by PBMC.Test should under various concentration Compound, and by being serially diluted in DMSO from 10mM stock solution, being then supplemented with 1%FBS and 1%Pen/ for 3 times 1:50 dilution step is carried out in 2 × M199 culture medium (Gibco, 21157-029) of Strep to prepare.It is final in the analysis Experimental concentration starts from 20 μM, then carries out 3 times and is serially diluted, and final DMSO concentration is 0.2%.In compound preculture After step, touched by being added the 10 μ g/mL CL097 solution (InvivoGen, tlrl-c97-5) of 20 μ L into each hole PBMC is sent out, final analysis volume is 200 holes μ L/, and final CL097 triggering agent concentration is 1 μ g/mL.It is dense with identical DMSO Degree (no CL097 triggers agent) adjusts negative control.Then, in 37 DEG C and 5%CO2Under, analysis plates are trained in humidified incubator It supports 4 hours.Then, cell supernatant is harvested by the way that cell culture medium to be transferred in 384 deep-well plates, and is shifted immediately Into elisa plate for being quantified to human TNF alpha.
3.4.3 TNF α is quantified by ELISA
Level with the TNF α secreted in antibody capture activity analysis (ELISA) quantitative cell supernatant.It will be white Greiner LumitracTM384 orifice plates diluted anti-human TNF α antibody-solutions (MAb1 of 1 μ g/mL in PBS with every 40 μ L of hole； BD Biosciences, 551220) in 4 DEG C of overnight incubations, it is coated with.After washing each hole with 100 μ L PBS, with 100 μ L blocks buffer (+5% sucrose of PBS+1% bovine serum albumin(BSA)) to block remaining binding site, and culture 4 is small at room temperature When.After blocking step, each hole washed once with the PBS (PBST) containing Tween 20, be subsequently added into sample and standard Product.1/3 sample of the dilution containing TNF α in dilution buffer, and 40 μ L overnight incubation at 4 DEG C is added.Then, adding Enter 35 μ L and antibody (MAb11 is detected for the two stage biological element anti-TNF alpha of 250ng/mL with 1/2000 dilution and ultimate density； BDBiosciences, 554511) after, 3 times (being washed twice with PBST, washed once with PBS) is washed into each hole.In room temperature It is lower culture 2 hours and washing step appropriate (2 × PBST, 1 × PBS) after, with the 1/4000 diluted horseradish peroxide of 35 μ L The solution of streptavidin (Life Technologies, SNN2004) that compound enzyme combines cultivates each hole, then keeps away at room temperature Optical culture 45 minutes.Then, 3 times (2 × PBST, 1 × PBS) is washed into each hole, it is then molten with 50 μ L chemiluminescence ELISA substrates Liquid (Roche, 11582950001) is cultivated 5 minutes.It is measured in 2104 multiple labeling plate reader of PerkinElmer EnVision The substrate luminous signal of conversion.
3.4.4 data are analyzed
All controls measure within the range of linearity of the human TNF alpha standard curve of ELISA.All data are directed to It analyzes mass parameter and checks validity (signal/background > 2, and Z > 0.3).
Using the sample that does not stimulate, (no triggering agent/solvent (0.2%DMSO) is as positive control (100% inhibit).It uses The sample (triggering agent/solvent (0.2%DMSO)) of stimulation is as negative control (0% inhibits).Use positive and negative control root Z ' and suppression percentage (PIN) value are calculated according to following formula:
RCLU=is with respect to chemiluminescence light unit.
It draws to the PIN value for the compound tested in concentration-response modes, and uses GraphPadSoftware application nonlinear regression (S-shaped) curve matching obtains IC50Value.
3.5 cell analysis: cancer cell assay
3.5.1 cell line
In 37 DEG C and 5%CO2Under, it is being supplemented with 10% fetal calf serum (Invitrogen, S7524) or 20% human serum (Invitrogen, 34005100) IMDM (21980-032) in culture from OCI-Ly3, OCI-Ly10, Human lymphocytes' oncocyte of OCI-Ly7 and OCI-Ly19 cell line (coming from DSMZ, Germany or ATTC, US).
3.5.2 cell growth analysis
By lymphoma cell (2 to 7 × 103) bed board is in 96 orifice plates, and with the test compound of various dose (from 30 μM start (1/3 dilution, 8 points)) it is handled.By the cell of processing in 37 DEG C and 5%CO2Lower culture 7 days.Use star spore bacterium (10 μM) of element are used as positive control.
Pass through use(Invitrogen, DAL 1025) is surveyed according to the cell that illustrates to cultivate of manufacturer Determine cell growth.Use PerkinElmerPlate reader measures fluorescence.Use DMSO solvent value as 0% suppression It makes and staurosporine value is used to inhibit to calculate growth inhibition percent as 100%.
3.5.3 the IL-1 in SW1353 cell and TNF α respond cell analysis
The purpose of the analysis be in vitro in people's cell analysis scene evaluation the compound of the present invention to the TLR/ of activation The selectivity of IRAK-4 approach.SW1353 cell comes from chondrocyte cell line, and to interleukin 1 (IL-1) and TNF α Cell factor triggering agent has response.Two kinds of cell factor triggering agent induce these cells to express interleukin-6 (IL-6) And MMP13.It uses IL-6 and MMP13 to discharge as reading in this analysis, and represents test compound to TLR/IRAK-4 The measurement of the suppression level of approach.IL-1 triggers agent and carries out signal transduction by IRAK-4 dependent pathway, and TNF α does not need IRAK-4 carries out signal transduction.The compound of IRAK-4 is optionally, therefore inhibited only to influence the IL-1 driving of SW1353 cell MMP13 or IL-6 expression, have no effect on TNF α driving these protein expression.
3.5.4 the harvest and inoculation of SW1353 cell
SW1353 cell is cultivated in the DMEM for being supplemented with 10%FBS and 1% penicillin/streptomycin.At 37 DEG C and 5% CO2Under humid atmosphere in cultivate cell, and carry out squamous subculture twice a week.During squamous subculture, tryptose is used Enzyme-EDTA separates cell, is then neutralized with cell culture medium.(1,000rpm, 5 minutes) after centrifugation, by sediment It is resuspended in cell culture medium, and uses automatic cell counter (Invitrogen CountessTM) cell is counted Number.
Using the cell in the 16th generation, and by it with the density bed board of 15,000 cells/wells in 96 well culture plates In 120 μ L cell culture mediums.Make cell attachment during being incubated overnight.
3.5.5 the compound processing and triggering in SW1353 analysis
In 37 DEG C and 5%CO2Under, it by SW1353 cell and is tested by the compound solution of 10 times of concentration of 15 μ L of addition Compound preculture 2 hours together.Test the compound under various concentration, and by from 10mM stock solution in DMSO 3 times are serially diluted, and carry out 1/50 dilution step in cell culture medium then to prepare.Final experimental concentration in the analysis from 20 μM of beginnings, then carry out 3 times and are serially diluted, and final DMSO concentration is 0.2%.Compound pre-culture step it Afterwards, by the way that the IL-1 β (Peprotech, 200-01B) of 10 times of concentration of 15 μ L or TNF α are triggered agent (Peprotech, 300- It 01A) is added in each hole and triggers SW1353 cell, wherein final analysis volume is 150 holes μ L/ and final triggering agent concentration point It Wei not 1ng/mL and 10ng/mL.Negative control is adjusted with identical MSO concentration (no triggering agent).Then, in 37 DEG C and 5%CO2 Under, analysis plates are cultivated in moist incubator.After 24 hours and 48 hours, by the way that cell culture medium is transferred to V-type bottom Cell supernatant is harvested in 96 orifice plate of polypropylene, and in -80 DEG C of storages until ELISA is read.
3.5.6 IL-6 is quantified by ELISA
The level of the IL-6 secreted in cell supernatant is quantified in Enzyme Linked Immunoadsorbent Assay (ELISA).With The diluted anti-human IL-6 mouse antibodies of 1 μ g/mL (R&D Systems, MAB206) solution will in PBS at 4 DEG C by every 40 μ L of hole White LumitracTM384 orifice plates coating is overnight.After hole is washed twice and washed once with PBS with 100 μ L PBST, Buffer (1%BSA and 5% sucrose in PBS) is blocked to block remaining binding site with 100 μ L, and culture 4 is small at room temperature When.After blocking step, each hole washed once with PBST, sample or the recombined human IL-6 as standard items is then added (R&D Systems,206-IL-050).1/20 dilute sample and 40 μ L of addition, were cultivated at 4 DEG C in dilution buffer Night.Then, in two stage biological elementization anti-IL-6 detection antibody (people's IL-6 biology that 35 μ L are added with the ultimate density of 50ng/mL Elementization goat polyclonal antibodies (R&D Systems, BAF206)) after, each hole is washed 3 times and (is washed twice, is used with PBST PBS washed once).2 hours and washing step appropriate (being washed twice with PBST, washed once with PBS) are cultivated at room temperature Later, with 35 μ L 1/2,000 diluted Streptavidin-HRP solution (Invitrogen, SNN2004) cultivates each hole, then It is protected from light culture 45 minutes at room temperature.Then, 3 times (being washed twice with PBST, washed once with PBS) is washed into each hole, then With 50 μ L chemiluminescence ELISA substrate solutions, (Roche, 001) 11 582 950 are cultivated 5 minutes.Use LuminoskanTM The substrate of Ascent photometer measurement conversion shines.
3.5.7 MMP13 is quantified by ELISA
The level of the MMP13 secreted in cell supernatant is quantified in antibody capture activity analysis.For the mesh , at 4 DEG C, with the anti-human MMP13 antibody-solutions of 1.5 μ g/mL of 35 μ L by black MaxiSorpTM384 orifice plates Coating is overnight.It, will be remaining with 5% skimmed milk power of the 100 μ L in PBS at 4 DEG C after being washed twice each hole with PBST Binding site blocks 24 hours.After blocking step, each hole is washed twice with PBST, is subsequently added into sample and standard items. 1/5 dilute sample and 35 μ L is added at room temperature 4 hours in dilution buffer.Then, each hole is washed twice with PBST. Then, by the way that 1.5mM APMA solution (Sigma-Aldrich, A9563) the activation MMP13 protein completely of 35 μ L is added, and And it is cultivated 1 hour at 37 DEG C.Then, each hole is washed twice with PBST, and be added 35 μ L MMP13 substrate (Fluorogenic substrate (BIOMOL, P-126)).After cultivating 1 hour at 37 DEG C, PerkinElmer is usedThe fluorescence of the substrate of (excitation wavelength: 320nm, launch wavelength: 405nm) measurement conversion.
3.5.8 data analysis and calculating
All controls measure within the range of linearity of mankind's IL-6 and MMP13 standard curve of ELISA.All generations Data be directed to analysis mass parameter check validity (signal/background > 2, and Z ' > 0.3).
Using the sample that does not stimulate, (no triggering agent/solvent (0.2%DMSO) is as positive control (100% inhibit).It uses The sample (triggering agent/solvent (0.2%DMSO)) of stimulation is as negative control (0% inhibits).Use positive and negative control meter Calculate Z ' and suppression percentage (PIN) value.
Suppression percentage (PIN)=((RU triggers agent/solvent-RU test compound)/(RU triggers agent/solvent-RU oncontacting Send out agent/solvent) * 100)；Wherein RU respectively refers to the opposite chemiluminescence light unit or relative fluorescence list of IL-6 and MMP13ELISA Position.It draws to the PIN value for the test compound tested in concentration-response, and uses GraphPadIt is soft Part application nonlinear regression (S-shaped) curve matching obtains IC50Value.
The SW1353 cell selective of the illustrative the compounds of this invention of table ix analyzes result
3.5.9 conclusion
The data of table ix show that the compound of formula I in the present composition effectively inhibits to be touched in SW1352 cell by IL-1 β The expression of the IL-6 and MMP13 of hair, and the effect of event that such compound triggers TNF α is in effect and peak swing side Face is limited.Compound of formula I in these data confirm that present compositions is selective to IRAK-4 driving approach, for The influence of TNF α signal transduction is very limited, this can limit treatment-related side effect such as neutrophils and reduce and feel The appearance of dye.
4 ADME of embodiment analysis
4.1 dynamics dissolubilities
Since the 3.3mM DMSO stock solution of compound, by carrying out 1/2 dilution prepare compound in DMSO Serial dilutions: 3.3,1.6,0.83,0.41 and 0.21mM.The dilution series are transferred to transparent 96 orifice plate of the bottom V- (Greiner, 651201), and in 0.1M phosphate buffer (pH 7.4) or 0.1M citrate buffer (pH 3.0) Further progress 1/33.5 dilutes.Final compound concentration is 99.5,49.7,24.9,12.4 and 6.22 μM.Final DMSO concentration No more than 3%.As the positive control of precipitating, pyrene (30mM) is added in the corner aperture of each 96 orifice plate.Analysis plates are close It seals and is cultivated 1 hour at 37 DEG C, while being vibrated with 230rpm.Then, each plate is scanned under white light microscope, is generated each The single image (50 × enlargement ratio) of sediment under concentration.Each hole, and report are analyzed by image analysis software It closes object and shows the maximum concentration being completely dissolved.
4.2 Microsomal Stability
By 10mM stock solution of the compound in DMSO, three times dilute in DMSO.Then, in 96 hole deep-well plates The prediluted compound solution is diluted to 2 μM in 105mM phosphate buffer (pH 7.4) in (Nunc, 278752), And it is preheated at 37 DEG C.
In 105mM phosphate buffer (pH 7.4), with the G-6-P of 1:700 multiple dilution 700U/mL Salt-dehydrogenase (G6PDH, Roche, 10127671001) working stock solution.In 105mM phosphate buffer (pH 7.4), Contain 0.528M MgCl with the dilution of 1:8 multiple2.6H2O (Sigma, M2670), 0.528M D-Glucose -6- phosphate (Sigma, G7879) and 0.208M NADP+The co-factor mixture of (Sigma, N0505).
1mg/mL hepatomicrosome (Tebu-bio) of the preparation containing interested species (for example, people, mouse, rat, dog), The G6PDH and co-factor mixture (6.6mM MgCl of 1.2U/mL2, 6.6mM G-6-P salt, 2.6mM NADP+) Working solution.At room temperature, by the mixture preculture 15 minutes, but it is no more than 20 minutes.
After preculture, it is added chemical compound diluted liquid and the mixture containing microsome together with equivalent, and with 300rpm is cultivated 30 minutes.For the 0th minute time point, add in the forward direction chemical compound diluted liquid that microsomal mixture is added Enter the MeCN of two volumes.Ultimate density in the training period are as follows: 1 μM of test compound or control compound, 0.2%DMSO, 0.5mg/mL microsome, 0.6U/mL G6PDH, 3.3mM MgCl2, 3.3mM G-6-P salt and 1.3mM NaDP+.
After cultivating 30 minutes at 37 DEG C, reaction is terminated using the MeCN of 2 volumes.
Sample is mixed, is centrifuged and harvests supernatant to be analyzed on LC-MS/MS.Instrument response (that is, peak height) ginseng Zero time point sample (being considered as 100%) is examined, to determine the percentage of remaining compound.It include general naphthalene in analysis design Luo Er and Verapamil are as object of reference.
Data about Microsomal Stability are indicated with the percentage of total amount of compound remaining after culture in 30 minutes.
Metabolic stability in 4.3 S9 subcellular fractions
The purpose of the analysis is the metabolic stability in vitro by measurement compound in S9 subcellular fraction to evaluate aldehyde Metabolism of the oxidizing ferment to compound.
Firstly, 10mM stock solution of the compound in DMSO is diluted (40 times) in DMSO, 250 μM of concentration are obtained. The compound solution is further diluted into (5 times) with water, obtains 50 μM of compound working solution (to obtain 1 μM of compound Ultimate density).The aqueous solution of hydralazine (selective depressant of aldehyde oxidase) is prepared (to obtain 100 μM with the concentration of 5mM Ultimate density).By at 37 DEG C, by liver S9 suspension (people, rat, mouse, monkey, the BD Gentest of 10 μ LTM, 20mg/ ML) be added in the 50mM kaliumphosphate buffer (pH 7.4) of 86 μ L prepare culture mix (ultimate density 2mg protein/ mL).The 5mM hydralazine that 2 μ L are added is used to cultivate in the case where selective depressant is added, or 2 μ L water are added and are used in unrestraint It is cultivated under agent.After 5 minutes pre- warms, started instead by the way that 50 μM of test compounds of 2 μ L are added in culture mix It answers.After culture 0 minute, 3 minutes, 6 minutes, 12 minutes, 18 minutes and 30 minutes, contain 1% acetate mixture with 300 μ L The MeCN:MeOH (2:1) of (warfarin comprising 10ng/mL is as analysis internal standard) terminates reaction (100 μ L).Sample is mixed, from The heart simultaneously analyzes supernatant by LC-MS/MS.Including phthalazines as positive control.
Instrument response (compound and interior target peak area ratio) refers to zero time point sample (being considered as 100%), so as to Determine the percentage of residue compound.It is drawn using remaining compound %, uses GraphPadSoftware determines that S9 is trained Support the half-life period in object and intrinsic clearance.External intrinsic clearance (μ L/min/mg) is calculated using following formula:
CLint(μ L/min/mg)=0.693/t1/2(min) * (culture of mL/mg protein) * 1000
If the clearance rate of S9 is inhibited by hydralazine, test compound can be classified as to the bottom of aldehyde oxidase Object.The species specificity clearance rate of test compound also can indicate that the metabolism of aldehyde oxidase.
Metabolic stability in 4.4 liver cells
Firstly, 10mM stock solution of the test compound in DMSO is diluted to 3mM in DMSO, then in modification 5 μM are diluted in Krebs-Henseleit buffer (Sigma, K3753).At 37 DEG C, in the case where gently vibrating by the compound Dilution is added in the suspension for collecting stored refrigerated liver cell (BioreclamationIVT).End reaction condition are as follows: 1 μM of test compound, 0.03% DMSO, 500,000 vigor liver cell/mL and 75 μ L volume of culture.Testosterone (1 μ is used respectively M it) is compareed with (1 μM) of umbelliferone as I phase and II phase metabolic response.
After culture 0 minute, 10 minutes, 20 minutes, 45 minutes, 90 minutes, 120 minutes and 180 minutes, with 225 μ L MeCN:MeOH (2:1) comprising 10ng/mL warfarin sodium (as analysis internal standard) terminates reaction.Sample is mixed, is centrifuged and passes through LC-MS/MS analyzes supernatant.
Instrument response (ratio of test compound and internal standard peak area) refers to zero time point sample (being considered as 100%), To determine the percentage of remaining compound.
It is drawn using the percentage of remaining compound, uses GraphPadSoftware determines hepatocyte cultures Half-life period and intrinsic clearance in object.
4.5 CYP inhibit
It is evaluated using the human-cytochrome P450 isodynamic enzyme of cDNA expression with the non-fluorescence substrate for being metabolized as fluorescent metabolite Inhibition potential of the test compound to human-cytochrome P450 isodynamic enzyme (CYP1A2,2C9,2C19,2D6 and 3A4).
Compound under 3.3 μM of test and 10 μM of concentration, final DMSO concentration are 0.3%.Compound is trained together with enzyme It supports 15 minutes, co-factor-substrate mixture is added later.For CYP3A4 (BD Biosciences, 456202), CYP2C9 (BD Biosciences, 456258), CYP2C19 (BD Biosciences, 456259) and CYP1A2 (BDBiosciences, 456203) it analyzes, the end reaction concentration in co-factor mixture are as follows: 0.4U/mL G-6-P salt-dehydrogenase (G6PDH,Roche,10165875001)、3.3mM MgCl2(Sigma, M2670), 3.3mM D-Glucose -6- phosphate (Sigma, G7879) and 1.3mM NADP+ (Sigma, N0505).It, should for CYP2D6 (BD Biosciences, 456217) End reaction concentration in analysis is 0.4U/mL G6PDH, 0.41mM MgCl2, 0.41mM D-Glucose -6- phosphate and 8.2μM NADP+.The concentration of enzyme and substrate is reported in Table X.After certain cultivation period, terminated by the way that stop bath is added Reaction.For using test of the DBF as substrate, using 2N NaOH stop bath, and for all other substrate, terminate liquid For 80%MeCN/20%0.5M Tris alkali.
In PerkinElmerOn reader, under suitable excitation and launch wavelength (reference table X) immediately (for CEC, AMMC, BFC) or (for using CYP2C9 and CYP3A4 of the DBF as substrate), reading is glimmering after 20 minutes Light.
Then, percentage CYP inhibited by the way that data normalization to blank sample to be calculated to test compound: 100% inhibits to be the blank sample terminated before enzyme/substrate mixture is added, and 0% inhibits to be that enzymatic reaction is occurring The blank sample terminated after (50 minutes).
Table X is used for the inhibition analysis condition of each CYP450 Studies on Isozymes
AMMC: amino-ethyl -7- methoxyl group -4- methylcoumarin CEC:3- cyano -7-ethoxy coumarin
BFC:7- benzyloxy -4- trifluoromethyl cumarin DBF: dibenzyl fluorescein
4.6.MDCKII-MDR1 permeability
MDCKII-MDR1 cell is to be overexpressed people's multi-drug resistance (MDR1) gene, coding P- glycoprotein (P-gp) Madin-Darby dog renal epithelial cell.Cell is from Teh Netherlands Cancer Inst (Netherlands Cancer Institute) It obtains, and in 24 holesCulture makes after 3 to 4 days in cell culture plate (Millipore, PSRP010R5) With.Two-way MDCKII-MDR1 permeability analysis is carried out as described below.
By 3 × 105A cell/mL (1.2 × 105A cells/well) it is seeded in by DMEM (Sigma, D5796)+1% Glutamax-100 (Sigma, G8541)+1% antibiotic/antimycotic agent (Sigma, A5955)+10%FBS (Sigma, F7524；Deactivate at 56 DEG C 30 minutes) composition plating medium in.By cell in CO2It is placed 3 to 4 days in incubator.? 24 hours and experimental day replacement culture medium after inoculation.
In Dulbecco phosphate buffered saline (PBS) (D-PBS, pH 7.4；Sigma, D8662) in prepare test compound and Reference compound (amphinate (Moravek Biochemicals, M-1613), Diclofenac (Sigma, D6889)), and The top room of Millicell tissue culture plate is added to the ultimate density of 10 μM (for 0.5 μM in the case of amphinate) In (400 μ L) or bottom side room (800 μ L), and final DMSO concentration is 1%.It will receive solution (D-PBS+1%DMSO) addition To Millicell tissue culture plate in side room.
100 μM of fluoresceins (Sigma, L0259) are added in all donor buffer solutions, will pass through monitoring fluorescence Yellow permeability evaluates the integrality of cell monolayer.Fluorescein is the fluorescent marker for Paracellular transport approach, and its As internal contrast with the integrality of combining closely of each cell monolayer during verifying analysis.
At 37 DEG C, respectively to take 75 from top room and basal compartment after 150rpm shaken cultivation 1 hour on orbital shaker μ L aliquot sample simultaneously adds it to the 225 μ L MeCN: aqueous solution in 96 orifice plates comprising analyzing internal standard (10ng/mL warfarin) In (2:1).Aliquots also are obtained from donor solution when testing and starting, to obtain initial concentration.
The concentration of compound in sample is measured by high performance liquid chromatography/mass spectrum (LC-MS/MS).
In 96 orifice plates comprising the 150 μ L liquid from all receiving orifices (bottom side or top side), Thermo is used Scientific Fluoroskan Ascent FL (excitation wavelength: 485nm measures wavelength: 530nm) measurement fluorescein.
5 whole blood of embodiment
5.1. in vitro human TNF alpha release inhibits (whole blood)
The purpose of the analysis is that the compound of the present invention is evaluated in vitro people's whole blood environment to the TLR/IRAK-4 of activation The activity of approach.Toll-like receptor (TLR) be identify multiple-microorganism molecule pattern recognition receptors (referred to as pathogen is relevant Molecular pattern (PAMP)).People TLR7 and TLR8 identify imidazoquinolie compounds (for example, CL097) and as its native ligands Single stranded RNA.The activation of TLR cause the cell handled through TLR agonist generate cytokine profiles (for example, TNF α, IL-8, IL-6).Use cytokine release as reading in this analysis, and it represents test compound to TLR/IRAK-4 approach Suppression level measurement.It should be noted that there are these cell factors independent of TLR/ in the case where intact organism Other sources of IRAK-4 approach, for example, macrophage (when activating Fc γ receptor (Yan et al., 2012)) or T cell ( When activating T cell receptor (Brehm et al., 2005)).
5.1.1. experimental design
By venipuncture, by the blood collection of healthy volunteer into heparin lithium pipe, then gently overturn for several times to prevent It only solidifies, is then cultivated on rocking mixer formula oscillator at least 15 minutes at 37 DEG C.Then, by the blood of 200 μ L point Be assigned in 2mL micro-pipe, and by its at 37 DEG C with the test compound of 0.3%DMSO or various concentration (10 μM to 0.01 μM, 3 times of dilutions are carried out in the RPMI 1640 (Life Technologies, 31870) of no glutamine) together in duplicate Preculture 15 minutes.After the preculture, at 37 DEG C, with CL097, (2 μ g/mL (come from 1mg/mL aqueous solution)； InvivoGen, tlrl-c97) or solvent (distilled water) by blood trigger 30 minutes 3 hours.It, will be micro- with 5000 × g at 4 DEG C Pipe be centrifuged 10 minutes, then will about 80 μ L plasma collections into 96 orifice plate of polystyrene.Can immediately to blood plasma carry out analysis or Blood plasma is freezed at -80 DEG C soon on triggering.Finally, by using humanTNF-α's DuoSet ELISA kit (R&D Systems, DY210) quantifying for TNF α carried out for 40 times of diluted plasma according to the explanation of manufacturer.In PerkinElmer In 2102 multiple labeling plate reader of EnVision, optical density (OD) (OD) is measured in 450nm.
5.1.2 data are analyzed
Standard curve is created by being drawn to the mean light absorbency in y-axis relative to the concentration in x-axis, and is led to Cross the point-rendering optimum fit curve on the figure.Linear regression analysis is carried out to determine equation (y=ax+b) and R square value. Calculate the TNF α concentration of each blood sample copy using following formula, when calculating considers extension rate:
TNF α concentrationSample 1=40* (ODSample 1-b)/a
Then, for each copy, data are expressed as suppression percentage (PIN) using following formula:
It is wherein the average TNF α concentration with the duplicate specimen of CL097 triggering ' with the average TNF α of CL097 '；' TNF α sample Product 1 ' are the TNF α concentration of sample 1；' with the average TNF α of solvent ' is the average TNF α concentration with the duplicate specimen of vehicle treated.
It is fitted using average PIN ± SEM formation curve.Use GraphPadSoftware obtains figure and IC50Meter Calculation value.
5.2. isolated rat TNF α release inhibits (whole blood)
The purpose of the analysis is that the compound of the present invention is evaluated in isolated rat whole blood environment to the TLR/IRAK- of activation The activity of 4 approach.Toll-like receptor (TLR) is to identify pattern recognition receptors (the referred to as pathogen correlation of multiple-microorganism molecule Molecular pattern (PAMP)).Although people TLR7 and TLR8 identify imidazoquinolie compounds (for example, CL097) and as them The single stranded RNA of native ligand, but rodent TLR8 needs other factors (such as oligodeoxynucleotide (for example, poly (dT))) It is activated.
5.2.1. experimental design
Sprague Dawley rat (male, 7 to 8 week old, 200g to 250g body are obtained from Janvier Labs (France) Weight).
By bloodletting by the blood collection obtained from least 2 rats into heparin lithium pipe, then at 37 DEG C, waving Preculture at least 15 minutes on mixer type oscillator.Blood from all rats is mixed in 50mL PA tube, is obtained To unique blood patch.Then, the blood of 200 μ L is distributed into 2mL micro-pipe, and by it at 37 DEG C with 0.3% DMSO or various concentration test compound (10 μM to 0.01 μM, in the 1640 (Life of RPMI of no glutamine Technologies, 31870) 3 times of dilutions are carried out in) it cultivates 15 minutes in duplicate together.After the preculture, 37 At DEG C, with CL097, (10 μ g/mL (come from 1mg/mL aqueous solution)；InvivoGen, tlrl-c97) and poly (dT) (1 μM (comes from 100 μM of aqueous solutions)；InvivoGen, tlrl-pt17) or solvent (distilled water) by blood trigger 30 minutes 3 hours.At 4 DEG C, By micro-pipe with 5000 × g centrifugation 10 minutes, then will about 80 μ L plasma collections into 96 orifice plate of polystyrene.It can be immediately to blood Slurry analyze or on triggering soon freeze blood plasma at -80 DEG C.Finally, using rat TNF-α Quantikine ELISA kit (R&D Systems, SRTA00) carries out TNF α to blood plasma (1:3 is diluted) according to the explanation of manufacturer and determines Amount.In 2102 multiple labeling plate reader of PerkinElmer EnVision, optical density (OD) (OD) is measured in 450nm.
5.2.2. data are analyzed
Standard curve is created by being drawn to the mean light absorbency in y-axis relative to the concentration in x-axis, and is led to Cross the point-rendering optimum fit curve on the figure.Linear regression analysis is carried out to determine equation (y=ax+b) and R square value. Calculate the TNF α concentration of each blood sample copy using following formula, when calculating considers extension rate:
TNF α concentrationSample 1=40* (ODSample 1-b)/a
Then, for each copy, data are expressed as suppression percentage (PIN) using following formula:
Wherein ' with the average TNF α of CL097 ' is dense with the average TNF α of duplicate specimen of CL097+poly (dT) triggering Degree；' TNF α sample 1' be sample 1 TNF α concentration；' with the average TNF α of solvent ' is flat with the duplicate specimen of vehicle treated Equal TNF α concentration.
It is fitted using average PIN ± SEM formation curve.Use GraphPadSoftware obtains figure and IC50Meter Calculation value.
Analysis in 6 body of embodiment
6.1. the psoriasiform epidermal hyperplasia Murine models induced by partial smearing TLR7/8 agonist imiquimod.
6.1.1. material
5% imiquimod cream agent is obtained from MEDA.
Anti-mouse IL-12/IL-23p40FG antibody purification (C17.8) is obtained from Affymetrix eBioscience (catalogue Number 16-7123-85).
6.1.2. animal
Balb/cJ mouse (female, 18-20g weight) obtains from Janvier Labs (France).Mouse is placed in 12 hours Under light/dark circulation (07:00-19:00).Temperature is maintained 22 ± 2 DEG C, arbitrarily obtains food and water.
6.1.3. researching and designing
Researching and designing is adapted from Van der Fits L. et al. (van der Fits et al., 2009).
At first day, under isoflurane light anaesthesia around two ears of mouse shaving.
By the commercially available imiquimod cream agent of 30mg (5% cream of Aldara) the continuous inside for being applied to every ear for 4 days On outer surface, it is then converted into the daily dose of 1.5mg reactive compound.Control-animal receives same amount of vaseline.
From the 1st day to the 5th day, twice daily to take orally the test compound (10mg/kg in 0.5% methylcellulose Or 30mg/kg) mouse is administered, later using imiquimod (at the 5th day, be only administered once to mouse, will be small after 2 hours Mouse euthanasia).
In positive reference group, on day 1 with the 1st day before 3 days, animal receives intraperitoneal injection anti-mouse IL- twice 12/IL-23p40 antibody (10mg/kg).
6.1.4. the evaluation of disease
The thickness of two ears is measured per daily thickness gauge (Mitutoyo, Absolute Digimatic, 547-321). Weight is measured when testing beginning and when putting to death animal.At the 5th day, 2 hours after last time is administered, mouse is put to death. Auricle is cut, cartilage is removed.It weighs to auricle, then, is immersed in comprising 1mLSolution it is small To carry out evaluation gene expression in bottle, or it is immersed in formalin and is used for Histological evaluation.
Every group has 14 mouse.As a result it is expressed as average value ± SEM, and uses single factor test ANOVA, is then used Dunnett post-hoc tests are for statistical analysis to imiquimod-solvent group.
6.1.5. histology
After execution, collects ear and be fixed in 3.7% formaldehyde, is embedded in paraffin later.Cut 2 μ m-thicks Slice, and dyed with hematoxylin and eosin.Ear epidermal thickness, every ear are measured by image analysis (SisNcom software) Piece 6 photos are shot with 20 times of enlargement ratios.Data are expressed as average value ± SEM, and use single factor test ANOVA, then use Dunnett post-hoc tests are for statistical analysis to imiquimod-solvent group.
6.1.6. gene expression analysis
FromEar is taken out in solution, and it is broken in Precellys device with 1.4mm ceramic bead It is broken to be placed onIn.Then it usesRNA kits total serum IgE.Preparation cDNA is simultaneously used SYBR Green technology is special using the gene from Qiagen in ViiA7 real-time PCR system (Applied Biosystems) Specific primer carries out quantitative PCR.Relative to cyclophilin A house-keeping gene expression calculate each gene (IL17A, IL1B, IL22, LCN2, S100A8 and S100A9) expression.Data are expressed as the average value ± SEM (RQ=2 of relative quantity-ΔC T, Middle Δ CT=CTSample-CTCyclophilin A).The statistical test used be to imiquimod-solvent group ANOVA variance analysis with And Dunnett post-hoc tests.
6.2. the Murine models of the psoriasiform epidermal hyperplasia of intracutaneous injection IL-23 induction
6.2.1. material
The carrier-free mouse recombinant il-2 3 (14-8231, CF) provided by e-Bioscience.
6.2.2. animal
Balb/c mouse (female, 18-20g weight) obtains from CERJ (France).Mouse is placed in 12 hours light/dark circulations Under (07:00-19:00).Temperature is maintained 22 DEG C, arbitrarily obtains food and water.
6.2.3. researching and designing
Researching and designing is adapted from Rizzo HL. et al. (Rizzo et al., 2011).
(D1) on day 1, the shaving around two ears of mouse.
It is 4 days continuous that (D1 to D4), under the anesthesia of isoflurane induction, the auris dextra exterior feature of mouse receives daily intradermal dosage Mouse recombinant il-2 3 (1 μ g/20 μ L, in PBS/0.1%BSA), left auricle receives the PBS/0.1%BSA of 20 μ L.
From D1 to D5, injected first 1 hour in IL-23, with test compound (10mg/kg, 30mg/kg or 100mg/kg, mouth Clothes, once a day, in 0.5% methylcellulose) or solvent to mouse be administered.
6.2.4. the evaluation of disease
The thickness of two ears of rule measurement per daily automatic measuring.Weight is measured in on-test and when putting to death animal.? 5th day, 2 hours after last time is administered, mouse is put to death.Auricle is cut, cartilage is removed.It weighs to auricle, so Afterwards, it places it in comprising 1mLIn the bottle of solution or formaldehyde.
In D4,1 hour, 3 hours, 6 hours after (T0) and administration before facing administration, also from retro-orbital sinus collection blood sample Product are analyzed for PK.
Every group has 8 mouse.As a result it is expressed as average value ± SEM, and uses single factor test ANOVA, then uses Dunnett Post-hoc tests are for statistical analysis to IL-23 solvent group.
6.2.5. histology
After execution, collects ear and be fixed in 3.7% formaldehyde, is embedded in paraffin later.Make 2 μ m-thicks Slice, and dyed with hematoxylin and eosin.Ear epidermal thickness, every ear are measured by image analysis (Sis ' Ncom software) Piece 6 photos are shot with 20 times of enlargement ratios.Data are expressed as average value ± SEM, and use single factor test ANOVA, then use Dunnett post-hoc tests are for statistical analysis to IL-23 solvent group.
6.2.6. gene expression analysis
FromHalf ear is taken out in solution, and it is ceramic with 1.4mm in Precellys device Pearl is broken to be placed onIn.Then it usesRNA kits total serum IgE.Preparation cDNA simultaneously makes The gene from Qiagen is utilized in ViiA7 real-time PCR system (Applied Biosystems) with SYBR Green technology Specific primer carries out quantitative PCR.Relative to cyclophilin A house-keeping gene expression calculate each gene (IL17A, IL1B, IL22, LCN2, S100A8 and S100A9) expression.Data are expressed as the average value ± SEM (RQ=2 of relative quantity-ΔC T, Middle Δ CT=CTSample-CTCyclophilin A).The statistical test used be to the ANOVA variance analysis of IL-23 solvent group and Dunnett post-hoc tests.
6.3.PK/PD it model: is discharged by the TNF α of specificity T LR7/8 agonist CL097 induction
The purpose of the analysis is the inhibition and the change of IRAK-4 dependence event in body when determining application the compounds of this invention Close the relationship between the circulation composition level of object.
6.3.1. material
CL097 (catalog number (Cat.No.) tlrl-c97) and poly (dT) (catalog number (Cat.No.) tlrl-pt17) are obtained from InvivoGen.
Mouse TNF α kit is obtained from Perkin-Elmer (catalog number (Cat.No.) AL505C).
6.3.2. animal
DBA/1J mouse (male, 18-20g weight) obtains from Janvier Labs (France).Mouse is placed in 12 hours Under light/dark circulation (07:00-19:00).Temperature is maintained 22 ± 2 DEG C, arbitrarily obtains food and water.
6.3.3. researching and designing
Mouse receives the test compound of oral dose.Use and does not receive one group of original sample animal of any administration as t=0 Time point.
30 minutes after administration, 1 hour, 3 hours, 8 hours or 24 hours, will be by sampling in heart (in isoflurane Under anesthesia) obtain two parts of Blood Sample Collections into heparin lithium pipe.A sample is analyzed for pharmacokinetics (PK), another Part is quantitative for pharmacodynamics (PD) marker.
6.3.4. chemical levels quantify in blood plasma
Whole blood sample is centrifuged 10 minutes with 5000rpm, and before analysis, obtained plasma sample is stored in -20 At DEG C.The plasma concentration of each test compound is measured by LC-MS/MS method.
6.3.5. the determination of pharmacokinetic parameter
It uses(The U.S.) calculate pharmacokinetic parameter.
6.3.6.PD marker quantifies
At 37 DEG C, each blood sample is stimulated 2 hours with CL097 and poly (dT).Then, blood plasma, and root are collected According to the explanation of manufacturer, TNF α is analyzed by AlphaLISA.
Every group has 6 mouse.As a result it is expressed as TNF α concentration (pg/mL), or is expressed as the suppression relative to t=0 time point Percentage (PIN) processed.Data are expressed as average value ± SEM, and use single factor test ANOVA, then use Dunnett post-hoc tests It is for statistical analysis to the solvent group at corresponding time point.
6.4. pass through the muroid prophylaxis model of the topical application MC903 atopic dermatitis induced
6.4.1. material
0.5% methylcellulose is obtained from VWR (catalog number (Cat.No.) AX021233).MC903 (Calcipotriol) is from Tocris Bioscience (catalog number (Cat.No.) 2700/50) is obtained.680 obtain from PerkinElmer (catalog number (Cat.No.) NEV10003) ?.It is obtained from Ambion (catalog number (Cat.No.) AM7021).1000 (Merial) and2% (Bayer) is obtained from Centravet (catalog number (Cat.No.) IMA004-6827812 and ROM001-6835444).
6.4.2. animal
Balb/cN mouse (female, 18-20g weight) or CD1/Swiss mouse (female, 24-26g weight) are from Janvier Labs (France) is obtained.Mouse is placed under 12 hours light/dark circulations (07:00-19:00).Temperature is maintained 22 ± 2 DEG C, Arbitrarily obtain food and water.
6.4.3. researching and designing
The design adaptations of research are from Li M. et al. (Li et al. people, 2006).
At first day (D1), intraperitoneal injection Imalgene and Rompun (7.5%/2.5%；0.1mL/10g) by mouse Anesthesia, and the shaving around two ears.
From D1, by MC903 (in the EtOH of the 20 μ L) topical application of the EtOH or 2nmol of 20 μ L at two of mouse On ear, continuous five days.
From D1 to D8, with test compound, (15mg/kg or 30mg/kg are taken orally, twice daily, in 0.5% Methyl cellulose In element) or dexamethasone (5mg/kg, take orally, once a day, in 0.5% methylcellulose) or with solvent to mouse administration.
6.4.4. chemical levels quantify in blood plasma
The plasma concentration of each test compound is measured by LC-MS/MS method, wherein with positivity or negativity electron spray mould Formula operates mass spectrograph.
6.4.5. the determination of pharmacokinetic parameter
It uses(The U.S.) calculate pharmacokinetic parameter.
6.4.6 the evaluation of disease
When studying beginning, every other day with animal is put to death, thickness gauge (Mitutoyo, Absolute are used Digimatic, 547-321) two ears of measurement thickness (by after isoflurane induced anesthesia).
When studying beginning, every other day and when the animals are executed measure weight.
In D4, all groups of mouse receives680 probes (0.8nmol/10g, IP).In D5, peritonaeum Interior injection Imalgene and Rompun (7.5%/2.5%；0.1mL/10g) by mouse anesthesia.Use internal molecular imaging (Bruker In-Vivo Xtreme imaging system, excitation wavelength: 630nm, launch wavelength: 700nm, acquisition time: 5 seconds) is surveyed Measure granulocyte infiltration.
In D8,2 hours after last time is administered, mouse is put to death, whole blood is collected in the pipe of coating EDTA and will Plasma freezing is for further measuring (including the compound in the circulatory system).A blood is also collected in the pipe of coating heparin Liquid sample.
Collect the auricle of ear and weighing.One ear is slit longitudinally into 2 half.Half is solid in 4% buffered formaldehyde liquid Surely it is used for histology；The other half is immersed inIn for evaluating gene expression.
Every group has 8 mouse.As a result it is expressed as average value ± SEM, and uses single factor test ANOVA, is then used Dunnett post-hoc tests are to the ear thickness and weight of MC903 solvent group, for statistical analysis to the weight of EtOH solvent group.
6.4.7. histology
After execution, collects half ear and fixed in 3.7% formaldehyde, is embedded in paraffin later.Pass through immune group Weave chemistry, with specific cell marker antibody (CD3 is used for T cell, and EPX is used for eosinophil) to 4 μm of slabs Carry out immunostaining.Every mouse is measured by image analysis (CaloPix software, TRIBVN Healthcare) to be entirely sliced Immunostaining cell area.Data are expressed as average value ± SEM, and use single factor test ANOVA, then use Dunnett post-hoc tests are for statistical analysis to MC903 solvent group.
6.4.8. gene expression analysis
FromEar is taken out in solution, and by it in Bertin Instruments It is placed in homogenizer with 1.4mm ceramic bead is brokenIn.Then, total serum IgE is extracted using phenol/chloroform scheme, and And with QIAcube, use96HT kit (Qiagen, catalog number (Cat.No.) 74171) purifying.System Standby cDNA is simultaneously come from using SYBR Green technology in middle utilize of ViiA7 real-time PCR system (Applied Biosystems) The gene-specific primer of Qiagen carries out quantitative PCR.Water is expressed relative to HPRT, GAPDH and beta-actin house-keeping gene It is flat calculate each gene (IL4, IL5, IL13, TSLP, IL33, ST2, IL25, IL31, IFN γ, IL6, IL10, LCN2, S100A8 and S100A9) expression.Data are expressed as the average value ± SEM (RQ=2- of relative quantityΔC T, wherein Δ CT=CT Sample-is averaged (CT HPRT、CT GAPDH、CTBeta-actin).The statistical test used is to EtOH/MC903 solvent group ANOVA variance analysis and Dunnett post-hoc tests.6.5. pass through the mouse of the topical application MC903 atopic dermatitis induced Class treats model
6.5.1. material
0.5% methylcellulose is obtained from VWR (catalog number (Cat.No.) AX021233).MC903 (Calcipotriol) is from Tocris Bioscience (catalog number (Cat.No.) 2700/50) is obtained.680 obtain from PerkinElmer (catalog number (Cat.No.) NEV10003) ?.It is obtained from Ambion (catalog number (Cat.No.) AM7021).1000 (Merial) and2% (Bayer) is obtained from Centravet (catalog number (Cat.No.) IMA004-6827812 and ROM001-6835444).
6.5.2. animal
Balb/cN mouse (female, 18-20g weight) or CD1/Swiss mouse (female, 24-26g weight) are from Janvier Labs (France) is obtained.Mouse is placed under 12 hours light/dark circulations (07:00-19:00).Temperature is maintained 22 ± 2 DEG C, Arbitrarily obtain food and water.
6.5.3. researching and designing
The design adaptations of the research are from Li M. et al. (Li et al. people, 2006).
At first day (D1), intraperitoneal injection Imalgene and Rompun (7.5%/2.5%；0.1mL/10g) by mouse Anesthesia, and the shaving around two ears.
From D1, the MC903 (in 20 μ L EtOH) of the EtOH or 2nmol of 20 μ L are applied topically to two of mouse Until D9, D11 or D15 on ear (in addition to during weekend).
From D5, with test compound, (15mg/kg or 30mg/kg are taken orally, twice daily, in 0.5% methylcellulose In) or dexamethasone (5mg/kg, take orally, once a day, in 0.5% methylcellulose) or with solvent to mouse administration, directly To D10, D12 or D16.
6.5.4. chemical levels quantify in blood plasma
The plasma concentration of each test compound is measured by LC-MS/MS method, wherein with positivity or negativity electron spray mould Formula operates mass spectrograph.
6.5.5. the determination of pharmacokinetic parameter
It uses(The U.S.) calculate pharmacokinetic parameter.
6.5.6. the evaluation of disease
Application MC903 before, study start when, three times a week with put to death animal when, use thickness gauge The thickness that (Mitutoyo, Absolute Digimatic, 547-321) measures two ears (is induced by isoflurane After anesthesia).
When studying beginning, weight is measured three times a week and when the animals are executed.
In D8, D10 or D11, all groups of mouse receives680 probes (0.8nmol/10g, peritonaeum It is interior).At second day (D9, D11 or D12), intraperitoneal injection Imalgene and Rompun (7.5%/2.5%；0.1mL/10g) will Mouse anesthesia.Then, using internal molecular imaging (Bruker In-Vivo Xtreme imaging system, excitation wavelength: 630nm, Launch wavelength: 700nm, acquisition time: 5 seconds) measurement granulocyte infiltration.
Mouse is put to death 2 hours after last time is administered in D10, D12 or D16, in the Guan Zhongshou of coating EDTA Collect whole blood, and plasma freezing is used to further to measure (including the compound in the circulatory system).
Collect the auricle of ear and weighing.One ear is slit longitudinally into 2 half.Half is solid in 4% buffered formaldehyde liquid Surely it is used for histology；The other half is immersed inIn for evaluating gene expression.
Every group has 8 mouse.As a result it is expressed as average value ± SEM, and uses single factor test ANOVA, is then used Dunnett post-hoc tests are to the ear thickness and weight of MC903 solvent group, for statistical analysis to the weight of EtOH solvent group.
6.5.7. histology
After execution, collects half ear and be fixed in 3.7% formaldehyde, be embedded in paraffin later.Pass through immune group 4 μm of slabs are carried out immunostaining with anti-cd 3 antibodies by weave chemistry.Pass through image analysis (CaloPix software, TRIBVN Healthcare the cell area for the immunostaining that every mouse is entirely sliced) is measured.Data are expressed as average value ± SEM, and And single factor test ANOVA is used, it is then for statistical analysis to MC903 matchmaker's solvent group using Dunnett post-hoc tests.
6.5.8. gene expression analysis
FromEar is taken out in solution, and by it in Bertin Instruments It is placed in homogenizer with 1.4mm ceramic bead is brokenIn.Then, total serum IgE is extracted using phenol/chloroform scheme, and And with QIAcube, use96HT kit (Qiagen, catalog number (Cat.No.) 74171) purifying.Preparation CDNA is simultaneously come from using SYBR Green technology in middle utilize of ViiA7 real-time PCR system (Applied Biosystems) The gene-specific primer of Qiagen carries out quantitative PCR.Water is expressed relative to HPRT, GAPDH and beta-actin house-keeping gene It is flat calculate each interested gene (GOI=IL4, IL5, IL13, TSLP, IL33, ST2, IL25, IL31, IFN γ, IL6, IL10, LCN2, S100A8 and S100A9) expression.
All qPCR data be represented as standardized relative quantity average value ± SEM (NRQ=2^ (Δ Cq GOI)/ Geomean (2^ (Δ Cq HPRT), 2^ (Δ Cq GAPDH), 2^ (Δ Cq beta-actin)), wherein Δ Cq=Cq average value- Cq sample.The statistical test used is the ANOVA variance analysis and Dunnett post-hoc tests to EtOH/MC903 solvent group.
6.6. the Murine models of the systemic loupus erythematosus induced by epidermis application imiquimod
6.6.1. material
5% imiquimod cream agent is obtained from MEDA.
Mouse anti-dsDNA antibody ELISA kit is from Alpha Diagnostic International (catalog number (Cat.No.) 5120) it obtains.Mouse urinary albumin ELISA kit is obtained from Abcam (catalog number (Cat.No.) ab108792).Urine creatinine assay kit It is obtained from Abnova (catalog number (Cat.No.) KA4344).
6.6.2. animal
Balb/cJ mouse (female, 18-20g weight) obtains from Janvier Labs (France).Mouse is placed in 12 hours Under light/dark circulation (07:00-19:00).Temperature is maintained 22 ± 2 DEG C, arbitrarily obtains food and water.
6.6.3. researching and designing
The researching and designing is adapted from Yokogawa M. et al. (Yokogawa et al., 2014).
(D1) on day 1, the shaving around the auris dextra piece of mouse.
Mouse receives the epidermis application of the imiquimod of 1.25mg, (D1 to D86) 12 weeks continuous in auris dextra exterior feature 3 times a week. Control group receives same amount of vaseline.
From D1 to D86, to mouse medicine-feeding test compound, (30mg/kg is taken orally, once a day, in 0.5% Methyl cellulose In element) or solvent (10mL/kg).
6.6.4. the evaluation of disease
Once a week with the thickness of automatic gauge (Mitutoyo, Absolute Digimatic, 547-321) measurement ear Degree.
Start when and once a week measurement weight until execution animal.In postmortem, spleen weight is also measured.Last Mouse was put to death in 2 hours after single administration.
In different time points (for example, at D28 days, D56 days and D84 days), mouse is individually placed in metabolic cage, to carry out Urinalysis and albuminuria assessment (the ratio between albumin and creatinine).
It is horizontal to evaluate anti-double-chain DNA IgG that serum is collected in different time points (for example, in D28, D56 and D86).
In D13,1 hour, 3 hours, 6 hours after (T0) and administration before facing administration, also from retro-orbital sinus collection blood sample It is analyzed for PK.
Every group has 8-19 mouse.As a result it is expressed as average value ± SEM, and uses single factor test ANOVA, is then used Dunnett post-hoc tests are for statistical analysis to imiquimod solvent group.
6.6.5. chemical levels quantify in blood plasma
The plasma concentration of each test compound is measured by LC-MS/MS method, wherein with positivity or negativity electron spray mould Formula operates mass spectrograph.
6.6.6. the determination of pharmacokinetic parameter
It uses(The U.S.) calculate pharmacokinetic parameter.
6.6.7. histology
After execution, collects left kidney and be slit longitudinally into 2 half.Half is fixed in 3.7% formaldehyde, is embedded in later In paraffin.It prepares 4 μm of slabs, and with Period acid-Schiff (PAS) carries out dyeing or with CD3 (T cell), CD20 (B cell) and F4/80 (macrophage) carry out immunostaining.
6.6.7.1. histopathology
In each glomerulus, by 0 to 2 grade to 4 different readings (including mesangial matrix *, capillary intraductal hyperplasia, Extracellular matrix extension and segmented sclerosis) it is classified, then it is added.For each kidney, about 50 glomerulus are carried out Scoring, then averages, obtains a glomerular injury score value (Yokogawa et al., 2014).Data are expressed as average value ± SEM, and examined using Kruskal-Wallis, then imiquimod solvent group is carried out using Dunnett post-hoc tests Statistical analysis.
6.6.7.2. cell quantification
For each cell type, using image analysis (CaloPix software, TRIBVN Healthcare) to entire group It knits slice and immunohistochemical analysis is carried out with 20 times of enlargement ratio.Data are expressed as average value ± SEM, and use Dan Yin Plain ANOVA, it is then for statistical analysis to imiquimod solvent group using Dunnett post-hoc tests.
6.6.7.3. gene expression analysis
When putting to death, the other half by left kidney is placed in the pipe containing 1.4mm ceramic bead, and uses Bertin Instruments Homogenizer is in 1%DTT RLT lysis buffer (Qiagen, catalog number (Cat.No.) 79216) by it It is broken.Then, it is used with QIAcube96HT kit (Qiagen, catalog number (Cat.No.) 74171) is pure Change total serum IgE.CDNA is prepared, and using SYBR Green technology at 7 real-time PCR system of ViiA (Applied Biosystems) It is middle to carry out quantitative PCR with the gene-specific primer from Qiagen.Relative to cyclophilin, GAPDH and beta-actin house keeper Gene expression dose calculate each interested gene (GOI=CD3, CD68, CD20, OAS1, Mx1, IFIT1, CXCL11 and Usp18 expression).
When putting to death, the spleen of one third is placed in the pipe containing 1.4mm ceramic bead, and use Bertin Instruments Homogenizer existsIt is middle to be crushed.Total serum IgE is extracted using phenol/chloroform method, so It is used afterwards with QIAcube96HT kit (Qiagen, catalog number (Cat.No.) 74171) purifying.Preparation CDNA, and using SYBR Green technology in ViiA7 real-time PCR system (Applied Biosystems), with coming from The gene-specific primer of Qiagen carries out quantitative PCR.It is expressed relative to cyclophilin, GAPDH and beta-actin house-keeping gene Level, calculate each interested gene (GOI=CD20, IRF7, OAS1, Mx1, IFIT1, CXCL11, Usp18, BCL6, CXCL13, CXCR5, MAF, ICOSL, PDCD1, SH2D1a) expression.
All qPCR data be represented as standardized relative quantity average value ± SEM (NRQ=2^ (Δ Cq GOI)/ Geomean (2^ (Δ Cq cyclophilin), 2^ (Δ Cq GAPDH), 2^ (Δ Cq beta-actin)), wherein Δ Cq=Cq average value- Cq sample.The statistical test used is the ANOVA variance analysis and Dunnett post-hoc tests to imiquimod solvent group.
6.7. pass through the Murine models of the psoriasis arthropathica of the overexpression induction of IL-23
6.7.1. material
The additional body expression vector (EEV) of hyperkine enhancing is from System Biosciences (catalog number (Cat.No.) EEV651A- 1) it obtains.Ringer's solution piece is obtained from Sigma-Aldrich (catalog number (Cat.No.) 96724-100TAB).Mouse IL- 23Quantikine ELISA kit is obtained from R&D Systems (catalog number (Cat.No.) M2300).680 Hes750EX is obtained from PerkinElmer (catalog number (Cat.No.) NEV10003 and NEV10053EX). It is obtained from Ambion (catalog number (Cat.No.) AM7021).1000 (Merial) and2% (Bayer) from Centravet (catalog number (Cat.No.) IMA004-6827812 and ROM001-6835444) is obtained.
6.7.2. animal
B10.RIII mouse (male, 8-20g week old) obtains from Charles River (France).It is small that mouse is placed in 12 When light/dark circulation (07:00-19:00) under.Temperature is maintained 22 ± 2 DEG C, arbitrarily obtains food and water.
6.7.3. researching and designing
The researching and designing is adapted from Sherlock JP. et al. (Sherlock et al., 2012).
At first day (D1), Ringer liquid or IL-23EEV (the 3 μ g/ in Ringer liquid are injected into mouse tail vein 2.1mL is injected intravenously 4-6 seconds).
From D5 days, twice a week, scoring is carried out to the clinical symptoms of mouse until off-test.
At D5 days, by puncturing the venous collection blood under jaw, to evaluate the concentration of serum IL-2 3.
At D9 days, all groups of mouse received680 probes (0.8nmol/10g, IP).At D10 days, Intraperitoneal injection Imalgene and Rompun (7.5%/2.5%；0.1mL/10g) by mouse anesthesia.Then, using internal molecule (Bruker In-Vivo Xtreme imaging system, excitation wavelength: 630nm, launch wavelength: 700nm, acquisition time: 5 is imaged Second) measurement granulocyte infiltration.
At D11 days, according to680 molecular imagings and scoring are randomized.
From D12 days, to mouse medicine-feeding test compound, (30mg/kg was taken orally, twice a day, in 0.5% Methyl cellulose In element) or solvent.
At D19 days, T1h, T3h and T6h after T0, last time administration were sampled blood.Separated plasma is simultaneously 20 DEG C are maintained at until carrying out bioanalysis.
At D36 days, all groups of mouse is put to death to 2 hours after drug compound in last time.Collect following the description:
It is immediately that tendon attachment point (without skin) around the heel of left hind is quick-frozen in Precellys pipe.By toe Collection is containingPipe in.Right hind is fixed in 4% buffered formaldehyde liquid immediately and is used for Histological evaluation. Progress X-ray measurement in 48 hours after fixation.
One ear collection is being containedPipe in be used for transcription analysis.
By whole blood collection in serum blood vessel, and mixed by gently overturning 8 to 10 times.After solidification, by blood Liquid sample was with 1800 × g centrifugation 10 minutes.After centrifugation, serum is stored at -80 DEG C.
It is a part of colon (1cm terminal colon) is quick-frozen in Precellys pipe immediately to be used for transcription analysis.By another portion Divide (1cm terminal colon) fixed in 4% buffered formaldehyde liquid immediately, is used for further histologic analysis.
6.7.4. the evaluation of disease
Weight is measured when studying and starting, then measures weight twice a week and when the animals are executed.
Twice a week, score the clinical sign of inflammation: normal pawl is 0 point；One toe swelling is 1 point；Two or Multiple toe swelling are 2 points；If entire pawl swelling, is 3 points.The score of all limbs is added and generates total score.
At D23 days, all groups of mouse received680 probes (0.8nmol/10g, IP).In D24 It, intraperitoneal injection Imalgene and Rompun (7.5%/2.5%；0.1mL/10g) by mouse anesthesia.Then, using internal Molecular imaging (Bruker In-Vivo Xtreme imaging system, excitation wavelength: 630nm, launch wavelength: 700nm, when acquisition Between: 5 seconds) measurement granulocyte infiltration.
At D32 days, all groups of mouse received680 probe (0.8nmol/10g；IP) and750EX probe (0.8nmol/10g, IP).At D33 days, intraperitoneal injection Imalgene and Rompun (7.5%/2.5%；0.1mL/10g) by mouse anesthesia.Using internal molecular imaging, (Bruker In-Vivo Xtreme is imaged System；For680 probes, excitation wavelength: 630nm, launch wavelength: 700nm, acquisition time: 5 seconds；For750EX probe, excitation wavelength: 720nm, launch wavelength: 790nm, acquisition time: 5 seconds) measurement granulocyte Infiltration and bone reconstruct.
Every group has 10 mouse.As a result it is expressed as average value ± SEM, and uses single factor test ANOVA, is then used Dunnett post-hoc tests are to the scoring and image analysis of illness solvent group, unite to the weight of pseudo- illness (sham) solvent group Meter analysis.
6.8.CIA model
6.8.1. material
Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) are all purchased from Difco.II type bovine collagen (CII), rouge Polysaccharide (LPS) and Enbrel are respectively from Chondrex (Isled ' Abeau, France)；Sigma (P4252, L ' Isled ' Abeau, France), Whyett (25mg syringe, France) AcrosOrganics (PaloAlto, CA) obtains.The all other examination used Agent is all SILVER REAGENT, and all solvents are all analysis levels.
6.8.2. animal
Black agouti (male, 7-8 week old) obtains from Harlan Laboratories (Maison-Alfort, France). Rat is placed in 12 hours light/dark periods (07:00-19:00).Temperature is maintained 22 DEG C, arbitrarily obtains food and water.
6.8.3. the arthritis (CIA) of collagen induction
It one day before the test, with 0.05M acetic acid preparation CII solution (2mg/mL) and is stored at 4 DEG C.Facing immunity inoculation Before, isometric adjuvant (IFA) and CII are passed through in the vial that homogenizer is pre-chilled in ice-water bath and is mixed.If no Lotion is formed, needs to add adjuvant and extends the homogenization time.At first day, in the tail base intracutaneous injection of every rat 0.2mL lotion, second of the booster intracutaneous injection of progress in the 9th day (the CII solution of 2mg/mL, in CFA 0.1mL salt water). The methods of vaccination be by disclosed method (Sims et al., 2004；Jou et al., 2005) improvement.
6.8.4. researching and designing
The therapeutic effect of compound is tested in rat CIA model.Rat is assigned at random in identical group, every group includes 10 rats.By all rats on day 1 immunity inoculation and the 9th day reinforce.Therapeutic administratp is from the 16th day to the 30th day. Negative control group is handled with solvent (MC 0.5%), positive controls Enbrel (10mg/kg, 3 × week, s.c.) processing.It is logical Often interested compound is tested under 4 dosage, such as 1,3,10,30mg/kg, twice daily.
6.8.5. arthritic clinical evaluation
According to Khachigian 2006, Lin et al., 2007 and Nishida et al., 2004 method carries out arthritis Scoring.Each swelling situation is sorted out according to following arthritis score in four claws: 0- does not have symptom；1- is slight, But there is apparent rubescent and swelling in a type of joint (such as ankle or wrist), or apparent rubescent and swelling is limited to list A finger (toe), no matter be affected finger (toe) number how much；It is rubescent and swollen that moderate occurs in the joint of two or more types of 2- It is swollen；There is severe redness and swelling in the entire pawl of 3- (including finger (toe))；The limb of 4- most severe inflammation is related to (every, multiple joints The clinical arthritis of animal highest accumulation is scored at 16) (Nishida et al., 2004).
In order to carry out the meta-analysis of multinomial research, clinical score value is standardized as follows:
The AUC (AUC scoring) of clinical score: each the 1st day to the 14th day area under the curve of independent rat is calculated (AUC).In this study, thus the AUC of every animal obtains the data of the animal divided by the average AUC obtained with solvent And multiplied by 100 (that is, percentages that AUC is expressed as to the average solvent AUC of each research).
Clinical score from the 1st day to the 14th day increases (end point scoring): in this study, the clinic of every animal is commented Divide difference poor divided by the mean clinical scores obtained with solvent, thus obtains the data of the animal and multiplied by 100 (that is, by poor table It is shown as the percentage of the average solvent clinical score difference of each research).
6.8.5.1. the changes of weight (%) after arthritis breaking-out
Clinically, weight loss and arthritis it is related (Shelton et al., 2005；Argiles et al., 1998；Rall, 2004；Walsmith et al., 2004).Therefore, it may be used as in evaluation rat model in the changes of weight after arthritis breaking-out Therapeutic effect non-specific endpoint (endpoint).The following variation (%) for calculating the weight after arthritis breaking-out:
Mouse:
Rat:
6.8.5.2. radiology
Shoot the X-ray photo of the rear solid end of each animal.To the identifier that every photo distribution is randomized, bone is invaded The severity of erosion is scored by two independent marking persons, and using following radiology Larsen points-scoring system: 0- is normal, has had Whole bone profile and normal joint space；1- mile abnormality, any one or two outside metatarsal show slight bone erosion； The apparent abnormal in early stage of 2-, any 3 to 5 external metatarsals show bone erosion；3- moderate damage sexual abnormality, all external metatarsals And metatarsal shows apparent bone erosion inside any one or two；4- seriously destroys sexual abnormality, and all metatarsals are shown obviously Bone erosion, and at least one internal plantar joint is corroded completely, leaves the Bones and joints profile of some parts reservation；Torturing property of 5- is different Often (mutilating abnormality), does not have sclerotin profile.The points-scoring system is from Salvemini et al. 2001；Bush Et al., 2002；Sims et al., 2004；Jou et al., 2005 points-scoring system improvement.
6.8.5.3. histology
It is after radiology analysis, the rear solid end of mouse is fixed in the formalin (pH7.4) of 10% phosphate-buffered, With quick decalcification of bone agent (Laboratories Eurobio) decalcification for delicate tissues and it is embedded in paraffin.In order to true The extensive evaluation to arthritis knuckle, cutting at least four series slice (5 μ m-thick) are protected, and is 100 between each slide series μm.Sections stained with hematoxylin and Yihong (H&E) are dyed.The histology double blind of Synovial inflammation and bone and cartilage damage into Row.In each pawl, 4 parameters are evaluated with 4- point grade.These parameters are Premeabilisation of cells, pannus seriousness, cartilage erosion And bone erosion.Scoring according to carrying out as follows: 1- is normal, and 2- is slight, 3- moderate, and 4- is significant.This 4 scores are added together And be expressed as additional score, i.e., " RA total score ".
6.8.5.4. micro- computed tomography (μ CT) analysis of calcaneum (calcaneus)
The bone degradation observed in RA especially occurs at cortex bone and can be analyzed by μ CT to show (Sims NA et al., Arthritis Rheum.50 (2004) 2338-2346:Targeting osteoclasts with zoledronic acid prevents bone destruction in collagen-induced arthritis；Oste L et al., ECTC Montreal 2007:A high throughput method of measuring bone architectural disturbance in a murine CIA model by micro-CT morphometry).With After bone scanning and 3 dimension volume reconstructions, computer simulation slice is carried out in the direction perpendicular to the bone longitudinal axis, and pass through each slice Present on dispersive target quantity come measure bone degradation.Bone is degraded more, and the dispersive target measured is more.Analyze 1000 It is a along the equally distributed slice (interval about 10.8 μm) of calcaneum.
6.8.5.5. stable state PK
At the 7th day or the 11st day, heparin lithium is used to collect retro-orbital sinus blood sample as anti-coagulants at following time point: before administration, 1,3 and 6 hours.Whole blood sample is centrifuged, obtained plasma sample is saved at -20 DEG C until analyzing.Pass through LC-MS/ MS method measures the plasma concentration of each test compound, and wherein mass spectrum is run with positive electrospray.It uses (The U.S.) pharmacokinetic parameter is calculated, and assume that the blood plasma level before administration is equal to 24 hours blood plasma It is horizontal.
6.8.6. result
Table X I clinical score
Table X II clinical score (p value)
6.8.7. conclusion
As above as it can be seen that the combination for taking IRAK and JAK inhibitor together obtains compared with taking every kind of compound respectively Better effect.
This causes the drug of available relatively low amount to obtain identical or better therapeutic effect again.This is particularly useful for being avoided taking Effect is kept while with unnecessary medication amount, to reduce drug-induced side effect.
Conclusion
It will be appreciated by those skilled in the art that the description of front its be exemplary in nature with it is explanatory, and be intended to Illustrate the present invention and its preferred embodiment.By routine experiment, those skilled in the art will appreciate that without departing substantially from this hair Apparent modification and accommodation can be made under bright spirit.All such modifications within the scope of claims attached hereto It is included in wherein.Therefore, the present invention is not intended to define by foregoing description, but passes through following the claims and its wait Jljl defines.
All publications (including but not limited to patents and patent applications) quoted in this specification are incorporated herein work For reference, as fully expounded each individually publication by specifically as individually pointing out to be incorporated herein by reference.
It should be appreciated that the factors such as difference of Premeabilisation of cells ability of various compounds are possible to that compound can be caused in vitro Activity in biochemistry and cell analysis is inconsistent.
At least some of chemical name for the compounds of this invention for providing and illustrating in the application may be to use commercially availableization It learns that name software program automatically generates and is not verified individually.Execute the function representative program include by The Lexichem of OpenEye Scientific Software, Inc. sale names tool and by MDL Information The Autonom Software tool that Systems, Inc are sold.It is inconsistent in indicated chemical name and the structure drawn In the case of, it is subject to drawn structure.
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Claims (17)

1. composition includes:
A) compound of Formulas I:
Wherein
Cy is
Optionally by the R of one or more independent choices3Substituted monocycle C3-7Naphthenic base, or
Optionally by the R of one or more independent choices3What is replaced includes one or two miscellaneous original independently selected from N, S and O The 4-7 unit monocycle Heterocyclylalkyl of son；
R1For
- H,
--SO3H,
-- P (=O) (OH)2,
-C1-4Alkyl,
-- C (=O)-(includes one or two heteroatomic 4-7 unit monocycle Heterocyclylalkyl independently selected from N, S and O), or
-- C (=O) C1-6Alkyl, wherein C1-6Alkyl is optionally by the R of one or more independent choices4Group replaces；
R2For H or C1-4Alkyl；
Each R3Independently selected from:
- OH,
=O,
Halogen, and
-C1-4Alkyl；
Each R4Independently selected from:
--NR5aR5b,
-- C (=O) OH,
Optionally by the C of one or more independent choices1-4Alkyl-substituted includes one or two independently selected from N, S and O Heteroatomic 4-7 unit monocycle Heterocyclylalkyl, and
-- NHC (=O)-C1-4Alkyl-NH2；And
R5aAnd R5bIt independently is H or C1-4Alkyl；
Or the salt of its officinal salt or solvate or solvate；With
B) with second of compound of JAK inhibitory.
2. composition according to claim 1, wherein Cy is by one or two in the compound or pharmaceutically acceptable salt thereof of Formulas I The R of independent choice3Substituted monocycle C3-7Naphthenic base.
3. composition according to claim 1 or 2, wherein Cy is THP trtrahydropyranyl in the compound or pharmaceutically acceptable salt thereof of Formulas I Or tetrahydro thiopyranyl, respectively optionally by the R of one or two independent choice3Replace.
4. composition as claimed in one of claims 1-3, wherein in the compound or pharmaceutically acceptable salt thereof of Formulas I, R3It is selected from OH ,=O, F and-CH3。
5. composition according to claim 1, wherein the compound or pharmaceutically acceptable salt thereof of Formulas I be Formula II a, IIb, IIc, IId, The compound of IIe or IIf:
6. composition as claimed in one of claims 1-5, wherein in the compound of any of Formulas I-IIf or its is pharmaceutically acceptable In salt, R1For H ,-CH3、-SO3H or-P (=O) (OH)2。
7. composition as claimed in one of claims 1-5, wherein in the compound of any of Formulas I-IIf or its is pharmaceutically acceptable In salt, R1For-C (=O) C1-6Alkyl, wherein C1-6Alkyl is by the R of one or two independent choice4Replace.
8. composition as claimed in one of claims 1-5, wherein in the compound of any of Formulas I-IIf or its is pharmaceutically acceptable In salt, R1For-C (=O) C1-6Alkyl, wherein C1-6Alkyl is by-C (=O) OH ,-NH of one or two independent choice2、-NHCH3 Or-N (CH3)2Replace.
9. composition as claimed in one of claims 1-8, wherein in the compound of any of Formulas I-IIf or its is pharmaceutically acceptable In salt, R2For H or-CH3。
10. composition according to claim 1, wherein the compound of the Formulas I is selected from
6- [6- [2- (2- Hydroxy-ethoxy)-ethyoxyl] -5- (ttetrahydro-pyran -4- base amino)-imidazo [4,5-b] pyridine - 3- yl]-nicotinic acid nitrile, and
(S) -2- amino -3- metliyl-butyric acid 2- { 2- [3- (5- Cyano-pyridin -2- base) -5- (ttetrahydro-pyran -4- base amino) - 3H- imidazo [4,5-b] pyridine -6- base oxygroup]-ethyoxyl }-ethyl ester.
11. composition as claimed in one of claims 1-10, wherein the JAK inhibiting compound is JAK1 inhibitor.
12. composition as claimed in one of claims 1-10, wherein the JAK inhibiting compound I is Formula X Xa's or XXb Compound:
13. pharmaceutical composition, the composition and pharmaceutical acceptable carrier of any one of claim 1-12 comprising pharmacy effective dose.
14. pharmaceutical composition according to claim 13 also includes other therapeutic agents.
15. pharmaceutical composition according to claim 14, wherein other therapeutic agents are for preventing and/or treating inflammatory disease Disease, proliferative diseases, allergic disease, graft rejection, is related to the disease, congenital that cartilage updates damage at autoimmune disease The reagent of cartilage deformity and/or disease relevant to IL6 or interferon hypersecretion.
16. being used for any one of -12 composition or according to claim 1 any one of 3-15 according to claim 1 of medicine Pharmaceutical composition.
17. for prevent and/or treat inflammatory disease, autoimmune disease, proliferative diseases, allergic disease, graft rejection, It is related to the root that cartilage updates the disease of damage, congenital cartilage deformity and/or disease relevant to IL6 or interferon hypersecretion According to the composition or the pharmaceutical composition of any one of 3-15 according to claim 1 of any one of claim 1-12.
CN201880012183.5A 2017-02-17 2018-02-15 Anti-inflammatory composition comprising IRAK and JAK inhibitor Pending CN110300586A (en)

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