HK1212975B

HK1212975B - Thiazole derivatives as inhibitors of bruton's tyrosine kinase

Info

Publication number: HK1212975B
Application number: HK16100856.5A
Authority: HK
Inventors: Nicholas John Silvester Huby; Francisco Javier Lopez-Tapia; Sung-Sau So
Original assignee: F. Hoffmann-La Roche Ag
Priority date: 2012-12-13
Filing date: 2013-12-09
Publication date: 2018-04-20

Description

Thiazole derivatives as inhibitors of bruton's tyrosine kinase

Technical Field

The present invention relates to the use of novel compounds that inhibit Btk and are useful in the treatment of autoimmune and inflammatory diseases caused by aberrant B-cell activation.

Background

Protein kinases constitute the largest family of human enzymes and regulate many different signaling processes by adding phosphate groups to proteins (T. Hunter, Cell 198750: 823-829). Specifically, tyrosine kinases phosphorylate the phenol moiety of tyrosine residues in proteins. The tyrosine kinase family includes members that control cell growth, migration, and differentiation. Abnormal kinase activity has been shown to be involved in a number of human diseases including cancer, autoimmune and inflammatory diseases. Since protein kinases are key regulators of cell signaling, they provide targets for the regulation of cellular function with small molecule kinase inhibitors, becoming good targets for drug design. In addition to treating kinase-mediated disease processes, potent and selective inhibitors of kinase activity can also be used to study cell signaling processes and identify other cellular targets of interest for treatment.

There is good evidence that B-cells play a key role in the pathogenesis of autoimmune and/or inflammatory diseases. Protein-based therapies that deplete B cells, such as rituximab (Rituxan), are effective against autoantibody driven inflammatory diseases such as rheumatoid arthritis (Rastetter et al, Annu Rev Med 200455: 477). Therefore, protein kinase inhibitors that play a role in B-cell activation would be useful in the treatment of B-cell mediated disease pathologies such as autoantibody production.

Signalling through the B-cell receptor (BCR) controls a range of B-cell responses including proliferation and differentiation into mature antibody producing cells. BCR is a key regulatory point of B-cell activity, and aberrant signaling can cause dysregulated B-cell proliferation and the formation of pathogenic autoantibodies leading to a variety of autoimmune and/or inflammatory diseases. Bruton's tyrosine kinase (Btk) is a non-BCR-associated kinase located in the membrane proximal region and directly downstream of BCR. It has been shown that Btk deficiency blocks BCR signaling, and therefore, inhibition of Btk would be a useful therapeutic approach to block B-cell mediated disease progression.

Btk is a member of the Tec family of tyrosine kinases that has been shown to be a key regulator of B-cell activation and survival in early B-cell development and maturation (Khan et al, Immunity 19953: 283; Ellmeier et al, J.Exp.Med.2000192: 1611). Btk mutations in humans result in X-linked agammaglobulinemia (XLA) (reviewed in Rosen et al, New Eng.J.Med.1995333: 431 and Lindval et al, Immunol.Rev.2005203: 200). These patients have low immune function, and exhibit impaired B-cell maturation, decreased levels of immunoglobulins and peripheral B-cells, a reduced T-cell independent immune response, and reduced calcium mobilization following BCR stimulation.

The Btk-deficient mouse model also provides evidence of a role for Btk in autoimmune and inflammatory diseases. Btk-deficient mice show significant disease progression improvement in preclinical murine models of Systemic Lupus Erythematosus (SLE). In addition, Btk-deficient mice are resistant to collagen-induced arthritis (Jansson and Holmdahl Clin. exp. Immunol.199394: 459). A selective Btk inhibitor has been shown to exhibit dose-dependent efficacy in a mouse model of arthritis (z.pan et al, chem.med chem.20072: 58-61).

Btk is also expressed by cells other than B-cells involved in disease progression. For example, Btk is expressed by mast cells, and mast cells derived from Btk-deficient bone marrow exhibit impaired antigen-induced degranulation (Iwaki et al, j.biol.chem.2005280: 40261). This suggests that Btk can be used to treat pathological mast cell responses such as allergy and asthma. Monocytes from XLA patients without Btk activity also showed reduced TNF α production following stimulation (Horwood et al, JExp Med 197:1603,2003). Thus, small molecule Btk inhibitors can be used to modulate TNF α -mediated inflammation. Btk has also been reported to play a role in apoptosis (Islam and Smith Immunol. Rev. 2000178: 49) and therefore Btk inhibitors would be useful in the treatment of certain B-cell lymphomas and leukemias (Feldhahn et al, J.exp. Med. 2005201: 1837).

Summary of The Invention

As described herein below, the present application provides Btk inhibitor compounds of formula I, methods of treatment, and compositions thereof:

the present application provides a compound of formula I or a pharmaceutically acceptable salt thereof,

wherein:

a is lower alkyl, phenyl, CH₂R¹、OR⁴、

R¹Is H,

R²Is H or halogen;

R³is halogen;

R⁴is a lower alkyl group;

R⁵is a lower alkyl group;

R⁶is H or halogen;

x is C (═ O) or S (═ O)₂(ii) a And is

Y is CH or N.

The present application provides a method of treating an inflammatory and/or autoimmune disorder comprising administering to a patient in need thereof a therapeutically effective amount of a compound of formula I.

The present application provides a pharmaceutical composition comprising a compound of formula I and at least one pharmaceutically acceptable carrier, excipient or diluent.

Detailed description of the invention

Definition of

The terms "a" or "an", as used herein, refer to one or more of such entities; for example, a compound refers to one or more compounds or at least one compound. Thus, the terms "a", "an", "one or more" and "at least one" are used interchangeably herein.

The phrase "as defined herein above" refers to the broadest definition of each group as provided in the summary of the invention or the broadest claims. In all other embodiments provided below, substituents that may be present in each embodiment and not explicitly defined retain the broadest definition provided in the summary of the invention.

The term "comprising" as used in this specification is to be interpreted in an open-ended fashion, whether in conjunctive or in the body of a claim. That is, the term is to be interpreted as synonymous with the word "having at least" or "including at least". When used in a method, the term "comprising" means that the method includes at least the recited steps, but may also include other steps. The term "comprising" when used in the context of a compound or composition means that the compound or composition includes at least the recited features or components, but may also include other features or components.

As used herein, unless otherwise specifically stated, the use of the word "or" is used in the "inclusive" sense of "and/or" and not in the "exclusive" sense of "either/or".

The term "independently" is used herein to indicate that a variable applies in any case, regardless of whether variables having the same or different definitions are present or absent in the same compound. Thus, in a compound where R "occurs twice and is defined as" independently carbon or nitrogen, "both R" can be both carbon, both R "can be nitrogen, or one R" can be carbon and the other can be nitrogen.

When any variable occurs more than one time in any moiety or formula depicting or describing a compound used or claimed in the present invention, its definition on each occurrence is independent of its definition at each other occurrence. Combinations of substituents and/or variables are permissible only if the compound is a stable compound.

The symbol "", or the symbol "-" located at the end of a bond, or through a bond, each refers to the point of attachment of a functional group or other chemical moiety to the rest of the molecule of which it is a part. Thus, for example:

MeC(＝O)OR⁴wherein

A bond drawn into a ring system (as opposed to a bond attached at a different vertex) means that the bond may be attached to any suitable ring atom.

The term "optional" or "optionally" as used herein means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "optionally substituted" means that the optionally substituted moiety may bind a hydrogen atom or a substituent.

The phrase "optional bond" means that the bond may or may not be present, and the description includes single, double, or triple bonds. If a substituent is defined as "bond" or "absent," the atom to which the substituent is attached is directly attached.

The term "about" as used herein means approximately, near, about, or around … …. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries upward and downward on the numerical values set forth. Generally, the term "about" as used herein varies the stated value by up to and down to 20%.

Certain compounds of formula I may exhibit tautomerism. Tautomeric compounds can exist in the form of two or more interconvertible species. Migration of a covalently bonded hydrogen atom between two atoms produces a proton tautomer. Tautomers are usually in equilibrium and when attempting to separate individual tautomers, a mixture is usually produced whose chemical and physical properties are consistent with a mixture of compounds. The position of equilibrium depends on the chemical characteristics within the molecule. For example, in many aliphatic aldehydes and ketones, such as acetaldehyde, the keto form predominates, while in phenol, the enol form predominates. Common proton tautomers include keto/enolAmide/imide acidAnd amidinesTautomers. The latter two tautomers are particularly common in heteroaryl and heterocyclic rings and the present invention includes all tautomeric forms of the compounds.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Reference is made herein to various methods and materials known to those skilled in the art. Standard references to state general principles of pharmacology include The Pharmacological Basis of Therapeutics, 10 th edition, McGraw Hill Companies Inc., New York, Goodman and Gilman (2001). The invention may be practiced with any suitable materials and/or methods known to those skilled in the art. However, preferred materials and methods are described. Unless otherwise indicated, materials, reagents and the like referred to in the following description and examples may be obtained from commercial sources.

The definitions described herein may be concatenated to form chemically related combinations such as "heteroalkylaryl," "haloalkyl heteroaryl," "arylalkyl heterocyclyl," "alkylcarbonyl," "alkoxyalkyl," and the like. When the term "alkyl" is used as a suffix following another term, as in "phenylalkyl" or "hydroxyalkyl", it is intended to refer to an alkyl group, as defined above, substituted with one to two substituents selected from the other specifically-named group. Thus, for example, "phenylalkyl" refers to an alkyl group having one to two phenyl substituents, and thus includes benzyl, phenylethyl, and biphenyl. "alkylaminoalkyl" is an alkyl group having one to two alkylamino substituents. "hydroxyalkyl" includes 2-hydroxyethyl, 2-hydroxypropyl, 1- (hydroxymethyl) -2-methylpropyl, 2-hydroxybutyl, 2, 3-dihydroxybutyl, 2- (hydroxymethyl), 3-hydroxypropyl and the like. Thus, as used herein, the term "hydroxyalkyl" is used to define a subset of heteroalkyl groups defined below. The term- (ar) alkyl refers to unsubstituted alkyl or aralkyl. The term (hetero) aryl refers to aryl or heteroaryl.

The term "spirocycloalkyl" as used herein means a spirocyclic cycloalkyl group, for example, spirocyclo [3.3] heptane. The term spiroheterocycloalkyl as used herein means a heterocycloalkyl of a spiro ring, for example, 2, 6-diazaspiro [3.3] heptane.

The term "acyl" as used herein denotes a group of formula-C (═ O) R, wherein R is hydrogen or lower alkyl as defined herein. The term "alkylcarbonyl" as used herein denotes a group of formula C (═ O) R where R is alkyl as defined herein. Term C_1-6Acyl refers to a group-C (═ O) R containing 6 carbon atoms. The term "arylcarbonyl" as used herein means a group of formula C (═ O) R where R is aryl; the term "benzoyl" as used herein is an "arylcarbonyl" group wherein R is phenyl.

The term "ester" as used herein denotes a group of formula-C (═ O) OR where R is lower alkyl as defined herein.

The term "alkyl" as used herein denotes a straight or branched chain saturated monovalent hydrocarbon residue containing 1 to 10 carbon atoms. The term "lower alkyl" denotes a straight or branched chain hydrocarbon residue comprising 1 to 6 carbon atoms. As used herein, "C₁-₁₀Alkyl "refers to an alkyl group consisting of 1 to 10 carbons. Examples of alkyl include, without limitation, lower alkyl groups including methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl or pentyl, isopentyl, neopentyl, hexyl, heptyl, and octyl.

When the term "alkyl" is used as a suffix following another term, as in "phenylalkyl" or "hydroxyalkyl", it is intended to refer to an alkyl group, as defined above, substituted with one to two substituents selected from the other specifically-named group. Thus, for example, "phenylalkyl" represents a group R 'R "-, where R' is phenyl and R" is alkylene as defined herein, it being understood that the point of attachment of the phenylalkyl moiety will be on the alkylene. Examples of arylalkyl groups include, but are not limited to, benzyl, phenylethyl, 3-phenylpropyl. The term "arylalkyl" or "aralkyl" has a similar interpretation, except that R' is aryl. The terms "(hetero) arylalkyl" or "(hetero) aralkyl" have similar interpretation except that R' is optionally aryl or heteroaryl.

The term "haloalkyl" or "halo-lower alkyl" or "lower haloalkyl" refers to a straight or branched chain hydrocarbon residue comprising 1 to 6 carbon atoms in which one or more carbon atoms are substituted with one or more halogen atoms.

The term "alkylene" as used herein, unless otherwise indicated, denotes a divalent saturated straight chain hydrocarbon radical of 1 to 10 carbon atoms (e.g., (CH)₂)_n) Or a divalent saturated branched hydrocarbon group of 2 to 10 carbon atoms (e.g., -CHMe-or-CH)₂CH(i-Pr)CH₂-). The open valences of the alkylene groups are not attached to the same atom, except in the case of methylene groups. Examples of alkylene include, without limitation, methylene, ethylene, propylene, 2-methyl-propylene, 1-dimethyl-ethylene, butylene, 2-ethylbutylene.

The term "alkoxy" as used herein means-O-alkyl, wherein alkyl is as defined above, such as methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, i-butoxy, t-butoxy, pentyloxy, hexyloxy, including isomers thereof. "lower alkoxy" as defined herein denotes alkoxy having "lower alkyl" as defined above. As used herein, "C₁-₁₀Alkoxy "means wherein alkyl is C_1-10an-O-alkyl group of (a).

The term "PCy₃"refers to a phosphine trisubstituted with three cyclic moieties.

The term "haloalkoxy" or "halo-lower alkoxy" or "lower haloalkoxy" refers to lower alkoxy wherein one or more carbon atoms are substituted with one or more halogen atoms.

The term "hydroxyalkyl" as used herein denotes an alkyl group as defined herein wherein one to three hydrogen atoms on different carbon atoms are replaced by a hydroxyl group.

As used hereinThe terms "alkylsulfonyl" and "arylsulfonyl" refer to the formula-S (═ O)₂R, wherein R is independently alkyl or aryl and alkyl and aryl are as defined herein. The term "heteroalkylsulfonyl" as used herein denotes a compound of formula-S (═ O)₂The group of R, wherein R is "heteroalkyl" as defined herein.

The terms "alkylsulfonylamino" and "arylsulfonylamino" as used herein refer to the formula-NR' S (═ O)₂R, wherein R is independently alkyl or aryl, R' is hydrogen or C_1-3Alkyl, and alkyl and aryl are as defined herein.

The term "cycloalkyl" as used herein refers to a saturated carbocyclic ring containing from 3 to 8 carbon atoms, i.e. cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl. As used herein, "C_3-7Cycloalkyl "refers to a cycloalkyl group consisting of 3 to 7 carbons in a carbocyclic ring.

The term "carboxy-alkyl" as used herein refers to an alkyl moiety wherein one hydrogen atom is replaced by a carboxy group, it being understood that the heteroalkyl group is attached through a carbon atom. The term "carboxy" refers to-CO₂And (4) a H part.

The term "heteroaryl" or "heteroaromatic" as used herein means a monocyclic or bicyclic group of 5 to 12 ring atoms having at least one aromatic or partially unsaturated ring, each ring containing four to eight atoms, incorporating one or more N, O or S heteroatoms, the remaining ring atoms being carbon, it being understood that the attachment point of the heteroaryl group will be on the aromatic or partially unsaturated ring. As is well known to those skilled in the art, heteroaryl rings have less aromatic character than their all-carbon counterparts. Thus, for the purposes of the present invention, heteroaryl groups need only have some degree of aromatic character. Examples of heteroaryl moieties include monocyclic aromatic heterocycles having 5 to 6 ring atoms and 1 to 3 heteroatoms, including, without limitation, pyridyl, pyrimidinyl, pyrazinyl, and the like,Oxazinyl, pyrrolyl, pyrazolyl, imidazolyl,Azolyl, 4, 5-dihydro-Azolyl, 5, 6-dihydro-4H- [1,3]Azolyl radical, isoOxazole, thiazole, isothiazole, triazoline, thiadiazole and oxadixoline, which may be optionally substituted with one or more, preferably one or two, substituents selected from the group consisting of hydroxy, cyano, alkyl, alkoxy, thio, lower haloalkoxy, alkylthio, halogen, lower haloalkyl, alkylsulfinyl, alkylsulfonyl, halogen, amino, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl and dialkylaminoalkyl, nitro, alkoxycarbonyl and carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, arylcarbamoyl, alkylcarbonylamino and arylcarbonylamino. Examples of bicyclic moieties include, without limitation, quinolinyl, isoquinolinyl, benzofuranyl, benzothienylAzolyl, benzisoylAzolyl, benzothiazolyl, naphthyridinyl, 5,6,7, 8-tetrahydro- [1,6]Naphthyridinyl and benzisothiazole. The bicyclic moiety may be optionally substituted on any one of the rings, however, its point of attachment is on the ring containing the heteroatom.

As used herein, unless otherwise specified, the term "heterocyclyl", "heterocycloalkyl" or "heterocycle" means a monovalent saturated cyclic group (including spiro ring systems) consisting of one or more rings, preferably one to two rings, such asAnd ionic forms thereof, each ring containing three to eight atoms, mixed with one or more ring heteroatoms (selected from N, O or S (O))_0-2) And may be optionally independently substituted with one or more, preferably one or two, substituents selected from hydroxy, oxo, cyano, lower alkyl, lower alkoxy, lower haloalkoxy, alkylthio, halogen, lower haloalkyl, hydroxyalkyl, nitro, alkoxycarbonyl, amino, alkylamino, alkylsulfonyl, arylsulfonyl, alkylaminosulfonyl, arylaminosulfonyl, alkylsulfonylamino, arylsulfonylamino, alkylaminocarbonyl, arylaminocarbonyl, alkylcarbonylamino, arylcarbonylamino. Examples of heterocyclic groups include, without limitation, morpholinyl, piperazinyl, piperidinyl, azetidinyl, pyrrolidinyl, hexahydroazepinylAn oxetanyl group, a tetrahydrofuryl group, a tetrahydrothienyl group,Oxazolidinyl, thiazolidinyl, isooxazolidinylOxazolidinyl, tetrahydropyranyl, thiomorpholinyl, quinuclidinyl, and imidazolinyl, as well as ionic forms thereof. Examples may also be bicyclic rings, such as, for example, 3, 8-diaza-bicyclo [3.2.1]Octane, 2, 5-diaza-bicyclo [2.2.2]Octane or octahydro-pyrazino [2,1-c][1,4]And (3) an oxazine.

Btk inhibitors

wherein:

a is lower alkyl, phenyl, CH₂R¹、OR⁴、

R¹Is H,

R²Is H or halogen;

R³is halogen;

R⁴is a lower alkyl group;

R⁵is a lower alkyl group;

R⁶is H or halogen;

x is C (═ O) or S (═ O)₂(ii) a And is

Y is CH or N.

Furthermore, it will be understood that reference to the specific residue A, R disclosed herein is made¹、R²、R³、R⁴、R⁵、R⁶Each embodiment of Y and X may be combined with any other residue A, R related to another residue disclosed herein¹、R²、R³、R⁴、R⁵、R⁶Embodiments of Y and X are combined.

The present application provides compounds of formula I, wherein X is C (═ O).

The present application provides compounds of formula I wherein A is

The present application provides compounds of formula I, wherein X is C (═ O) and a is

The present application provides compounds of formula I, wherein R⁶Is H.

The present application provides compounds of formula I, wherein R⁶Is Cl.

The present application provides compounds of formula I, wherein R⁶Is H, X is C (═ O) and A is

The present application provides compounds of formula I, wherein R⁶Is Cl, X is C (═ O) and A is

The present application provides compounds of formula I, wherein A is methyl OR OR⁴And R is⁴Is a tert-butyl group.

The present application provides compounds of formula I, wherein a is methyl.

The present application provides compounds of formula I wherein X is C (═ O) and a is methyl.

The present application provides compounds of formula I, wherein X is C (═ O), a is methyl OR⁴And R is⁴Is a tert-butyl group.

The present application provides compounds of formula I wherein A is

And R is⁵Is a tert-butyl group.

The present application provides compounds of formula I, wherein X is C (═ O), a is

And R is⁵Is a tert-butyl group.

The present application provides compounds of formula I, wherein Y is N.

The present application provides compounds of formula I, wherein Y is CH.

The present application provides compounds of formula I wherein Y is N, X is C (═ O), a is

And R is⁵Is a tert-butyl group.

The present application provides compounds of formula I wherein Y is CH, X is C (═ O), a is

And R is⁵Is a tert-butyl group.

The present application provides compounds of formula I wherein A is

The present application provides compounds of formula I, wherein R²Is H.

This applicationThere is provided a compound of formula I wherein R²Is a tert-butyl group.

The present application provides compounds of formula I, wherein X is C (═ O), R²Is H and A is

The present application provides compounds of formula I, wherein X is C (═ O), R²Is tert-butyl and A is

The present application provides compounds of formula I, wherein X is S (═ O)₂And A is methyl, isopropyl or phenyl.

The present application provides compounds of formula I, wherein X is S (═ O)₂And A is methyl.

The present application provides compounds of formula I, wherein X is S (═ O)₂And a is isopropyl.

The present application provides compounds of formula I, wherein X is S (═ O)₂And A is phenyl.

The present application provides compounds of formula I wherein A is

And R is³Is Cl.

The present application provides a compound of formula I selected from:

1, 3-dihydro-isoindole-2-carboxylic acid { (R) -1- [ 4-carbamoyl-5- (pyridin-2-ylamino) -thiazol-2-yl ] -piperidin-3-yl } -amide;

5-chloro-1, 3-dihydro-isoindole-2-carboxylic acid { (R) -1- [ 4-carbamoyl-5- (pyridin-2-ylamino) -thiazol-2-yl ] -piperidin-3-yl } -amide;

{ (R) -1- [ 4-carbamoyl-5- (pyridin-2-ylamino) -thiazol-2-yl ] -piperidin-3-yl } -carbamic acid tert-butyl ester;

6-tert-butyl-N- { (R) -1- [ 4-carbamoyl-5- (pyridin-2-ylamino) -thiazol-2-yl ] -piperidin-3-yl } -nicotinamide;

2- ((R) -3-phenylacetylamino-piperidin-1-yl) -5- (pyridin-2-ylamino) -thiazole-4-carboxylic acid amide;

5- (pyridin-2-ylamino) -2- [ (R) -3- (2-m-tolyl-acetylamino) -piperidin-1-yl ] -thiazole-4-carboxylic acid amide;

2- [ (R) -3- (4-tert-butyl-benzoylamino) -piperidin-1-yl ] -5- (pyridin-2-ylamino) -thiazole-4-carboxylic acid amide;

2- ((R) -3-methanesulfonylamino-piperidin-1-yl) -5- (pyridin-2-ylamino) -thiazole-4-carboxylic acid amide;

2- ((R) -3-benzenesulfonylamino-piperidin-1-yl) -5- (pyridin-2-ylamino) -thiazole-4-carboxylic acid amide;

2- [ (R) -3- (propane-2-sulfonylamino) -piperidin-1-yl ] -5- (pyridin-2-ylamino) -thiazole-4-carboxylic acid amide;

2- { (R) -3- [2- (3-chloro-phenylamino) -acetylamino ] -piperidin-1-yl } -5- (pyridin-2-ylamino) -thiazole-4-carboxylic acid amide; and

2- ((R) -3-acetylamino-piperidin-1-yl) -5- (pyridin-2-ylamino) -thiazole-4-carboxylic acid amide.

The present application provides a method of treating rheumatoid arthritis comprising administering to a patient in need thereof a therapeutically effective amount of a compound of formula I.

The present application provides a method of treating asthma comprising administering to a patient in need thereof a therapeutically effective amount of a compound of formula I.

The present application provides a pharmaceutical composition comprising a compound of formula I.

The present application provides the use of a compound as described above as therapeutically active substance.

The application provides the use of a compound of formula I for the preparation of a medicament for the treatment of inflammation.

The present application provides the use of a compound of formula I for the preparation of a medicament for the treatment of an autoimmune disorder.

The application provides application of a compound shown in a formula I in preparing a medicament for treating rheumatoid arthritis.

The application provides the use of a compound of formula I in the manufacture of a medicament for the treatment of asthma.

The present application provides the use of a compound as described above for the treatment of an inflammatory and/or autoimmune disorder.

The present application provides the use of a compound as described above for the treatment of rheumatoid arthritis.

The present application provides the use of a compound as described above for the treatment of asthma.

The present application provides a compound as described above for use in the treatment of an inflammatory and/or autoimmune disorder.

The present application provides a compound as described above for use in the treatment of rheumatoid arthritis or asthma.

The present application provides compounds, methods, or compositions as described herein.

Compounds and preparation

Examples of typical compounds encompassed by the present invention within the scope of the present invention are provided in the following table. The following examples and preparations are provided so that those skilled in the art may more clearly understand and practice the present invention. They are merely illustrative and representative of the present invention and should not be considered as limiting the scope of the invention.

Generally, the nomenclature used in this application is based on AUTONOMTM v.4.0, a Beilstein Institute computer system used to generate IUPAC system nomenclature. The shown structure is weighted higher if there is a discrepancy between the structure and the name given to the structure. In addition, a structure or a portion of a structure is to be construed as including all stereoisomers of it if the stereochemistry of the structure or portion of the structure is not indicated, for example, by bold or dashed lines.

Table I describes some examples of compounds of general formula I:

TABLE I

General synthetic scheme

The compounds of the present invention may be prepared by any conventional method. Suitable methods for synthesizing these compounds are provided in the examples. In general, the compounds of the present invention can be prepared according to the following scheme.

Scheme 1

In the first step of the synthesis procedure, commercially available 2-bromothiazole-4-carbonitrile 2 is mixed with an appropriately substituted piperidine derivative such as piperidin-3-yl-carbamic acid tert-butyl ester 3 to form a 2-piperidinyl substituted thiazole derivative of formula 4. Coupling of the piperidine cyclic secondary amine to the 2-bromothiazole can be accomplished under palladium (0) catalysis in the presence of a suitable phosphine ligand that can promote efficient cross-coupling between the two organic substrates. Many variations of reaction conditions effective to couple the heteroaromatic halide and the amine are known in the chemical literature (chem.sci.,2011,2,27) and include variations in the stoichiometry and concentration of the palladium (0) catalyst precursor, phosphine ligand, base, reaction temperature, solvent, reagents. Preferred conditions for accomplishing the coupling of 2-bromothiazole 2 and substituted piperidine 3 are to use tris- (dibenzylideneacetone) dipalladium (0) (CAS number 51364-51-3) as the palladium (0) source, 2- (dicyclohexylphosphino) -3, 6-dimethoxy-2 ', 4', 6 '-tri-isopropyl-1, 1' -biphenyl (BrettPhos, CAS number 1070663-78-3) as the phosphine ligand, cesium carbonate as the base and to carry out the reaction in t-butanol heated to 95 ℃.

In the second step of the synthesis process, one bromine atom is introduced into the 5-position of the thiazole ring in compound 4. The preferred conditions for accomplishing this conversion are the use of a slight stoichiometric excess of N-bromosuccinimide in DMF. Under these conditions, pure thiazole bromide 5 can be obtained in good yield in a short reaction time. Other bromination conditions using different bromine atom transfer reagents and/or reaction conditions may be equally effective.

In the third step of the synthesis process, thiazole bromide 5 is reacted with an amine to replace the bromine atom with a substituted nitrogen atom and form an amino-substituted thiazole. When the amine involved in the reaction is 2-aminopyridine, the product is 2-aminopyridine substituted thiazole 6. The conversion is preferably catalyzed by palladium (0) in the presence of a suitable phosphine ligand which promotes efficient cross-coupling between the two organic substrates. Many variations of reaction conditions effective to couple the heteroaromatic halide and the amine are known in the chemical literature (chem.sci.,2011,2,27) and include variations in the stoichiometry and concentration of the palladium (0) catalyst precursor, phosphine ligand, base, reaction temperature, solvent, reagents. Preferred conditions for accomplishing the coupling of 5-bromothiazole 5 and 2-aminopyridine are to use tris- (dibenzylideneacetone) dipalladium as the palladium (0) source, 4, 5-bis (diphenylphosphino) -9, 9-dimethylxanthene (Xantphos, CAS No. 161265-03-8) as the phosphine ligand, potassium phosphate as the base and to carry out the reaction in p-dioxane heated to 110 ℃.

In the fourth step of the synthesis procedure, the nitrile at position 4 of the thiazole ring in compound 6 is converted to the primary carboxamide. Various conditions have been reported for the hydrolysis of nitriles to form primary formamides, including the use of aqueous bases or acids. A preferred condition for carrying out this reaction that is compatible with the other functional groups present in the starting material 6 and product 7 is the use of hydrogenation (dimethylphosphonic acid-kP) [ hydrogen bis (dimethylphosphino-kP ] platinum (II) (Parkins catalyst, CAS No. 173416-05-2, J.mol.Catal.A: chem.2000,160, 249; Organic Letters 2007,9,227) in a heated mixture of tetrahydrofuran and water.

To functionalize the exocyclic nitrogen on the piperidine ring, the tert-butoxycarbonyl group was removed in the fifth step of the synthesis. The tert-butoxycarbonyl group is usually removed under acidic conditions. The preferred conditions for removing the tert-butoxycarbonyl group from the exocyclic nitrogen in compound 7 are treatment with a 1:1 mixture of trifluoroacetic acid in dichloromethane at room temperature. Under these reaction conditions, the primary amine 8 is first formed in the form of the corresponding trifluoroacetate salt, which can be converted into the free base in a further step, for example by partitioning between an organic solvent and an aqueous solution of a suitable base, for example sodium bicarbonate. Alternatively, neutralization of 8 can be carried out in situ with functionalization of the primary amine by addition of an organic tertiary amine base such as triethylamine or diisopropylethylamine.

The compounds claimed in the present invention, compounds of formula 1, are prepared by derivatizing the primary amine present in compound 8. If a primary amine is to be converted to an amide, the amine can be reacted with an acylating agent such as an acid chloride in the presence of a tertiary amine base to scavenge the liberated acid, or condensed with a carboxylic acid in the presence of a peptide coupling agent such as O- (benzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (HBTU, CAS number 94790-37-1). Alternatively, primary amines can also be converted to sulfonamides by reaction with sulfonyl chlorides. Alternatively, it is also possible to convert primary amines into ureas, which can be carried out in the following manner: reacting with a preformed isocyanate, by converting the amine to an isocyanate and then reacting with a secondary amine or a reagent comprising a secondary amine, or by reacting with a carbonate comprising two labile groups attached to a carbonyl group to react the amine 8 with the carbonate to first form a reactive carbamate, which can react with a 2 nd equivalent of a primary or secondary amine to form a urea. If the 2 nd equivalent of amine is replaced with an alcohol, the product formed is a carbamate. Alternatively, primary amines can be converted directly to carbamates by reacting the primary amine with a substituted chloroformate or equivalent reagent.

Those skilled in the art of organic chemistry will appreciate that the synthetic procedure shown in scheme 1 is only one of the routes to prepare the compounds claimed herein, and that the reagents and conditions set forth with respect to the conversion reactions involved in the various steps may not be optimal for a particular derivative. It is expected that some of the skilled in the art of organic chemistry will be able to deduce the conditions under which the transformations described in scheme 1 are carried out or find suitable reagents by reference to the examples provided herein or to the appropriate examples disclosed in the chemical literature.

Pharmaceutical compositions and administration

The compounds of the present invention may be formulated into a variety of oral administration dosage forms and carriers. Oral administration can be in the form of tablets, coated tablets, dragees, hard and soft gelatin capsules, solutions, emulsions, syrups or suspensions. The compounds of the present invention are effective when administered by other routes of administration, including continuous (intravenous drip) topical parenteral, intramuscular, intravenous, subcutaneous, transdermal (which may include a permeation enhancer), buccal, nasal, inhalation, and suppository administration. The preferred mode of administration is generally oral administration using a convenient daily dosing regimen which may be adjusted according to the degree of affliction and the patient's response to the active ingredient.

One or more compounds of the present invention, and pharmaceutically acceptable salts thereof, may be formulated with one or more conventional excipients, carriers or diluents into pharmaceutical compositions and unit dosage forms. The pharmaceutical compositions and unit dosage forms can contain conventional ingredients in conventional proportions, with or without additional active compounds or ingredients, and the unit dosage form can contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be employed. The pharmaceutical compositions may be applied in the form of solids such as tablets or filled capsules, semisolids, powders, sustained release formulations, or liquids such as solutions, suspensions, emulsions, elixirs, or filled capsules for oral use; or may be in the form of suppositories for rectal or vaginal administration; or may be applied in the form of a sterile injectable solution for parenteral application. Typical formulations will contain from about 5% to about 95% of one or more active compounds (w/w). The term "formulation" or "dosage form" is intended to include both solid and liquid formulations of the active compound, and one skilled in the art will appreciate that the active ingredient may be present in different formulations depending on the target organ or tissue and the desired dosage and pharmacokinetic parameters.

The term "excipient" as used herein refers to a compound used in the preparation of pharmaceutical compositions, which are generally safe, non-toxic and not biologically or otherwise undesirable, and include veterinary as well as human pharmaceutical excipients. The compounds of the invention may be administered alone, but will generally be administered in admixture with one or more suitable pharmaceutical excipients, diluents or carriers selected with regard to the desired route of administration and standard pharmaceutical practice.

"pharmaceutically acceptable" means useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and not biologically or otherwise undesirable, and includes materials that are acceptable for veterinary use as well as for human pharmaceutical use.

The "pharmaceutically acceptable salt" form of the active ingredient may firstly confer the desired pharmacokinetic properties not present in the non-salt form of the active ingredient and may even have a positive effect on the pharmacodynamics of the active ingredient with respect to its therapeutic activity in vivo. The phrase "pharmaceutically acceptable salt" of a compound means a salt that is pharmaceutically acceptable and that possesses the pharmacological activity of the desired parent compound. Such salts include (1) acid addition salts with inorganic or organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3- (4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1, 2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo [2.2.2] -oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, laurylsulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or (2) salts formed when the acidic proton present in the parent compound is replaced by a metal ion, for example an alkali metal ion, an alkaline earth metal ion or an aluminium ion; or salts of acidic protons present in the parent compound with organic bases such as ethanolamine, diethanolamine, triethanolamine, aminotributol, N-methylglucamine, and the like.

Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances which also act as diluents, flavoring agents, stabilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material. In powders, the carrier is usually a finely divided solid which is a mixture with the finely divided active component. In tablets, the active ingredient is usually mixed with a carrier having the desired binding capacity in suitable proportions and compacted in the shape and size desired. Suitable carriers include, without limitation, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. Formulations in solid form may contain, in addition to the active ingredient, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.

Liquid preparations are also suitable for oral administration, including emulsions, syrups, elixirs, aqueous solutions, and aqueous suspensions. These include solid form preparations which are intended to be converted, shortly before use, to liquid form preparations. Emulsions may be prepared in solution, for example, in aqueous propylene glycol, or may contain emulsifying agents such as lecithin, sorbitan monooleate, or acacia. Aqueous solutions can be prepared by dissolving the active ingredient in water and adding suitable colorants, flavors, stabilizers, and thickening agents. Aqueous suspensions may be prepared by dispersing the finely divided components in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents.

The compounds of the invention may be formulated for parenteral administration (e.g., by injection, such as bolus injection or continuous infusion) and may be presented in unit dosage form in ampoules, pre-filled syringes, small volume infusion containers or in multi-dose containers with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous bases, for example, solutions in aqueous polyethylene glycol. Examples of oily or nonaqueous carriers, diluents, solvents or bases include propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate), and which may contain formulatory agents such as preservatives, wetting, emulsifying or suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form, for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use, by isolation of a sterile solid or by lyophilization of solution.

The compounds of the invention may be formulated for topical administration to the epidermis as an ointment, cream or lotion, or as a transdermal patch. For example, ointments and creams may be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents. Formulations suitable for topical administration to the oral cavity include: lozenges comprising the active agent in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

The compounds of the invention may be formulated for administration as suppositories. The low melting wax (e.g. a mixture of fatty acid glycerides or cocoa butter) is first melted and the active ingredient is then dispersed homogeneously, for example by stirring. The molten homogeneous mixture is then poured into a suitably sized mold, allowed to cool and solidify.

The compounds of the invention may be formulated for vaginal administration. Suitable formulations are pessaries, tampons, creams, gels, pastes, foams or sprays containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

The compounds of the present invention may be formulated for intranasal administration. The solution or suspension is applied directly to the nasal cavity by conventional means, such as with a dropper, pipette or spray. The formulations may be provided in single or multiple dose forms. In the case of multiple doses in a dropper or pipette, administration is accomplished by administering to the patient an appropriate, predetermined volume of solution or suspension. In the case of a spray, administration is accomplished by means of, for example, a metered spray pump.

The compounds of the invention may be formulated for aerosol administration, particularly for use in the respiratory tract, and include intranasal administration. The compounds typically have a very small particle size, for example five (5) microns or less. Such particle sizes may be obtained in a manner known in the art, for example, by micronization. The active ingredient is provided in pressurized packs with a suitable propellant, such as a chlorofluorocarbon (CFC), for example dichlorodifluoromethane, trichlorofluoromethane or dichlorotetrafluoroethane, or carbon dioxide or other suitable gas. The aerosol may also conveniently contain a surfactant such as lecithin. The dosage of the drug may be controlled by a metering valve. Alternatively, the active ingredient may be in the form of a dry powder, for example a powder mix of the compound in a suitable powder base such as lactose, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidone (PVP). The powder carrier can form a gel within the nasal cavity. The powder composition may be in unit dosage form, such as a capsule or cartridge of gelatin or an aluminium-plastic package from which the powder can be administered using an inhaler.

When desired, the formulations can be prepared with enteric coatings suitable for sustained or controlled release administration of the active ingredient. For example, the compounds of the present invention may be formulated into transdermal or subcutaneous drug delivery devices. These delivery systems are advantageous when sustained release of the compound is necessary and patient compliance with the treatment regimen is critical. The compounds in transdermal delivery systems are typically attached to a skin-adherent solid carrier. The compound of interest may also be combined with a permeation enhancer, such as azone (1-dodecylaza-cycloheptan-2-one). The sustained release delivery system is implanted into the subcutaneous layer by surgery or injection. Subcutaneous implants embed compounds in a lipid-soluble membrane (e.g., silicone rubber) or a biodegradable polymer (e.g., polylactic acid).

Suitable formulations are described in Remington, The Science and Practice of Pharmacy 1995, edited by E.W. Martin, Mack Publishing Company, 19 th edition, Easton, Pennsylvania, together with pharmaceutically acceptable carriers, diluents and excipients. The skilled formulator can, under the teaching of this specification, modify such formulations to provide a wide variety of formulations for a particular route of administration without destabilizing or compromising the therapeutic activity of the compositions of the present invention.

For example, the compounds of the invention may be modified to render them more soluble in water or other vehicles by minor modifications (salt formation, esterification, etc.) well known to those skilled in the art. It is also well known to those skilled in the art that the route of administration and dosage regimen of a particular compound can be varied to control the pharmacokinetics of the compounds of the present invention in order to maximize their beneficial effects in the patient.

The term "therapeutically effective amount" as used herein means the amount required to alleviate the symptoms of a disease in an individual. The dosage will be adjusted according to the individual needs of each particular case. The dosage may vary widely depending on a number of factors, such as the severity of the disease being treated, the age and general health of the patient, the other drugs being used by the patient being treated, the route and form of administration, and the preferences and experience of the physician involved. For oral administration, a daily dose of about 0.01 to about 1000mg/kg body weight/day will be appropriate in monotherapy and/or in combination therapy. Preferred daily dosages are from about 0.1 to about 500mg/kg body weight per day, more preferably from 0.1 to about 100mg/kg body weight, and most preferably from 1.0 to about 10mg/kg body weight. Thus, for administration to a human of 70kg body weight, the dosage will range from about 7mg to 0.7g per day. The daily dose may be administered in a single dose or in divided doses, typically 1 to 5 doses per day. Treatment is usually initiated with smaller doses than the optimal dose of the compound. Thereafter, the dose is increased in small increments until the optimum effect is achieved for each patient. One of ordinary skill in the art of treating the diseases described herein will be able to determine, without undue experimentation, a therapeutically effective amount of a compound of the invention for a given disease and patient, depending on personal knowledge, experience, and disclosure of the application.

The pharmaceutical preparation is preferably in unit dosage form. In such forms, the formulations are subdivided into unit doses containing appropriate quantities of the active ingredient. The unit dosage form may be a packaged preparation, the package containing a discrete quantity of the preparation, for example, packaged tablets, capsules, and powders in vials or ampoules. The unit dosage form can also be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of the above in packaged form.

Indications and treatment methods

The compounds of formula I inhibit bruton's tyrosine kinase (Btk). Activation of Btk by upstream kinases results in the activation of phosphatase-C γ, which in turn stimulates the release of pro-inflammatory mediators. The compounds of formula I are useful in the treatment of arthritis and other anti-inflammatory and autoimmune diseases. Accordingly, the compounds of formula I are useful in the treatment of arthritis. The compounds of formula I are useful for inhibiting Btk in cells and for modulating B-cell development. The invention further comprises a pharmaceutical composition comprising a compound of formula I and a pharmaceutically acceptable carrier, excipient or diluent.

The compounds described herein are kinase inhibitors, particularly Btk inhibitors. These inhibitors are useful for treating one or more diseases in a mammal that respond to inhibition of kinases, including diseases that respond to inhibition of Btk and/or inhibition of B-cell proliferation. Without wishing to be bound by any particular theory, it is believed that the interaction of the compounds of the present invention with Btk results in the inhibition of Btk activity and thus in the pharmaceutical use of these compounds. Accordingly, the invention includes a method of treating a mammal, e.g., a human, having a disease responsive to inhibition of Btk activity and/or inhibition of B-cell proliferation, comprising administering to a mammal having such a disease an effective amount of at least one chemical entity provided herein. The effective concentration may be determined experimentally, for example by measuring the blood concentration of the compound, or may be determined theoretically by calculating bioavailability. In addition to Btk, other kinases that may be affected include, without limitation, other tyrosine kinases and serine/threonine kinases.

Kinases play an important role in the signaling pathways that control basic cellular processes such as proliferation, differentiation and death (apoptosis). Abnormal kinase activity has been shown to be involved in a number of diseases including various cancers, autoimmune and/or inflammatory diseases, and acute inflammatory responses. The multifaceted role of kinases in key cellular signaling pathways provides an important opportunity to identify new drugs that target kinases and signaling pathways.

One embodiment includes a method of treating a patient suffering from an autoimmune and/or inflammatory disease, or an acute inflammatory response responsive to inhibition of Btk activity and/or B-cell proliferation.

Autoimmune and/or inflammatory diseases that may be affected by the compounds and compositions of the present invention include, without limitation, psoriasis, allergies, Crohn's disease, irritable bowel syndrome, Sjogren's disease, tissue transplant rejection and hyperacute rejection of transplanted organs, asthma, systemic lupus erythematosus (and associated glomerulonephritis), dermatomyositis, multiple sclerosis, scleroderma, vasculitis (ANCA-associated and other vasculitis), autoimmune hemolysis and thrombocytopenia states, Goodpasture's syndrome (and associated glomerulonephritis and pulmonary hemorrhage), atherosclerosis, rheumatoid arthritis, chronic Idiopathic Thrombocytopenic Purpura (ITP), Addison's disease, Parkinson's disease, Alzheimer's disease, diabetes, septic shock and myasthenia gravis.

Included herein are methods of treatment wherein at least one chemical entity provided herein is administered in combination with an anti-inflammatory agent. Anti-inflammatory agents include, but are not limited to, NSAIDs, non-specific and COX-2 specific cyclooxygenase inhibitors, gold compounds, corticosteroids, methotrexate, tumor necrosis factor receptor (TNF) antagonists, immunosuppressive agents, and methotrexate.

Examples of NSAIDs include, without limitation, ibuprofen, flurbiprofen, naproxen and naproxen sodium, diclofenac, a combination of diclofenac sodium and misoprostol, sulindac, oxaprozin, diflunisal, piroxicam, indomethacin, etodolac, fenoprofen calcium, ketoprofen, naproxone sodium, sulfasalazine, tolytidine sodium acetate, and hydroxychloroquine. Examples of NSAIDs also include COX-2 specific inhibitors such as celecoxib, valdecoxib, lumiracoxib and/or etoricoxib.

In some embodiments, the anti-inflammatory agent is a salicylate. Salicylates include, but are not limited to, acetylsalicylic acid or aspirin, sodium salicylate, and choline and magnesium salicylate.

The anti-inflammatory agent may also be a corticosteroid. For example, the corticosteroid can be cortisone, dexamethasone, methylprednisolone, prednisolone sodium phosphate, or prednisone.

In other embodiments, the anti-inflammatory agent is a gold compound such as gold sodium thiomalate or auranofin.

The invention also includes embodiments wherein the anti-inflammatory agent is a metabolic inhibitor such as a dihydrofolate reductase inhibitor, e.g., methotrexate or a dihydroorotate dehydrogenase inhibitor, e.g., leflunomide.

Other embodiments of the invention relate to combinations wherein at least one anti-inflammatory compound is an anti-C5 monoclonal antibody (such as eculizumab or peclizumab), a TNF antagonist such as etanercept, or infliximab (which is an anti-TNF α monoclonal antibody).

Further embodiments of the present invention relate to further combinations wherein at least one active agent is an immunosuppressive compound such as an immunosuppressive compound selected from the group consisting of methotrexate, leflunomide, cyclosporine, tacrolimus, azathioprine and mycophenolate mofetil.

Btk-expressing B-cells and B-cell precursors have been shown to be involved in the pathology of B-cell malignancies, including, without limitation, B-cell lymphomas, lymphomas (including Hodgkin's and non-Hodgkin's lymphomas), hairy cell lymphomas, multiple myeloma, chronic and acute myelogenous leukemias, and chronic and acute lymphocytic leukemias.

BTK has been shown to be an inhibitor of the Fas/APO-1(CD-95) death-inducing signaling complex (DISC) in B-lineage lymphocytes. The fate of leukemia/lymphoma cells may lie in the balance between the anti-pro-apoptotic action of caspases activated by DISC and upstream anti-apoptotic regulatory mechanisms involved in BTK and/or its substrates (Vassilev et al, j.biol.chem.1998,274, 1646-1656).

It has also been found that BTK inhibitors can be used as chemosensitizers and, therefore, in combination with other chemotherapeutic agents, particularly agents that induce apoptosis. Examples of other chemotherapeutic agents that may be used in combination with chemosensitizing BTK inhibitors include topoisomerase I inhibitors (camptothecin or topotecan), topoisomerase II inhibitors (e.g., daunomycin and etoposide), alkylating agents (e.g., cyclophosphamide, melphalan, and BCNU), agents active against tubulin (e.g., paclitaxel and vinblastine), and biologically active agents (e.g., antibodies such as anti-CD 20 antibodies, IDEC 8, immunotoxins, and cytokines).

Btk activity has been shown to be associated with some leukemias that express the bcr-abl fusion gene resulting from translocation of parts of chromosomes 9 and 22. This abnormality is commonly observed in chronic myelogenous leukemia. Btk is structurally phosphorylated by bcr-abl kinase, which initiates downstream survival signals that prevent apoptosis in bcr-abl cells. (N.Feldhahn et al, J.exp.Med.2005201 (11): 1837-1852).

Method of treatment

The present application provides a method of treating an inflammatory disorder comprising administering to a patient in need thereof a therapeutically effective amount of a compound of formula I.

The present application provides a method of treating asthma comprising administering to a patient in need thereof a therapeutically effective amount of formula I.

The present application provides a method of treating an inflammatory and/or autoimmune disorder comprising administering to a patient in need thereof a therapeutically effective amount of a Btk inhibitor compound of formula I.

The present application provides a method of treating arthritis comprising administering to a patient in need thereof a therapeutically effective amount of a Btk inhibitor compound of formula I.

The present application provides a method of treating asthma comprising administering to a patient in need thereof a therapeutically effective amount of a Btk inhibitor compound of formula I.

The present application provides a method of inhibiting B-cell proliferation comprising administering to a patient in need thereof a therapeutically effective amount of a Btk inhibitor compound of formula I.

The present application provides a method of inhibiting Btk activity comprising administering any of the Btk inhibitor compounds of formula I, wherein the Btk inhibitor compound exhibits an IC of 50 micromolar or less in an in vitro biochemical assay of Btk activity₅₀。

In a variation of the above method, the Btk inhibitionThe agent compounds exhibit an IC of 100 nanomolar or less in an in vitro biochemical assay of Btk activity₅₀。

In another variation of the above method, the compound exhibits an IC of 10 nanomolar or less in an in vitro biochemical assay of Btk activity₅₀。

The present application provides a method of treating an inflammatory disorder comprising co-administering to a patient in need thereof a therapeutically effective amount of an anti-inflammatory compound in combination with a Btk inhibitor compound of formula I.

The present application provides a method of treating arthritis comprising co-administering to a patient in need thereof a therapeutically effective amount of an anti-inflammatory compound in combination with a Btk inhibitor compound of formula I.

The present application provides a method of treating lymphoma or BCR-ABL1 by administering to a patient in need thereof a therapeutically effective amount of a Btk inhibitor compound of formula I⁺Methods of leukemia cells.

Examples

General abbreviations

Common abbreviations include acetyl (Ac), Azobisisobutyronitrile (AIBN), atmospheric pressure (Atm), 9-borabicyclo [3.3.1]Nonane (9-BBN or BBN), 2 '-bis (diphenylphosphino) -1,1' -Binaphthyl (BINAP), tert-butoxycarbonyl (Boc), di-tert-butyl pyrocarbonate or Boc anhydride (BOC)₂O), benzyl (Bn), butyl (Bu), chemical Abstract registry number (CASRN), benzyloxycarbonyl (CBZ or Z), Carbonyldiimidazole (CDI), 1, 4-diazabicyclo [2.2.2]Octane (DABCO), diethylaminosulfur trifluoride (DAST), dibenzylideneacetone (dba), 1, 5-diazabicyclo [4.3.0]Non-5-ene (DBN), 1, 8-diazabicyclo [5.4.0]Undec-7-ene (DBU), N' -Dicyclohexylcarbodiimide (DCC), 1, 2-Dichloroethane (DCE), Dichloromethane (DCM), 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone (DDQ), diethyl azodicarboxylate (DEAD), diisopropyl azodicarboxylate (DIAD), di-isobutyl aluminum hydride (DIBAL or DIBAL-H), di-isopropylEthylamine (DIPEA), N-Dimethylacetamide (DMA), 4-N, N-Dimethylaminopyridine (DMAP), N-Dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1 '-bis- (diphenylphosphino) ethane (dppe), 1' -bis- (diphenylphosphino) ferrocene (dppf), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI), 2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline (EEDQ), ethyl (Et), ethyl acetate (EtOAc), ethanol (EtOH), ethyl 2-ethoxy-2H-quinoline-1-carboxylate (EEDQ), diethyl ether (Et)₂O), ethyl isopropyl ether (EtOiPr), O- (7-azabenzotriazol-1-yl) -N, N, N 'N' -tetramethyluronium Hexafluorophosphate (HATU), acetic acid (HOAc), 1-N-hydroxybenzotriazole (HOBt), High Pressure Liquid Chromatography (HPLC), isopropyl alcohol (IPA), isopropyl magnesium chloride (iPrMgCl), Hexamethyldisilazane (HMDS), Liquid Chromatography Mass Spectrometry (LCMS), lithium hexamethyldisilazane (LiHMDS), m-chloroperoxybenzoic acid (m-CPBA), methanol (MeOH), melting point (mp), MeSO₂- (methylsulfonyl or Ms), methyl (Me), acetonitrile (MeCN), m-chloroperbenzoic acid (MCPBA), mass spectrometry (Ms), methyl tert-butyl ether (MTBE), methyltetrahydrofuran (MeTHF), N-bromosuccinimide (NBS), N-butyllithium (nBuLi), N-carboxyanhydride (NCA), N-chlorosuccinimide (NCS), N-methylmorpholine (NMM), N-methylpyrrolidone (NMP), pyridinium chlorochromate (PCC), dichloro- ((di-diphenylphosphino) ferrocenyl) palladium (II) (Pd (dppf) Cl₂) Palladium (II) acetate (Pd (OAc)₂) Tris (dibenzylideneacetone) dipalladium (0) (Pd)₂(dba)₃) Pyridine Dichromate (PDC), phenyl (Ph), propyl (Pr), iso-propyl (i-Pr), pounds per square inch (psi), pyridine (pyr), 1,2,3,4, 5-pentaphenyl-1' - (di-tert-butylphosphino) ferrocene (Q-Phos), room temperature (ambient temperature, RT or RT), sec-butyl lithium (sBuLi), tert-butyldimethylsilyl, or t-BuMe₂Si (TBDMS), tetra-n-butylammonium fluoride (TBAF), triethylamine (TEA or Et)₃N), 2,6, 6-tetramethylpiperidine 1-oxyl (TEMPO), trimethylsilylethoxymethyl (SEM), triflate or CF₃SO₂- (Tf), trifluoroacetic acid (TFA), 1' -di-2, 2,6, 6-tetramethylheptane-2, 6-dione (TMHD), O-benzotriazol-1-yl-N, N, N ', N ' -tetramethyluronium tetrafluoroborate (TBTU), Thin Layer Chromatography (TLC), Tetrahydrofuran (THF), Trimethylammoniumtetrafluorosilane (TMDA), trimethylammoniumtSilyl or Me₃Si (TMS), p-toluenesulfonic acid monohydrate (TsOH or pTsOH), 4-Me-C₆H₄SO₂-or tosyl (Ts), N-urethane-N-carboxy anhydride (UNCA). When used with alkyl moieties, the conventional nomenclature, including the prefixes n (n), iso (i-), secondary (sec-), tertiary (t-), and neo, has its conventional meaning. (J.Rigaudy and D.P.Klesney, Nomenclature in Organic Chemistry, IUPAC 1979Pergamon Press, university of Oxford).

General conditions

The compounds of the present invention can be prepared starting from commercially available starting materials using general synthetic techniques and procedures known to those skilled in the art. The following summary is a reaction scheme suitable for preparing such compounds. Further exemplifications may be found in the specific embodiments.

Specific abbreviations

boc tert-butoxycarbonyl

CH₂Cl₂Methylene dichloride

Cs₂CO₃Cesium carbonate

DCM dichloromethane

DMF N, N-dimethylformamide

DMSO dimethyl sulfoxide

EtOAc ethyl acetate

HATU O- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethyl

Urea hexafluorophosphates

Hunig's base N, N-diisopropylethylamine

HCl hydrogen chloride

LC-MS liquid chromatography-mass spectrometry

HPLC high pressure liquid chromatography

MeOH methanol

MgSO₄Magnesium sulfate

nBuLi n-butyllithium

NaCl sodium chloride

Na₂CO₃Sodium carbonate

NaOMe sodium methoxide

Na₂SO₄Sodium sulfate

NH₄OH ammonium hydroxide

NMP 1-methyl-2-pyrrolidone

NMR nuclear magnetic resonance

Pd(OAc)₂Palladium acetate (II)

TFA trifluoroacetic acid

THF tetrahydrofuran

TLC thin layer chromatography

TMSCl trimethylchlorosilane

General experimental details

Reagents were purchased from Aldrich, Oakwood, Matrix or other suppliers and used without further purification. The reaction heated by microwave irradiation is carried out using a Personal Chemistry Emrys Optimizer System or CEMDiscovery System. Purification on the microgram scale to the gram scale is carried out by methods known to those skilled in the art, such as elution on silica gel flash columns; in some cases, preparative flash column purification using a disposable prepacked gram-grade silica gel column (RediSep) was also performed, eluting with the Combiflash system. Biotage^TMAnd ISCO^TMIs also a rapid column apparatus useful in the present invention for purifying intermediates.

To identify the identity and purity of the compound, LC/MS (liquid chromatography/mass spectrometry) spectra were recorded using the following system. For mass spectrometry measurements, the system was operated with a Micromass Platform II spectrometer with ES ionization in positive ion mode (mass range: 150-. Simultaneous chromatographic separation is achieved using an HPLC system employing ES Industries Chromegabond WRC-183 u(3.2x30mm) short column; mobile phase a water (0.02% TFA) and mobile phase B acetonitrile (0.02% TFA); gradient 10% B to 90% B, 3 min; equilibration time 1 minute; the flow rate was 2 mL/min.

Many of the compounds of formula 1 are also purified by reverse phase HPLC using methods known to those skilled in the art. In some cases, preparative HPLC purification was performed using PE Sciex 150EX Mass Spec, which controls a Gilson 215 trap connected to a Shimadzu preparative HPLC system and a Leap autoinjector. Compounds were collected from the elution stream using LC/MS detection (positive ion detection) using solvent (A) 0.05% TFA/H over 10 min₂Appropriate linear gradient profile of O and solvent (B) 0.035% TFA/acetonitrile Compound was eluted from a C-18 column (2.0X 10cm, elution 20 mL/min). For injection onto the HPLC system, the crude sample was dissolved in a mixture of methanol, acetonitrile and DMSO.

Passing the compound through¹H-NMR was characterized using a Bruker 400MHz NMR spectrometer.

The compounds of the invention can be synthesized according to known techniques. The following examples and references are provided to aid in the understanding of the present invention. The examples are not intended, however, to be limiting and the true scope of the invention is indicated by the appended claims. The name of the final product in the examples was generated using Isis AutoNom 2000.

Preparation examples

Example 1

{ (R) -1- [ 4-carbamoyl-5- (pyridin-2-ylamino) -thiazol-2-yl ] -piperidin-3-yl } -carbamic acid tert-butyl ester

Step 1 preparation of [ (R) -1- (4-cyano-thiazol-2-yl) -piperidin-3-yl ] -carbamic acid tert-butyl ester

To a 500mL round bottom flask was added (R) -piperidin-3-yl-carbamic acid tert-butyl ester (10.3g,51.6mmol), cesium carbonate (33.6g,103mmol), 2- (dicyclohexylphosphino) -3, 6-dimethoxy-2 ', 4', 6 '-tri-isopropyl-1, 1' -biphenyl (Brettphos, CAS No. 1070663-78-3) (369mg, 688. mu. mol), and 2-bromothiazole-4-carbonitrile (6.5g,34.4mmol), followed by tert-butanol (225 mL). The mixture was degassed in vacuo (-50 mmHg) while sonicating, and then the flask was purged with argon. Degassing was repeated twice. Tris- (dibenzylideneacetone) dipalladium (0) (CAS number 51364-51-3) (315mg,344 μmol) was added, the flask evacuated and refilled with argon. This was repeated twice. The reaction flask was transferred to an aluminum block heating mantle and heated to reflux under argon for 5 hours. The reaction mixture was cooled to room temperature and poured into water (1L), extracted with ethyl acetate (3 × 200mL), the combined organic extracts were washed with brine (2 × 200mL), dried over sodium sulfate, filtered and concentrated in vacuo. The residue was chromatographed on 330g silica gel, eluting with a gradient of 10% to 40% ethyl acetate in hexane at a flow rate of 100 mL/min. Fractions containing the product were combined and concentrated in vacuo to afford [ (R) -1- (4-cyano-thiazol-2-yl) -piperidin-3-yl ] -carbamic acid tert-butyl ester as a beige solid (8.75g, 78%).

LC/MS calculated m/z C₁₄H₂₀N₄O₂S([M+H-Boc]⁺):209.3. Found 209.1 (positive ion mode electrospray ionization).

Step 2 preparation of [ (R) -1- (5-bromo-4-cyano-thiazol-2-yl) -piperidin-3-yl ] -carbamic acid tert-butyl ester

To a solution of [ (R) -1- (4-cyano-thiazol-2-yl) -piperidin-3-yl ] -carbamic acid tert-butyl ester (13.0g,42.2mmol) in N, N-dimethylformamide (250mL) was added N-bromosuccinimide (8.25g,46.mmol) and the mixture was stirred at room temperature for 30 minutes under argon atmosphere. Thin layer chromatography (silica gel stationary phase eluting with 25% v/v ethyl acetate in hexane) showed that [ (R) -1- (4-cyano-thiazol-2-yl) -piperidin-3-yl ] -carbamic acid tert-butyl ester was clearly converted to the less polar compound. The reaction mixture was poured into water (1L) and extracted with ethyl acetate (3x 250mL), the combined organic extracts were washed with brine (2x 200mL), dried over sodium sulfate, filtered and concentrated in vacuo to give a beige solid. The crude product was triturated with 1:1v/v diethyl ether/petroleum ether (300mL total volume). Filtration afforded [ (R) -1- (5-bromo-4-cyano-thiazol-2-yl) -piperidin-3-yl ] -carbamic acid tert-butyl ester as a colorless solid, washed with 1:2v/v diethyl ether/petroleum ether and dried under air (14.33g, 88%). The residue from concentrating the mother liquor was purified by column chromatography on silica gel eluting with a gradient of 5% to 40% v/v ethyl acetate in hexane to give additional [ (R) -1- (5-bromo-4-cyano-thiazol-2-yl) -piperidin-3-yl ] -carbamic acid tert-butyl ester. The product containing fractions were combined and concentrated in vacuo to afford additional [ (R) -1- (5-bromo-4-cyano-thiazol-2-yl) -piperidin-3-yl ] -carbamic acid tert-butyl ester as a colorless solid (1.18g, 7%).

LC/MS calculated m/z C₁₄H₁₉BrN₄O₂S([M+H-Boc]⁺):288.2. Found 287 and 289(1:1 ratio, consistent with the presence of 1 bromine atom) (positive ion mode electrospray ionization).

Step 3 preparation of { (R) -1- [ 4-cyano-5- (pyridin-2-ylamino) -thiazol-2-yl ] -piperidin-3-yl } -carbamic acid tert-butyl ester

A pressurized reaction vessel with a threaded port was evacuated and filled with argon. To a reaction vessel were added [ (R) -1- (5-bromo-4-cyano-thiazol-2-yl) -piperidin-3-yl ] -carbamic acid tert-butyl ester (1.00g,2.58mmol), 2-aminopyridine (384mg,3.87mmol), 4, 5-bis (diphenylphosphino) -9, 9-dimethylxanthene (Xantphos, CAS number 161265-03-8) (462mmol, 775. mu. mol) and potassium phosphate (1.64g,7.75mmol), followed by p-dioxane (20 mL). The reaction vessel was degassed by sonication under vacuum (-50 mmHg) and then the vessel was refilled with argon. Degassing was repeated twice. Tris- (dibenzylideneacetone) dipalladium (0) (CAS number 51364-51-3) (241mg,258 μmol) was added, the flask evacuated and refilled with argon. This was repeated twice. The vessel was sealed and placed in an oil bath preheated to 110 ℃ for 16 hours. The reaction mixture was cooled to room temperature, poured into water (200mL) and extracted with 1:1v/v ethyl acetate/tetrahydrofuran (3x 100mL), the combined organic extracts were washed with brine (2x75mL), dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by chromatography on silica gel eluting with a gradient from 0% to 10% v/v methanol in dichloromethane. The product containing fractions were combined and concentrated in vacuo to afford the product as a partially purified red/brown solid (429 mg). To the partially purified material was added 2:1v/v isopropanol/ethyl acetate (150mL total volume) and activated carbon was added. After standing at room temperature for 10 minutes, the mixture was filtered with celite filter aid and the filtrate was concentrated in vacuo to give { (R) -1- [ 4-cyano-5- (pyridin-2-ylamino) -thiazol-2-yl ] -piperidin-3-yl } -carbamic acid tert-butyl ester (0.38g, 35%) as a light brown/yellow solid.

LC/MS calculated m/z C₁₉H₂₄N₆O₂S([M+H]⁺):401.5. Found 401.2 (positive ion mode electrospray ionization).

Step 4 preparation of { (R) -1- [ 4-carbamoyl-5- (pyridin-2-ylamino) -thiazol-2-yl ] -piperidin-3-yl } -carbamic acid tert-butyl ester.

A solution of { (R) -1- [ 4-cyano-5- (pyridin-2-ylamino) -thiazol-2-yl ] -piperidin-3-yl } -carbamic acid tert-butyl ester (455mg,1.14mmol) in 10:1v/v tetrahydrofuran/water (16.5mL total volume) was degassed by bubbling argon under stirring for 10 minutes. Hydrogenation (dimethylphosphonic acid-kP) [ hydro bis (dimethylphosphino-kP ] platinum (II) (II) (CAS number 173416-05-2) (78mg, 183. mu. mol) was added and the mixture was heated by microwave to 150 ℃ for 45 minutes, the reaction mixture was cooled to room temperature, poured into water (150mL) and extracted with 1:1v/v tetrahydrofuran/ethyl acetate (4X 50mL), the combined organic extracts were washed with brine (2X 40mL), dried over sodium sulfate, filtered and concentrated in vacuo to a yellow solid, the crude product was purified by silica gel chromatography, eluted with a gradient of 30% to 70% v/v ethyl acetate in hexane, the fractions containing the product were combined and concentrated in vacuo to give { (R) -1- [ 4-carbamoyl-5- (pyridin-2-ylamino) -thiazol-2-yl ] -piperi ne as a beige solid Pyridin-3-yl } -carbamic acid tert-butyl ester (362mg, 76%).

LC/MS calculated m/z C₁₉H₂₆N₆O₃S([M-H]^-):417.5. Found 417.4 (negative ion mode electrospray ionization).

Example 2

1, 3-dihydro-isoindole-2-carboxylic acid { (R) -1- [ 4-carbamoyl-5- (pyridin-2-ylamino) -thiazol-2-yl ] -piperidin-3-yl } -amide

Step 1 preparation of 2- ((R) -3-amino-piperidin-1-yl) -5- (pyridin-2-ylamino) -thiazole-4-carbonitrile

To a solution of { (R) -1- [ 4-cyano-5- (pyridin-2-ylamino) -thiazol-2-yl ] -piperidin-3-yl } -carbamic acid tert-butyl ester (prepared as described in example 1, step 3) (20.2mg, 50.4. mu. mol) in dichloromethane (0.5mL) was added trifluoroacetic acid (1mL,13mmol) and the mixture was stirred at room temperature for 30 minutes under argon atmosphere. The reaction mixture was concentrated in vacuo and the crude 2- ((R) -3-amino-piperidin-1-yl) -5- (pyridin-2-ylamino) -thiazole-4-carbonitrile was used in the next synthetic step without further purification.

LC/MS calculated m/z C₁₄H₁₆N₆S([M+H]⁺):301.4. Found 301.2 (positive ion mode electrospray ionization).

Step 2 preparation of { (R) -1- [ 4-cyano-5- (pyridin-2-ylamino) -thiazol-2-yl ] -piperidin-3-yl } -carbamic acid pentafluorophenyl ester

To a solution of crude 2- ((R) -3-amino-piperidin-1-yl) -5- (pyridin-2-ylamino) -thiazole-4-carbonitrile (. about.15 mg,. about.50. mu. mol) in tetrahydrofuran (1mL) at 0 ℃ under argon was added diisopropylethylamine (18. mu.L, 100. mu. mol) followed by di-pentafluorophenyl carbonate (22mg, 55. mu. mol). The reaction mixture was slowly warmed to room temperature and stirring was continued for 1 hour. Samples were taken for LC-MS analysis to ensure completion of the reaction before the reaction mixture was used for the subsequent synthesis step.

LC/MS calculated m/z C₂₁H₁₅F₅N₆O₂S([M+H]⁺):511.5. Found 511.2 (positive ion mode electrospray ionization).

Step 3 preparation of 1, 3-dihydro-isoindole-2-carboxylic acid { (R) -1- [ 4-cyano-5- (pyridin-2-ylamino) -thiazol-2-yl ] -piperidin-3-yl } -amide

A solution of crude { (R) -1- [ 4-cyano-5- (pyridin-2-ylamino) -thiazol-2-yl ] -piperidin-3-yl } -carbamic acid pentafluorophenyl ester (. about.50. mu. mol) in tetrahydrofuran (1mL) was cooled to 0 ℃ under argon and isoindoline (12.6mg, 106. mu. mol) and diisopropylethylamine (20. mu.L, 115. mu. mol) were added. The reaction mixture was stirred at 0 ℃ for 1 hour, then warmed to room temperature and stirred for an additional 1 hour. The reaction was quenched by the addition of saturated aqueous ammonium chloride (2mL), poured into water (10mL) and extracted with ethyl acetate (4 × 5 mL). The combined organic extracts were washed with brine (2 × 5mL), dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by reverse phase chromatography using a 12g C-18 column eluting with a gradient of 10% acetonitrile in water to 100% acetonitrile. The product containing fractions were combined and concentrated in vacuo to a yellow solid. To the yellow solid was added dichloromethane, dried over sodium sulfate, filtered and concentrated in vacuo to afford 1, 3-dihydro-isoindole-2-carboxylic acid { (R) -1- [ 4-cyano-5- (pyridin-2-ylamino) -thiazol-2-yl ] -piperidin-3-yl } -amide (11.3mg, 50%) as a yellow viscous oil.

LC/MS calculated m/z C₂₃H₂₃N₇OS([M+H]⁺):446.6. Found 446.3 (electrospray ionization in positive ion mode).

Step 4 preparation of 1, 3-dihydro-isoindole-2-carboxylic acid { (R) -1- [ 4-carbamoyl-5- (pyridin-2-ylamino) -thiazol-2-yl ] -piperidin-3-yl } -amide

To a 10:1v/v tetrahydrofuran/water (2.75mL total volume) solution of 1, 3-dihydro-isoindole-2-carboxylic acid { (R) -1- [ 4-cyano-5- (pyridin-2-ylamino) -thiazol-2-yl ] -piperidin-3-yl } -amide (11.3mg, 25.4. mu. mol) was added hydrogenation (dimethylphosphonic acid-kP) [ hydrogen bis (dimethylphosphino-kP ] platinum (II) (CAS number 173416-05-2) (1.1mg, 2.5. mu. mol) and the mixture was heated to reflux under argon atmosphere after 8 hours, LC/MS showed about-30% conversion to a single main product, showing the molecular ion of the desired product II) (1mg, 2.3. mu. mol) and the tube was heated at 80 ℃ overnight. LC-MS showed the expected product to be the major component in the reaction mixture, but some starting material was still present. Add one additional portion of hydrogenated (dimethylphosphonic acid-kP) [ Hydrobis (dimethylphosphino-kP ] platinum (II) (1mg, 2.3. mu. mol) and heat the reaction vessel at 100 ℃ for 3.5 h, cool the reaction mixture to room temperature and concentrate in vacuo, purify the residue by reverse phase chromatography on a C-18 column, eluting with a gradient of 10% to 100% acetonitrile in water, combine the fractions containing the product and concentrate in vacuo to give a partially purified 1, 3-dihydro-isoindole-2-carboxylic acid { (R) -1- [ 4-carbamoyl-5- (pyridin-2-ylamino) -thiazol-2-yl ] -piperidin-3-yl } -amide as a yellow solid, purify further by reverse phase HPLC on a C-18 column, eluting with a gradient of 30% to 100% acetonitrile in water to remove similarly polar impurities The fractions were combined and concentrated in vacuo, and the residue was lyophilized with acetonitrile/water to give 1, 3-dihydro-isoindole-2-carboxylic acid { (R) -1- [ 4-carbamoyl-5- (pyridin-2-ylamino) -thiazol-2-yl ] -piperidin-3-yl } -amide (2.1mg, 18%) as a light yellow solid.

LC/MS calculated m/z C₂₃H₂₅N₇O₂S([M+H]⁺):464.6. Found 464.3 (positive ion mode electrospray ionization).

Example 3

(R) -2- (3- (5-chloroisoindoline-2-carboxamido) piperidin-1-yl) -5- (pyridin-2-ylamino) thiazole-4-carboxamide

Prepared by the same synthetic route as described in example 2, except that (i) in step 3 the isoindoline and 1.1 equivalents of diisopropylethylamine were replaced with 5-chloroisoindoline hydrobromide and 3 equivalents of diisopropylethylamine, and (II) in step 4, 0.2 equivalents of hydrogenated (dimethylphosphonic acid-kP) [ hydrogen bis (dimethylphosphino-kP ] platinum (II) were reacted and heated by microwave in a sealed tube at 150 ℃.

LC/MS calculated m/z C₂₃H₂₄ClN₇O₂S([M+H]⁺):499.0. Observed values are 498.3 and 500.3(3:1 intensity ratio corresponds to the presence of 1 chlorine atom) (positive ion mode electrospray ionization).

Example 4

(R) -2- (3- (6-tert-butylnicotinamido) piperidin-1-yl) -5- (pyridin-2-ylamino) thiazole-4-carboxamide

Step 1 preparation of 6-tert-butyl nicotinic acid

To a suspension of nicotinic acid (2.00g,16.2mmol) in water (250mL) was added concentrated sulfuric acid (1mL,18.8mmol) and the mixture was stirred under nitrogen to form a clear solution. Tert-pentanoic acid (1.83g,17.9mmol) was added and stirring continued at room temperature under nitrogen for 10 min. Silver nitrate (125mg,0.74mmol) was added followed by ammonium persulfate (295mg,1.3mmol), the flask was wrapped with aluminum foil protected from light and the mixture was heated to 90 ℃ under nitrogen. After 2 hours, the reaction mixture was cooled to room temperature and the aqueous mixture was concentrated in vacuo to a colorless solid. The solid was triturated with tetrahydrofuran and filtered. The solid residue was triturated again with methanol and filtered. The two filtrates were combined and concentrated in vacuo. The crude product was purified by reverse phase chromatography using 85g C-18 column eluting with a gradient of 10% acetonitrile in water to 100% acetonitrile. The product containing fractions were combined and concentrated in vacuo. The residue was lyophilized in water to give 6-tert-butylnicotinic acid (139mg, 4%) as a colorless solid.

LC/MS calculated m/z C₁₀H₁₃NO₂([M+H]⁺):180.2. Found 180.1 (electrospray ionization in positive ion mode).

Step 2 preparation of 2- ((R) -3-amino-piperidin-1-yl) -5- (pyridin-2-ylamino) -thiazole-4-carboxylic acid amide

To a suspension of {1- [ 4-carbamoyl-5- (pyridin-2-ylamino) -thiazol-2-yl ] -piperidin-3-yl } -carbamic acid tert-butyl ester (prepared as described in example 1) (359mg,0.86mmol) in dichloromethane (10mL) was added trifluoroacetic acid (10mL,130mmol) to form a yellow solution. The mixture was stirred at room temperature for 30 minutes and then evaporated to dryness in vacuo. Dichloromethane (25mL) was added to the residue and washed with saturated aqueous sodium bicarbonate (100 mL). The organic layer was separated and the aqueous layer was extracted with dichloromethane (5x 20mL) until no more yellow color was extracted into the organic layer. The combined organic extracts were dried over sodium sulfate, filtered and concentrated in vacuo to give 2- ((R) -3-amino-piperidin-1-yl) -5- (pyridin-2-ylamino) -thiazole-4-carboxylic acid amide (280mg) as a yellow solid.

LC/MS calculated m/z C₁₄H₁₈N₆OS([M+H]⁺):319.4. Found 319.1 (electrospray ionization in positive ion mode).

Step 3 preparation of (R) -2- (3- (6-tert-butylnicotinamido) piperidin-1-yl) -5- (pyridin-2-ylamino) thiazole-4-carboxamide

To a solution of 6-tert-butylnicotinic acid (23.5mg, 131. mu. mol) and 2- ((R) -3-amino-piperidin-1-yl) -5- (pyridin-2-ylamino) -thiazole-4-carboxylic acid amide (38mg, 119. mu. mol) in N, N-dimethylformamide (1mL) was added diisopropylethylamine (35. mu.L, 200. mu. mol), followed by O- (benzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (61mg, 161. mu. mol). After stirring overnight at room temperature under argon, the reaction was quenched by addition of aqueous ammonium chloride (2mL), the mixture was poured into water (5mL) and extracted with ethyl acetate (3 × 3 mL). The combined organic extracts were washed with brine (2 × 2mL), dried over sodium sulfate, filtered and concentrated in vacuo to a yellow solid. The crude product was purified by chromatography on a 13g C-18 column eluting with a gradient of 10% acetonitrile in water to 100% acetonitrile. The product containing fractions were combined and concentrated in vacuo and the residue was lyophilized in acetonitrile/water to give (R) -2- (3- (6-tert-butylnicotinamido) piperidin-1-yl) -5- (pyridin-2-ylamino) thiazole-4-carboxamide (37.8mg, 66%) as a pale yellow solid.

LC/MS calculated m/z C₂₄H₂₉N₇O₂S([M+H]⁺):480.6. Found 480.3 (electrospray ionization in positive ion mode).

Example 5

(R) -2- (3- (2-phenylacetylamino) piperidin-1-yl) -5- (pyridin-2-ylamino) thiazole-4-carboxamide

Prepared by the same synthetic route as described in example 4, except that 2-phenylacetic acid (17.9mg, 131. mu. mol) was used instead of 6-tert-butylnicotinic acid. This gave (R) -2- (3- (2-phenylacetamido) piperidin-1-yl) -5- (pyridin-2-ylamino) thiazole-4-carboxamide as a pale yellow solid (59%).

LC/MS calculated m/z C₂₂H₂₄N₆O₂S([M+H]⁺):437.5. Found 437.3 (electrospray ionization in positive ion mode).

Example 6

(R) -5- (pyridin-2-ylamino) -2- (3- (2-m-tolylacetylamino) piperidin-1-yl) thiazole-4-carboxamide

Prepared by the same synthetic route as described in example 4, except that 2-m-tolylacetic acid (19.7mg, 131. mu. mol) was used instead of 6-tert-butylnicotinic acid. This gave (R) -5- (pyridin-2-ylamino) -2- (3- (2-m-tolylacetylamino) piperidin-1-yl) thiazole-4-carboxamide as a pale yellow solid (49%).

LC/MS calculated m/z C₂₃H₂₆N₆O₂S([M+H]⁺):451.6. Found 451.3 (electrospray ionization in positive ion mode).

Example 7

(R) -2- (3- (4-tert-butylbenzoylamino) piperidin-1-yl) -5- (pyridin-2-ylamino) thiazole-4-carboxamide

Prepared by the same synthetic route as described in example 4, except that 4-tert-butylbenzoic acid (23.4mg, 131. mu. mol) was used instead of 6-tert-butylnicotinic acid. This gave (R) -2- (3- (4-tert-butylbenzoylamino) piperidin-1-yl) -5- (pyridin-2-ylamino) thiazole-4-carboxamide as a pale yellow solid (68%).

LC/MS calculated m/z C₂₅H₃₀N₆O₂S([M+H]⁺):479.6. Found 479.3 (positive ion mode electrospray ionization).

Example 8

(R) -2- (3- (methylsulfonylamino) piperidin-1-yl) -5- (pyridin-2-ylamino) thiazole-4-carboxamide

To a suspension of 2- ((R) -3-amino-piperidin-1-yl) -5- (pyridin-2-ylamino) -thiazole-4-carboxylic acid amide (prepared as described in example 4) (5.6mg,171 μmol) in tetrahydrofuran (1mL) was added N, N-dimethylformamide (0.5mL) to form a clear solution. Diisopropylethylamine (59.9. mu.L, 343. mu. mol) was added, followed by methanesulfonyl chloride (20. mu.L, 257. mu. mol) and the mixture was stirred at room temperature under argon overnight. The reaction was quenched by the addition of saturated aqueous ammonium chloride (2mL), poured into water (5mL) and extracted with ethyl acetate (5x 2mL), the combined organic extracts were washed with brine (2x2mL), dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by reverse phase chromatography on a C-18 column eluting with a gradient of 10% acetonitrile in water to 100% acetonitrile. The product containing fractions were combined and concentrated in vacuo. The residue was lyophilized in water/ethanol to give (R) -2- (3- (methylsulfonylamino) piperidin-1-yl) -5- (pyridin-2-ylamino) thiazole-4-carboxamide as a beige lyophilized powder (41mg, 60%).

LC/MS calculated m/z C₁₅H₂₀NO₃S₂([M+H]⁺):397.5. Found 397.2 (electrospray ionization in positive ion mode).

Example 9

(R) -2- (3- (phenylsulfonylamino) piperidin-1-yl) -5- (pyridin-2-ylamino) thiazole-4-carboxamide

Prepared by the same synthetic route as described in example 8, except that benzenesulfonyl chloride (33 μ L,257 μmol) was used instead of methanesulfonyl chloride. This gave (R) -2- (3- (phenylsulfonylamino) piperidin-1-yl) -5- (pyridin-2-ylamino) thiazole-4-carboxamide as a pale yellow solid (23%).

LC/MS calculated m/z C₂₀H₂₂N₆O₃S₂([M+H]⁺):459.6. Found 459.3 (positive ion mode electrospray ionization).

Example 10

(R) -2- (3- (1-methylethylsulfonylamino) piperidin-1-yl) -5- (pyridin-2-ylamino) thiazole-4-carboxamide

Prepared by the same synthetic route as described in example 8, except propane-2-sulfonyl chloride (30 μ L,257 μmol) was used instead of methanesulfonyl chloride. This gave (R) -2- (3- (1-methylethylsulfonylamino) piperidin-1-yl) -5- (pyridin-2-ylamino) thiazole-4-carboxamide (4%) as a pale yellow solid.

LC/MS calculated m/z C₁₇H₂₄N₆O₃S₂([M+H]⁺):425.6. Found 425.3 (positive ion mode electrospray ionization).

Example 11

(R) -2- (3- (2- (3-chlorophenylamino) acetylamino) piperidin-1-yl) -5- (pyridin-2-ylamino) thiazole-4-carboxamide

Step 1 preparation of (3-chloro-phenylamino) -acetic acid methyl ester

Methyl bromoacetate (8.3g,54.3mmol), 3-chloroaniline (8.31g,65.1mmol) and diisopropylethylamine (7.01g,54.3mmol) were dissolved in N, N-dimethylformamide (20mL) to give a pale yellow solution, and the reaction mixture was heated at 60 ℃ overnight. The reaction mixture was cooled to room temperature, diluted with ethyl acetate and washed with brine. The organic layer was dried over sodium sulfate, filtered and concentrated in vacuo to give (3-chloro-phenylamino) -acetic acid methyl ester as a solid, which was washed with hexane, filtered and dried (10.2g, 96%).

LC/MS calculated m/z C₉H₁₀ClNO₂([M+H]⁺):200.6. Found 200.0 (electrospray ionization in positive ion mode).

Step 2 preparation of (3-chloro-phenylamino) -acetic acid

(3-chloro-phenylamino) -acetic acid methyl ester (4g,20.0mmol) and sodium hydroxide (4.00g,100mmol) were combined in ethanol (60.0mL) and water (10mL) to give a pale yellow solution, which was heated at 60 ℃ for 4 hours. The reaction mixture was cooled to room temperature, diluted with ethyl acetate and washed with 1M hydrochloric acid. The organic layer was separated, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give (3-chloro-phenylamino) -acetic acid (3.20g, 86%) as a brown solid.

LC/MS calculated m/z C₈H₈ClNO₂([M+H]⁺):186.0. Found 186.0 (positive ion mode electrospray ionization).

Step 3 preparation of (R) -2- (3- (2- (3-chlorophenylamino) acetylamino) piperidin-1-yl) -5- (pyridin-2-ylamino) thiazole-4-carboxamide

Prepared by the same synthetic route as described in example 4, except that (3-chloro-phenylamino) -acetic acid is used instead of 6-tert-butylnicotinic acid. Obtained (R) -2- (3- (2- (3-chlorophenylamino) acetylamino) piperidin-1-yl) -5- (pyridin-2-ylamino) thiazole-4-carboxamide as a light yellow solid (63%).

LC/MS calculated m/z C₂₂H₂₄ClN₇O₂S([M+H]⁺):487.0. Observed values 486.3 and 488.3 (intensity ratio of 3:1 is consistent with the presence of one chlorine atom) (positive ion mode electrospray ionization).

Example 12

(R) -2- (3-Acylaminopiperidin-1-yl) -5- (pyridin-2-ylamino) thiazole-4-carboxamide

Prepared by the same synthetic route as described in example 4, except that acetic acid was used instead of 6-tert-butylnicotinic acid. (R) -2- (3-Acylaminopiperidin-1-yl) -5- (pyridin-2-ylamino) thiazole-4-carboxamide was obtained as a pale yellow solid (75%).

LC/MS calculated m/z C₁₆H₂₀N₆O₂S([M+H]⁺):361.4. Found 361.3 (positive ion mode electrospray ionization).

Biological examples

Bruton's tyrosine kinase (Btk) inhibition assay

The test being carried out by capturing radioactivity by filtration³³P phosphorylation product. Btk, biotinylated SH₂The interaction of the peptide substrate (Src homology) and ATP leads to phosphorylation of the peptide substrate. The biotinylated product was bound streptavidin agarose beads. All bound radiolabeled products were detected with a scintillation counter.

The assay plates were 96-well polypropylene (Greiner) and 96-well1.2 μm hydrophilic PVDF filter plate (Millipore). The concentrations reported here are the final assay concentrations of 10-100. mu.M compound in DMSO (Burdick and Jackson), 5-10nM Btk enzyme (His-tagged, full-length), 30. mu.M peptide substrate (biotin-Aca-AAAEEIYGEI-NH)₂) 100 μ M ATP (Sigma), 8mM imidazole (Sigma, pH 7.2), 8mM glycerol-2-phosphate (Sigma), 200 μ M EGTA (Roche diagnostics), 1mM MnCl₂(Sigma)、20mM MgCl₂(Sigma)、0.1mg/ml BSA(Sigma)、2mM DTT(Sigma)、1μCi³³P ATP (Amersham), 20% streptavidin agarose beads (Amersham), 50mM EDTA (Gibco), 2M NaCl w/1% phosphate (Gibco), microscint-20 (PerkinElmer).

IC₅₀The assay was calculated from 10 data points per compound using data generated from standard 96-well plate assay templates. One control compound and seven unknown inhibitors were tested on each plate, which was run twice. In general, compounds are diluted in half log starting at 100 μ M and ending at 3 nM. The control compound was staurosporine. The background was calculated in the absence of peptide substrate. The total activity was determined in the presence of the peptide substrate. The following protocol was used to determine Btk inhibition.

1) Sample preparation in half log increments with assay buffer (imidazole, glycerol-2-phosphate, EGTA, MnCl)₂，MgCl₂BSA) to dilute the test compound.

2) Preparation of beads

a.) washing the beads by centrifugation at 500g

b.) reconstituting the beads with PBS and EDTA to produce a 20% bead slurry

3) The reaction mixture without substrate (assay buffer, DTT, ATP,³³p ATP) and a reaction mixture containing the substrate (assay buffer, DTT, ATP,³³p ATP, peptide substrate) was preincubated at 30 ℃ for 15 minutes.

4) To start the assay, 10 μ L of Btk in enzyme buffer (imidazole, glycerol-2-phosphate, BSA) and 10 μ L of test compound were preincubated for 10 min at room temperature.

5) To Btk and compound was added 30 μ L of reaction mixture without or with substrate.

6) A total of 50. mu.L of the test mixture was incubated at 30 ℃ for 30 minutes.

7) The reaction was stopped by transferring 40. mu.L of the test sample to 150. mu.L of a slurry of beads on a filter plate.

8) After 30 minutes, the filter plate was washed by the following procedure

a.3x 250μL NaCl

b.3X 250. mu.L NaCl containing 1% phosphoric acid

c.1x 250μL H₂O

9) The plates were dried at 65 ℃ for 1 hour or at room temperature overnight

10) Add 50. mu.L microscint-20 and pair on scintillation counter³³P cpm were counted.

Percent Activity calculation from raw data in cpm

Percent activity ═ (sample-bgg)/(total activity-bgg) x 100

Calculation of IC from percent Activity Using a Single Point dose response sigmoidal model₅₀

y＝A+((B-A)/(1+((x/C)^D))))

x is compound concentration, y is% activity, a is min, B is max, C is IC₅₀D ═ 1 (hill slope)

Bruton's Tyrosine Kinase (BTK) inhibition of TR-FRET (time resolved FRET) assay

The BTK competition assay utilizes FRET (fluorescence resonance energy transfer) technology to determine the potency of a compound for the inactivated state of bruton's tyrosine kinase (IC 50). Compounding BTK-EuThe material was incubated on ice for 1 hour, then 50 nMBK-Bioease at the starting concentration^Tm10nM Eu-streptavidin (Perkin-Elmer Cat No. AD 0062). The assay buffer was composed of 20mM HEPES (pH 7.15), 0.1mM DTT, 10mM MgCl₂0.5mg/ml BSA and 3% kinase stabilizer (Fremontbiosolutions, cat # STB-K02). After 1 hour, the above reaction mixture was diluted 10-fold in assay buffer to make 5nM BTK:1nM Eu-streptavidin complex (donor fluorophore). Mu.l of a mixture of 0.11nM BTK-Eu and 0.11nM Kinase Tracer178(Invitrogen, Cat. No. PV5593) was then dispersed into 384-well flat bottom plates (Greiner,784076) with BTK-Eu alone as a negative control. Test compounds in the assay were prepared at 10x concentration and serially diluted in DMSO at half log increments to generate a curve of 10 data points. To start the FRET reaction, a DMSO solution of compound prepared as a 10x stock solution was added to the plate and the plate was incubated at 14 ℃ for 18-24 hours.

After incubation, the plates were read on a BMG Pherastar fluorescence plate reader (or equivalent instrument) and used to determine the emission energy from the europium donor fluorophore (620nm emission) and FRET (665nm emission). The values of the negative control wells were averaged to give the average lowest value. Positive "no inhibitor" control wells were averaged to give a mean maximum. The percentage of maximum FRET is calculated using the following equation:

% maximum FRET of 100x [ (FSR)_{Compound (I)}–FSR_{Mean minimum value})/(FSR_{Mean maximum value}–FSR_{Mean minimum value})]Wherein FSR is FRET signal ratio. The% Max FRET curves were plotted in Activity Base (Excel) and IC50 (%), Hill slope, z' and% CV were determined. Mean IC50 and standard deviation were derived from duplicate curves (single inhibition curves from two independent dilutions) using Microsoft Excel.

Data for representative compounds in this assay are set forth in table II below.

Table II.

Inhibition of B cell activation in whole blood as measured by CD69 expression

One procedure for testing the ability of Btk inhibitors to inhibit B cell receptor-mediated B cell activation in human blood is as follows:

human Whole Blood (HWB) was obtained from healthy volunteers under the following limiting conditions that no drug was taken within 24 hours and no smoking was observed. Blood was collected by venipuncture into Vacutainer tubes anticoagulated with sodium heparin. Test compounds were diluted ten-fold with PBS (20x) to the desired starting drug concentration, followed by a three-fold serial dilution with 10% DMSO in PBS to generate a nine-point dose-response curve. Mu.l of each compound dilution was added in duplicate to 2ml of a 96-well V-plate (analytical salts and Services, # 59623-23); 5.5 μ l of 10% DMSO in PBS was added to control and non-irritant wells. HWB (100. mu.l) was added to each well and after mixing, the plates were placed at 37C, 5% CO₂Incubate for 30 minutes at 100% humidity. With mixing, Goat F (ab') 2 anti-human IgM (Southern Biotech, #2022-14) (10. mu.l of 500. mu.g/ml solution at a final concentration of 50. mu.g/ml) (except for the non-irritant wells) was added to each well and the plates were incubated for an additional 20 hours.

At the end of the 20-hour incubation, these samples were combined with a fluorescent-probe-labeled antibody (15. mu.l PE mouse anti-human CD20, BD Pharmingen, #555623 and/or 20ul APC mouse anti-human CD69, BD Pharmingen #555533) at 37 ℃, 5% CO₂Incubate for 30 minutes at 100% humidity. To compensate for the adjustments and initial voltage settings, control, unstained and single stained were included. Then, the sample was lysed with 1ml of 1X Pharmingen lysis buffer (BDPharmingen #555899) and the plates were centrifuged at 1800rpm for 5 minutes. Removing the supernatant by aspiration and removing the remainingThe pellet was again dissolved with another 1ml 1X Pharmingen lysis buffer and the plates were centrifuged as before. The supernatant was aspirated and the remaining sediment was washed in FACs buffer (PBS + 1% FBS). After the final centrifugation, the supernatant was removed and the pellet was resuspended in 180. mu.l of FACs buffer. The samples were transferred to 96-well plates suitable for running on an HTS 96-well system on a BDLSR II flow cytometer.

Data were acquired using excitation and emission wavelengths appropriate to the fluorescein used and percent positive Cell values were obtained using Cell Quest Software. The results were initially analyzed using FACS analysis software (Flow Jo). The IC50 of the test compound was defined as the concentration at which the percentage of CD 69-positive (also CD 20-positive) cells decreased by 50% after stimulation with anti-IgM (mean of 8 control wells after subtraction of the mean of 8 wells on the non-stimulated background). IC50 values were calculated using XLfit software version 3, equation 201.

Inhibition of B-cell activation-B-cell FLIPR assay in Lamus cells

Inhibition of B-cell activation by the compounds of the invention was demonstrated by measuring the effect of test compounds on anti-IgM stimulated B-cell responses.

The B cell FLIPR assay is a cell-based functional method of determining the effect of potential inhibitors on intracellular calcium increase caused by anti-IgM antibody stimulation. Lamus cells (human Burkitt's lymphoma cell line, ATCC-No. CRL-1596) were cultured in growth medium (described below). On the day before the experiment, the ramus cells were resuspended in fresh growth medium (supra) and set to a concentration of 0.5x 10 in tissue culture flasks⁶and/mL. On the day of the experiment, cells were counted and set to a concentration of 1X10 in growth medium supplemented with 1. mu.M FLUO-3AM (TefLabs Cat: 0116, prepared in anhydrous DMSO and 10% pluronic acid) in tissue culture flasks⁶mL, which was treated at 37 deg.C (4% CO)₂) The cells were incubated for 1 hour. To remove extracellular dye, cells were harvested by centrifugation (5min,1000rpm), which was performedAt 1x10⁶cells/mL were resuspended in FLIPR buffer (described below) and then 1 × 10 per well⁵Number of individual cells it was dispersed into a 96-well poly-D-lysine coated black/transparent plate (BD catalog No.: 356692). Test compounds were added at various concentrations (7 concentrations, details below) ranging from 100 μ M to 0.03 μ M and allowed to incubate with the cells for 30 minutes at room temperature. Lames cells Ca were stimulated by the addition of 10. mu.g/mL anti-IgM (Southern Biotech, Cat. No.: 2020-01)²⁺Signals were conducted and measured on a FLIPR (Molecular Devices, using a CCD camera with an argon laser to capture images of 96-well plates at 480nm excitation).

Media/buffer:

growth medium containing L-glutamine (Invitrogen, Cat: 61870-010), 10% fetal bovine serum (FBS, Summit Biotechnology Cat: FP-100-05); RPMI 1640 medium with 1mM sodium pyruvate (Invitrogen Cat. No. 11360-070).

FLIPR buffer HBSS (Invitrogen, Cat. No. 141175-079), 2mM CaCl₂(Sigma Cat No. C-4901), HEPES (Invitrogen, Cat No. 15630-080), 2.5mM probenecid (Sigma, Cat No. P-8761), 0.1% BSA (Sigma, Cat No. A-7906), 11mM glucose (Sigma, Cat-No. G-7528)

Compound dilution details:

to obtain the highest final assay concentration of 100. mu.M, 24. mu.L of a 10mM compound stock solution (prepared in DMSO) was added directly to 576. mu.L of FLIPR buffer. Test compounds were diluted with FLIPR buffer (using a Biomek 2000 auto pipettor) to give the following dilution series: substrate, 1.00X 10^-4M、1.00x 10^-5、3.16x 10^-6、1.00x 10^-6、3.16x 10^-7、1.00x 10^-7、3.16x 10^-8。

Testing and analysis:

Max-min statistics (subtraction of resting baseline from peak due to addition of stimulatory antibody), Moleculrudevies FLIPR control and statistics output software reported the intramolecular increase in calcium. IC determination by nonlinear Curve fitting (GraphPadprism software)₅₀。

Mouse collagen-induced arthritis (mCIA)

On day 0, mice were injected with an emulsion of type II collagen in Complete Freund's Adjuvant (CFA) at some point on the roots or back of the tail (i.d.). Following collagen immunization, animals develop arthritis at about 21-35 days. On day 21, the onset of arthritis was synchronized (challenged) by systemic administration of collagen in incomplete Freund's adjuvant (IFA; i.d.). After day 20, the animals were examined daily for the presence of mild arthritis (score 1 or 2; see scoring below), which is the signal for stimulation. After challenge, mice are scored and dosed with candidate therapeutic agents once daily (QD) or twice daily (BID) for a prescribed time (typically 2-3 weeks) and dosing frequency.

Rat collagen-induced arthritis (rCIA)

On day 0, rats were injected intradermally (i.d.) with an emulsion of bovine type II collagen in incomplete freund's adjuvant (CFA) at several sites on the back. On approximately day 7, booster injections of collagen emulsion were performed at the root of the tail or other sites on the back (i.d.). Arthritis is usually observed on days 12-14 after the initial collagen injection. From day 14 onwards, animals were evaluated for arthritis development as described below (evaluation of arthritis). Animals are administered with the candidate therapeutic agent in a prophylactic manner starting from the second challenge, once daily (QD) or twice daily (BID) for a prescribed time (typically 2-3 weeks) and frequency of administration.

Arthritis assessment:

In both models, the development of inflammation in the paw and limb joints was quantified using a scoring system involving the evaluation of 4 paws according to the following criteria:

score 1 ═ paw or one finger (toe) head swelling and/or redness

Swelling of two or more joints

Swelling of the entire paw with more than two joints involved

Severe arthritis of the entire paw and toe

Basal measurement assessments were performed on day 0 and started again at the first sign or swelling, up to three times per week until the end of the experiment. The arthritis index for each mouse was obtained by adding the four scores for each paw, giving a score of up to 16 per animal.

Rat in vivo asthma model

Male Brown-Norway rats were i.p. dosed weekly with 100 μ g OA (ovalbumin) in 0.2ml alum (alum) for three weeks (days 0, 7 and 14). On day 21 (one week after the last sensitization), rats were dosed with either the vehicle or compound formulation (q.d.), subcutaneously 0.5 hours prior to OA aerosol challenge (1% OA for 45 minutes), and terminated 4 or 24 hours after challenge. At sacrifice, serum and plasma were collected from all animals for serology and PK, respectively. The trachea cannula was inserted and the lungs were lavaged 3 times with PBS. The BAL fluid was subjected to total white blood cell count and differential white blood cell count. Total white blood cell counts in cell aliquots (20-100. mu.l) were determined using a Coulter counter. For differential white blood cell counts, 50-200. mu.l of the sample was centrifuged in Cytospin and the slides were stained with Diff-Quik. The proportion of monocytes, eosinophils, centrogranulocytes and lymphocytes was counted and expressed as a percentage under light microscopy using standard morphological criteria. Representative inhibitors of Btk exhibited reduced total leukocyte counts in BAL of OA sensitized and challenged rats compared to control levels.

For the purposes of clarity and clarity, some of the details of the foregoing invention have been described in terms of illustration and example. It will be apparent to those skilled in the art that changes and modifications may be made within the scope of the appended claims. Accordingly, it is to be understood that the above description is intended to be illustrative, and not restrictive. The scope of the invention should, therefore, be determined not with reference to the above description, but instead should be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled.

All patents, patent applications, and publications cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual patent, patent application, or publication were individually indicated to be so incorporated.

Claims

1. A compound of formula I or a pharmaceutically acceptable salt thereof,

wherein:

a is C_1-6Alkyl, phenyl, OR⁴、Wherein R is³Is Cl;

R²is H or tert-butyl;

R⁴is C_1-6An alkyl group;

R⁵is C_1-6An alkyl group;

R⁶is H or halogen;

x is C (═ O) or S (═ O)₂(ii) a And is

Y is CH or N.

2. The compound of claim 1, wherein X is C (═ O).

3. The compound of claim 1 or 2, wherein a is

4. The compound of any one of claims 1-3, wherein R⁶Is H.

5. The compound of any one of claims 1-3, wherein R⁶Is Cl.

6. The compound of claim 2, wherein a is methyl OR⁴And R is⁴Is a tert-butyl group.

7. The compound of claim 2, wherein A is

And R is⁵Is a tert-butyl group.

8. The compound of claim 7, wherein Y is N.

9. The compound of claim 7, wherein Y is CH.

10. The compound of claim 2, wherein A is

11. The compound of claim 10, wherein R²Is H.

12. The compound of claim 10, wherein R²Is a tert-butyl group.

13. The compound of claim 1, wherein X is S (═ O)₂And A is methyl, isopropyl or phenyl.

14. The compound of claim 2, wherein A is

And R is³Is Cl.

15. A compound selected from:

16. Use of a compound according to any one of claims 1 to 15 for the manufacture of a medicament for the treatment of an inflammatory and/or autoimmune disorder.

17. Use of a compound according to any one of claims 1 to 15 for the manufacture of a medicament for the treatment of rheumatoid arthritis.

18. The use of a compound according to any one of claims 1 to 15 for the manufacture of a medicament for the treatment of asthma.

19. A pharmaceutical composition comprising a compound according to any one of claims 1 to 15 and at least one pharmaceutically acceptable carrier, excipient or diluent.

20. A compound according to any one of claims 1 to 15 for use in the treatment of an inflammatory and/or autoimmune disorder.