# Patent

## Title
Inhibitors of bruton's tyrosine kinase

## Abstract

This application discloses compounds according to generic Formula (I): wherein all variables are defined as described herein, which inhibit BTK. The compounds disclosed herein are useful to modulate the activity of BTK and treat diseases associated with excessive BTK activity. The compounds are further useful to treat inflammatory and auto immune diseases associated with aberrant B-cell proliferation such as rheumatoid arthritis. Also disclosed are compositions containing compounds of Formula I and at least one carrier, diluent or excipient.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a National Stage Application of PCT/EP2014/054018 filed Mar. 3, 2014, which claims priority from U.S. Provisional Patent Application No. 61/772,954, filed on Mar. 5, 2013. The priority of both said PCT and U.S. Provisional Patent Application are claimed. Each of the prior mentioned applications is hereby incorporated by reference herein in its entirety.


FIELD OF THE INVENTION
The present invention relates to the use of novel compounds which inhibit BTK and are useful for the treatment of auto-immune and inflammatory diseases caused by aberrant B-cell activation.
BACKGROUND OF THE INVENTION
Protein kinases constitute one of the largest families of human enzymes and regulate many different signaling processes by adding phosphate groups to proteins (T. Hunter, Cell 1987 50:823-829). Specifically, tyrosine kinases phosphorylate proteins on the phenolic moiety of tyrosine residues. The tyrosine kinase family includes members that control cell growth, migration, and differentiation. Abnormal kinase activity has been implicated in a variety of human diseases including cancers, autoimmune and inflammatory diseases. Since protein kinases are among the key regulators of cell signaling they provide a target to modulate cellular function with small molecular kinase inhibitors and thus make good drug design targets. In addition to treatment of kinase-mediated disease processes, selective and efficacious inhibitors of kinase activity are also useful for investigation of cell signaling processes and identification of other cellular targets of therapeutic interest.
There is good evidence that B-cells play a key role in the pathogenesis of autoimmune and/or inflammatory disease. Protein-based therapeutics that deplete B cells such as Rituxan are effective against autoantibody-driven inflammatory diseases such as rheumatoid arthritis (Rastetter et al. Annu Rev Med 2004 55:477). Therefore inhibitors of the protein kinases that play a role in B-cell activation should be useful therapeutics for B-cell mediated disease pathology such as autoantibody production.
Signaling through the B-cell receptor (BCR) controls a range of B-cell responses including proliferation and differentiation into mature antibody producing cells. The BCR is a key regulatory point for B-cell activity and aberrant signaling can cause deregulated B-cell proliferation and formation of pathogenic autoantibodies that lead to multiple autoimmune and/or inflammatory diseases. Bruton's Tyrosine Kinase (BTK) is a non-BCR associated kinase that is membrane proximal and immediately downstream from BCR. Lack of BTK has been shown to block BCR signaling and therefore inhibition of BTK could be a useful therapeutic approach to block B-cell mediated disease processes.
BTK is a member of the Tec family of tyrosine kinases, and has been shown to be a critical regulator of early B-cell development and mature B-cell activation and survival (Khan et al. Immunity 1995 3:283; Ellmeier et al. J. Exp. Med. 2000 192:1611). Mutation of BTK in humans leads to the condition X-linked agammaglobulinemia (XLA) (reviewed in Rosen et al. New Eng. J. Med. 1995 333:431 and Lindvall et al. Immunol. Rev. 2005 203:200). These patients are immunocompromised and show impaired maturation of B-cells, decreased immunoglobulin and peripheral B-cell levels, diminished T-cell independent immune responses as well as attenuated calcium mobilization following BCR stimulation.
Evidence for a role for BTK in autoimmune and inflammatory diseases has also been provided by BTK-deficient mouse models. In preclinical murine models of systemic lupus erythematosus (SLE), BTK-deficient mice show marked amelioration of disease progression. In addition, BTK-deficient mice are resistant to collagen-induced arthritis (Jansson and Holmdahl Clin. Exp. Immunol. 1993 94:459). A selective BTK inhibitor has been demonstrated dose-dependent efficacy in a mouse arthritis model (Z. Pan et al., Chem. Med Chem. 2007 2:58-61).
BTK is also expressed by cells other than B-cells that may be involved in disease processes. For example, BTK is expressed by mast cells and BTK-deficient bone marrow derived mast cells demonstrate impaired antigen induced degranulation (Iwaki et al. J. Biol. Chem. 2005 280:40261). This shows BTK could be useful to treat pathological mast cells responses such as allergy and asthma. Also monocytes from XLA patients, in which BTK activity is absent, show decreased TNF alpha production following stimulation (Horwood et al. J Exp Med 197:1603, 2003). Therefore TNF alpha mediated inflammation could be modulated by small molecular BTK inhibitors. Also, BTK has been reported to play a role in apoptosis (Islam and Smith Immunol. Rev. 2000 178:49) and thus BTK inhibitors would be useful for the treatment of certain B-cell lymphomas and leukemias (Feldhahn et al. J. Exp. Med. 2005 201:1837).
SUMMARY OF THE INVENTION
The present application provides the BTK inhibitor compounds of Formula I, methods of use thereof, as described herein below:
The application provides a compound of Formula I,




wherein:

    n is 1 or 2;
    R1 is —C(═O)R1, —S(═O)2R1, or —OC(R1)2CH2OH;
    
        R1′ is methyl or morpholine;
    
    
    R2 is H or F;
    R3 is chloro or C(CH2)2R3′;
    
        R3′ is methyl, cyano, or hydroxymethyl;
    
    
    X is CH, CH2, or N;
    X2 is CH or N;
    X3 is CH or N; and
    Y is CH or O;
    with the proviso that when n is 2, both X are CH2;
    or a pharmaceutically acceptable salt thereof.


The application provides a method for treating an inflammatory and/or autoimmune condition comprising administering to a patient in need thereof a therapeutically effective amount of the compound of Formula I.
The application provides a pharmaceutical composition comprising the compound of Formula I, admixed with at least one pharmaceutically acceptable carrier, excipient or diluent.


DETAILED DESCRIPTION OF THE INVENTION
Definitions
The phrase “a” or “an” entity as used herein refers to one or more of that entity; for example, a compound refers to one or more compounds or at least one compound. As such, the terms “a” (or “an”), “one or more”, and “at least one” can be used interchangeably herein.
The phrase “as defined herein above” refers to the broadest definition for each group as provided in the Summary of the Invention or the broadest claim. In all other embodiments provided below, substituents which can be present in each embodiment and which are not explicitly defined retain the broadest definition provided in the Summary of the Invention.
As used in this specification, whether in a transitional phrase or in the body of the claim, the terms “comprise(s)” and “comprising” are to be interpreted as having an open-ended meaning. That is, the terms are to be interpreted synonymously with the phrases “having at least” or “including at least”. When used in the context of a process, the term “comprising” means that the process includes at least the recited steps, but may include additional steps. When used in the context of a compound or composition, the term “comprising” means that the compound or composition includes at least the recited features or components, but may also include additional features or components.
As used herein, unless specifically indicated otherwise, the word “or” is used in the “inclusive” sense of “and/or” and not the “exclusive” sense of “either/or”.
The term “independently” is used herein to indicate that a variable is applied in any one instance without regard to the presence or absence of a variable having that same or a different definition within the same compound. Thus, in a compound in which R′′ appears twice and is defined as “independently carbon or nitrogen”, both R′′s can be carbon, both R′′s can be nitrogen, or one R′′ can be carbon and the other nitrogen.
When any variable occurs more than one time in any moiety or formula depicting and describing compounds employed or claimed in the present invention, its definition on each occurrence is independent of its definition at every other occurrence. Also, combinations of substituents and/or variables are permissible only if such compounds result in stable compounds.
The symbols “*” at the end of a bond or “- - - ” drawn through a bond each refer 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:




A bond drawn into ring system (as opposed to connected at a distinct vertex) indicates that the bond may be attached to any of the suitable ring atoms.
The term “optional” or “optionally” as used herein means that a subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not. For example, “optionally substituted” means that the optionally substituted moiety may incorporate a hydrogen atom or a substituent.
The phrase “optional bond” means that the bond may or may not be present, and that the description includes single, double, or triple bonds. If a substituent is designated to be a “bond” or “absent”, the atoms linked to the substituents are then directly connected.
The term “about” is used herein to mean approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20%.
Certain compounds of Formulae I may exhibit tautomerism. Tautomeric compounds can exist as two or more interconvertable species. Prototropic tautomers result from the migration of a covalently bonded hydrogen atom between two atoms. Tautomers generally exist in equilibrium and attempts to isolate an individual tautomers usually produce a mixture whose chemical and physical properties are consistent with a mixture of compounds. The position of the equilibrium is dependent on chemical features within the molecule. For example, in many aliphatic aldehydes and ketones, such as acetaldehyde, the keto form predominates while; in phenols, the enol form predominates. Common prototropic tautomers include keto/enol (—C(═O)—CH—⇄—C(—OH)═CH—), amide/imidic acid (—C(═O)—NH—⇄—C(—OH)═N—) and amidine (—C(═NR)—NH—⇄—C(—NHR)═N—) tautomers. The latter two are particularly common in heteroaryl and heterocyclic rings and the present invention encompasses all tautomeric forms of the compounds.
Technical and scientific terms used herein have the meaning commonly understood by one of skill in the art to which the present invention pertains, unless otherwise defined. Reference is made herein to various methodologies and materials known to those of skill in the art. Standard reference works setting forth the general principles of pharmacology include Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th Ed., McGraw Hill Companies Inc., New York (2001). Any suitable materials and/or methods known to those of skill can be utilized in carrying out the present invention. However, preferred materials and methods are described. Materials, reagents and the like to which reference are made in the following description and examples are obtainable from commercial sources, unless otherwise noted.
The definitions described herein may be appended to form chemically-relevant combinations, such as “heteroalkylaryl”, “haloalkylheteroaryl”, “arylalkylheterocyclyl”, “alkylcarbonyl”, “alkoxyalkyl”, and the like. When the term “alkyl” is used as a suffix following another term, as in “phenylalkyl”, or “hydroxyalkyl”, this is intended to refer to an alkyl group, as defined above, being 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. An “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 so forth. Accordingly, as used herein, the term “hydroxyalkyl” is used to define a subset of heteroalkyl groups defined below. The term -(ar)alkyl refers to either an unsubstituted alkyl or an aralkyl group. The term (hetero)aryl or (het)aryl refers to either an aryl or a heteroaryl group.
The term “spirocycloalkyl”, as used herein, means a spirocyclic cycloalkyl group, such as, for example, spiro[3.3]heptane. The term spiroheterocycloalkyl, as used herein, means a spirocyclic heterocycloalkyl, such as, for example, 2,6-diaza spiro[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 or “alkylcarbonyl” as used herein denotes a group of formula C(═O)R wherein R is alkyl as defined herein. The term C1-6 acyl refers to a group —C(═O)R contain 6 carbon atoms. The term “arylcarbonyl” as used herein means a group of formula C(═O)R wherein R is an aryl group; the term “benzoyl” as used herein an “arylcarbonyl” group wherein R is phenyl.
The term “ester” as used herein denotes a group of formula —C(═O)OR wherein R is lower alkyl as defined herein.
The term “alkyl” as used herein denotes an unbranched 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 containing 1 to 6 carbon atoms. “C1-10 alkyl” as used herein refers to an alkyl composed of 1 to 10 carbons. Examples of alkyl groups include, but are not limited to, lower alkyl groups include methyl, ethyl, propyl, i-propyl, n-butyl, i-butyl, t-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”, this is intended to refer to an alkyl group, as defined above, being substituted with one to two substituents selected from the other specifically-named group. Thus, for example, “phenylalkyl” denotes the radical R′R′′—, wherein R′ is a phenyl radical, and R′′ is an alkylene radical as defined herein with the understanding that the attachment point of the phenylalkyl moiety will be on the alkylene radical. Examples of arylalkyl radicals include, but are not limited to, benzyl, phenylethyl, 3-phenylpropyl. The terms “arylalkyl” or “aralkyl” are interpreted similarly except R′ is an aryl radical. The terms “(het)arylalkyl” or “(het)aralkyl” are interpreted similarly except R′ is optionally an aryl or a heteroaryl radical.
The terms “haloalkyl” or “halo-lower alkyl” or “lower haloalkyl” refers to a straight or branched chain hydrocarbon residue containing 1 to 6 carbon atoms wherein one or more carbon atoms are substituted with one or more halogen atoms.
The term “alkylene” or “alkylenyl” as used herein denotes a divalent saturated linear hydrocarbon radical of 1 to 10 carbon atoms (e.g., (CH2)n) or a branched saturated divalent hydrocarbon radical of 2 to 10 carbon atoms (e.g., —CHMe- or —CH2CH(i-Pr)CH2—), unless otherwise indicated. Except in the case of methylene, the open valences of an alkylene group are not attached to the same atom. Examples of alkylene radicals include, but are not limited to, methylene, ethylene, propylene, 2-methyl-propylene, 1,1-dimethyl-ethylene, butylene, 2-ethylbutylene.
The term “alkoxy” as used herein means an —O-alkyl group, wherein alkyl is as defined above such as methoxy, ethoxy, n-propyloxy, i-propyloxy, n-butyloxy, i-butyloxy, t-butyloxy, pentyloxy, hexyloxy, including their isomers. “Lower alkoxy” as used herein denotes an alkoxy group with a “lower alkyl” group as previously defined. “C1-10 alkoxy” as used herein refers to an —O-alkyl wherein alkyl is C1-10.
The term “PCy3” refers to a phosphine trisubstituted with three cyclic moieties.
The terms “haloalkoxy” or “halo-lower alkoxy” or “lower haloalkoxy” refers to a lower alkoxy group, wherein one or more carbon atoms are substituted with one or more halogen atoms.
The term “hydroxyalkyl” as used herein denotes an alkyl radical as herein defined wherein one to three hydrogen atoms on different carbon atoms is/are replaced by hydroxyl groups.
The terms “alkylsulfonyl” and “arylsulfonyl” as used herein refers to a group of formula —S(═O)2R wherein R is alkyl or aryl respectively and alkyl and aryl are as defined herein. The term “heteroalkylsulfonyl” as used herein refers herein denotes a group of formula —S(═O)2R wherein R is “heteroalkyl” as defined herein.
The terms “alkylsulfonylamino” and “arylsulfonylamino” as used herein refers to a group of formula —NR′S(═O)2R wherein R is alkyl or aryl respectively, R′ is hydrogen or C1-3 alkyl, and alkyl and aryl are as defined herein.
The term “cycloalkyl” as used herein refers to a saturated carbocyclic ring containing 3 to 8 carbon atoms, i.e. cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl. “C3-7 cycloalkyl” as used herein refers to a cycloalkyl composed of 3 to 7 carbons in the carbocyclic ring.
The term “carboxy-alkyl” as used herein refers to an alkyl moiety wherein one, hydrogen atom has been replaced with a carboxyl with the understanding that the point of attachment of the heteroalkyl radical is through a carbon atom. The term “carboxy” or “carboxyl” refers to a —CO2H moiety.
The term “heteroaryl” or “heteroaromatic” as used herein means a monocyclic or bicyclic radical of 5 to 12 ring atoms having at least one aromatic or partially unsaturated ring containing four to eight atoms per ring, incorporating one or more N, O, or S heteroatoms, the remaining ring atoms being carbon, with the understanding that the attachment point of the heteroaryl radical will be on an aromatic or partially unsaturated ring. As well known to those skilled in the art, heteroaryl rings have less aromatic character than their all-carbon counter parts. Thus, for the purposes of the invention, a heteroaryl group 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 include, but is not limited to, pyridinyl, pyrimidinyl, pyrazinyl, oxazinyl, pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, 4,5-Dihydro-oxazolyl, 5,6-Dihydro-4H-[1,3]oxazolyl, isoxazole, thiazole, isothiazole, triazoline, thiadiazole and oxadiaxoline which can optionally be substituted with one or more, preferably one or two substituents selected from hydroxy, cyano, alkyl, alkoxy, thio, lower haloalkoxy, alkylthio, halo, 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, but are not limited to, quinolinyl, isoquinolinyl, benzofuryl, benzothiophenyl, benzoxazole, benzisoxazole, benzothiazole, naphthyridinyl, 5,6,7,8-Tetrahydro-[1,6]naphthyridinyl, and benzisothiazole. Bicyclic moieties can be optionally substituted on either ring, however the point of attachment is on a ring containing a heteroatom.
The term “heterocyclyl”, “heterocycloalkyl” or “heterocycle” as used herein denotes a monovalent saturated cyclic radical, consisting of one or more rings, preferably one to two rings, including spirocyclic ring systems, of three to eight atoms per ring, incorporating one or more ring heteroatoms (chosen from N, O or S(O)0-2), and which can optionally be independently substituted with one or more, preferably one or two substituents selected from hydroxy, oxo, cyano, lower alkyl, lower alkoxy, lower haloalkoxy, alkylthio, halo, lower haloalkyl, hydroxyalkyl, nitro, alkoxycarbonyl, amino, alkylamino, alkylsulfonyl, arylsulfonyl, alkylaminosulfonyl, arylaminosulfonyl, alkylsulfonylamino, arylsulfonylamino, alkylaminocarbonyl, arylaminocarbonyl, alkylcarbonylamino, arylcarbonylamino, and ionic forms thereof, unless otherwise indicated. Examples of heterocyclic radicals include, but are not limited to, morpholinyl, piperazinyl, piperidinyl, azetidinyl, pyrrolidinyl, hexahydroazepinyl, oxetanyl, tetrahydrofuranyl, tetrahydrothiophenyl, oxazolidinyl, thiazolidinyl, isoxazolidinyl, tetrahydropyranyl, thiomorpholinyl, quinuclidinyl and imidazolinyl, and ionic forms thereof. Examples may also be bicyclic, 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]oxazine.
Inhibitors of BTK
The application provides a compound of Formula I,




wherein:

    n is 1 or 2;
    R1 is —C(═O)R1′, —S(═O)2R1′, or —OC(R1′)2CH2OH;
    
        R1 is methyl or morpholine;
    
    
    R2 is H or F;
    R3 is chloro or C(CH2)2R3′;
    
        R3′ is methyl, cyano, or hydroxymethyl;
    
    
    X is CH, CH2, or N;
    X2 is CH or N;
    X3 is CH or N; and
    Y is CH or O;
    with the proviso that when n is 2, both X are CH2;
    or a pharmaceutically acceptable salt thereof.


The application provides a compound of Formula I, wherein R2 is H.
The application provides the above compound of Formula I, wherein R3 is tert-butyl.
The application provides the above compound of Formula I, wherein X3 is CH
The application provides the above compound of Formula I, wherein X2 is N.
The application provides the above compound of Formula I, wherein R1 is —C(═O)R1′ and R′ is morpholine.
The application provides the above compound of Formula I, wherein n is 1.
The application provides the above compound of Formula I, wherein X is CH or CH2.
The application provides the above compound of Formula I, wherein Y is CH or CH2.
The application provides a compound of Formula I, wherein n is 2 and Y is O.
The application provides a compound of Formula I, wherein n is 2, Y is O, R2 is H, R3 is tert-butyl, X3 is CH, and X2 is N.
The application provides the above compound of Formula I, wherein R1 is —S(═O)2R′′ and R′ is methyl or R1 is —C(═O)R1′ and R′ is morpholine.
The application provides a compound of Formula I, selected from the group consisting of:

    2-[2-[2-(hydroxymethyl)-3-[1-methyl-5-[[5-(morpholine-4-carbonyl)pyridin-2-yl]amino]-6-oxopyridazin-3-yl]phenyl]-1-oxo-3,4-dihydroisoquinolin-6-yl]-2-methylpropanenitrile;
    2-[8-fluoro-2-[2-(hydroxymethyl)-3-[5-[[5-(1-hydroxy-2-methylpropan-2-yl)oxypyridin-2-yl]amino]-1-methyl-6-oxopyridazin-3-yl]phenyl]-1-oxoisoquinolin-6-yl]-2-methylpropanenitrile;
    4-[2-(hydroxymethyl)-3-[1-methyl-5-[(5-methylsulfonylpyridin-2-yl)amino]-6-oxopyridazin-3-yl]phenyl]-8-(1-hydroxy-2-methylpropan-2-yl)-2,3-dihydro-1,4-benzoxazepin-5-one;
    5-chloro-N-[2-(hydroxymethyl)-3-[1-methyl-5-[[5-(morpholine-4-carbonyl)pyridin-2-yl]amino]-6-oxopyridin-3-yl]phenyl]-1,3-dihydroisoindole-2-carboxamide;
    6-tert-butyl-2-[2-(hydroxymethyl)-3-[1-methyl-5-[[5-(morpholine-4-carbonyl)pyridin-2-yl]amino]-6-oxopyridin-3-yl]phenyl]-3,4-dihydro-2,7-naphthyridin-1-one;
    6-tert-butyl-2-[2-(hydroxymethyl)-3-[1-methyl-5-[[5-(morpholine-4-carbonyl)pyridin-2-yl]amino]-6-oxopyridazin-3-yl]phenyl]-3,4-dihydro-2,7-naphthyridin-1-one;
    6-tert-butyl-8-fluoro-2-[2-methyl-3-[1-methyl-5-[[5-(morpholine-4-carbonyl)pyridin-2-yl]amino]-6-oxopyridazin-3-yl]phenyl]phthalazin-1-one;
    8-tert-butyl-4-[2-(hydroxymethyl)-3-[1-methyl-5-[[5-(morpholine-4-carbonyl)pyridin-2-yl]amino]-6-oxopyridazin-3-yl]phenyl]-2,3-dihydro-1,4-benzoxazepin-5-one; and
    8-tert-butyl-4-[2-(hydroxymethyl)-3-[1-methyl-5-[(5-methylsulfonylpyridin-2-yl)amino]-6-oxopyridazin-3-yl]phenyl]-2,3-dihydro-1,4-benzoxazepin-5-one.


The application provides a method for treating an inflammatory and/or autoimmune condition comprising administering to a patient in need thereof a therapeutically effective amount of the compound of Formula I.
The application provides a method for treating rheumatoid arthritis comprising administering to a patient in need thereof a therapeutically effective amount of the compound of Formula I.
The application provides a method for treating asthma comprising administering to a patient in need thereof a therapeutically effective amount of the compound of Formula I.
The application provides a pharmaceutical composition comprising the compound of Formula I.
The application provides a pharmaceutical composition comprising the compound of Formula I, admixed with at least one pharmaceutically acceptable carrier, excipient or diluent.
The application provides a use of the compound of formula I in the manufacture of a medicament for the treatment of an inflammatory disorder.
The application provides a use of the compound of formula I in the manufacture of a medicament for the treatment of an autoimmune disorder.
The application provides a use of the compound of formula I in the manufacture of a medicament for the treatment of rheumatoid arthritis.
The application provides a use of the compound of formula I in the manufacture of a medicament for the treatment of asthma.
The application provides the use of a compound as described above for the treatment of inflammatory and/or autoimmune condition.
The application provides the use of a compound as described above for the treatment of rheumatoid arthritis.
The application provides the use of a compound as described above for the treatment of asthma.
The application provides a compound, method, or composition as described herein.
Compounds and Preparation
Examples of representative compounds encompassed by the present invention and within the scope of the invention are provided in the following Table. These examples and preparations which follow are provided to enable those skilled in the art to more clearly understand and to practice the present invention. They should not be considered as limiting the scope of the invention, but merely as being illustrative and representative thereof.
In general, the nomenclature used in this Application is based on AUTONOMTM v.4.0, a Beilstein Institute computerized system for the generation of IUPAC systematic nomenclature. If there is a discrepancy between a depicted structure and a name given that structure, the depicted structure is to be accorded more weight. In addition, if the stereochemistry of a structure or a portion of a structure is not indicated with, for example, bold or dashed lines, the structure or portion of the structure is to be interpreted as encompassing all stereoisomers of it.
TABLE I depicts examples of compounds according to generic Formula I. These compounds were designed to contain a more polar (cLogP<2) selectivity group (for example, where the tert-butyl benzamide is located) combined with a pyridazinone core group to minimize liver toxicity found with similar compounds that do not contain this benefitial combination.









TABLE I





Compound
Nomenclature
Structure







1
2-[2-[2- (hydroxymethyl)-3-[1- methyl-5-[[5- (morpholine-4- carbonyl)pyridin-2- yl]amino]-6- oxopyridazin-3- yl]phenyl]-1-oxo-3,4- dihydroisoquinolin-6- yl]-2- methylpropanenitrile






 


2
2-[8-fluoro-2-[2- (hydroxymethyl)-3-[5- [[5-(1-hydroxy-2- methylpropan-2- yl)oxypyridin-2- yl]amino]-1-methyl-6- oxopyridazin-3- yl]phenyl]-1- oxoisoquinolin-6-yl]- 2-methylpropanenitrile






 


3
4-[2- (hydroxymethyl)-3-[1- methyl-5-[(5- methylsulfonylpyridin- 2-yl)amino]-6- oxopyridazin-3- yl]phenyl]-8-(1- hydroxy-2- methylpropan-2-yl)- 2,3-dihydro-1,4- benzoxazepin-5-one






 


4
5-chloro-N-[2- (hydroxymethyl)-3-[1- methyl-5-[[5- (morpholine-4- carbonyl)pyridin-2- yl]amino]-6- oxopyridin-3- yl]phenyl]-1,3- dihydroisoindole-2- carboxamide






 


5
6-tert-butyl-2-[2- (hydroxymethyl)-3-[1- methyl-5-[[5- (morpholine-4- carbonyl)pyridin-2- yl]amino]-6- oxopyridin-3- yl]phenyl]-3,4- dihydro-2,7- naphthyridin-1-one






 


6
6-tert-butyl-2-[2- (hydroxymethyl)-3-[1- methyl-5-[[5- (morpholine-4- carbonyl)pyridin-2- yl]amino]-6- oxopyridazin-3- yl]phenyl]-3,4- dihydro-2,7- naphthyridin-1-one






 


7
6-tert-butyl-8-fluoro- 2-[2-methyl-3-[1- methyl-5-[[5- (morpholine-4- carbonyl)pyridin-2- yl]amino]-6- oxopyridazin-3- yl]phenyl]phthalazin- 1-one






 


8
8-tert-butyl-4-[2- (hydroxymethyl)-3-[1- methyl-5-[[5- (morpholine-4- carbonyl)pyridin-2- yl]amino]-6- oxopyridazin-3- yl]phenyl]-2,3- dihydro-1,4- benzoxazepin-5-one






 


9
8-tert-butyl-4-[2- (hydroxymethyl)-3-[1- methyl-5-[(5- methylsulfonylpyridin- 2-yl)amino]-6- oxopyridazin-3- yl]phenyl]-2,3- dihydro-1,4- benzoxazepin-5-one













General Synthetic Schemes

The compounds of the present invention may be prepared by any conventional means. Suitable processes for synthesizing these compounds are provided in the examples. Generally, compounds of the invention may be prepared according to the schemes below.




5-Fluoro-indan-1-one 10 was subjected to a Schmidt reaction to afford dihydro-isoquinolinone 11, which was treated with excess isobutyronitrile potassium salt to furnish cyano compound 12. Buckwald-Hartwig coupling with dibromobenzaldehyde 13 gave mono-bromide 14, which through a Suzuki coupling with the preformed boronate of 15 afforded pyridazinone 16. Final reduction to the benzylic alcohol gave compound of interest 1.










1-Bromo-3,5-difluorobenzene 17 was subjected to an aromatic nucleophilic substitution with the lithium salt of isobutyronitrile to afford selectively cyano compound 18, which was converted to its lithium salt with lithium diisopropylamide and quenched with carbon dioxide to furnish acid 19. After treatment with carbonyl diimidazole and ammonium hydroxide amide 20 was obtained. Suzuki coupling with the ethoxy-vinyl boronate 21, followed by acid treatment gave isoquinolinone 22, which was subjected to a nucleophilic aromatic substitution with 2-bromo-6-fluoro-benzaldehyde and potassium carbonate to afford intermediate 23. On the other hand, 6-amino-pyridin-3-ol 24 was alkylated with ethyl bromo-isobutyrate 25 to afford aromatic ether 26, which was subjected to a Buckwald-Hartwig coupling with the bromo-chloro-pyridazinone 27 to give 28. Reduction of ester with LAH afforded alcohol 29. Transformation of 29 to its boronate ester, followed by Suzuki coupling with bromide 23 furnished aldehyde 30, which was reduced to the corresponding alcohol to obtain compound of interest 2.







O-methyl-2-chloro-benzaldoxime 32, obtained from chlorobenzaldehyde 31, was selectively ortho-brominated under Pd-catalyzed C—H activation (J. Org. Chem. 2011, 76, 6414-6420) to afford 33, which was hydrolyzed to intermediate 34. On the other hand, 5-bromo-pyridin-2-ylamine 35 was treated with sodium methanesulfinate to give sulfone 36, which was coupled with bromo-pyridazinone 27 to afford intermediate 37. p-Methoxybenzaldehyde 38 was reductively aminated to obtain aminoalcohol 39, which was transformed to the amide 41 after condensation with 4-bromo-2-fluorobenzoic acid 40. Intramolecular cyclization to the benzo-oxazepinone 42 was achieved after treatment with sodium hydride. Palladium catalyzed condensation of isobutyraldehyde with 42 under basic conditions gave aldehyde 43. Debenzylation followed by Buckwald-Hartwig coupling with intermediate 34 afforded bis-aldehyde 45. Suzuki coupling with chloropyridazinone intermediate 37 gave 46, which was reduced to obtain the compound of interest 3.




Nitroalcohol 47 was O-protected and reduced to aniline 49, which was transformed to boronate ester 50 and coupled with bromide 51 to afford 52, which through its isocyanate was condensed with chloro-isoindoline 53 to afford urea 54. Final deprotection gave compound of interest 4.




Nicotinamide 55 was treated with 2,2-dimethyl-propionic acid in the presence of silver nitrate and ammonium persulfate to obtain tert-butylnicotinamide 56, which was treated with phthalimidopropanoic acid 57 in the presence again of silver nitrate and ammonium persulfate to afford 58 (Journal of Heterocyclic Chemistry 1989, 26, 45). Treatment of 58 with hydrazine deprotected the phthalimide to afford after intramolecular cyclization the dihydro-naphthyridinone 59, which was coupled with dibromobenzaldehyde 13 under Buckwald-Hartwig conditions to furnish bromide 60. Suzuki coupling with the halide 61 gave 62, which after reduction gave compounds of interest 5 and 6.




Fluorophthalazinone 63 (US 20100222325 A1) was coupled with dibromotoluene 64 under palladium (0) conditions to afford monobromide 65, which was transformed to the boronate ester 66 and coupled with 15 under Suzuki conditions to furnished compound of interest 7.




3-tert-Butylphenol 67 was alkylated with bromide 68 to afford aromatic ether 69, which by acid treatment cyclized to chromanol 70. Oxidation with PCC gave chromanone 71. Schmidt reaction with sodium azide and methanesulfonic acid afforded dihydro-benzoxazepinone 72, which was subjected to a Buckwald-Hartwig coupling with dibrobenzaldehyde 13 to furnish bromide 73. Suzuki coupling with the corresponding chloropyridazinone 15 or 37 gave 74, which was reduced to compounds of interest 8 and 9.
Pharmaceutical Compositions and Administration
The compounds of the present invention may be formulated in a wide variety of oral administration dosage forms and carriers. Oral administration can be in the form of tablets, coated tablets, dragées, hard and soft gelatin capsules, solutions, emulsions, syrups, or suspensions. Compounds of the present invention are efficacious when administered by other routes of administration including continuous (intravenous drip) topical parenteral, intramuscular, intravenous, subcutaneous, transdermal (which may include a penetration enhancement agent), buccal, nasal, inhalation and suppository administration, among other routes of administration. The preferred manner of administration is generally oral using a convenient daily dosing regimen which can be adjusted according to the degree of affliction and the patient's response to the active ingredient.
A compound or compounds of the present invention, as well as their pharmaceutically useable salts, together with one or more conventional excipients, carriers, or diluents, may be placed into the form of pharmaceutical compositions and unit dosages. The pharmaceutical compositions and unit dosage forms may be comprised of conventional ingredients in conventional proportions, with or without additional active compounds or principles, and the unit dosage forms may contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be employed. The pharmaceutical compositions may be employed as 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 in the form of suppositories for rectal or vaginal administration; or in the form of sterile injectable solutions for parenteral use. A typical preparation will contain from about 5% to about 95% active compound or compounds (w/w). The term “preparation” 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 an active ingredient can exist in different preparations depending on the target organ or tissue and on the desired dose and pharmacokinetic parameters.
The term “excipient” as used herein refers to a compound that is useful in preparing a pharmaceutical composition, generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes excipients that are acceptable for veterinary use as well as human pharmaceutical use. The compounds of this invention can 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 intended route of administration and standard pharmaceutical practice.
“Pharmaceutically acceptable” means that which is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable and includes that which is acceptable for veterinary as well as human pharmaceutical use.
A “pharmaceutically acceptable salt” form of an active ingredient may also initially confer a desirable pharmacokinetic property on the active ingredient which were absent in the non-salt form, and may even positively affect the pharmacodynamics of the active ingredient with respect to its therapeutic activity in the body. The phrase “pharmaceutically acceptable salt” of a compound means a salt that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. Such salts include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids 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, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like.
Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier may be one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material. In powders, the carrier generally is a finely divided solid which is a mixture with the finely divided active component. In tablets, the active component generally is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired. Suitable carriers include but are not limited to magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. Solid form preparations may contain, in addition to the active component, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.
Liquid formulations also are suitable for oral administration include liquid formulation including emulsions, syrups, elixirs, aqueous solutions, aqueous suspensions. These include solid form preparations which are intended to be converted to liquid form preparations shortly before use. Emulsions may be prepared in solutions, for example, in aqueous propylene glycol solutions or may contain emulsifying agents such as lecithin, sorbitan monooleate, or acacia. Aqueous solutions can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizing, and thickening agents. Aqueous suspensions can be prepared by dispersing the finely divided active component 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 present invention may be formulated for parenteral administration (e.g., by injection, for example bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, for example solutions in aqueous polyethylene glycol. Examples of oily or nonaqueous carriers, diluents, solvents or vehicles include propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate), and may contain formulatory agents such as preserving, wetting, emulsifying or suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution for constitution before use with a suitable vehicle, e.g., sterile, pyrogen-free water.
The compounds of the present invention may be formulated for topical administration to the epidermis as ointments, creams or lotions, or as a transdermal patch. Ointments and creams may, for example, 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 containing one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents. Formulations suitable for topical administration in the mouth include lozenges comprising active agents in a flavored 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 present invention may be formulated for administration as suppositories. A low melting wax, such as a mixture of fatty acid glycerides or cocoa butter is first melted and the active component is dispersed homogeneously, for example, by stirring. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool, and to solidify.
The compounds of the present invention may be formulated for vaginal administration. 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 nasal administration. The solutions or suspensions are applied directly to the nasal cavity by conventional means, for example, with a dropper, pipette or spray. The formulations may be provided in a single or multidose form. In the latter case of a dropper or pipette, this may be achieved by the patient administering an appropriate, predetermined volume of the solution or suspension. In the case of a spray, this may be achieved for example by means of a metering atomizing spray pump.
The compounds of the present invention may be formulated for aerosol administration, particularly to the respiratory tract and including intranasal administration. The compound will generally have a small particle size for example of the order of five (5) microns or less. Such a particle size may be obtained by means known in the art, for example by micronization. The active ingredient is provided in a pressurized pack 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 conveniently also contain a surfactant such as lecithin. The dose of drug may be controlled by a metered valve. Alternatively the active ingredients may be provided in a form of a dry powder, for example a powder mix of the compound in a suitable powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidine (PVP). The powder carrier will form a gel in the nasal cavity. The powder composition may be presented in unit dose form for example in capsules or cartridges of e.g., gelatin or blister packs from which the powder may be administered by means of an inhaler.
When desired, formulations can be prepared with enteric coatings adapted for sustained or controlled release administration of the active ingredient. For example, the compounds of the present invention can be formulated in transdermal or subcutaneous drug delivery devices. These delivery systems are advantageous when sustained release of the compound is necessary and when patient compliance with a treatment regimen is crucial. Compounds in transdermal delivery systems are frequently attached to an skin-adhesive solid support. The compound of interest can also be combined with a penetration enhancer, e.g., Azone (1-dodecylaza-cycloheptan-2-one). Sustained release delivery systems are inserted subcutaneously into to the subdermal layer by surgery or injection. The subdermal implants encapsulate the compound in a lipid soluble membrane, e.g., silicone rubber, or a biodegradable polymer, e.g., polyactic acid.
Suitable formulations along with pharmaceutical carriers, diluents and excipients are described in Remington: The Science and Practice of Pharmacy 1995, edited by E. W. Martin, Mack Publishing Company, 19th edition, Easton, Pa. A skilled formulation scientist may modify the formulations within the teachings of the specification to provide numerous formulations for a particular route of administration without rendering the compositions of the present invention unstable or compromising their therapeutic activity.
The modification of the present compounds to render them more soluble in water or other vehicle, for example, may be easily accomplished by minor modifications (salt formulation, esterification, etc.), which are well within the ordinary skill in the art. It is also well within the ordinary skill of the art to modify the route of administration and dosage regimen of a particular compound in order to manage the pharmacokinetics of the present compounds for maximum beneficial effect in patients.
The term “therapeutically effective amount” as used herein means an amount required to reduce symptoms of the disease in an individual. The dose will be adjusted to the individual requirements in each particular case. That dosage can vary within wide limits depending upon numerous factors such as the severity of the disease to be treated, the age and general health condition of the patient, other medicaments with which the patient is being treated, the route and form of administration and the preferences and experience of the medical practitioner involved. For oral administration, a daily dosage of between about 0.01 and about 1000 mg/kg body weight per day should be appropriate in monotherapy and/or in combination therapy. A preferred daily dosage is between about 0.1 and about 500 mg/kg body weight, more preferred 0.1 and about 100 mg/kg body weight and most preferred 1.0 and about 10 mg/kg body weight per day. Thus, for administration to a 70 kg person, the dosage range would be about 7 mg to 0.7 g per day. The daily dosage can be administered as a single dosage or in divided dosages, typically between 1 and 5 dosages per day. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect for the individual patient is reached. One of ordinary skill in treating diseases described herein will be able, without undue experimentation and in reliance on personal knowledge, experience and the disclosures of this application, to ascertain a therapeutically effective amount of the compounds of the present invention for a given disease and patient.
The pharmaceutical preparations are preferably in unit dosage forms. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.
Indications and Methods of Treatment
The compounds of generic Formula I inhibit Bruton's tyrosine kinase (BTK). Activation of BTK by upstream kinases results in activation of phospholipase-Cγ which, in turn, stimulates release of pro-inflammatory mediators. Compounds of Formula I are useful in the treatment of arthritis and other anti-inflammatory and auto-immune diseases. Compounds according to Formula I are, accordingly, useful for the treatment of arthritis. Compounds of Formula I are useful for inhibiting BTK in cells and for modulating B-cell development. The present invention further comprises pharmaceutical compositions containing compounds of Formula I admixed with pharmaceutically acceptable carrier, excipients or diluents.
The compounds described herein are kinase inhibitors, in particular BTK inhibitors. These inhibitors can be useful for treating one or more diseases responsive to kinase inhibition, including diseases responsive to BTK inhibition and/or inhibition of B-cell proliferation, in mammals. Without wishing to be bound to any particular theory, it is believed that the interaction of the compounds of the invention with BTK results in the inhibition of BTK activity and thus in the pharmaceutical utility of these compounds. Accordingly, the invention includes a method of treating a mammal, for instance a human, having a disease responsive to inhibition of BTK activity, and/or inhibiting B-cell proliferation, comprising administrating to the mammal having such a disease, an effective amount of at least one chemical entity provided herein. An effective concentration may be ascertained experimentally, for example by assaying blood concentration of the compound, or theoretically, by calculating bioavailability. Other kinases that may be affected in addition to BTK include, but are not limited to, other tyrosine kinases and serine/threonine kinases.
Kinases play notable roles in signaling pathways controlling fundamental cellular processes such as proliferation, differentiation, and death (apoptosis). Abnormal kinase activity has been implicated in a wide range of diseases, including multiple cancers, autoimmune and/or inflammatory diseases, and acute inflammatory reactions. The multifaceted role of kinases in key cell signaling pathways provides a significant opportunity to identify novel drugs targeting kinases and signaling pathways.
An embodiment includes a method of treating a patient having an autoimmune and/or inflammatory disease, or an acute inflammatory reaction responsive to inhibition of BTK activity and/or B-cell proliferation.
Autoimmune and/or inflammatory diseases that can be affected using compounds and compositions according to the invention include, but are not limited to: psoriasis, allergy, Crohn's disease, irritable bowel syndrome, Sjogren's disease, tissue graft rejection, and hyperacute rejection of transplanted organs, asthma, systemic lupus erythematosus (and associated glomerulonephritis), dermatomyositis, multiple sclerosis, scleroderma, vasculitis (ANCA-associated and other vasculitides), autoimmune hemolytic and thrombocytopenic 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 in which 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 cyclooxgenase enzyme inhibitors, gold compounds, corticosteroids, methotrexate, tumor necrosis factor receptor (TNF) receptors antagonists, immunosuppressants and methotrexate.
Examples of NSAIDs include, but are not limited to, ibuprofen, flurbiprofen, naproxen and naproxen sodium, diclofenac, combinations of diclofenac sodium and misoprostol, sulindac, oxaprozin, diflunisal, piroxicam, indomethacin, etodolac, fenoprofen calcium, ketoprofen, sodium nabumetone, sulfasalazine, tolmetin sodium, 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 by are not limited to acetylsalicylic acid or aspirin, sodium salicylate, and choline and magnesium salicylates.
The anti-inflammatory agent may also be a corticosteroid. For example, the corticosteroid may be cortisone, dexamethasone, methylprednisolone, prednisolone, prednisolone sodium phosphate, or prednisone.
In additional embodiments the anti-inflammatory agent is a gold compound such as gold sodium thiomalate or auranofin.
The invention also includes embodiments in which the anti-inflammatory agent is a metabolic inhibitor such as a dihydrofolate reductase inhibitor, such as methotrexate or a dihydroorotate dehydrogenase inhibitor, such as leflunomide.
Other embodiments of the invention pertain to combinations in which at least one anti-inflammatory compound is an anti-C5 monoclonal antibody (such as eculizumab or pexelizumab), a TNF antagonist, such as entanercept, or infliximab, which is an anti-TNF alpha monoclonal antibody.
Still other embodiments of the invention pertain to combinations in which at least one active agent is an immunosuppressant compound such as an immunosuppressant compound chosen from methotrexate, leflunomide, cyclosporine, tacrolimus, azathioprine, and mycophenolate mofetil.
B-cells and B-cell precursors expressing BTK have been implicated in the pathology of B-cell malignancies, including, but not limited to, B-cell lymphoma, lymphoma (including Hodgkin's and non-Hodgkin's lymphoma), hairy cell lymphoma, multiple myeloma, chronic and acute myelogenous leukemia and chronic and acute lymphocytic leukemia.
BTK has been shown to be an inhibitor of the Fas/APO-1 (CD-95) death inducing signaling complex (DISC) in B-lineage lymphoid cells. The fate of leukemia/lymphoma cells may reside in the balance between the opposing proapoptotic effects of caspases activated by DISC and an upstream anti-apoptotic regulatory mechanism involving BTK and/or its substrates (Vassilev et al., J. Biol. Chem. 1998, 274, 1646-1656).
It has also been discovered that BTK inhibitors are useful as chemosensitizing agents, and, thus, are useful in combination with other chemotherapeutic drugs, in particular, drugs that induce apoptosis. Examples of other chemotherapeutic drugs that can 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), tubulin directed agents (e.g. taxol and vinblastine), and biological agents (e.g. antibodies such as anti CD20 antibody, IDEC 8, immunotoxins, and cytokines).
BTK activity has also be associated with some leukemias expressing the bcr-abl fusion gene resulting from translocation of parts of chromosome 9 and 22. This abnormality is commonly observed in chronic myelogenous leukemia. BTK is constitutively phosphorylated by the bcr-abl kinase which initiates downstream survival signals which circumvents apoptosis in bcr-abl cells. (N. Feldhahn et al. J. Exp. Med. 2005 201(11):1837-1852).
Methods of Treatment
The application provides a method for treating an inflammatory and/or autoimmune condition comprising administering to a patient in need thereof a therapeutically effective amount of the compound of Formula I.
The application provides a method for treating an inflammatory condition comprising administering to a patient in need thereof a therapeutically effective amount of the compound of Formula I.
The application provides a method for treating rheumatoid arthritis comprising administering to a patient in need thereof a therapeutically effective amount of the compound of Formula I.
The application provides a method for treating asthma comprising administering to a patient in need thereof a therapeutically effective amount of Formula I.
The application provides a method for treating an inflammatory and/or autoimmune condition comprising administering to a patient in need thereof a therapeutically effective amount of the BTK inhibitor compound of Formulae I.
The application provides a method for treating arthritis comprising administering to a patient in need thereof a therapeutically effective amount of the BTK inhibitor compound of Formula I.
The application provides a method for treating asthma comprising administering to a patient in need thereof a therapeutically effective amount of the BTK inhibitor compound of Formula I.
The application provides a method of inhibiting B-cell proliferation comprising administering to a patient in need thereof a therapeutically effective amount of the BTK inhibitor compound of Formula I.
The application provides a method for inhibiting BTK activity comprising administering the BTK inhibitor compound of any one of Formula I, wherein the BTK inhibitor compound exhibits an IC50 of 50 micromolar or less in an in vitro biochemical assay of BTK activity.
In one variation of the above method, the BTK inhibitor compound exhibits an IC50 of 100 nanomolar or less in an in vitro biochemical assay of BTK activity.
In another variation of the above method, the compound exhibits an IC50 of 10 nanomolar or less in an in vitro biochemical assay of BTK activity.
The application provides a method for treating an inflammatory condition comprising co-administering to a patient in need thereof a therapeutically effective amount of an anti-inflammatory compound in combination with the BTK inhibitor compound of Formula I.
The application provides a method for treating arthritis comprising co-administering to a patient in need thereof a therapeutically effective amount of an anti-inflammatory compound in combination with the BTK inhibitor compound of Formula I.
The application provides a method for treating a lymphoma or a BCR-ABL1+ leukemia cells by administering to a patient in need thereof a therapeutically effective amount of the BTK inhibitor compound of Formula I.
The invention provides the use of a compound as described above for use as therapeutically active substance.
The invention provides the use of a compound as described above in the treatment of an inflammatory and/or autoimmune condition.
The invention provides the use of a compound as described above for the preparation of a medicament for the treatment of an inflammatory and/or autoimmune condition.
The invention provides a compound as described above for use in the treatment of an inflammatory and/or autoimmune condition.
The invention provides a compound as described above for use in the treatment of rheumatoid arthritis.
The invention provides a compound as described above for use in the treatment of asthma.
The invention provides the invention as hereinbefore described.
EXAMPLES
General Abbreviations
Commonly used abbreviations include: acetyl (Ac), azo-bis-isobutyrylnitrile (AIBN), atmospheres (Atm), 9-borabicyclo[3.3.1]nonane (9-BBN or BBN), 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (BINAP), tert-butoxycarbonyl (Boc), di-tert-butyl pyrocarbonate or boc anhydride (BOC2O), benzyl (Bn), butyl (Bu), Chemical Abstracts Registration Number (CASRN), benzyloxycarbonyl (CBZ or Z), carbonyl diimidazole (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,N′-dicyclohexylcarbodiimide (DCC), 1,2-dichloroethane (DCE), dichloromethane (DCM), 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), diethyl azodicarboxylate (DEAD), di-iso-propylazodicarboxylate (DIAD), di-iso-butylaluminumhydride (DIBAL or DIBAL-H), di-iso-propylethylamine (DIPEA), N,N-dimethyl acetamide (DMA), 4-N,N-dimethylaminopyridine (DMAP), N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,1′-bis-(diphenylphosphino)ethane (dppe), 1,1′-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), 2-ethoxy-2H-quinoline-1-carboxylic acid ethyl ester (EEDQ), diethyl ether (Et2O), ethyl isopropyl ether (EtOiPr), O-(7-azabenzotriazole-1-yl)-N, N,N′N′-tetramethyluronium hexafluorophosphate acetic acid (HATU), acetic acid (HOAc), 1-N-hydroxybenzotriazole (HOBt), high pressure liquid chromatography (HPLC), iso-propanol (IPA), isopropylmagnesium chloride (iPrMgCl), hexamethyl disilazane (HMDS), liquid chromatography mass spectrometry (LCMS), lithium hexamethyl disilazane (LiHMDS), meta-chloroperoxybenzoic acid (m-CPBA), methanol (MeOH), melting point (mp), MeSO2-(mesyl or Ms), methyl (Me), acetonitrile (MeCN), m-chloroperbenzoic acid (MCPBA), mass spectrum (ms), methyl t-butyl ether (MTBE), methyl tetrahydrofuran (MeTHF), N-bromosuccinimide (NBS), n-Butyllithium (nBuLi), N-carboxyanhydride (NCA), N-chlorosuccinimide (NCS), N-methylmorpholine (NMM), N-methylpyrrolidone (NMP), pyridinium chlorochromate (PCC), Dichloro-((bis-diphenylphosphino)ferrocenyl) palladium(II) (Pd(dppf)Cl2), palladium(II) acetate (Pd(OAc)2), tris(dibenzylideneacetone)dipalladium(0) (Pd2(dba)3), pyridinium 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-Butyllithium (sBuLi), tert-butyldimethylsilyl or t-BuMe2Si (TBDMS), tetra-n-butylammonium fluoride (TBAF), triethylamine (TEA or Et3N), 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO), trimethylsilylethoxymethyl (SEM), triflate or CF3SO2— (Tf), trifluoroacetic acid (TFA), 1,1′-bis-2,2,6,6-tetramethylheptane-2,6-dione (TMHD), 0-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU), thin layer chromatography (TLC), tetrahydrofuran (THF), trimethylsilyl or Me3Si (TMS), p-toluenesulfonic acid monohydrate (TsOH or pTsOH), 4-Me-C6H4SO2— or tosyl (Ts), and N-urethane-N-carboxyanhydride (UNCA). Conventional nomenclature including the prefixes normal (n), iso (i-), secondary (sec-), tertiary (tert-) and neo have their customary meaning when used with an alkyl moiety. (J. Rigaudy and D. P. Klesney, Nomenclature in Organic Chemistry, IUPAC 1979 Pergamon Press, Oxford.).
General Conditions
Compounds of the present invention can be prepared beginning with the commercially available starting materials by utilizing general synthetic techniques and procedures known to those skilled in the art. Outlines below are reaction schemes suitable for preparing such compounds. Further exemplification can be found in the specific examples.
SPECIFIC ABBREVIATIONS


    boc tert-butoxycarbonyl
    CH2Cl2 dichloromethane
    Cs2CO3 cesium carbonate
    DCM Dichloromethane
    DMF N,N-dimethylformamide
    DMSO Dimethylsulfoxide
    EtOAc ethyl acetate
    HATU O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate
    Hunig's Base N,N-diisopropylethylamine
    HCl hydrogen chloride
    LC-MS liquid chromatography mass spectrometry
    HPLC high pressure liquid chromatography
    MeOH methyl alcohol
    MgSO4 magnesium sulfate
    nBuLi n-butyl lithium
    NaCl sodium chloride
    Na2CO3 sodium carbonate
    NaOMe sodium methoxide
    Na2SO4 sodium sulfate
    NH4OH ammonium hydroxide
    NMP 1-methyl-2-pyrrolidinone
    NMR nuclear magnetic resonance
    Pd(OAc)2 palladium(II) acetate
    TFA trifluoroacetic acid
    THF tetrahydrofuran
    TLC thin layer chromatography
    TMSCl trimethylsilyl chloride

General Experimental Details



Reagents were purchased from Aldrich, Oakwood, Matrix or other suppliers and used without further purification. Reactions using microwave irradiation for heating were conducted using either a Personal Chemistry Emrys Optimizer System or a CEM Discovery System. The purification of multi-milligram to multi-gram scale was conducted by methods known know to those skilled in the art such as elution of silica gel flash column; preparative flash column purifications were also effected in some cases by use of disposal pre-packed multigram silica gel columns (RediSep) eluted with a CombiFlash system. BiotageTM and ISCOTM are also flash column instruments that may have been used in this invention for purification of intermediates.
For the purpose of judging compound identity and purity, LC/MS (liquid chromatography/mass spectroscopy) spectra were recorded using the following system. For measurement of mass spectra, the system consists of a Micromass Platform II spectrometer: ES Ionization in positive mode (mass range: 150-1200). The simultaneous chromatographic separation was achieved with the following HPLC system: ES Industries Chromegabond WR C-18 3u 120 Å (3.2×30 mm) column cartridge; Mobile Phase A: Water (0.02% TFA) and Phase B: Acetonitrile (0.02% TFA); gradient 10% B to 90% B in 3 minutes; equilibration time of 1 minute; flow rate of 2 mL/minute.
Many compounds of Formula 1 were also purified by reversed phased HPLC, using methods well known to those skilled in the art. In some cases, preparative HPLC purification was conducted using PE Sciex 150 EX Mass Spec controlling a Gilson 215 collector attached to a Shimadzu preparative HPLC system and a Leap autoinjector. Compounds were collected from the elution stream using LC/MS detection in the positive ion detection: The elution of compounds from C-18 columns (2.0×10 cm eluting at 20 mL/min) was effected using appropriate linear gradation mode over 10 minutes of Solvent (A) 0.05% TFA/H2O and Solvent (B) 0.035% TFA/acetonitrile. For injection on to HPLC systems, the crude samples were dissolved in mixtures of methanol, acetonitrile and DMSO.
Compounds were characterized either by 1H-NMR using a Bruker 400 MHz NMR Spectrometer.
The compounds of the present invention may be synthesized according to known techniques. The following examples and references are provided to aid the understanding of the present invention. The examples are not intended, however, to limit the invention, the true scope of which is set forth in the appended claims. The names of the final products in the examples were generated using Isis AutoNom 2000.
Preparative Examples




To a stirred solution of 5-fluoro-indan-1-one (15 g, 104.83 mmol) in DCM (168 mL) was added mesic acid (120 mL) at 0° C. To this clear solution was added sodium azide (9.5 g, 146.76 mmol) at −10° C. portion wise over 40 min. The mixture was stirred for 2 h at −10° C., 20% aqueous NaOH solution was added drop wise at 0° C., stirred for 15 min (TLC, silica; 50% Ethyl acetate in hexane, Rf=0.3). After separating the DCM, aqueous part was extracted with DCM (2×50 mL), organic part was dried and concentrated, crude was purified by normal column chromatography (silica gel 100-200, product was eluted at 60% ethyl acetate in hexane) to get 6-fluoro-3,4-dihydro-2H-isoquinolin-1-one (12.0 g, 69.31%) as white solid. LC-MS: 166.0 (M+H).




To a stirred solution of 6-fluoro-3,4-dihydro-2H-isoquinolin-1-one (2.5 g, 15.14 mmol) in dry THF (30 mL) was added isobutyronitrile (3) (5.4 mL, 60.54 mmol). To this solution was added 0.5 (M) KHMDS in toluene (104 ml, 52 mmol) slowly at 0° C., it was turned into yellowish viscous liquid. The mixture was heated at 70° C. for 4 h (monitored by LCMS). Cooled to RT 2nd lot of isobutyronitrile (3 mL, 33.42 mmol) was added, again heated at 70° C. and stirred for 16 h (TLC, silica; 50% ethyl acetate in hexane, Rf=0.25). Reaction mixture was cooled; quenched with ice cold water (50 mL) and extracted with ethyl acetate (3×50 mL). Combined organic part was concentrated, obtained crude was purified by CombiFlash column (40-60% ethyl acetate in hexane was used as eluent) to get 2-methyl-2-(1-oxo-1,2,3,4-tetrahydro-isoquinolin-6-yl)-propionitrile (2.5 g, 77.09%) as white solid. LC-MS: 215.2 (M+H).




To a stirred solution of 2-methyl-2-(1-oxo-1,2,3,4-tetrahydro-isoquinolin-6-yl)-propionitrile (2.5 g, 11.68 mmol) in dry dioxane (50 mL) were added 2,6-dibromo-benzaldehyde (4.6 g, 17.52 mmol), Cs2CO3 (7.6 g, 23.36 mmol). The reaction mixture was degassed with argon, Pd(dba)2 (134 mg, 0.234 mmol) and Xantphos (206 mg, 0.35 mmol) were added and again degassed. The mixture was heated at 100° C. for 3 h, cooled to RT, filtered through a pad of celite; washed with ethyl acetate (3×50 mL). The mixture was concentrated and purified by CombiFlash column (eluted at 40% ethyl acetate in hexane) to get 2-[2-(3-bromo-2-formyl-phenyl)-1-oxo-1,2,3,4-tetrahydro-isoquinolin-6-yl]-2-methyl-propionitrile (2.5 g, 53.87%) as light brown solid. LC-MS: 397.2 (M+H).




The reaction mixture of 6-chloro-2-methyl-4-[5-(morpholine-4-carbonyl)-pyridin-2-ylamino]-2H-pyridazin-3-one (1.5 g, 4.29 mmol) (US2012/40949 A1), bis(pinacolato)diboron (1.4 g, 5.57 mmol) and KOAc (dry) (1.3 g, 12.86 mmol) in dioxane (35 mL), was degassed under argon, X-Phos (307 mg, 0.64 mmol) and Pd(OAc)2 (96 mg, 0.43 mmol) were added and stirred at 98° C. for 15 min. The brown reaction mixture was turned greenish brown solution (monitored by LCMS). The flask was raised out of the heating bath, cooled, filtered through a short plug of celite under argon into a second flask which contained a degassed suspension of 2-[2-(3-bromo-2-formyl-phenyl)-1-oxo-1,2,3,4-tetrahydro-isoquinolin-6-yl]-2-methyl-propionitrile (1.53 g, 3.86 mmol) and K2CO3 (2.37 g, 17.15 mmol) in dioxane-water (1:1; 90 mL), n-BuOH (10.5 mL). The reaction mixture was degassed with argon, ticyclohexylphosphine (359 mg, 1.28 mmol), and Pd(dba)2 (369 mg, 0.642 mmol) were added, again degassed, and heated at 110° C. for 1 h. Reaction was cooled to RT, filtered through a plug of celite; washed with ethylacetate (3×100 mL). Filtrate was concentrated, combined crude was purified by normal column chromatography (normal silica gel 100-200 mesh, eluted at 2 to 2.5% MeOH in DCM) to get 2-[2-(2-formyl-3-{1-methyl-5-[5-(morpholine-4-carbonyl)-pyridin-2-ylamino]-6-oxo-1,6-dihydro-yridazin-3-yl}-phenyl)-1-oxo-1,2,3,4-tetrahydro-isoquinolin-6-yl]-2-methyl-propionitrile as light brown solid (1.5 g, 55.56%). LC-MS: 632.6 (M+H).




To a stirred solution of 2-[2-(2-formyl-3-{1-methyl-5-[5-(morpholine-4-carbonyl)-pyridin-2-ylamino]-6-oxo-1,6-dihydro-yridazin-3-yl}-phenyl)-1-oxo-1,2,3,4-tetrahydro-isoquinolin-6-yl]-2-methyl-propionitrile (3 g, 4.75 mmol) in DCM (55 mL) and MeOH (36 mL) was added solution of sodium borohydride (360 mg, 9.51 mmol) in water (3.6 mL) at 0° C. The reaction mixture was stirred for 20 min at the same temperature. Reaction was monitored by TLC (Silica, UV/DNP, 5% MeOH in DCM, Rf=0.35). Reaction was quenched with ice, organic part was separated, dried and concentrated). Crude was purified by normal silica gel column chromatography (silica, 100-200, eluted at 2-2.5% MeOH in DCM). Purified material was re-crystallized with DCM and hexane to get 2-[2-(2-hydroxymethyl-3-{1-methyl-5-[5-(morpholine-4-carbonyl)-pyridin-2-ylamino]-6-oxo-1,6-dihydro-pyridazin-3-yl}-phenyl)-1-oxo-1,2,3,4-tetrahydro-isoquinolin-6-yl]-2-methyl-propionitrile as white solid (2.1 g, 69.7%). LC-MS: 634.6 (M+H).




Charged 1-bromo-3,5-difluorobenzene (300 g, 1.55 mol, Eq: 1.00), isobutyronitrile (215 g, 3.11 mol, Eq: 2) and THF (1.2 l) into 5 L 3-neck flask at room temperature. Added 1 M LiHMDS (1.71 l, 1.71 mol, Eq: 1.1) solution dropwise at temperature range 15-22° C. under cold water bath; took 2.5 hr for addition. After 2.5 hr, conversion 91% by HPLC (9% SM and 12% dialkylated). Added additional 0.1 eq. of LiHMDS solution. Stirred overnight when the conversion was 97% (3% SM and 21% dialkylated). Quenched reaction with 6N HCl to pH  ̃3-4. Extracted with toluene (twice); then the combined organics were concentrated down; used more toluene to remove water and also excess isobutyronitrile. Dialkylated byproduct was coming out during concentration; around 600 mL n-heptane was added, then stirred overnight at rt. Filtered the solution to remove most of byproduct as solid; washed with heptane. Concentrated down the filtrate to afford 2-(3-bromo-5-fluorophenyl)-2-methylpropanenitrile (337.9 g, 1.26 mol, 80.8% yield) with 90% HPLC purity (no SM and 9% dialkylated).




2-(3-bromo-5-fluorophenyl)-2-methylpropanenitrile (172 g, 710 mmol, Eq: 1.00) was dissolved in THF (1.03 L), then the solution was cooled down to −75° C. Added 2M LDA (373 ml, 746 mmol, Eq: 1.05) into the solution dropwise, and kept temperature below −70° C. Additional stirring for 2 hr, and an aliquot was tested with dryice; full conversion. To another 3 L rbf, dryice and THF were added. Reverse quenched with dryice; took 30 min to transfer whole anion solution and kept temperature below −50° C. (−60 ̃−50° C.). Allowed to warm up to room temperature after additional 1 hr stirring. Added water and acidified with 6 N HCl solution (pH 3-4).
Phase separation and extracted with MTBE and DCM. Concentrated the combined organics. Obtained 2-bromo-4-(2-cyanopropan-2-yl)-6-fluorobenzoic acid (246 g, 765 mmol, 108% yield) as an oil ( ̃89% HPLC purity). No further purification and moved on to the next reaction.




CDI (3.33 g, 20.6 mmol, Eq: 1.4) was added into a THF (42 ml) solution of 2-bromo-4-(2-cyanopropan-2-yl)-6-fluorobenzoic acid (4.2 g, 14.7 mmol, Eq: 1.00) portion-wise at room temperature. Stirred for 3 hr. Added ammonium hydroxide (17.1 g, 19.1 ml, 147 mmol, Eq: 10) to the mixture. Stirred for 2 hr. Washed with 20% K2CO3 solution to remove unreacted starting material, then washed with 1 N HCl. Crystallization from EtOAc/Heptane to give 2-bromo-4-(2-cyanopropan-2-yl)-6-fluorobenzamide (3.4 g, 11.9 mmol, 81.2% yield).




To a stirred solution of 2-bromo-4(cyano-dimethyl-methyl)-6-fluoro-benzamide (2 g, 7.018 mmol) and 2-((E)-2-ethoxy-vinyl)-4,4,5,5-tetramethyl-[1,3,2]dioxaborolane (1.783 mL, 8.421 mmol) in dioxane-water (7:1; 16 mL) was added K2CO3 (1.94 gm, 14.04 mmol) and followed by tricyclohexylphosphine (157 mg, 0.561 mmol). The reaction mixture was well degassed with nitrogen. To this reaction mixture was added Pd(dba)2 (161 mg, 0.281 mmol), again degassed with nitrogen and heated at 90° C. for 16 h and 6N HCl (6 mL) was added and heated to 40° C. for 5 h (silica TLC; 50% ethyl acetate in hexane, Rf=0.3). From the reaction mixture dioxane was removed under reduced pressure, obtained crude residue was purified by washings with 10% EtOAc in hexane to get 2-(8-fluoro-1-oxo-1,2-dihydro-isoquinolin-6-yl)-2-methyl-propionitrile (1.26 g, 77.98%) as a brown solid. LC-MS: 229.2 (M−H).




To a stirred solution of 2-(8-fluoro-1-oxo-1,2-dihydro-isoquinolin-6-yl)-2-methyl-propionitrile (1.26 gm, 5.478 mmol) in DMA (28 mL), was added 2-bromo-6-fluoro-benzaldehyde (1.66 gm, 8.217 mmol), potassium carbonate (1.512 gm, 10.957 mmol) and followed by tetraethyl ammonium chloride (118 mg, 0.712 mmol) was added under argon. The reaction mixture heated at 78° C. for 24 h (silica TLC; 50% ethyl acetate in hexane, Rf=0.50), water was added and extracted with DCM (2×90 mL). The combined extract was concentrated to get crude product, which was purified by column chromatography (normal silica gel 100-200 mesh; 25% EtOAc in hexane used as eluent) to get 2-[2-(3-bromo-2-formyl-phenyl)-8-fluoro-1-oxo-1,2-dihydro-isoquinolin-6-yl]-2-methyl-propionitrile (840 mg, 37.11%) as a light yellow solid. LC/MC:
412.8 (M+), 414.8 (M+2).




The reaction mixture of 6-chloro-4-[5-(2-hydroxy-1,1-dimethyl-ethoxy)-pyridin-2-ylamino]-2-methyl-2H-pyridazin-3-one (1 g, 3.08 mmol) (WO 2012020008 A1), bis(pinacolato)diboron (1.06 g, 4.00 mmol) and KOAc (dry) (907 mg, 9.23 mmol) in dioxane (50 mL), was degassed under argon. X-Phos (220 mg, 0.46 mmol) and Pd(OAc)2 (69 mg, 0.31 mmol) were added and stirred at 98° C. for 15 min (monitored by LCMS). The flask was raised out of the heating bath, cooled, filtered through a short plug of celite under argon into a second flask which contained a degassed suspension of 2-[2-(3-bromo-2-formyl-phenyl)-8-fluoro-1-oxo-1,2-dihydro-isoquinolin-6-yl]-2-methyl-propionitrile (1.14 g, 2.77 mmol) and K2CO3 (1.7 g, 12.31 mmol), dioxane (15.6 mL), water (15 mL), n-BuOH (3.7 mL). The reaction mixture was degassed with argon again and ticyclohexylphosphine (257 mg, 0.92 mmol), Pd(dba)2 (264 mg, 0.46 mmol) were added under inert atmosphere, the reaction mixture was heated at 110° C. for 1 h. Reaction was cooled to RT, filtered through a plug of celite, washed with ethylacetate (3×100 mL). Combined filtrate was concentrated, obtained crude was purified by normal silica column chromatography (silica gel 100-200 mesh, eluted at 2-2.5% MeOH in DCM) to get 2-[8-fluoro-2-(2-formyl-3-{5-[5-(2-hydroxy-1,1-dimethyl-ethoxy)-pyridin-2-ylamino]-1-methyl-6-oxo-1,6-dihydro-pyridazin-3-yl}-phenyl)-1-oxo-1,2-dihydro-isoquinolin-6-yl]-2-methyl-propionitrile (1.1 g, 57.38%, 400 mg mixture) as brown sticky solid. LC-MS: 622.2 (M+H).




To a stirred solution of 2-[8-fluoro-2-(2-formyl-3-{5-[5-(2-hydroxy-1,1-dimethyl-ethoxy)-pyridin-2-ylamino]-1-methyl-6-oxo-1,6-dihydro-pyridazin-3-yl}-phenyl)-1-oxo-1,2-dihydro-isoquinolin-6-yl]-2-methyl-propionitrile (2.4 g, 3.85 mmol) in DCM (46.7 mL) and MeOH (30.8 mL) was added solution of sodium borohydride (292 mg, 7.70 mmol) in water (3.4 mL) at 0° C. The reaction mixture was stirred for 15 min at the same temperature (Silica TLC; 5% MeOH in DCM, Rf=0.4). Reaction was quenched with ice (100 g), organic part was diluted with DCM (200 mL) and separated, dried and concentrated. Obtained crude was purified by normal silica column chromatography (silica, 100-200, eluted at 2% MeOH in DCM). Purified material was re-crystallized with DCM and ether to get 2-[8-fluoro-2-(3-{5-[5-(2-hydroxy-1,1-dimethyl-ethoxy)-pyridin-2-ylamino]-1-methyl-6-oxo-1,6-dihydro-pyridazin-3-yl}-2-hydroxymethyl-phenyl)-1-oxo-1,2-dihydro-soquinolin-6-yl]-2-methyl-propionitrile (1.6 g, 66.45%) as white solid. LC-MS: 625.3 (M+H).




To a stirred solution of 6-amino-pyridin-3-ol (4 gm, 36.364 mmol) in ACN (80 mL) was added cesium carbonate (47.41 gm, 145.45 mmol), stirred for 1 h at RT, 2-bromo-2-methyl-propionic acid ethyl ester (5.66 gm, 38.18 mmol) was added and stirred for 16 h (silica TLC; 50% EtOAc in hexane, Rf=0.5). To the reaction water (200 mL) was added and extracted with ethyl acetate (3×100 mL). Combined extracts was concentrated, obtained crude was purified by column chromatography (normal silica gel 100-200 mesh, 18 to 22% EtOAc in hexane was used as eluent) to get 2-(6-amino-pyridin-3-yloxy)-2-methyl-propionic acid ethyl ester (4.4 g, 53.96%) as a light yellow solid, LC/MS: 224.8 (M+H).




To a stirred solution of 2-(6-amino-pyridin-3-yloxy)-2-methyl-propionic acid ethyl ester (2 g, 8.929 mmol) and 4-bromo-6-chloro-2-methyl-2H-pyridazin-3-one (2.588 gm, 11.607 mmol) in dry dioxane (80 mL) was added Cs2CO3 (10.188 gm, 31.25 mmol). The reaction mixture was well degassed with argon, xantphos (774 mg, 1.339 mmol) and Pd2(dba)3 (654 mg, 0.714 mmol) were added under argon atmosphere, heated at 90° C. for 18 h. Cooled the reaction mixture, filtered through a plug of celite; washed with dioxane (50 mL). Combined extracts was concentrated to get the crude, which was purified by normal silica gel column chromatography using 18% EtOAc in hexane as eluent to get 2-[6-(6-chloro-2-methyl-3-oxo-2,3-dihydro-pyridazin-4-ylamino)-pyridin-3-yloxy]-2-methyl-propionic acid ethyl ester (2.7 gm, 82.44%) as a yellow solid, LC-MS: 367.0 (M+H).




To a stirred solution of 2-[6-(6-chloro-2-methyl-3-oxo-2,3-dihydro-pyridazin-4-ylamino)-pyridin-3-yloxy]-2-methyl-propionic acid ethyl ester (1 g, 2.73 mmol) in dry THF (25 mL) was added LAH [3.8 mL, 3.8 mmol, 1(M) solution in THF] drop wise (over 15 min via syringe) at −40° C. The reaction mixture was stirred for 1 h at the same temperature (silica TLC; 50% EtOAc in hexane used as eluent; Rf=0.2). quench by adding water (0.1 mL), stirred for 10 min, added 5% NaOH solution (0.2 mL), stirred for 10 min, again added water (0.2 mL), stirred for 10 min, clear organic part was separated, dried and concentrated, crude was purified by normal silica column chromatography (silica gel 100-200 mesh, eluted at 40-50% ethyl acetate in hexane) to get 6-chloro-4-[5-(2-hydroxy-1,1-dimethyl-ethoxy)-pyridin-2-ylamino]-2-methyl-2H-pyridazin-3-one as off white solid (650 mg, 73.42%). LC-MS: 325.2 (M+H).




To a stirred solution of 4-methoxybenzaldehyde (20 g, 147.05 mmol) in methanol (150 mL), was added amino ethanol (35.88 g, 588.23 mmol), acetic acid (40.58 g, 676.47 mmol) and stirred at RT for 20 h, to reaction sodium cyanoborohydride (5.54 g, 88.23 mmol) was added and stirred at RT for 36 h (silica TLC; 5% MeOH in DCM used as eluent; Rf=0.2). To the reaction mixture saturated NaHCO3 in water (500 mL) was added and extracted with EtOAc (3×300 mL). Combined extracts was dried, concentrated under vacuum to get 2-(4-methoxy-benzylamino)-ethanol (6.0 g, 22.51%) as reddish oil LC-MS: 182.2 (M+H).




To a stirred solution of, 2-(4-methoxybenzylamino)ethanol (1.0 g, 5.52 mmol) in DCM (20 mL), were added 4-bromo-2-fluorobenzoic acid (1.21 g, 5.52), DIPEA (1.18 mL, 7.18 mmol) and HATU (2.1 g, 5.52 mmol), stirred at RT for 16 h (silica TLC; 5% MeOH in DCM as mobile phase; Rf=0.5). Reaction mixture was concentrated; to the residue, saturated aqueous NH4Cl (50 mL) was added and extracted with EtOAc (3×20 mL). Combined organic phase was washed with 1% aqueous HCl (30 mL) and water (20 mL). Separated organic part was dried, concentrated to get the crude mass, which was purified by flash chromatography (silica gel, eluted with 40% EtOAc in hexane), to get 4-bromo-2-fluoro-N-(2-hydroxy-ethyl)-N-(4-methoxy-benzyl)-benzamide (0.5 g, 23.68%) as yellow liquid. LC-MS: 382.2 (M+), 384.2 (M+2).




To a stirred solution of, 4-bromo-2-fluoro-N-(2-hydroxyethyl)-N-(4-methoxybenzyl)-benzamide (1.0 g, 2.61 mmol) in DMF (10 mL), was added NaH [(60% in paraffin oil); 0.125 g, 3.14 mmol] at RT and stirred under argon for 16 h (silica TLC; 50% EtOAc in hexane; Rf=0.5). To the reaction mixture aqueous saturated NH4Cl (30 mL) was added and extracted with EtOAc (3×25 mL). Combined extracts was washed with water (50 mL) followed by brine (30 mL); dried and concentrated to get the crude mass, which was purified by flash chromatography (normal silica gel, 25% EtOAc in hexane used as eluent), to get 8-bromo-4-(4-methoxy-benzyl)-3,4-dihydro-2H-benzo[f][1,4]oxazepin-5-one (0.6 g, 63.28%) as white solid LC-MS: 362.2 (M+), 264.2 (M+2).




In a sealed tube, 8-bromo-4-(4-methoxy-benzyl)-3,4-dihydro-2H-benzo[f][1,4]oxazepin-5-one (2 g, 5.52 mmol), BINAP (0.206 g, 0.33 mmol) and Cs2CO3 (4.67 g, 14.36 mmol) were taken in dioxane (35 mL). Reaction mixture was de-gassed with argon for 5 min, isobutyraldehyde (27) (1.03 g, 14.36 mmol) and Pd(OAc)2 (50 mg, 0.22 mmol) were added under argon atmosphere. Reaction mixture was slowly heated to 80° C. and stirred for 16 h (silica TLC; 40% EtOAC/hexane; Rf=0.4). To the reaction mixture aqueous saturated NH4Cl (50 mL) was added at RT, and extracted with EtOAc (2×40 mL), combined extracts was washed with water (50 mL), dried and concentrated. Obtained crude product mixture was purified by flash chromatography (normal silica gel, 25% EtOAc in hexane used as mobile phase) to get pure 2-[4-(4-methoxy-benzyl)-5-oxo-2,3,4,5-tetrahydro-benzo[f][1,4]oxazepin-8-yl]-2-methyl-propionaldehyde (1.0 g, 51.22%) as white solid. LC-MS: 354.0 (M+H).




To a stirred solution of 2-[4-(4-methoxy-benzyl)-5-oxo-2,3,4,5-tetrahydro-benzo[f][1,4]oxazepin-8-yl]-2-methyl-propionaldehyde (1.5 g, 4.24 mmol) in TFA (66.77 mL, 866.85 mmol) was taken in a sealed tube, reaction mixture was heated at 100° C. for 1 h (silica TLC; 60% EtOAc in hexane; Rf=0.2). Volatiles were removed under reduced pressure; residue was neutralized with aqueous saturated NaHCO3 and extracted with EtOAc (2×200 mL). Combined extracts were dried, concentrated to get the crude residue which was purified by flash chromatography (normal silica gel, 60% EtOAc in hexanes used as eluent) to get 2-methyl-2-(5-oxo-2,3,4,5-tetrahydro-benzo[f][1,4]oxazepin-8-yl)-propionaldehyde (0.7 g, 70.6%) as white solid. LC-MS: 234.4 (M+H).




To a stirred solution of 2-methyl-2-(5-oxo-2,3,4,5-tetrahydro-benzo[f][1,4]oxazepin-8-yl)-propionaldehyde (0.5 g, 2.14 mmol) and 2-bromo-6-chloro-benzaldehyde (30)(1.175 g, 5.36 mmol) in dioxane (15 mL), Cs2CO3 (1.74 g, 5.36 mmol) was added and degassed for 5 min with argon. To the degassed reaction mixture, Xantphos (0.124 g, 0.215 mmol) and Pd(dba)2 (0.062 g, 0.107 mmol) were added under argon. Reaction was heated to 105 oC in sealed condition for 6 h followed by 16 h at RT under stirring. Reaction mixture was diluted with EtOAc (300 mL) and washed with water (100 mL). Organic layer was dried, concentrated under vacuum. Obtained crude residue was purified by flash chromatography (normal silica gel, 30% EtOAc in hexanes) to get 2-chloro-6-[8-(1,1-dimethyl-2-oxo-ethyl)-5-oxo-2,3-dihydro-5H-benzo[f][1,4]oxazepin-4-yl]-benzaldehyde (0.45 g, 56.4%) as Off white solid. LC-MS: 372.2 (M+), 374.2 (M+2).




To a stirred solution of 6-chloro-4-(5-methanesulfonyl-pyridin-2-ylamino)-2-methyl-2H-pyridazin-3-one (0.75 g, 2.38 mmol), bis(pinacolato)diboron (1.2 g, 4.76 mmol), (X-phos) (0.17 g, 0.357 mmol) and KOAc (0.7 g, 7.14 mmol) were taken up in dry dioxane (75 mL) and placed under vacuum and back-filled with argon. To this was added palladium acetate (0.053 g, 0.238 mmol) and the flask was evacuated and back-filled with argon. The mixture was heated in an oil bath to 100° C. and stirred for 30 min (monitored by LCMS). A small amount of des-Cl material was observed. Heating was turned down to 80° C. Flask was raised out of the heating bath but continued stirring, the following reagents: 2-chloro-6-[8-(1,1-dimethyl-2-oxo-ethyl)-5-oxo-2,3-dihydro-5H-benzo[f][1,4]oxazepin-4-yl]-benzaldehyde (0.883 g, 2.38 mmol), K2CO3 (1.64 g, 11.90 mmol), water (5 mL; degassed separately), tricyclohexylphosphine (200 mg, 0.71 mmol) and Pd(dba)2 (205 mg, 0.36 mmol) was added. The flask was stirred and heated for 2 h at 80° C. and then cooled to RT. Poured in to water (100 mL) and extracted with EtOAc (3×150 mL), combined extracts was washed with brine (200 mL), aqueous part was re-extracted with 5% MeOH in DCM (200 mL). Both the organic extracts were combined, dried and concentrated under reduced pressure. Obtained crude was purified by flash chromatography (silica gel, 70% EtOAc in hexanes), to get 2-[8-(1,1-dimethyl-2-oxo-ethyl)-5-oxo-2,3-dihydro-5H-benzo[f][1,4]oxazepin-4-yl]-6-[5-(5-methanesulfonyl-pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydro-pyridazin-3-yl]-benzaldehyde (0.55 g, 37.5%) as off white solid. LC-MS: 616.2 (M+H).




To a stirred solution of 2-[8-(1,1-dimethyl-2-oxo-ethyl)-5-oxo-2,3-dihydro-5H-benzo[f][1,4]oxazepin-4-yl]-6-[5-(5-methanesulfonyl-pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydro-pyridazin-3-yl]-benzaldehyde (2.5 g, 4.065 mmol) in methanol (90 mL) and dichloromethane (150 mL) was cooled in an ice bath. To this was added sodium borohydride (0.92 g, 24.39 mmol). The mixture was stirred for 10 min (silica TLC; only EtOAc used as eluent; Rf=0.2), diluted with water (600 mL) and extracted with dichloromethane (2×600 mL). Combined organic layer was washed with brine (600 mL), dried concentrated under vacuum. Obtained residue was washed with 80% EtOAc in hexane to get 8-(2-hydroxy-1,1-dimethyl-ethyl)-4-{2-hydroxymethyl-3-[5-(5-methanesulfonyl-pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydro-pyridazin-3-yl]-phenyl}-3,4-dihydro-2H-benzo[f][1,4]oxazepin-5-one (2 g, 79.39%) as off white solid. LC-MS: 620.4 (M+H).




To a stirred solution of 2-chloro-benzaldehyde (10 g, 71.42 mmol) in DCM (220 mL), O-methyl-hydroxylamine hydrochloride (35) (7.11 g, 85.71 mmol) and pyridine (23 mL) were added. The reaction mixture was stirred for 1 h at RT (silica TLC; 15% EtOAc in hexane; Rf=0.6). Reaction mixture was concentrated, obtained residue was purified by flash chromatography using 10% EtOAc in hexanes as eluent to get [(2-chlorophenyl)methylidene](methoxy)amine (9 g, 74.3%) as colorless oil LC-MS: 170.4 (M+H).




In sealed tube [(2-chlorophenyl)methylidene](methoxy)amine (5 g, 29.58 mmol) was taken in DCE (100 mL), followed by N-bromosuccinimide (5.23 g, 29.58 mmol), silver trifluoroacetate (0.65 g, 2.95 mmol), palladium acetate (0.66 g, 2.95 mmol) and acetic acid (1.77 g, 29.58 mmol) were added. The resulted mixture was heated to 120° C. for 24 h. At RT, water (150 mL) and DCM (300 mL) was added, and the mixture was filtered through a pad of celite. Organic layer was separated and the aqueous layer was re-extracted with dichloromethane (200 mL). The combined organic layer was dried, concentrated, obtained crude mass was purified by flash chromatography using only hexane, to get [(2-bromo-6-chlorophenyl)methylidene](methoxy)amine (4.0 g, 54.4%) as yellow colored liquid.




In a sealed tube [(2-bromo-6-chlorophenyl)methylidene](methoxy)amine (4.0 g, 16.12 mmol) was taken in THF—H2O (10:1; 110 mL). To the reaction mixture PTSA (6.12 g, 32.25 mmol) followed by para formaldehyde (4.83 g, 161.29 mmol), was added and heated to 100° C. for 30 min (silica TLC; only hexane used as eluent; Rf=0.3). Reaction mixture was diluted with EtOAc (500 mL) and washed with water (200 mL). Organic layer was dried, concentrated under vacuum. Obtained crude residue was purified by flash chromatography using 5% EtOAc in hexanes as eluent to get 2-bromo-6-chloro-benzaldehyde (3.0 g, 84.75%) as yellow solid.




In a round bottom flask under argon placed 5-bromo-pyridin-2-ylamine (10 g, 57.8 mmol), sodium methanesulfinate (9.4 g, 92.48 mmol), copper (I) triflate-benzene complex (1.74 g, 3.468 mmol), racemic trans 1,2-diaminocyclohexane (40) (1.41 mL, 11.742 mmol) and DMSO (50 mL). Reaction mixture was heated at 110° C. for 16 h. After completion of the reaction, mixture was poured into ice cooled water and aqueous was extracted with EtOAc (3×100 mL). Combined organic layer was washed with saturated brine solution, dried over anhydrous Na2SO4 and concentrated under reduced pressure. Crude mass was purified over normal silica gel column chromatography eluted with EtOAc-hexane to get 5-methanesulfonyl-pyridin-2-ylamine as brown solid (4.0 g, 40.18%).




To a solution of 5-methanesulfonyl-pyridin-2-ylamine (1.9 g, 11.047 mmol) in THF (40 mL) was added potassium tert-pentoxide (2M solution in THF) (4.2 mL, 8.4 mmol) drop wise. The mixture was stirred for 10 min at RT, then cooled in an ice bath and 4-bromo-6-chloro-2-methyl-2H-pyridazin-3-one (2.96 g, 13.256 mmol) was dissolved in THF (20 mL) and added drop wise to the previous mixture. The whole mixture was again warmed to RT and stirred for 1.5 h. After completion of the reaction, it was quenched with 1M HCl and the mixture was taken up in DCM and filtered through a sintered funnel. Filtrate was washed with saturated brine solution, dried over anhydrous Na2SO4 and concentrated under reduced pressure to get crude 6-chloro-4-(5-methanesulfonyl-pyridin-2-ylamino)-2-methyl-2H-pyridazin-3-one as off white solid (1.0 g, 28.76%), which was directly used for the next step without further purification.




To a solutions of (2-bromo-6-nitro-phenyl)-methanol (15.0 g, 64.65 mmol) in DCM (150 mL) were added sequentially tertiary butyl dimethyl sillyl chloride (29.233 g, 193.94 mmol) and TEA (19.6 mL, 193.94 mmol). The resulting mixture was stirred at 60° C. for 40 h. Reaction was monitored by TLC (ethyl acetate:hexane=1:9; Rf=0.7). Solvent was removed under reduced pressure. The reaction mixture was partitioned between ethyl acetate (1.5 L) and water (1.0 L). The organic layer was dried, filtered and concentrated. The crude was purified by silica gel (normal, 100-200 mesh) column chromatography eluting with hexane to 5% ethyl acetate in hexane to get pure (2-bromo-6-nitro-benzyloxy)-tert-butyl-dimethyl-silane (21.0 gm, 93.81%) as light yellow liquid). LC-MS: 346.4 (M+), 348.4 (M+2).




A mixture of (2-bromo-6-nitro-benzyloxy)-tert-butyl-dimethyl-silane (7.0 gm, 20.29 mmol), iron powder (3.83 gm, 68.58 mmol) and ammonium chloride (6.121 gm, 114.44 mmol) in ethanol-water (1:1; 340 mL) was heated at 90° C. for 4 h (silica TLC; ethyl acetate:hexane=1:19, Rf=0.4). The reaction mixture was filtered through a pad of celite in sintered funnel and washed with ethyl acetate (2×20 mL). The combined filtrate and washing was concentrated, ethyl acetate (1.5 L) was added to the crude and washed with water (700 mL). The organic layer was dried, filtered and concentrated, obtained crude was purified by neutral alumina column chromatography eluting with hexane to 2% ethyl acetate in hexane to get pure 3-bromo-2-(tert-butyl-dimethyl-silanyloxymethyl)-phenylamine (5.8 gm, 90.37%) as light orange liquid.




To a solution of 3-bromo-2-(tert-butyl-dimethyl-silanyloxymethyl)-phenylamine (1.0 gm, 3.164 mmol) in dioxane (60.0 mL) were added 4,4,5,5,4′,4′,5′,5′-octamethyl-[2,2′]bi[[1,3,2]dioxaborolanyl](4.018 gm, 15.821 mmol) and KOAc (0.932 gm, 9.493 mmol). The mixture was degassed with argon and to this mixture was added Pd(OAc)2 (71 mg, 0.316 mmol) and X-Phos (211 mg, 0.443 mmol), degassing was continued with argon for another 5 min then heated at 90° C. for 3 h (silica TLC; ethyl acetate:hexane=1:19, Rf=0.4). Reaction mixture was filtered through a pad of celite, washed with dioxane (2×5 mL) and combined with the filtrate. Combined filtrates was concentrated under reduced pressure, obtained crude residue was purified by neutral alumina column chromatography eluting with hexane to 2% ethyl acetate in hexane to get pure 2-(tert-butyl-dimethyl-silanyloxymethyl)-3-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenylamine (350 mg, 30.44%) as light orange solid. LC-MS: 364.2 (M+H).




To a stirred mixture of 2-(tert-butyl-dimethyl-silanyloxymethyl)-3-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenylamine (1.3 g, 3.58 mmol) and 5-bromo-1-methyl-3-[5-(morpholine-4-carbonyl)-pyridin-2-ylamino]-1H-pyridin-2-one (1.407 g, 3.58 mmol) in dioxane (52.0 mL) in a sealed tube was added 1(M) aqueous K2CO3 solution (10.7 mL) and degassed with argon. S-Phos (220 mg, 0.54 mmol) and Pd(PPh3)4 (207 mg, 0.18 mmol) were added subsequently and again degassed with argon; sealed and heated at 90° C. for 2.5 h (silica TLC; only ethyl acetate, Rf=0.5). Reaction mixture was cooled to RT and was added ethyl acetate (250 mL). The organic part was washed with water (120 mL) and brine (120 mL), dried, filtered and concentrated in vacuum to get crude mass which was purified by normal silica gel (normal, 100-200 mesh) column chromatography using gradient polarity eluent 50% ethyl acetate in hexane to pure ethyl acetate to get pure 5-[3-amino-2-(tert-butyl-dimethyl-silanyloxymethyl)-phenyl]-1-methyl-3-[5-(morpholine-4-carbonyl)-pyridin-2-ylamino]-1H-pyridin-2-one (1.31 g, 66.61%) as off white solid. LC-MS: 550.4 (M+H).




To a solution of 5-[3-amino-2-(tert-butyl-dimethyl-silanyloxymethyl)-phenyl]-1-methyl-3-[5-(morpholine-4-carbonyl)-pyridin-2-ylamino]-1H-pyridin-2-one (700 mg, 1.27 mmol) in DCM (70.0 mL) was added triethylamine (0.44 mL, 3.18 mmol) and cooled to 0° C. in a ice bath, followed by the addition of phosgene solution[0.8 mL, 1.53 mmol (20% in toluene)] and stirred for 10 min at 0° C., followed by 5-chloro-2,3-dihydro-1H-isoindole (489 mg, 3.18 mmol) was added and stirred for another 30 min (silica TLC; ethyl acetate 100%, Rf=0.45). Solvent was removed under vacuum, obtained crude was purified by silica gel (normal, 100-200 mesh) column chromatography using gradient eluent 50% ethyl acetate in hexane to 100% ethyl acetate to get 5-chloro-1,3-dihydro-isoindole-2-carboxylic acid (2-(tert-butyl-dimethyl-silanyloxymethyl)-3-[1-methyl-5-[5-(morpholine-4-carbonyl)-pyridin-2-ylamino]-6-oxo-1,6-dihydro-pyridin-3-yl]-phenyl)-amide (910 mg, 97.99%) as brown sticky solid. LC-MS: 729.4 (M+H).




To a solution of 5-chloro-1,3-dihydro-isoindole-2-carboxylic acid (2-(tert-butyl-dimethyl-silanyloxymethyl)-3-{1-methyl-5-[5-(morpholine-4-carbonyl)-pyridin-2-ylamino]-6-oxo-1,6-dihydro-pyridin-3-yl}-phenyl)-amide (900 mg, 1.23 mmol) in methanol (45.0 mL) and DCM (18.0 mL) was added 12(N) HCl (0.9 mL) at RT and stirred for 10 min (silica TLC; methanol:DCM=1:19 used as eluent, Rf=0.4). Saturated NaHCO3 solution was added drop wise till bubbling ceases. Reaction mass was diluted with DCM (600 mL) and washed with water (100 mL), brine (100 mL), dried and concentrated in vacuum to get sticky crude mass which was purified by re-crystallization from DCM-hexane mixture to get pure 5-chloro-1,3-dihydro-isoindole-2-carboxylic acid (2-hydroxymethyl-3-{1-methyl-5-[5-(morpholine-4-carbonyl)-pyridin-2-ylamino]-6-oxo-1,6-dihydro-pyridin-3-yl}-phenyl)-amide (620 mg, 81.69%) as ash color solid. LC-MS: 615.2 (M+H).




To a stirred solution of nicotinamide (10.0 g, 81.88 mmol) in 10% H2SO4 (82 mL), 2,2-dimethyl-propionic acid (41.8 g, 409.4 mmol) and AgNO3 (4.2 g, 24.564 mmol) were added and the mixture was heated at 70° C. with stirring. A solution of (NH4)2S2O8 (56.1 g, 245.64 mmol) in H2O (122 mL) was added until the CO2 evolution was ceased. Reaction was stirred for 1 h more at the same temperature under argon atmosphere. After completion of the reaction, monitored by TLC, the mixture was cooled to RT, adjusted the pH ̃9 by addition of aqueous NH3 and extracted with EtOAc (3×200 mL). Combined organic layer was washed with water and brine, dried over anhydrous Na2SO4, concentrated under vacuum. The crude compound was purified by CombiFlash column chromatography using EtOAc-hexane as eluting solvent to get 6-tert-butyl-nicotinamide as off white solid (9.6 g, 66%). LCMS: 179.0 (M+H).




To a stirred solution of 6-tert-butyl-nicotinamide (2 g, 11.221 mmol) in 10% H2SO4 (90 mL), 3-phthalimidopropanoic acid (9.84 g, 44.883 mmol), AgNO3 (1.91 g, 11.221 mmol), (NH4)2S2O8 (20.5 g, 89.767 mmol) and cetyltrimethylammonium bromide (1.8 g, 4.937 mmol) were added respectively and the whole mixture was heated at 90° C. for 30 min. This mixture was cooled to 70° C. and a solution of (NH4)2S2O8 (20.5 g, 89.767 mmol) in ACN—H2O(3:7) (100 mL) was added and the mixture was again heated at 90° C. for 3 h. Mixture was again cooled to 70° C. and a solution of (NH4)2S2O8 (20.5 g, 89.767 mmol) in ACN—H2O(3:7) (100 mL) was added and kept at 90° C. for 16 h. RM was monitored by SiO2 TLC, a little SM was still present so the mixture was again cooled to 70° C. and a solution of (NH4)2S2O8 (20.5 g, 89.767 mmol) in ACN—H2O(3:7) (100 mL) was added and kept at 90° C. for another 6 h. Some amount starting material was present in the mixture which was not consumed even after prolonged heating. Reaction mixture was cooled to RT, neutralized to pH ̃7 by addition of aqueous NH3 and extracted with DCM (2×300 mL). Combined organic layer was washed with water and brine, dried over anhydrous Na2SO4 and concentrated under vacuum to get crude mass, which was passed through a small bed of normal silica gel column to remove unwanted impurities to get 6-tert-butyl-4-[2-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-ethyl]-nicotinamide as brown viscous oil (1.7 g crude) along with some other impurities. This impure material was directly used for the next step without further purification. LCMS: 353.0 (M+H).




To a stirred solution of (2.55 g crude, 7.256 mmol) in CH3CN (40 mL), NH2NH2.H2O (0.68 mL, 14.513 mmol) was added and the mixture was refluxed for 3 h. After completion of the reaction, monitored by SiO2 TLC, the mixture was cooled to RT, filtered through a sinter funnel and the filtrate was concentrated under reduced pressure. The crude compound was purified over normal silica gel column chromatography using EtOAc-hexane as eluting solvent to get 6-tert-butyl-3,4-dihydro-2H-[2,7]naphthyridin-1-one as off white solid (165 mg, 4.5% over 2 steps). LCMS: 205.0 (M+H).




6-tert-Butyl-3,4-dihydro-2H-[2,7]naphthyridin-1-one (0.46 g, 2.252 mmol), 2,6-dibromo-benzaldehyde (2.97 g, 11.26 mmol), xanthphos (65.2 mg, 0.113 mmol) and Cs2CO3 (1.03 g, 3.153 mmol) were taken up in 1,4-dioxane (4.5 mL) and argon gas was bubbled through the mixture for 10 min Pd(dba)2 (38.8 mg, 0.068 mmol) was added to it and stirred at 100° C. for 4 h. After completion of the reaction, (monitored by TLC), resultant mixture was cooled to RT and this was partitioned between EtOAc and water. Organic layer was collected and dried over anhydrous Na2SO4 and concentrated under reduced pressure. Crude mass was purified over normal silica gel column chromatography using EtOAc-hexane to get 2-bromo-6-(6-tert-butyl-1-oxo-3,4-dihydro-1H-[2,7]naphthyridin-2-yl)-benzaldehyde as off white solid (416.0 mg, 47.7%). LCMS: 387.0 (M) & 389.0 (M+2).




A solution of 5-chloro-1-methyl-3-[5-(morpholine-4-carbonyl)-pyridin-2-ylamino]-1H-pyridin-2-one (506 mg, 1.29 mmol), bis(pinacolato)diboron (424.7 mg, 1.67 mmol), X-phos (92 mg, 0.193 mmol) and KOAc (379 mg, 3.86 mmol) in dry 1,4-dioxane (22 mL) was placed under vacuum and back-filled with argon for 15 min. Pd(OAc)2 (31.7 mg, 0.142 mmol) was added to it and the flask was evacuated and back-filled with argon again for 5 min. This was refluxed for 0.5 h and then cooled to RT. Solids were filtered off through celite bed and the filtrate was directly used for the next reaction. In a separate RB flask a mixture of 2-bromo-6-(6-tert-butyl-1-oxo-3,4-dihydro-1H-[2,7]naphthyridin-2-yl)-benzaldehyde (0.5 g, 1.29 mmol), K2CO3 (888 mg, 6.44 mmol) and Cy3P (108 mg, 0.39 mmol) in dioxane-water-n-butanol (1:1:0.4) (13.6 mL) was degassed and back filled with argon for 15 min. Pd(dba)2 (111 mg, 0.193 mmol) followed by the crude solution of the previous reaction was added to it respectively and the whole mixture was heated at 110° C. for 1 h. After completion of the reaction, monitored by TLC and LC-MS, reaction was filtered and the filtrate was concentrated under reduced pressure to get crude over normal silica gel column chromatography using MeOH-EtOAc as eluting solvent to get 2-(6-tert-butyl-1-oxo-3,4-dihydro-1H-[2,7]naphthyridin-2-yl)-6-{1-methyl-5-[5-(morpholine-4-carbonyl)-pyridin-2-ylamino]-6-oxo-1,6-dihydro-pyridin-3-yl}-benzaldehyde as off white solid (488 mg, 61% over 2 steps). LCMS: 621.2 (M+H).




A solution of 2-(6-tert-butyl-1-oxo-3,4-dihydro-1H-[2,7]naphthyridin-2-yl)-6-{1-methyl-5-[5-(morpholine-4-carbonyl)-pyridin-2-ylamino]-6-oxo-1,6-dihydro-pyridin-3-yl}-benzaldehyde (1130 mg, 1.82 mmol) in methanol-DCM (2:3) (15.2 mL) was cooled to 0° C. and a solution of NaBH4 (344 g, 9.11 mmol) in water (3.8 mL) was added to it drop wise. Resulting mixture was stirred for 5 min and additional solid sodium borohydride (344 g, 9.11 mmol) was added portion wise, stirred for 10 min After completion of the reaction, monitored by LCMS, diluted with water, extracted with DCM, dried over Na2SO4 and concentrated under reduced pressure. Crude over flash silica gel using basic solution (DCM-MeOH—NH4OH in 60:10:1) in DCM to get 6-tert-butyl-2-(2-hydroxymethyl-3-{1-methyl-5-[5-(morpholine-4-carbonyl)-pyridin-2-ylamino]-6-oxo-1,6-dihydro-pyridin-3-yl}-phenyl)-3,4-dihydro-2H-[2,7]naphthyridin-1-one which was further purified by washing with diethyl ether and n-pentane to get as off white solid 6-tert-Butyl-2-(2-hydroxymethyl-3-{1-methyl-5-[5-(morpholine-4-carbonyl)-pyridin-2-ylamino]-6-oxo-1,6-dihydro-pyridin-3-yl}-phenyl)-3,4-dihydro-2H-[2,7]naphthyridin-1-on (215 mg, 18.96%). LCMS: 623.2 (M+H).




A solution of 6-chloro-2-methyl-4-[5-(morpholine-4-carbonyl)-pyridin-2-ylamino]-2H-pyridazin-3-one (280 mg, 0.801 mmol), bis(pinacolato)diboron (264.3 mg, 1.041 mmol), X-phos (57.2 mg, 0.12 mmol) and KOAc (235.7 mg, 2.402 mmol) in dry 1,4-dioxane (12.5 mL) was placed under vacuum and back-filled with argon for 15 min Pd(OAc)2 (19.7 mg, 0.088 mmol) was added to it and the flask was evacuated and back-filled with argon again for 5 min. This was refluxed for 16 min and then cooled to RT. Solids were filtered off through celite bed and the filtrate was directly used for the next reaction. In a separate RB flask a mixture of 2-bromo-6-(6-tert-butyl-1-oxo-3,4-dihydro-1H-[2,7]naphthyridin-2-yl)-benzaldehyde (56) (305.0 mg, 0.788 mmol), K2CO3 (543.4 mg, 3.938 mmol) and Cy3P (66.3 mg, 0.236 mmol) in dioxane-water-n-butanol (1:1:0.25) (8.1 mL) was degassed and back filled with argon for 15 min Pd(dba)2 (67.9 mg, 0.118 mmol) followed by the crude solution of the previous reaction was added to it respectively and the whole mixture was heated at 110° C. for 1 h. After completion of the reaction, monitored by TLC and LC-MS, reaction was filtered and the filtrate was concentrated under reduced pressure to get crude mass which was purified over normal silica gel column chromatography using EtOAc as eluting solvent to get 2-(6-tert-butyl-1-oxo-3,4-dihydro-1H-[2,7]naphthyridin-2-yl)-6-{1-methyl-5-[5-(morpholine-4-carbonyl)-pyridin-2-ylamino]-6-oxo-1,6-dihydro-pyridazin-3-yl}-benzaldehyde as yellow solid (291.0 mg, 59.44% over 2 steps). LCMS: 622.6 (M+H).




A solution of 2-(6-tert-butyl-1-oxo-3,4-dihydro-1H-[2,7]naphthyridin-2-yl)-6-{1-methyl-5-[5-(morpholine-4-carbonyl)-pyridin-2-ylamino]-6-oxo-1,6-dihydro-pyridazin-3-yl}-benzaldehyde (700 mg, 1.13 mmol) in methanol-DCM (2:3) (22.2 mL) was cooled to 0° C. and a solution of NaBH4 (213 mg, 5.63 mmol) in water (4 mL) was added to it drop wise. Resulting mixture was stirred for 5 min and additional solid sodium borohydride (213 mg, 5.63 mmol) was added portion wise and again stirred for 10 min at that temperature. After completion of the reaction, monitored by LCMS, water was added and extracted with DCM. Combined organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. Crude was purified over flash silica gel using 4-6% magic solution (DCM-MeOH—NH4OH in 60:10:1 ratio) in DCM to get 6-tert-butyl-2-(2-hydroxymethyl-3-{1-methyl-5-[5-(morpholine-4-carbonyl)-pyridin-2-ylamino]-6-oxo-1,6-dihydro-pyridazin-3-yl}-phenyl)-3,4-dihydro-2H-[2,7]naphthyridin-1-one, which was further purified by washing with diethyl ether and n-pentane to get off white solid (322 mg, 46.00%). LCMS: 624.4 (M+H).




A suspension of 6-tert-butyl-8-fluoro-2H-phthalazin-1-one (58) (1 g, 4.545 mmol), 1,3-dibromo-2-methyl-benzene (2.27 g, 9.091 mmol), CuI (0.26 g, 1.364 mmol) and K2CO3 (0.63 g, 4.545 mmol) in DMSO (10 mL) was degassed followed by back-filled with argon for 15 min in a sealed tube and the mixture was heated at 140° C. for 18 h. After completion of the reaction, monitored by LC-MS, mixture was cooled to RT and diluted with EtOAc—H2O. Organic layer was washed with saturated brine solution, dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude mass was purified over normal silica gel using EtOAc-hexane as eluting solvent to get 2-(3-bromo-2-methyl-phenyl)-6-tert-butyl-8-fluoro-2H-phthalazin-1-one (4.65 g, 43%) as a light yellow solid. LCMS: 389.0 (M+), 391.2 (M+2).




A mixture of 2-(3-bromo-2-methyl-phenyl)-6-tert-butyl-8-fluoro-2H-phthalazin-1-one (1.55 g, 3.985 mmol), bis(pinacolato)diboron (2.02 g, 7.969 mmol), X-phos (0.23 g, 0.398 mmol) and KOAc (0.9 g, 9.165 mmol) was taken in dioxane (12 mL) in a sealed tube and degassed followed by back-filled with argon for 15 min Pd(OAc)2 (54 mg, 0.239 mmol) was added to it and was heated at 80° C. for 6 h. Reaction was not completed according to LC-MS and TLC so half amount of all the reagents were added again and continued heating at same temperature for 16 h. After completion of the reaction, monitored by TLC & LC-MS, mixture was cooled to RT and filtered over a bed of celite. Filtrate was concentrated under reduced pressure and crude mass was passed through a small bed of normal silica gel column chromatography using EtOAc-hexane to get semi-pure 6-tert-butyl-8-fluoro-2-[2-methyl-3-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-2H-phthalazin-1-one (0.8 g,  ̃46.01%) which was directly used for the next reaction without further purification. LCMS: 437.0 (M+H).




To a solution of 6-tert-butyl-8-fluoro-2-[2-methyl-3-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenyl]-2H-phthalazin-1-one (2.4 g, 5.51 mmol) and 6-chloro-2-methyl-4-[5-(morpholine-4-carbonyl)-pyridin-2-ylamino]-2H-pyridazin-3-one (1.54 g, 4.4 mmol) in 10% aq-dioxane (69 mL), were added Cs2CO3 (6.75 g, 20.8 mmol) and this mixture was heated at 120° C. for 3 h under microwave condition. After completion of the reaction, mixture was filtered through a bed of celite and washed with EtOAc. Filtrate was concentrated under reduced pressure and the crude was purified over flash silica gel column chromatography using 10% magic solution (DCM:MeOH:NH4OH in 60:10:1 ratio) in DCM to get 6-tert-butyl-8-fluoro-2-(2-methyl-3-{1-methyl-5-[5-(morpholine-4-carbonyl)-pyridin-2-ylamino]-6-oxo-1,6-dihydro-pyridazin-3-yl}-phenyl)-2H-phthalazin-1-one as off white solid (1.5 g, 43.7%). LC-MS: 624.2 (M+H).




To stirred solution of sodium hydroxide (3.46 g, 86.667 mmol) in water (20 mL) 3-tert-butylphenol (10 g, 66.667 mmol) was added and resulting mixture was stirred at RT for 30 min 2-(2-bromo-ethyl)-[1,3]dioxane (14.3 g, 73.33 mmol) was added to it and resulting mixture was heated to reflux for 41 h. The reaction mixture was cooled to RT and then added EtOAc (150 mL) and with stirring acetic acid was added to pH-4, followed by dilution with water (100 mL) and EtOAc (150 mL). After partitioning, the layers were separated and the organic layer was washed with water (3×100 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure to get 2-[2-(3-tert-butyl-phenoxy)-ethyl]-[1,3]dioxane (16 g crude,  ̃90.79%), which was directly used for the next step without further purification. GC-MS: 264.0 (M+).




Concentrated HCl (80 mL) was cooled in an ice bath and THF (40 mL) was added to it. 2-[2-(3-tert-butyl-phenoxy)-ethyl]-[1,3]dioxane (10 g crude, 37.879 mmol) in THF (40 mL) slowly at that temperature with stirring. After completion of addition, ice bath was removed and resulting mixture was stirred at RT for 3 h. After complete consumption of starting material, ether (100 mL) was added to the mixture and layers were separated. Combined organic layer was washed with water and dried over anhydrous Na2SO4 and concentrated to get crude 7-tert-butyl-chroman-4-ol as light yellow oil (7.0 g crude), which was carried to next step without further purification.




7-tert-butyl-chroman-4-ol (7 g crude, 33.981 mmol) was taken up in DCM (300 mL) and with stirring was added PCC (14.65 g, 67.961 mmol) portion wise. After completion of addition, the reaction mixture was stirred at RT for 3 h. After completion of the reaction, (monitored by LCMS), hexane was added to the reaction mixture and filtered through celite bed. Filtrate was concentrated under reduced pressure and crude mass was purified over normal silica gel column chromatography using EtOAc-hexane as eluting solvent to get 7-tert-butyl-chroman-4-one as light brown solid (3.8 g, 49.18% over 2 steps). LCMS: 205.2 (M+H).




7-tert-Butyl-chroman-4-one (5.0 g, 24.51 mmol) and methane sulfonic acid (7.5 mL) were taken up in DCM (12 mL) and cooled to 0° C. Solid sodium azide (3.2 g, 49.02 mmol) was added to this mixture and the resulting mixture was stirred at that temperature for 3 h. The reaction mixture was poured into 20% aqueous solution of NaOH at 0° C. and stirred again for 10 min. The whole aqueous part was extracted with CH2Cl2 (3×50 mL) and combined organic layer was washed with brine, dried over anhydrous Na2SO4 and concentrated under reduced pressure. Crude mass was purified over normal silica gel column chromatography using EtOAc-hexane to get 8-tert-butyl-3,4-dihydro-2H-benzo[f][1,4]oxazepin-5-one as off white solid (5.0 g, 93.03%). LCMS: 220.2 (M+H).




8-tert-Butyl-3,4-dihydro-2H-benzo[f][1,4]oxazepin-5-one (0.92 g, 4.201 mmol), 2,6-dibromo-benzaldehyde (4.99 g, 18.904 mmol), xanthphos (109.4 mg, 0.189 mmol) and Cs2CO3 (1.91 g, 5.881 mmol) were taken up in 1,4-dioxane (15 mL) and argon gas was bubbled through the mixture for 10 min Pd(dba)2 (72.5 mg, 0.126 mmol) was added to it and stirred at 100° C. for 3 h. After completion of the reaction, monitored by TLC, resultant mixture was cooled to RT and this was partitioned between EtOAc and water. Organic layer collected and dried over anhydrous Na2SO4 and concentrated under reduced pressure. Crude mass was purified over normal silica gel column chromatography using EtOAc-hexane to get 2-bromo-6-(8-tert-butyl-5-oxo-2,3-dihydro-5H-benzo[f][1,4]oxazepin-4-yl)-benzaldehyde as light brown solid (1.1 g, 65.09%). LCMS: 402.0 (M−H) & 404.0 (M+H).




A solution of 6-chloro-2-methyl-4-[5-(morpholine-4-carbonyl)-pyridin-2-ylamino]-2H-pyridazin-3-one (0.86 g, 2.464 mmol), bis(pinacolato)diboron (1001.4 mg, 3.943 mmol), X-phos (176.2 mg, 0.37 mmol) and KOAc (725.6 mg, 7.393 mmol) in dry 1,4-dioxane (30 mL) was placed under vacuum and back-filled with argon for 15 min Pd(OAc)2 (55.3 mg, 0.246 mmol) was added to it and the flask was evacuated and back-filled with argon again for 5 min. This was heated at 100° C. for 30 min and then cooled to RT. Solids were filtered off through celite bed and the filtrate was directly used for the next reaction (M+H:316.2). In a separate RB flask a mixture of 2-bromo-6-(8-tert-butyl-5-oxo-2,3-dihydro-5H-benzo[f][1,4]oxazepin-4-yl)-benzaldehyde (1 g, 2.481 mmol), K2CO3 (1.03 g, 7.444 mmol) and Cy3P (0.21 g, 0.744 mmol) in dioxane-water-n-butanol (1:1:0.4) (24 mL) was degassed and back filled with argon for 15 min Pd(dba)2 (0.21 g, 0.372 mmol) followed by the crude filtrate of the previous reaction was added to it respectively and the whole mixture was heated at 110° C. for 1.5 h. After completion of the reaction, monitored by TLC and LC-MS, reaction was filtered and the filtrate was concentrated under reduced pressure to get crude mass which was purified by CombiFlash column chromatography using MeOH-EtOAc as eluting solvent to get 2-(8-tert-butyl-5-oxo-2,3-dihydro-5H-benzo[f][1,4]oxazepin-4-yl)-6-{1-methyl-5-[5-(morpholine-4-carbonyl)-pyridin-2-ylamino]-6-oxo-1,6-dihydro-pyridazin-3-yl}-benzaldehyde as white solid (1.0 g, 63.3% over 2 steps). LCMS: 637.6 (M+H).




A solution of 2-(8-tert-butyl-5-oxo-2,3-dihydro-5H-benzo[f][1,4]oxazepin-4-yl)-6-{1-methyl-5-[5-(morpholine-4-carbonyl)-pyridin-2-ylamino]-6-oxo-1,6-dihydro-pyridazin-3-yl}-benzaldehyde (4.6 g, 3.145 mmol) in methanol-DCM (2:3) (69 mL) was cooled to 0° C. and a solution of NaBH4 (1.38 g, 15.723 mmol) in water (6.9 mL) was added to it drop wise. Resulting mixture was stirred for 10 min and additional solid sodium borohydride (2.7 g, 31.447 mmol) was added portion wise and again stirred for 1 h at that temperature. After completion of the reaction, (monitored by LCMS), water was added and extracted with DCM. Combined organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. Crude mass was purified over flash silica gel using 10-30% magic solution (DCM-MeOH—NH4OH in 60:10:1 ratio) in DCM to get 8-tert-butyl-4-(2-hydroxymethyl-3-{1-methyl-5-[5-(morpholine-4-carbonyl)-pyridin-2-ylamino]-6-oxo-1,6-dihydro-pyridazin-3-yl}-phenyl)-3,4-dihydro-2H-benzo[f][1,4]oxazepin-5-one, which was further purified by washing with diethyl ether and n-pentane to get off white solid (2.12 g, 45.87%). LCMS: 639.2 (M+H).




A solution of 6-chloro-4-(5-methanesulfonyl-pyridin-2-ylamino)-2-methyl-2H-pyridazin-3-one (0.78 g, 2.484 mmol), bis(pinacolato)diboron (1009.5 mg, 3.975 mmol), X-phos (177.6 mg, 0.373 mmol) and KOAc (731.4 mg, 7.452 mmol) in dry 1,4-dioxane (70 mL) was placed under vacuum and back-filled with argon for 15 min. Pd(OAc)2 (55.8 mg, 0.248 mmol) was added to it and the flask was evacuated and back-filled with argon again for 5 min. This was heated at 100° C. for 1 h and then cooled to RT. This solution was directly used for the next reaction (M+H:323.6). In a separate RB flask a mixture of 2-bromo-6-(8-tert-butyl-5-oxo-2,3-dihydro-5H-benzo[f][1,4]oxazepin-4-yl)-benzaldehyde (1 g, 2.481 mmol), K2CO3 (1.71 g, 12.407 mmol) and Cy3P (0.21 g, 0.744 mmol) in dioxane-water-n-butanol (1:1:0.25) (27 mL) was degassed and back filled with argon for 15 min Pd(dba)2 (0.21 g, 0.372 mmol) followed by the crude solution of the previous reaction was added to it respectively and the whole mixture was heated at 110° C. for 1.5 h. After completion of the reaction, monitored by TLC and LC-MS, reaction was filtered and the filtrate was concentrated under reduced pressure to get crude mass which was purified by CombiFlash column chromatography using MeOH-EtOAc as eluting solvent to get 2-(8-tert-butyl-5-oxo-2,3-dihydro-5H-benzo[f][1,4]oxazepin-4-yl)-6-[5-(5-methanesulfonyl-pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydro-pyridazin-3-yl]-benzaldehyde as light yellow solid (510 mg, 34.16% over 2 steps). LCMS: 602.2 (M+H).




A solution of 2-(8-tert-butyl-5-oxo-2,3-dihydro-5H-benzo[f][1,4]oxazepin-4-yl)-6-[5-(5-methanesulfonyl-pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydro-pyridazin-3-yl]-benzaldehyde (1 g, 1.572 mmol) in methanol-DCM (2:3) (30 mL) was cooled to 0° C. and a solution of NaBH4 (0.3 g, 7.862 mmol) in water (3 mL) was added to it drop wise. Resulting mixture was stirred for 10 min and additional solid sodium borohydride (0.59 g, 15.723 mmol) was added portion wise and again stirred for 1 h at that temperature. After completion of the reaction, monitored by LCMS, water was added and extracted with DCM. Combined organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. Crude mass was purified over flash silica gel using 10-30% magic solution (DCM-MeOH—NH4OH in 60:10:1 ratio) in DCM to get 8-tert-butyl-4-[2-hydroxymethyl-3-[5-(5-methanesulfonyl-pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydro-pyridazin-3-yl]-phenyl]-3,4-dihydro-2H-benzo[f][1,4]oxazepin-5-one, which was further purified by washing with diethyl ether and n-pentane to get off white solid (540 mg, 56.89%). LCMS: 604.4 (M+H).
Biological Examples
Bruton's Tyrosine Kinase (BTK) Inhibition Assay
The assay is a capture of radioactive 33P phosphorylated product through filtration. The interactions of BTK, biotinylated SH2 peptide substrate (Src homology), and ATP lead to phosphorylation of the peptide substrate. Biotinylated product is bound streptavidin sepharose beads. All bound, radiolabeled products are detected by scintillation counter.
Plates assayed are 96-well polypropylene (Greiner) and 96-well 1.2 μm hydrophilic PVDF filter plates (Millipore). Concentrations reported here are final assay concentrations: 10-100 μM compounds in DMSO (Burdick and Jackson), 5-10 nM BTK enzyme (His-tagged, full-length), 30 μM peptide substrate (Biotin-Aca-AAAEEIYGEI-NH2), 100 μM ATP (Sigma), 8 mM imidazole (Sigma, pH 7.2), 8 mM glycerol-2-phosphate (Sigma), 200 μM EGTA (Roche Diagnostics), 1 mM MnCl2 (Sigma), 20 mM MgCl2 (Sigma), 0.1 mg/ml BSA (Sigma), 2 mM DTT (Sigma), 1 μCi 33P ATP (Amersham), 20% streptavidin sepharose beads (Amersham), 50 mM EDTA (Gibco), 2 M NaCl (Gibco), 2 M NaCl w/1% phosphoric acid (Gibco), microscint-20 (Perkin Elmer).
IC50 determinations are calculated from 10 data points per compound utilizing data produced from a standard 96-well plate assay template. One control compound and seven unknown inhibitors were tested on each plate and each plate was run twice. Typically, compounds were diluted in half-log starting at 100 μM and ending at 3 nM. The control compound was staurosporine. Background was counted in the absence of peptide substrate. Total activity was determined in the presence of peptide substrate. The following protocol was used to determine BTK inhibition.
1) Sample preparation: The test compounds were diluted at half-log increments in assay buffer (imidazole, glycerol-2-phosphate, EGTA, MnCl2, MgCl2, BSA).
2) Bead preparation

    
    
        a.) rinse beads by centrifuging at 500 g
        b.) reconstitute the beads with PBS and EDTA to produce a 20% bead slurry
    
    


3) Pre-incubate reaction mix without substrate (assay buffer, DTT, ATP, 33P ATP) and mix with substrate (assay buffer, DTT, ATP, 33P ATP, peptide substrate) 30° C. for 15 min
4) To start assay, pre-incubate 10 μL BTK in enzyme buffer (imidazole, glycerol-2-phosphate, BSA) and 10 μL of test compounds for 10 min at RT.
5) Add 30 μL reaction mixture without or with substrate to BTK and compounds.
6) Incubate 50 μL total assay mix for 30 min at 30° C.
7) Transfer 40 μL of assay to 150 μL bead slurry in filter plate to stop reaction.
8) Wash filter plate after 30 min, with following steps

    
    
        a. 3×250 μL NaCl
        b. 3×250 μL NaCl containing 1% phosphoric acid
        c. 1×250 μL H2O
    
    


9) Dry plate for 1 h at 65° C. or overnight at RT
10) Add 50 μL microscint-20 and count 33P cpm on scintillation counter.

    
    
        Calculate percent activity from raw data in cpm

percent activity=(sample−bkg)/(total activity−bkg)×100

        Calculate IC50 from percent activity, using one-site dose response sigmoidal model

y=A+((B−A)/(1+((x/C)D))))

        x=cmpd conc, y=% activity, A=min, B=max, C=IC50, D=1 (hill slope)

Bruton's Tyrosine Kinase (BTK) Inhibition TR-FRET (Time Resolved FRET) Assay

    
    


This BTK competition assay measures compound potency (IC50) for the inactivated state of Bruton's Tyrosine Kinase using FRET (Förster/Flouresence Resonance Energy Transfer) technology. The BTK-Eu complex was incubated on ice one hour prior to use at a starting concentration of 50 nM BTK-BioeaseTM: 10 nM Eu-streptavidin (Perkin-Elmer Catalog# AD0062). The assay buffer consisted of 20 mM HEPES (pH 7.15), 0.1 mM DTT, 10 mM MgCl2, 0.5 mg/ml BSA with 3% Kinase Stabilizer (Fremont Biosolutions, Catalog # STB-K02). After 1 h, the reaction mixture from above was diluted 10 fold in assay buffer to make 5 nM BTK: 1 nM Eu-Streptavidin complex (donor fluorophore). 18 μl of a mixture of 0.11 nM BTK-Eu and 0.11 nM Kinase Tracer 178 (Invitrogen, Catalog # PV5593) with BTK-Eu alone as no negative control, was then dispensed into 384-well flat bottom plates (Greiner, 784076). Compounds to be tested in assay were prepared as 10× concentrations and serial dilution in half-log increments was performed in DMSO so as to generate 10 point curves. To initiate the FRET reaction, compounds prepared as 10× stock in DMSO was added to the plates and the plates were incubated 18-24 h at 14° C.
After the incubation the plates were read on a BMG Pherastar Fluorescent plate reader (or equivalent) and used to measure the emission energy from the europium donor fluorophore (620 nm emission) and the FRET (665 nm emission). The negative control well values were averaged to obtain the mean minimum. The positive “no inhibitor” control wells were averaged to obtain the mean maximum. Percent of maximal FRET was calculated using following equation:

% max FRET=100×[(FSRcmpd−FSRmean min)/(FSRmean max−FSRmean min)]

where FSR=FRET Signal ratio. % Max FRET curves were plotted in Activity Base (Excel) and the IC50(%), hill slope, z′ and % CV were determined. The mean IC50 and standard deviation will be derived from duplicate curves (singlet inhibition curves from two independent dilutions) using Microsoft Excel.

Representative compound data for this assay are listed below in Table II.










TABLE II







Compound
FRET IC50 (μmol)



















1
0.01118



2
0.00112



3
0.0526



4
0.03195



5
0.0137



6
0.01462



7
0.0065



8
0.00177



9
0.00424










Inhibition of B Cell Activation in Whole Blood Measured by CD69 Expression

A procedure to test the ability of BTK inhibitors to suppress B cell receptor-mediated activation of B cells in human blood is as follows:
Human whole blood (HWB) is obtained from healthy volunteers, with the following restrictions: 24 hr drug-free, non-smokers. Blood is collected by venipuncture into Vacutainer tubes anticoagulated with sodium heparin. Test compounds are diluted to ten times the desired starting drug concentration in PBS (20×), followed by three-fold serial dilutions in 10% DMSO in PBS to produce a nine point dose-response curve. 5.5 μl of each compound dilution is added in duplicate to a 2 ml 96-well V bottom plate (Analytical Sales and Services, #59623-23); 5.5 μl of 10% DMSO in PBS is added to control and no-stimulus wells. HWB (100 μl) is added to each well, and after mixing the plates are incubated at 37C, 5% CO2, 100% humidity for 30 minutes. Goat F(ab′)2 anti-human IgM (Southern Biotech, #2022-14) (10 μl of a 500 μg/ml solution, 50 μg/ml final concentration) is added to each well (except the no-stimulus wells) with mixing and the plates are incubated for an additional 20 hours.
At the end of the 20 hour incubation, samples are incubated with florescent-probe-labeled antibodies (15 μl PE Mouse anti-Human CD20, BD Pharmingen, #555623, and/or 20 μl APC Mouse anti-Human CD69, BD Pharmingen #555533) for 30 minutes, at 37C, 5% CO2, 100% humidity. Included are induced control, unstained and single stains for compensation adjustments and initial voltage settings. Samples are then lysed with 1 ml of 1× Pharmingen Lyse Buffer (BD Pharmingen #555899), and plates are centrifuged at 1800 rpm for 5 minutes. Supernatants are removed via suction and the remaining pellets are lysed again with another 1 ml of 1× Pharmingen Lyse Buffer, and plates are spun down as before. Supernatants are aspirated and remaining pellets are washed in FACs buffer (PBS+1% FBS). After a final spin, the supernantants are removed and pellets are resuspended in 180 μl of FACs buffer. Samples are transferred to a 96 well plate suitable to be run on the HTS 96 well system on the BD LSR II flow cytometer.
Using appropriate excitation and emission wavelengths for the fluorophores used, data are acquired and percent positive cell values are obtained using Cell Quest Software. Results are initially analyzed by FACS analysis software (Flow Jo). The IC50 for test compounds is defined as the concentration which decreases by 50% the percentage of CD69-positive cells that are also CD20-positive after stimulation by anti-IgM (average of 8 control wells, after subtraction of the average of 8 wells for the no-stimulus background). The IC50 values are calculated using XLfit software version 3, equation 201.
Inhibition of B-Cell Activation-B Cell FLIPR Assay in Ramos Cells
Inhibition of B-cell activation by compounds of the present invention is demonstrated by determining the effect of the 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 of the intracellular calcium increase from stimulation by an anti-IgM antibody. Ramos cells (human Burkitt's lymphoma cell line. ATCC-No. CRL-1596) were cultivated in Growth Media (described below). One day prior to assay, Ramos cells were resuspended in fresh growth media (same as above) and set at a concentration of 0.5×106/mL in tissue culture flasks. On day of assay, cells are counted and set at a concentration of 1×106/mL1 in growth media supplemented with 1 μM FLUO-3AM (TefLabs Cat-No. 0116, prepared in anhydrous DMSO and 10% Pluronic acid) in a tissue culture flask, and incubated at 37° C. (4% CO2) for one h. To remove extracellular dye, cells were collected by centrifugation (5 min, 1000 rpm), resuspended in FLIPR buffer (described below) at 1×106 cells/mL and then dispensed into 96-well poly-D-lysine coated black/clear plates (BD Cat-No. 356692) at 1×105 cells per well. Test compounds were added at various concentrations ranging from 100 μM to 0.03 μM (7 concentrations, details below), and allowed to incubate with cells for 30 min at RT. Ramos cell Ca2+ signaling was stimulated by the addition of 10 μg/mL anti-IgM (Southern Biotech, Cat-No. 2020-01) and measured on a FLIPR (Molecular Devices, captures images of 96 well plates using a CCD camera with an argon laser at 480 nM excitation).
Media/Buffers:
Growth Medium: RPMI 1640 medium with L-glutamine (Invitrogen, Cat-No. 61870-010), 10% Fetal Bovine Serum (FBS, Summit Biotechnology Cat-No. FP-100-05); 1 mM Sodium Pyruvate (Invitrogen Cat. No. 11360-070).
FLIPR buffer: HBSS (Invitrogen, Cat-No. 141175-079), 2 mM CaCl2 (Sigma Cat-No. C-4901), HEPES (Invitrogen, Cat-No. 15630-080), 2.5 mM Probenecid (Sigma, Cat-No. P-8761), 0.1% BSA (Sigma, Cat-No.A-7906), 11 mM Glucose (Sigma, Cat-No.G-7528)
Compound Dilution Details:
In order to achieve the highest final assay concentration of 100 μM, 24 μL of 10 mM compound stock solution (made in DMSO) is added directly to 576 μL of FLIPR buffer. The test compounds are diluted in FLIPR Buffer (using Biomek 2000 robotic pipettor) resulting in the following dilution scheme: vehicle, 1.00×10−4 M, 1.00×10−5, 3.16×10−6, 1.00×10−6, 3.16×10−7, 1.00×10−7, 3.16×10−8.
Assay and Analysis:
Intracellular increases in calcium were reported using a max-min statistic (subtracting the resting baseline from the peak caused by addition of the stimulatory antibody using a Molecular Devices FLIPR control and statistic exporting software. The IC50 was determined using a non-linear curve fit (GraphPad Prism software).
Mouse Collagen-Induced Arthritis (mCIA)
On day 0 mice are injected at the base of the tail or several spots on the back with an emulsion of Type II Collagen (i.d.) in Complete Freund's adjuvant (CFA). Following collagen immunization, animals will develop arthritis at around 21 to 35 days. The onset of arthritis is synchronized (boosted) by systemic administration of collagen in Incomplete Freund's adjuvant (IFA; i.d.) at day 21 Animals are examined daily after day 20 for any onset of mild arthritis (score of 1 or 2; see score description below) which is the signal to boost. Following boost, mice are scored and dosed with candidate therapeutic agents for the prescribed time (typically 2-3 weeks) and dosing frequency, daily (QD) or twice-daily (BID).
Rat Collagen-Induced Arthritis (rCIA)
On day 0, rats are injected with an emulsion of Bovine Type II Collagen in Incomplete Freund's adjuvant (IFA) is injected intradermally (i.d.) on several locations on the back. A booster injection of collagen emulsion is given around day 7, (i.d.) at the base of the tail or alternative sites on the back. Arthritis is generally observed 12-14 days after the initial collagen injection. Animals may be evaluated for the development of arthritis as described below (Evaluation of arthritis) from day 14 onwards. Animals are dosed with candidate therapeutic agents in a preventive fashion starting at the time of secondary challenge and for the prescribed time (typically 2-3 weeks) and dosing frequency, daily (QD) or twice-daily (BID).
Evaluation of Arthritis:
In both models, developing inflammation of the paws and limb joints is quantified using a scoring system that involves the assessment of the 4 paws following the criteria described below:
Scoring: 1=swelling and/or redness of paw or one digit.

    
    
        2=swelling in two or more joints.
        3=gross swelling of the paw with more than two joints involved.
        4=severe arthritis of the entire paw and digits.
    
    


Evaluations are made on day 0 for baseline measurement and starting again at the first signs or swelling for up to three times per week until the end of the experiment. The arthritic index for each mouse is obtained by adding the four scores of the individual paws, giving a maximum score of 16 per animal.
Rat In Vivo Asthma Model
Male Brown-Norway rats are sensitized i.p. with 100 μg of OA (ovalbumin) in 0.2 ml alum once every week for three weeks (day 0, 7, and 14). On day 21 (one week following last sensitization), the rats are dosed q.d. with either vehicle or compound formulation subcutaneously 0.5 hour before OA aerosol challenge (1% OA for 45 minutes) and terminated 4 or 24 hours after challenge. At time of sacrifice, serum and plasma are collected from all animals for serology and PK, respectively. A tracheal cannula is inserted and the lungs are lavaged 3× with PBS. The BAL fluid is analyzed for total leukocyte number and differential leukocyte counts. Total leukocyte number in an aliquot of the cells (20-100 μl) is determined by Coulter Counter. For differential leukocyte counts, 50-200 μl of the sample is centrifuged in a Cytospin and the slide stained with Diff-Quik. The proportions of monocytes, eosinophils, neutrophils and lymphocytes are counted under light microscopy using standard morphological criteria and expressed as a percentage. Representative inhibitors of BTK show decreased total leucocyte count in the BAL of OA sensitized and challenged rats as compared to control levels.
The foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity and understanding. It will be obvious to one of skill in the art that changes and modifications may be practiced within the scope of the appended claims. Therefore, 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 should instead be determined with reference to the following 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 hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual patent, patent application or publication were so individually denoted.

## Claims

1. A compound of Formula I,



wherein:
n is 1 or 2;
R1 is —C(═O)R1′, —S(═O)2R1′, or —OC(R1′)2CH2OH;
R1′ is methyl or morpholine;
R2 is H or F;
R3 is chloro or C(CH2)2R3′;
R3′ is methyl, cyano, or hydroxymethyl;
X is CH, CH2, or N;
X2 is N;
X3 is CH or N; and
Y is CH or O;
with the proviso that when n is 2, both X are CH2;
or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1, wherein R2 is H.

3. The compound of claim 1, wherein R3 is tert-butyl.

4. The compound of claim 1, wherein X3 is CH.

5. The compound of claim 1, wherein X2 is N.

6. The compound of claim 1, wherein R1 is —C(═O)R1′ and R1′ is morpholine.

7. The compound of claim 1, wherein n is 1.

8. The compound of claim 1, wherein X is CH or CH2.

9. The compound of claim 1, wherein Y is CH or CH2.

10. The compound of claim 5, wherein n is 2 and Y is O.

11. The compound of claim 10, wherein R1 is —S(═O)2R1′ and R1′ is methyl or R1 is —C(═O)R1′ and R1′ is morpholine.

12. The compound of claim 1, selected from the group consisting of:
2-[2-[2-(hydroxymethyl)-3-[1-methyl-5-[[5-(morpholine-4-carbonyl)pyridin-2-yl]amino]-6-oxopyridazin-3-yl]phenyl]-1-oxo-3,4-dihydroisoquinolin-6-yl]-2-methylpropanenitrile;
2-[8-fluoro-2-[2-(hydroxymethyl)-3-[5-[[5-(1-hydroxy-2-methylpropan-2-yl)oxypyridin-2-yl]amino]-1-methyl-6-oxopyridazin-3-yl]phenyl]-1-oxoisoquinolin-6-yl]-2-methylpropanenitrile;
4-[2-(hydroxymethyl)-3-[1-methyl-5-[(5-methylsulfonylpyridin-2-yl)amino]-6-oxopyridazin-3-yl]phenyl]-8-(1-hydroxy-2-methylpropan-2-yl)-2,3-dihydro-1,4-benzoxazepin-5-one;
6-tert-butyl-2-[2-(hydroxymethyl)-3-[1-methyl-5-[[5-(morpholine-4-carbonyl)pyridin-2-yl]amino]-6-oxopyridazin-3-yl]phenyl]-3,4-dihydro-2,7-naphthyridin-1-one;
6-tert-butyl-8-fluoro-2-[2-methyl-3-[1-methyl-5-[[5-(morpholine-4-carbonyl)pyridin-2-yl]amino]-6-oxopyridazin-3-yl]phenyl]phthalazin-1-one;
8-tert-butyl-4-[2-(hydroxymethyl)-3-[1-methyl-5-[[5-(morpholine-4-carbonyl)pyridin-2-yl]amino]-6-oxopyridazin-3-yl]phenyl]-2,3-dihydro-1,4-benzoxazepin-5-one; and
8-tert-butyl-4-[2-(hydroxymethyl)-3-[1-methyl-5-[(5-methylsulfonylpyridin-2-yl)amino]-6-oxopyridazin-3-yl]phenyl]-2,3-dihydro-1,4-benzoxazepin-5-one.

13. A pharmaceutical composition, comprising a therapeutically effective amount of a compound of claim 1, or a pharmaceutically acceptable salt thereof, admixed with at least one pharmaceutically acceptable carrier, excipient or diluent.