HK1194381B

HK1194381B - Inhibitors of bruton's tyrosine kinase

Info

Publication number: HK1194381B
Application number: HK14107956.1A
Authority: HK
Inventors: Roland J. Billedeau; Rama K. Kondru; Francisco Javier Lopez-Tapia; Yan Lou; Timothy D. Owens; Yimin Qian; Sung-Sau So; Kshitij Chhabilbhai Thakkar; Jutta Wanner; Omar Jose Morales
Original assignee: F. Hoffmann-La Roche Ag
Priority date: 2011-05-17
Filing date: 2012-05-14
Publication date: 2016-04-15

Description

Tyrosine kinase inhibitors

Technical Field

The present invention relates to the use of novel compounds that inhibit Btk and can be used to treat autoimmune and inflammatory diseases caused by aberrant B-cell activation.

Background

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 198750: 823-829). In particular, tyrosine kinases phosphorylate proteins on the phenol portion of tyrosine residues. The tyrosine kinase family includes members that control cell growth, migration, and differentiation. Abnormal kinase activity has been implicated in a number of human diseases, including cancer, autoimmune disorders and inflammatory diseases. Since protein kinases belong to key regulators of cell signaling, they provide the goal of regulating cellular function with small molecule kinase inhibitors and are therefore good drug design targets. In addition to the treatment of kinase-mediated disease processes, selective and potent inhibitors of kinase activity may also be useful in studying cellular signaling processes and in identifying other therapeutically significant cellular targets.

There is good evidence for the critical role of B cells in the pathogenesis of autoimmune and/or inflammatory diseases. Protein-based therapeutics that deplete B cells, such as Rituxan, are effective against autoantibody-induced inflammatory diseases such as rheumatoid arthritis (Rastetter et al Annu Rev Med 200455: 477). Therefore, inhibitors of protein kinases that play a role in B-cell activation should be useful therapeutic agents for B-cell mediated disease pathologies 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. BCR is a key regulatory point for B-cell activity and aberrant signaling can lead to deregulated B-cell proliferation and the formation of pathogenic autoantibodies, which lead to a variety of autoimmune and/or inflammatory diseases. Tyrosine protein kinases (Btk) are non-BCR-related kinases that are proximal and immediately downstream of the membrane of BCR. The lack of Btk has been shown to block BCR signaling, and thus inhibition of Btk can be an effective 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 key regulator of early B-cell formation and mature B-cell activation and survival (Khan et al Immunity 19953: 283; Ellmeier et al J.exp.Med.2000192: 1611). Btk mutations in humans lead to the disorder x-chromosome-linked gammoproteinemia (XLA) (reviewed in Rosen et al New Eng.J.Med.1995333: 431 and Lindval et al Immunol. Rev.2005203: 200). These patients are immunocompromised and show impaired B cell maturation, reduced immunoglobulin and peripheral B cell levels, reduced T cell-independent immune responses and impaired calcium mobilization following BCR stimulation.

Evidence for a role for Btk in autoimmune disorders and inflammatory diseases has been provided by Btk-deficient mouse models. In a preclinical murine model of Systemic Lupus Erythematosus (SLE), Btk deficient mice show significant improvement in disease progression. Furthermore, Btk-deficient mice are resistant to collagen-induced arthritis (Jansson and Holmdahl Clin. exp. Immunol. 199394: 459). Dose-dependent efficacy of selective Btk inhibitors in mouse models of arthritis has been demonstrated (z.pan et al, chem.med chem.20072: 58-61).

Btk is also substituted by other than B_-Extracellular cellular expression may be involved in the disease process. For example, Btk is expressed by mast cells and Btk-deficient bone marrow-derived mast cells exhibit impaired antigen-induced degranulation (Iwaki et al j.bi0l.chem.2005280: 40261). This shows that Btk can be used to treat pathological mast cell responses such as allergy and asthma. Furthermore, monocytes from XLA patients, where Btk activity is absent, show reduced TNF α production following stimulation (Horwood et al JExp Med 197: 1603, 2003). Thus, TNF α -mediated inflammation can be modulated by small molecule Btk inhibitors. In addition, Btk has been reported to play a role in apoptosis (Islam and Smith immunol. rev.2000178: 49) and therefore Btk inhibitors would be effective for the treatment of certain B-cell lymphomas and leukemias (Feldhahn et al j.exp.med.2005201: 1837).

Summary of The Invention

The present application provides Btk inhibitor compounds of formula I, methods of use thereof, as described herein below:

the present application provides a compound of formula I,

wherein:

is a single or double bond;

a is 5-membered heteroaryl or 5, 6-membered bicyclic heteroaryl, wherein CONH₂Linking 5-membered heteroaryl, each of which is optionally substituted with one or more a';

a' is-NHR or R⁴；

[0017] R is H, -R¹、-R¹-R²-R³、-R¹-R³or-R²-R³；

[0018] R¹Is aryl, heteroaryl, cycloalkyl, heterocycloalkyl or heteroaryl fused with heterocycloalkyl, each of which is optionally substituted with one or more R^1’Or R^1”Substitution;

R^1’each independently is halogen, nitro, cyano, lower alkylsulfonylamino, -S (O)₂Or oxo;

R^1”each independently is lower alkyl, cycloalkyl, heterocycloalkyl, lower alkoxy, amino or amido, each of which is optionally substituted with one or more R^1”’Substitution;

R^1”’each independently is hydroxy, halogen, amino, alkylamino, dialkylamino, or heterocycloalkyl;

R²is-C (═ O), -C (═ O) O, -C (═ O) NR^2’、-NHC(＝O)O、-C(R^2’)₂、-O、-C(＝NH)NR^2’or-S (═ O)₂；

R^2’Each independently is H or lower alkyl;

R³is H or R⁴；

R⁴Is lower alkyl, lower haloalkyl, lower alkoxy, amino, lower alkylamino, lower dialkylamino, aryl, arylalkyl, alkylaryl, heteroaryl, alkylheteroaryl, heteroarylalkyl, cycloalkyl, alkylcycloalkyl, optionally substituted with one or more substituents selected from the group consisting of lower alkyl, lower haloalkyl, lower alkoxy, amino, lower alkylamino, lower dialkylamino, aryl, arylalkyl, alkylaryl, heteroaryl, alkylheteroaryl, heteroarylalkyl, cycloalkyl, alkylcycloalkyl, heteroarylalkyl, cycloalkyl,cycloalkylalkyl, heterocycloalkyl, alkylheterocycloalkyl, heterocycloalkylalkyl, bicyclocycloalkyl, bicycloheterocycloalkyl, spirocycloalkyl or spiroheterocycloalkyl, each of which is optionally substituted with one or more lower alkyl, halogen, lower alkylamino, lower dialkylamino, hydroxy lower alkyl, lower alkoxy, halogen, nitro, amino, amido, acyl, cyano, oxo, guanidino, hydroxyamino, carboxy, carbamoyl, carbamate, halogenated lower alkoxy or halogenated lower alkyl, wherein two lower alkyl groups may together form a ring;

q is CH or N;

x is CH, N or N (X');

x' is lower alkyl;

Y⁰is H, halogen or lower alkyl;

Y¹is Y^1a、Y^1b、Y^1cOr Y^1d；

Y^1aIs H or halogen;

Y^1bis lower alkyl, optionally substituted with one or more substituents selected from the group consisting of lower haloalkyl, halogen, hydroxy, amino, cyano and lower alkoxy;

Y^1cis lower cycloalkyl, optionally substituted by one or more substituents selected from the group consisting of lower alkyl, lower haloalkyl, halogen, hydroxy, amino, cyano and lower alkoxy;

Y^1dis amino, optionally substituted with one or more lower alkyl, alkoxy lower alkyl or hydroxy lower alkyl;

Y²is H, halogen or lower alkyl;

Y³is H, halogen, lower alkyl, lower haloalkyl, lower alkoxy or lower hydroxyalkyl; and is

Y⁴Is H, lower alkyl or lower hydroxyalkyl;

or a pharmaceutically acceptable salt thereof.

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

The present application provides pharmaceutical compositions comprising a compound of formula I in combination with at least one pharmaceutically acceptable carrier, excipient or diluent.

Detailed Description

Definition of

As used herein, the phrase "an" or "an" entity refers to one or more of that entity; for example, a compound refers to one or more compounds or at least one compound. Thus, the terms "a" (or "an)", "one or more" and "at least one" may be used interchangeably herein.

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

As used in this specification, the terms "comprises" and "comprising," whether in transitional phrases or in the main body of the claims, should be construed to have an open-ended meaning. That is, the term should be construed as synonymous with the phrase "having at least" or "including at least". When used in the context of a method, the term "comprising" means that the method includes at least the recited steps, but may include additional steps. The term "comprising" when used in the context of a compound or composition means that the compound or composition includes at least the recited features or components, but may also include additional features or components.

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

The term "independently" as used herein means that the variables are used in either case regardless of the presence or absence of variables having the same or different definitions within the same compound. Thus, in a compound where R "occurs twice and is defined as" independently carbon or nitrogen ", two R" can be carbon, two R "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 or describing compounds employed or claimed in the present invention, its definition on each occurrence is independent of its definition at every other occurrence. Furthermore, combinations of substituents and/or variables are permissible only if such compounds result in stable compounds.

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

MeC(=O)OR⁴wherein

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

The term "optional" or "optionally" as used herein means that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "optionally substituted" means that the optionally substituted moiety may 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 as "bond" or "absent," the atoms attached to that substituent are directly attached.

The term "about" as used herein means approximately, near. 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. Generally, the term "about" as used herein changes a numerical value to a variance of 20% above and below the stated value.

Certain compounds of formula I may exhibit tautomerism. Tautomeric compounds may exist as two or more species that can be interconverted. Tautomers of proton transfer result from the migration of a covalently bound hydrogen atom between two atoms. Tautomers generally exist in equilibrium and attempts to isolate individual tautomers generally produce mixtures whose chemical and physical properties are consistent with mixtures of compounds. The position of equilibrium depends on the chemical characteristics within the molecule. For example, in the case of many aliphatic aldehydes and ketones such as acetaldehyde, the keto form predominates, while in the phenols, the enol form predominates common prototropic tautomers include keto/enolAmide/imide acidAnd amidinesTautomers. The latter two are particularly common in heteroaryl and heterocyclic rings, and the present invention includes all tautomeric forms of the compounds.

Technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention belongs, unless otherwise defined. Reference is made herein to various methods and materials known to those skilled in the art. Standard reference works listing general principles of pharmacology include Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10 th edition, McGraw Hill Companies Inc., New York (2001). Any suitable materials and/or methods known to those skilled in the art may be used in the practice of the present invention. However, preferred materials and methods are described. Materials, reagents, etc. referred to in the following description and examples are available from commercial sources unless otherwise indicated.

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

The term "spirocycloalkyl" as used herein refers to a spiro cycloalkyl group such as, for example, spiro [3.3] heptane. The term spiroheterocycloalkyl as used herein refers to a spirocyclic heterocycloalkyl group such as, for example, 2, 6-diazaspiro [3.3] heptane.

The term "acyl" as used herein denotes a group of formula-C (= O) R, wherein R is hydrogen or lower alkyl as defined herein. The term "alkylcarbonyl" as used herein, denotes a group of formula-C (═ O) R, wherein R is alkyl as defined herein. Term C_1-6Acyl means that the group-C (═ O) R contains 1 to 6 carbon atoms. The term "arylcarbonyl" as used herein denotes a group of formula C (═ O) R, wherein R is aryl; the term "benzoyl" as used herein is an "arylcarbonyl" group wherein R is phenyl.

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

The term "alkyl" as used herein denotes an unbranched or branched saturated monovalent hydrocarbon residue containing from 1 to 10 carbon atoms. The term "lower alkyl" denotes a straight or branched chain hydrocarbon residue containing 1 to 6 carbon atoms. "C" as used herein_1-10Alkyl "refers to an alkyl group consisting of 1 to 10 carbons. Examples of alkyl groups include, but are not limited to, lower alkyl groups including methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl or pentyl, isopentyl, neopentyl, hexyl, heptyl, and octyl.

When the term "alkyl" is used as a suffix following another term, as in "phenylalkyl" or "hydroxyalkyl", it is intended to indicate an alkyl group, as defined above, substituted with one to two substituents selected from the group of the additional specified nomenclature. Thus, for example, "phenylalkyl" denotes the group R 'R "-, where R' is phenyl and R" is alkylene as defined herein, with the understanding that the attachment point of the phenylalkyl moiety will be on the alkylene. Examples of arylalkyl groups include, but are not limited to, benzyl, phenylethyl, 3-phenylpropyl. The term "arylalkyl" or "aralkyl" is similarly construed, except that R' is aryl. The terms "(hetero) arylalkyl" or "(hetero) aralkyl" are similarly construed, except that R' is optionally aryl or heteroaryl.

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

The term "alkylene (or alkylene 1)" as used herein means a divalent saturated straight-chain hydrocarbon group of 1 to 10 carbon atoms (e.g., (CH)₂)_n) Or a branched saturated divalent hydrocarbon radical of 2 to 10 carbon atoms (e.g. -CHMe-or-CH)₂CH(i-Pr)CH₂-) unless otherwise indicated. Except in the case of methylene, the open valences of the alkylene groups are not attached to the same atom. Examples of alkylene groups include, but are not limited to: methylene, ethylene, propylene, 2-methyl-propylene, 1-dimethyl-ethylene, butylene, 2-ethylbutylene.

The term "alkoxy" as used herein denotes-O-alkyl, wherein alkyl is as defined above, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentyloxy, hexyloxy, including isomers thereof. "lower alkoxy" as used herein denotes alkoxy having a "lower alkyl" group as defined above. "C" as used herein_1-10Alkoxy "means an-O-alkyl group wherein alkyl is C_1-10。

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

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

The term "hydroxyalkyl" as used herein denotes an alkyl group as defined herein wherein 1 to 3 hydrogen atoms on different carbon atoms are replaced by a hydroxyl group.

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

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

The term "cycloalkyl" as used herein refers to a saturated carbocyclic ring containing 3 to 8 carbon atoms, i.e. cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl. "C" as used herein_3-7Cycloalkyl "or" lower alkyl "refers to cycloalkyl consisting of 3 to 7 carbons in a carbocyclic ring.

The term carboxy-alkyl as used herein refers to an alkyl moiety in which one hydrogen atom has been replaced by a carboxy group, wherein it is understood that the point of attachment of the heteroalkyl group is through a carbon atom. The term "carboxy (or carboxyl)" means-CO₂And (4) a H part.

The term "heteroaryl" or "heteroaromatic" as used herein refers to a monocyclic or bicyclic group of 5 to 12 ring atoms having at least one aromatic or partially unsaturated ring, each ring containing 4 to 8 atoms, to which one or more N, O or S heteroatoms are attached, the remaining ring atoms being carbon, wherein it is understood that the attachment point of the heteroaryl group will be on the aromatic or partially unsaturated ring. As is well known to those skilled in the art, heteroaryl rings have less aromatic character than their all-carbon counterparts. Thus, for the purposes of the present invention, 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, including but not limited to pyridyl, pyrimidinyl, pyrazinyl, oxazinyl, pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, 4, 5-dihydro-oxazolyl, 5, 6-dihydro-4H- [1, 3] oxazolyl, isoxazole, thiazole, isothiazole, triazoline, thiadiazole and oxadiazoline (oxaxoline), which may be optionally substituted with 1 or more, preferably 1 or 2 substituents selected from hydroxy, cyano, alkyl, alkoxy, thio, lower haloalkoxy, alkylthio, halogen, lower haloalkyl, alkylsulfinyl, alkylsulfonyl, halogen, amino, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl and dialkylaminoalkyl, amino, alkylamino, dialkylamino, alkylthio, and dialkylamino, Nitro, alkoxycarbonyl and carbamoyl radicals, alkylcarbamoyl radicals, dialkylcarbamoyl radicals, arylcarbamoyl radicals, alkylcarbonylamino radicals and arylcarbonylamino radicals. Examples of bicyclic moieties include, but are not limited to, quinolinyl, isoquinolinyl, benzofuranyl, benzothienyl, benzoxazole, benzisoxazole, benzothiazole, naphthyridinyl, 5,6,7, 8-tetrahydro- [1, 6] naphthyridinyl, and benzisothiazole. The bicyclic moiety may be optionally substituted on either ring, but the point of attachment is on the ring containing the heteroatom.

The terms "heterocyclyl", "heterocycloalkyl" or "heterocycle" as used herein denote a monovalent saturated cyclic group consisting of one or more rings, preferably 1 to 2 rings (including spiro ring systems), with 3 to 8 atoms per ring, to which one or more ring heteroatoms (selected from N, O or S (O))_0-2) And which may be optionally independently substituted with one or more, preferably 1 or 2, substituents selected from hydroxy, oxo, cyano, lower alkyl, lower alkoxy, lower haloalkoxy, alkylthio, halogen, lower haloalkyl, hydroxyalkyl, nitro, alkoxycarbonyl, amino, alkylamino, alkylsulfonyl, arylsulfonyl, alkylaminosulfonyl, arylaminosulfonyl, alkylsulfonylamino, arylsulfonylamino, alkylaminocarbonyl, arylaminocarbonyl, alkylcarbonylamino, arylcarbonylamino and ionic forms thereof, unless otherwise indicated. Examples of heterocyclyl groups include, but are not limited to, morpholinyl, piperazinyl, piperidinyl, azetidinyl, pyrrolidinyl, hexahydroazepinylAlkyl, oxetanyl, tetrahydrofuryl, tetrahydrothienyl, 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]Oxazines.

Btk inhibitors

The present application provides a compound of formula I,

wherein:

is a single or double bond;

a' is-NHR or R⁴；

[0082] R is H, -R¹、-R¹-R²-R³、-R¹-R³or-R²-R³；

[0083] R¹Is aryl, heteroaryl, cycloalkyl, heterocycloalkyl or heteroaryl fused with heterocycloalkyl, each of which is optionally substituted with one or more R^1’Or R^1”Substitution;

R^2’Each independently is H or lower alkyl;

R³is H or R⁴；

R⁴Is lower alkyl, lower haloalkyl, lower alkoxy, amino, lower alkylamino, lower dialkylamino, aryl, arylalkyl, alkylaryl, heteroaryl, alkylheteroaryl, heteroarylalkyl, cycloalkyl, alkylcycloalkyl, cycloalkylalkyl, heterocycloalkyl, alkylheterocycloalkyl, heterocycloalkylalkyl, bicycloalkylcycloalkyl, bicycloheteroalkyl, spirocycloalkyl or spiroheterocycloalkyl, each of which is optionally substituted by one or more lower alkyl groups, halogen, lower alkylamino, lower dialkylamino, hydroxy lower alkyl, lower alkoxy, halogen, nitro, amino, amido, acyl, cyano, oxo, guanidino, hydroxyamino, carboxy, carbamoyl, carbamate, halogenated lower alkoxy, or halogenated lower alkyl, wherein two lower alkyl groups may together form a ring;

q is CH or N;

x is CH, N or N (X');

x' is lower alkyl;

Y⁰is H, halogen or lower alkyl;

Y¹is Y^1a、Y^1b、Y^1cOr Y^1d；

Y^1aIs H or halogen;

Y²is H, halogen or lower alkyl;

Y⁴Is H, lower alkyl or lower hydroxyalkyl;

or a pharmaceutically acceptable salt thereof.

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

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

The application provides compounds of formula I, wherein Y¹Is Y^1a。

The application provides compounds of formula I, wherein Y^1aIs H.

The application provides compounds of formula I, wherein Y¹Is lower alkyl or lower cycloalkyl.

The application provides compounds of formula I, wherein Y¹Is tert-butyl, isopropyl, cyclopropyl or isopropylnitrile.

The application provides compounds of formula I, wherein Y¹Is a tert-butyl group.

The application provides compounds of formula I, wherein X is N, Y¹Is tert-butyl or cyclopropyl.

The application provides compounds of formula I, wherein Y²Is H.

The application provides compounds of formula I, wherein Y³Is H or halogen.

The application provides compounds of formula I, wherein Y³Is H or F.

The application provides compounds of formula I, wherein Y⁰Is H, Y²Is H, Y³Is F or H, Y⁴Is hydroxymethyl.

The application provides compounds of formula I, wherein Q is CH, X is N, andis a double bond.

The present application provides compounds of formula I wherein a is furyl, imidazolyl, thiazolyl, pyrrolyl, pyrazolyl, phenyl, indolyl, pyrrolo [2, 3-b ] pyridinyl, or oxazolyl.

The application provides compounds of formula I, wherein Y¹Is tert-butyl or cyclopropyl.

The application provides compounds of formula I, wherein Y³Is F.

The application provides compounds of formula I, wherein Y³Is H or F, and X is N.

The application provides compounds of formula I, wherein Y³Is H or F, Y¹Is tert-butyl or cyclopropyl.

The application provides compounds of formula I, wherein Y³Is H or F, X is N, Y¹Is tert-butyl or cyclopropyl.

The application provides compounds of formula IA compound of formula (I) wherein Y⁴Is a lower hydroxyalkyl.

The application provides compounds of formula I, wherein Y⁴Is hydroxymethyl.

The present application provides compounds of formula I, wherein a is furyl optionally substituted with one or more a'.

The application provides compounds of formula I, wherein Y³Is H or F, X is N, Y¹Is tert-butyl or cyclopropyl, A is furyl optionally substituted by one or more A'.

The present application provides compounds of formula I wherein a is imidazolyl optionally substituted with one or more a'.

The application provides compounds of formula I, wherein Y³Is H or F, X is N, Y¹Is tert-butyl or cyclopropyl, A is imidazolyl optionally substituted with one or more A'.

The present application provides compounds of formula I, wherein a is thiazolyl optionally substituted with one or more a'.

The application provides compounds of formula I, wherein Y³Is H or F, X is N, Y¹Is tert-butyl or isopropyl, A is thiazolyl optionally substituted with one or more A'.

The present application provides compounds of formula I, wherein a is pyrrolyl optionally substituted with one or more a'.

The application provides compounds of formula I, wherein Y³Is H or F, X is N, Y¹Is tert-butyl or isopropyl, A is pyrrolyl optionally substituted with one or more A'.

The present application provides compounds of formula I, wherein a is pyrazolyl optionally substituted with one or more a'.

The application provides compounds of formula I, wherein Y³Is H or F, X is N, Y¹Is tert-butyl or isopropyl, A is optionally substituted by one or more A'A substituted pyrazolyl group.

The present application provides compounds of formula I, wherein a is optionally substituted with one or more a'.

The application provides compounds of formula I, wherein Y³Is H or F, X is N, Y¹Is tert-butyl or isopropyl, A is phenyl optionally substituted with one or more A'.

The present application provides compounds of formula I, wherein a is indolyl optionally substituted with one or more a'.

The application provides compounds of formula I, wherein Y³Is H or F, X is N, Y¹Is tert-butyl or isopropyl, and A is indolyl optionally substituted with one or more A'.

The present application provides compounds of formula I wherein a is pyrrolo [2, 3-b ] pyridinyl optionally substituted with one or more a'.

The application provides compounds of formula I, wherein Y³Is H or F, X is N, Y¹Is tert-butyl or isopropyl, A is pyrrolo [2, 3-b ] optionally substituted by one or more A]A pyridyl group.

The present application provides compounds of formula I wherein a is oxazolyl optionally substituted with one or more a'.

The application provides compounds of formula I, wherein Y³Is H or F, X is N, Y¹Is tert-butyl or isopropyl, a is oxazolyl optionally substituted with one or more a'.

The application provides compounds of formula I, wherein Y³Is hydroxymethyl.

The application provides compounds of formula I, wherein Y³Is hydroxymethyl, X is N, Y¹Is tert-butyl or isopropyl.

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

4- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -1-methyl-1H-imidazole-2-carboxamide;

2- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -thiazole-4-carboxamide;

4- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -1H-pyrrole-2-carboxamide;

4- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -1-methyl-1H-pyrrole-2-carboxamide;

5- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -1-methyl-1H-pyrrole-2-carboxamide;

2- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -4-methyl-oxazole-5-carboxylic acid amide;

2- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -thiazole-4-carboxamide;

4- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -1-methyl-1H-imidazole-2-carboxamide;

4- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -1-methyl-1H-pyrrole-2-carboxamide;

5- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -furan-2-carboxamide;

4- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -furan-2-carboxamide;

4- [3- (6-tert-butyl-8-hydroxymethyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -1-methyl-1H-pyrrole-2-carboxamide;

4- [3- (6-cyclopropyl-8-fluoro-1-oxo-1H-isoquinolin-2-yl) -2-hydroxymethyl-phenyl ] -1-methyl-1H-pyrrole-2-carboxamide;

4- [3- (6-tert-butyl-3-methyl-1-oxo-3, 4-dihydro-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -1-methyl-1H-pyrrole-2-carboxamide;

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -1H-pyrrolo [2, 3-b ] pyridine-3-carboxamide;

4- [2- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -3-hydroxymethyl-pyridin-4-yl ] -1-methyl-1H-pyrrole-2-carboxamide;

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -1H-indole-3-carboxamide;

1- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -1H-indole-3-carboxamide;

1- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -3- [4- (morpholine-4-carbonyl) -phenylamino ] -1H-pyrazole-4-carboxamide;

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -3- [4- (morpholine-4-carbonyl) -phenylamino ] -1H-pyrazole-4-carboxamide;

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -phenyl ] -3- [4- (morpholine-4-carbonyl) -phenylamino ] -1H-pyrazole-4-carboxamide;

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -3- [4- (1-hydroxy-1-methyl-ethyl) -phenylamino ] -1H-pyrazole-4-carboxamide;

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -3- (5-chloro-pyridin-2-ylamino) -1H-pyrazole-4-carboxamide;

3- [5- (2-azetidin-1-yl-1, 1-dimethyl-ethoxy) -pyridin-2-ylamino ] -1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -1H-pyrazole-4-carboxamide;

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -3- (5-methyl-4, 5,6, 7-tetrahydro-pyrazolo [1, 5-a ] pyrazin-2-ylamino) -1H-pyrazole-4-carboxamide;

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -3- (pyridin-2-ylamino) -1H-pyrazole-4-carboxamide;

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -3- (4-methanesulfonyl-phenylamino) -1H-pyrazole-4-carboxamide;

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -3- (1-methyl-1H-pyrazol-3-ylamino) -1H-pyrazole-4-carboxamide;

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -3- (pyrazin-2-ylamino) -1H-pyrazole-4-carboxamide;

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -3- (5-fluoro-pyridin-2-ylamino) -1H-pyrazole-4-carboxamide;

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -3- (1, 5-dimethyl-1H-pyrazol-3-ylamino) -1H-pyrazole-4-carboxamide;

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -3- (5-trifluoromethyl-pyridin-2-ylamino) -1H-pyrazole-4-carboxamide;

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -3- (5-methyl-pyridin-2-ylamino) -1H-pyrazole-4-carboxamide;

1- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -3- (1, 5-dimethyl-1H-pyrazol-3-ylamino) -1H-pyrazole-4-carboxamide;

1- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -3- (5-fluoro-pyridin-2-ylamino) -1H-pyrazole-4-carboxamide;

1- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -3- (pyrazin-2-ylamino) -1H-pyrazole-4-carboxamide;

1- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -3- (5-methyl-pyridin-2-ylamino) -1H-pyrazole-4-carboxamide;

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -3- (5-methanesulfonyl-pyridin-2-ylamino) -1H-pyrazole-4-carboxamide;

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -3- (5-cyano-pyridin-2-ylamino) -1H-pyrazole-4-carboxamide;

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -1H-pyrazole-3-carboxamide;

1- [3- (6-tert- [ yl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -1H-pyrazole-4-carboxamide;

7- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -7H-pyrrolo [2, 3-d ] pyrimidine-5-carboxamide;

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -6- (4-methyl-piperazin-1-yl) -1H-pyrrolo [2, 3-b ] pyridine-3-carboxamide;

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -6-morpholin-4-yl-1H-pyrrolo [2, 3-b ] pyridine-3-carboxamide;

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl]-6- (6-ethoxy)_-Pyridin-3-yl) -1H-pyrrolo [2, 3-b]Pyridine-3-carboxamide;

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -6- (2-fluoro-phenyl) -1H-pyrrolo [2, 3-b ] pyridine-3-carboxamide;

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -6- (2-chloro-phenyl) -1H-pyrrolo [2, 3-b ] pyridine-3-carboxamide;

6-bromo-1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -1H-pyrrolo [2, 3-b ] pyridine-3-carboxamide;

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl]-6- (1, 2-dihydroxy)_-Ethyl) -1H-pyrrolo [2, 3-b]Pyridine-3-carboxamide;

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl]-6- (1, 1-dioxo-1. lamda.)⁶-thiomorpholin-4-yl) -1H-pyrrolo [2, 3-b]Pyridine-3-carboxamide;

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -6- (2-dimethylamino-ethylamino) -1H-pyrrolo [2, 3-b ] pyridine-3-carboxamide;

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -6-dimethylaminomethyl-1H-pyrrolo [2, 3-b ] pyridine-3-carboxamide;

3- (4-acetyl-phenylamino) -1- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -1H-pyrazole-4-carboxamide;

1- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -3- (pyridin-2-ylamino) -1H-pyrazole-4-carboxamide;

1- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -3- (5-dimethylaminomethyl-pyridin-2-ylamino) -1H-pyrazole-4-carboxamide; and

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -phenyl ] -1H-pyrazole-4-carboxamide.

The present application provides the use of compounds of formula I as therapeutically active substances.

The present application provides the use of a compound of formula I in the treatment of inflammatory diseases and/or autoimmune disorders.

The present application provides the use of a compound of formula I in the treatment of an inflammatory disorder.

The present application provides the use of a compound of formula I in the treatment of rheumatoid arthritis or asthma.

The present application provides compounds of formula I for use in treating inflammatory and/or autoimmune disorders.

The present application provides compounds of formula I for use in treating inflammatory disorders.

The present application provides compounds of formula I for use in the treatment of rheumatoid arthritis or asthma.

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

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

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

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

The present application provides the use of a compound of formula I in the manufacture of a medicament for the treatment of an inflammatory disorder.

The present application provides the use of a compound of formula I for the manufacture of a medicament for the treatment of autoimmune disorders.

The present application provides the use of a compound of formula I in the manufacture of a medicament for the treatment of an inflammatory condition and/or an autoimmune disorder.

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

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

Compounds and preparation

Examples of representative compounds encompassed by and within the scope of the present invention are provided in the following tables. The following examples and preparations are provided to enable those skilled in the art to more clearly understand and practice the present invention. They should not be considered as limiting the scope of the invention, but merely as being exemplary and representative thereof.

Generally, the nomenclature used in this application is based on AUTONOM^TMv.4.0, a Beilstein Institute computerized system for generating IUPAC systematic nomenclature. If there is a difference between the drawn structure and the name giving it, the drawn structure will be given more weight. Further, 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 describes examples of pyridazinone compounds of formula I:

table I.

Synthesizing: general synthetic schemes

As depicted in scheme 1, bromo-substituted 5-membered heterocyclic aromatic carboxamides (1) can be prepared from the corresponding esters or carboxylic acids by amide formation using ammonia. In structure (1), X may be NH, NR, O, or S, where R may be a lower alkyl group, such as methyl. In structure (1), Y may be CH or a carbon or nitrogen atom substituted with a lower alkyl group such as methyl. In structure (2), Z may be nitrogen or CH and R may be a mono-or di-substituent. The substituents may be fluorine, chlorine, alkyl, substituted alkyl or cycloalkyl. (2) As described in the literature (US 2010/0222325). Coupling of compounds (1) and (2) under palladium-catalyzed coupling conditions (US 2010/0222325) can yield compound (3), which can be hydrolyzed to the desired compound (4).

Alternatively, 5-membered formamide (4) can be prepared according to scheme (2). The bromo-substituted 5-membered heterocyclic aromatic carboxamide (1) may be prepared from the corresponding ester or carboxylic acid by amide formation using ammonia. In structure (1), X may be NH, NR, O, or S, where R may be a lower alkyl group, such as methyl. In structure (1), Y may be CH or a carbon or nitrogen atom substituted with a lower alkyl group such as methyl. Bromo-substituted 5-membered heterocyclic aromatic carboxamides (1) can be converted into the corresponding pinacolato boronates (5) by reaction with bis-pinacolborane under palladium-catalyzed aryl borate formation conditions (US 2010/0222325). In structure (6), Z may be nitrogen or CH and R may be a mono-or di-substituent. The substituents may be fluorine, chlorine, alkyl, substituted alkyl or cycloalkyl. The preparation of compound (6) is described in scheme 9. Aryl bromides (6) can be coupled with 5-membered heteroaromatic boronic acid esters (5) under palladium catalyzed coupling conditions (US 2010/0222325) to afford the desired compounds (4).

The 5-membered heterocyclic carboxamides can be fused to an aromatic ring to give bicyclic heteroaromatic carboxamides as shown in scheme 3. Indole or aza-indole derivatives (7) can be coupled with aryl boronic acids (8) under palladium-catalyzed coupling conditions to give aryl-substituted indoles or aza-indoles (9) according to similar conditions in the literature (tretetrahedron Letters2009, 50, 15-18). The preparation of arylboronic acids (8) is as described in scheme 9. In structure (7), X may be CH or nitrogen and Y may be alkyl, alkyl substituted with a heteroatom, or a heterocycle. In structure (8), R may be mono-or di-substituted. The substituents may be fluorine, chlorine, alkyl, substituted alkyl or cycloalkyl. In structure (8), Z may be nitrogen or CH. The cyano group on compound (9) can be hydrolyzed in the presence of palladium catalysis under neutral conditions (Journal of Organic Chemistry2004, 69, 2327-2331) to give the corresponding carboxamide (10).

Alternatively, formamide (10) may be prepared according to scheme 4. In structure (7), X may be CH or nitrogen and Y may be alkyl, alkyl substituted with a heteroatom, or a heterocycle. Under basic conditions, such as potassium tert-butoxide, compound (7) reacts with aryl fluoride (11) by nucleophilic aromatic substitution to give compound (12), where the halogen on compound (11) can be a bromine or iodine atom. Compound (14) can be obtained by treating compound (12) (US 2010/0222325) with (13) in the presence of cuprous iodide. The preparation of compound (13) is described in the literature (US 2010/0222325), wherein R may be mono-or di-substituted. The substituents may be fluorine, chlorine, alkyl, substituted alkyl or cycloalkyl. In structure (13), Z may be nitrogen or CH. The aldehyde group on compound (14) can be reduced to an alcohol by using a reducing agent such as sodium borohydride. The cyano group on compound (14) can be hydrolyzed in the presence of palladium catalysis under neutral conditions (Journal of Organic Chemistry2004, 69, 2327-2331) to give the corresponding carboxamide (10).

For compounds in which the 5-membered heteroaromatic carboxamide is an aminopyrazole carboxamide, the preparation is described in scheme 5. Commercially available aminopyrazole nitrile (15) can be reacted with potassium trifluoroborate salt (16) in the presence of copper acetate to give compound (17). For mixed regioisomers that are N-arylated during conversion of (15) to (17), the desired compound (17) can be isolated under Supercritical Fluid Chromatography (SFC) conditions. In structure (16), R1 can be hydrogen, alkyl, and acetoxy. In structure (16), R can be mono_-Or a di-substituent. The substituents may be fluorine, chlorine, alkyl, substituted alkyl or cycloalkyl, and Z may be nitrogen or carbon. The preparation of compound (16) is depicted in scheme 10. N-arylation of compound (17) with an aryl halide such as aryl bromide under palladium-catalyzed conditions can provide compound (18), wherein X and Y can be CH or one of X and Y can be nitrogen. The R2 group on the aryl halide can be dialkylaminocarbonyl, aminoalkyl, heterocycloalkyl, methylsulfonyl, mono-or di-hydroxy substituted alkyl. (17) The reaction agent for converting the aryl halide in (18) is not limited to 6-membered aromatic halide and it may be 5_-A 5-membered aromatic halide fused with a heterocycle. Hydrolysis of the cyano group on (18) affords the desired carboxamide (19).

An alternative route to 5-membered aromatic carboxamides containing an aminopyrazole moiety is depicted in scheme 6. Protection of the NH group on compound (15) with trimethylsilylethoxymethylene chloride (SEM-Cl) in the presence of a base such as sodium hydride can give two regioisomers. These two regioisomers can be separated to give (20). N-arylation of (20) with an aryl halide in the presence of palladium catalysisTo compound (21) wherein X and Y may be CH or one of X and Y may be nitrogen. The radical on the aryl halide may be dialkylaminocarbonyl, aminoalkyl, heterocycloalkyl, methylsulfonyl, mono_-Or two_-A hydroxy-substituted alkyl group. (20) The aryl halide reactant to be converted into (21) is not limited to 6-membered aromatic halide and it may be 5-membered aromatic halide or 5-membered aromatic halide condensed with a heterocyclic ring. The SEM group on (21) may be deprotected under acidic conditions such as dilute hydrochloric acid or basic conditions such as tetrabutylammonium fluoride to provide compound (22). Compound (23) can be obtained by treating compound (22) with 2-fluoro-6-iodobenzaldehyde in the presence of a base such as potassium tert-butoxide. Compound (23) can be N-arylated with compound (13) under Cu (I) catalytic conditions, such as cuprous iodide, to give compound (24), wherein Z and R1 on compound (24) have the same definitions as Z and R on compound (13). Reduction of the aldehyde on the compound (24) followed by nitrile hydrolysis gives the desired carboxamide (25).

An alternative route to 5-membered aromatic carboxamides containing an aminopyrazole moiety is depicted in scheme 7. Commercially available compound (15) can be converted to the corresponding iodide (26) according to literature procedures (WO 2005/005414). Protection of the NH group on (26) with SEM-Cl according to literature procedures (WO 2005/005414) gave compound (27). Compound (27) may be reacted with an arylamine in the presence of a palladium catalyst such as bis (tri-tert-butylphosphine) palladium (0) to provide an N-arylated product (28) in which the aryl group may be a heteroaromatic moiety and which may be substituted with dialkylaminocarbonyl, aminoalkyl, heterocycloalkyl, methylsulfonyl, mono-or di-hydroxy substituted alkyl. The SEM group on (28) can be deprotected under acidic conditions, e.g., dilute hydrochloric acid, or basic conditions, e.g., tetrabutylammonium fluoride, to provide compound (29). Compound (30) can be obtained by treating compound (29) with potassium trifluoroborate (16) in the presence of cu (ii) catalysis, e.g., copper acetate, wherein the R and R1 groups on compound (16) are as defined in scheme 5. Hydrolysis of the cyano group on compound (30) affords the desired carboxamide (31).

During the conversion of (29) to (30) in scheme 7, the desired regioisomer of the N-arylation product can be obtained by purification of the mixed regioisomers.

Alternatively, the desired aminopyrazole derivatives can be prepared according to scheme 8. The compound (32) can be obtained by treating R1-substituted 2, 6-dibromobenzene with the compound (13) in the presence of cuprous iodide. N-arylating a commercially available hydrazone (33) with a compound (32) under palladium-catalyzed conditions, followed by hydrolysis under acidic conditions, can give a hydrazine (34). Compound (34) can be treated with commercially available compound (35) by using a similar literature method (Journal of Organic Chemistry2005, 70, 9222) to give the desired aminopyrazole (36). Compound (36) can be N-arylated with an aryl halide as described for the preparation of compound (18) in scheme 5. Hydrolysis of the nitrile on compound (30) affords the desired carboxamide (31).

The preparation of the desired aryl borate ester (2), arylboronic acid (8) and intermediate (6) is described in scheme 9. Compound (37) may be substituted with compound (13) in the presence of cuprous iodide to give N-arylate product (38). Hydrolysis (38) under mildly alkaline conditions can yield the desired hydroxy derivative (6). Conversion of (38) to (2) can be achieved by using bis (pinacolato) borane under palladium catalyzed conditions. The boric acid ester (2) is hydrolyzed to obtain boric acid (8).

Finally, the preparation of the desired potassium trifluoroborate salt (16) is depicted in scheme 10. N-arylating compound (13) with aryl bromide (39) in the presence of cuprous iodide can give compound (40), wherein R can be mono-or di-substituted. Conversion of (40) to (41) can be carried out using bis (pinacolato) borane under palladium catalysis. The boronic acid pinacol ester (41) can be converted to the corresponding trifluoroborate (16) according to literature methods (Tetrahedron Letters 2005, 46, 7899).

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

The compound or compounds of the present invention and their pharmaceutically acceptable salts, together with one or more conventional excipients, carriers or diluents, may be placed in the form of pharmaceutical compositions and unit dosages. The pharmaceutical compositions and unit dosage forms may comprise the usual ingredients in conventional proportions, with or without additional active compounds or principles, and may contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range of use. The pharmaceutical compositions may be used in solid form such as tablets or filled capsules, semi-solid, powder, sustained release formulations or liquid form 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 a sterile injectable solution for parenteral use. Typical formulations contain from about 5% to about 95% of the active compound or compounds (w/w). The term "formulation" or "dosage form" is intended to include both solid and liquid formulations of the active compound and those skilled in the art will understand that the active ingredient may be present in different formulations depending on the target organ or tissue and depending on the desired dosage and pharmacokinetic parameters.

The term "excipient" as used herein refers to a compound that is generally safe, non-toxic, and biologically or otherwise undesirable for use in preparing a pharmaceutical composition, and includes excipients that are useful for veterinary use as well as human pharmaceutical applications. The compounds of the invention may be administered alone, but will generally be administered in admixture with one or more suitable pharmaceutical excipients, diluents or carriers selected with regard to the intended route of administration and standard pharmaceutical practice.

By "pharmaceutically acceptable" it is meant that it can be used to prepare pharmaceutical compositions that are generally safe, non-toxic, and not biologically or otherwise undesirable, and includes that it is acceptable for veterinary as well as human pharmaceutical use.

The "pharmaceutically acceptable salt" form of the active ingredient may also initially impart desirable pharmacokinetic properties to the active ingredient which are lacking in the non-salt form and may even positively affect the pharmacodynamics of the active ingredient with respect to its in vivo therapeutic activity. The phrase "pharmaceutically acceptable salt" of a compound refers to a salt that is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound. The salt comprises: (1) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or 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-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo [2.2.2] -oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, laurylsulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or (2) a salt formed when an acid proton present in the parent compound is replaced with a metal ion, such as an alkali metal ion, an alkaline earth ion, or an aluminum ion; or salts coordinated with organic bases such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, etc.

Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances that may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material. In powders, the carrier is typically a finely divided solid which is in admixture with the finely divided active ingredient. In tablets, the active ingredient is usually mixed with a 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. Formulations in solid form may contain, in addition to the active ingredient, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.

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

The compounds of the invention may be formulated for parenteral administration (e.g., by injection, e.g., bolus injection, or continuous infusion) and may be presented in unit dosage form in ampoules, pre-filled syringes, small volume infusion solutions 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 preservatives, wetting agents, emulsifying or suspending agents, stabilizing agents and/or dispersing agents. Alternatively, the active ingredient may be presented in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

The compounds of the present invention may be formulated for topical administration to the epidermis as an ointment, cream or lotion, 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 contain 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 the active ingredient in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

The compounds of the 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 ingredient is dispersed homogeneously, for example by stirring. The molten homogeneous mixture is then poured into a suitably sized mold, allowed to cool and solidify.

The compounds of the 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 suitable.

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

The compounds of the invention may be formulated for aerosol administration, particularly to the respiratory tract, and include intranasal administration. The compounds typically have small particle sizes, for example about five (5) microns or less. Such particle sizes may be obtained by means known in the art, for example by micronization. The active ingredient is provided in a pressurized container with a suitable propellant, such as a chlorofluorocarbon (CFC), for example dichlorodifluoromethane, trichlorofluoromethane or dichlorotetrafluoroethane, or carbon dioxide or other suitable gas. The aerosol may also conveniently contain a surfactant such as lecithin. The dosage of the medicament may be controlled by a metering valve. Alternatively, the active ingredient may be provided in the form of a dry powder, for example a powder mix of the compound in a suitable powder base such as lactose, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidone (PVP). The powder carrier will form a gel in the nasal cavity. The powder compositions may be presented in unit dosage form, for example in capsules or cartridges of, for example, gelatin, or in blister packs, from which the powder may be administered by means of an inhaler.

When desired, the formulations can be prepared with an enteric coating suitable for sustained or controlled release administration of the active ingredient. For example, the compounds of the present invention may be formulated in a transdermal or subcutaneous drug delivery device. These delivery systems are advantageous when sustained release of the compound is desired and when patient compliance with a treatment regimen is important. Compounds in transdermal delivery systems are often attached to a skin-adherent solid support. The compound of interest may also be combined with a penetration enhancer such as lauryl nitrogenA ketone (1-decadialkylaza-cyclohept-2-one) combination. The sustained release delivery system is inserted subcutaneously into the subcutaneous layer by surgery or injection. Subcutaneous implants encapsulate compounds in a lipid soluble film such as silicone rubber or a biodegradable polymer such as polylactic acid (polylactic acid).

In Remington: suitable formulations are described in pharmaceutical Science and Practice (Remington: The Science and Practice of Pharmacy 1995, edited by e.w. martin, Mack publishing company, 19 th edition, Easton, pa) together with pharmaceutical carriers, diluents and excipients. The skilled formulation scientist is able to modify the formulation within the scope of the present specification to provide a variety of formulations for the specific route of administration without destabilizing the compositions of the present invention or compromising their therapeutic activity.

Modifications thereof to increase the solubility of the compounds of the invention in water or other excipients, such as can be readily achieved by minor modifications (salt formation, esterification, etc.), are within the ordinary skill in the art. It is also within the ordinary skill in the art to vary the route of administration and dosage regimen of a particular compound in order to maximize the beneficial effect of the pharmacokinetics of the compounds of the present invention in a patient.

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

The pharmaceutical preparation is preferably in unit dosage form. In this form, the preparation is subdivided into unit doses containing appropriate quantities of the active ingredient. The unit dosage form may be a packaged preparation, the package containing discrete quantities of preparation, such as tablets, capsules, and powders in vials or ampoules. In addition, 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 treatment methods

The compounds of formula I inhibit tyrosine kinases (Btk). Activation of Btk with upstream kinases results in phosphatase-C γ activation, thereby stimulating the release of proinflammatory mediators. The compounds of formula I incorporating the 1-oxo-1H-phthalazin-2-yl side chain show surprisingly enhanced inhibitory activity compared to analogues with other side chains. Notably, the fluorine substitution on the unsaturated side chain produced a surprising about 5-10-fold increase in potency in human whole blood. Hydroxymethyl substitution on the phenyl ring further provides a surprising increase in potency over analogues bearing an optional substitution at that position. The compounds of formula I are useful in the treatment of arthritis and other anti-inflammatory and autoimmune diseases. The compounds of formula I are thus useful for the treatment of arthritis. The compounds of formula I are useful for inhibiting Btk in cells and modulating B-cell development. The invention also encompasses pharmaceutical compositions comprising a compound of formula I in combination with a pharmaceutically acceptable carrier, excipient or diluent.

The main carboxamide derivatives described herein are kinase inhibitors, in particular Btk inhibitors. These inhibitors may be used to treat one or more diseases in a mammal that respond to kinase inhibition, including diseases that respond to inhibition of Btk and/or inhibition of B-cell proliferation. Without wishing to be bound by any particular theory, it is believed that the interaction of the compounds of the present invention with Btk results in the inhibition of Btk activity and thus the pharmaceutical use of these compounds. Accordingly, the invention includes a method for treating a mammal, e.g., a human, having a disease that responds to inhibition of Btk activity and/or inhibition of B-cell proliferation, the method comprising: administering to a mammal having such a disease an effective amount of at least one chemical entity provided herein. Effective concentrations can be determined experimentally, for example, by measuring the blood concentration of the compound, or theoretically, by calculating bioavailability. In addition to Btk, other kinases that may also be affected include, but are not limited to, other tyrosine kinases and serine/threonine kinases.

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

One embodiment includes a method of treating a patient having an autoimmune and/or inflammatory disease or an acute inflammatory response in response to inhibition of Btk activity and/or B cell proliferation.

Autoimmune and/or inflammatory diseases that may be affected using the compounds and compositions according to the invention include, but are not limited to: psoriasis, allergies, 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 vasculitis), autoimmune hemolytic and thrombocytopenic symptoms, goodpasture's syndrome (and associated glomerulonephritis and pulmonary hemorrhage), atherosclerosis, rheumatoid arthritis, chronic Idiopathic Thrombocytopenic Purpura (ITP), addison's disease, parkinson's disease, alzheimer's disease, diabetes, septic shock and myasthenia gravis.

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

Examples of NSAIDs include, but are not limited to, ibuprofen, flurbiprofen, naproxen and naproxen sodium, diclofenac, a combination of diclofenac sodium and misoprostol, sulindac, oxaprozin, diflunisal, piroxicam, indomethacin, etodolac, fenoprofen calcium, ketoprofen, nabumetone sodium, 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, but are not limited to, acetylsalicylic acid or aspirin, sodium salicylate, and choline salicylate and magnesium salicylate.

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

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

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

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

Still other embodiments of the present invention relate to combinations wherein at least one of the active agents is an immunosuppressant compound such as an immunosuppressant compound selected from the group consisting of methotrexate, leflunomide, cyclosporin, tacrolimus, azathioprine and mycophenolate mofetil.

BTK-expressing B-cells and B-cell precursors have been implicated in the pathology of B-cell malignancies, including but not limited to B-cell lymphomas, lymphomas (including hodgkin and non-hodgkin lymphomas), hairy cell lymphomas, multiple myeloma, chronic and acute myeloid leukemias, and chronic and acute lymphocytic leukemias.

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

It has also been found that BTK inhibitors can be used as chemosensitizers and thus in combination with other chemotherapeutic agents, in particular agents that induce apoptosis. Examples of other chemotherapeutic agents that may be used in combination with the chemosensitizing BTK inhibitor include topoisomerase I inhibitors (camptothecin or topotecan), topoisomerase II inhibitors (such as daunomycin and etoposide), alkylating agents (such as cyclophosphamide, melphalan and BCNU), tubulin-directed agents (such as taxol and vinblastine), and biologies (e.g., antibodies such as anti-CD 20 antibodies, IDEC8, immunotoxins and cytokines).

Btk activity is also associated with some leukemias that express a bcr-abl fusion gene resulting from translocation of part of chromosomes 9 and 22. This abnormality is commonly observed in chronic myelogenous leukemia. Btk is essentially phosphorylated by bcr-abl kinase, which triggers a downstream survival signal that prevents apoptosis in bcr-abl cells (N.Feldhahn et al J.exp.Med.2005201 (11): 1837-1852).

Method of treatment

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

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

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

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

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

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

In one variation of the above method, the Btk inhibitor compound exhibits an IC of 100 nanomolar or less in an in vitro biochemical assay of Btk activity₅₀。

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

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

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

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

Examples

Abbreviations

Abbreviations commonly used include: acetyl (Ac), Azobisisobutyronitrile (AIBN), atmospheric pressure (Atm), 9-borabicyclo [3.3.1 ]]Nonane (9-BBN or BBN), 2,2 '-bis (diphenylphosphino) -1, 1' -Binaphthyl (BINAP), tert-butyloxycarbonyl (Boc), di-tert-butyl pyrocarbonate (di-tert-butyl pyrocarbonate) or Boc anhydride (BOC)₂O), benzyl (Bn), butyl (Bu), chemical abstracts accession number (CASRN), benzyloxycarbonyl (CBZ or Z), Carbonyldiimidazole (CDI), 1, 4-diazabicyclo [2.2.2]Octane (DABCO), diethylaminosulfur trifluoride (DAST), dibenzylideneacetone (dba), 1, 5-diazabicyclo [4.3.0]Non-5-ene (DBN), 1, 8-diazabicyclo [5.4.0]Undec-7-ene (DBU), N, N ' -Dicyclohexylcarbodiimide (DCC), 1, 2-Dichloroethane (DCE), Dichloromethane (DCM), 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone, diethyl azodicarboxylate (DEAD), diisopropyl azodicarboxylate (DIAD), diisobutylaluminum hydride (DIBAL or DIBAL-H), Diisopropylethylamine (DIPEA), N, N-Dimethylacetamide (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 (ea, EtOAc), ethanol (EtOH), ethyl 2-ethoxy-2H-quinoline-1-carboxylate (EEDQ), diethyl ether (Et)₂O), ethyl isopropyl ether (EtOiPr), O- (7-azabenzotriazol-1-yl) -N, N, N 'N' -tetramethylureaHexafluorophosphate acetic acid (HATU), acetic acid (HOAc), 1-N-hydroxybenzotriazole (HOBt), High Pressure Liquid Chromatography (HPLC), Isopropanol (IPA), isopropylmagnesium chloride (iPrMgCl), lithium hexamethyldisilazide (LiHMDS), Hexamethyldisilylamine (HMDS), Liquid Chromatography Mass Spectrometry (LCMS), hexamethyllithium disilylamine (LiHMDS), m-chloroperoxybenzoic acid (m-CPBA), methanol (MeOH), melting point (mp), MeSO₂- (methylsulfonyl or Ms), methyl (Me), acetonitrile (MeCN), m-chloroperbenzoic acid (MCPBA), mass spectrometry (Ms), methyl tert-butyl ether (MTBE), methyltetrahydrofuran (MeTHF), N-bromosuccinimide (NBS), N-butyllithium (nBuLi), N-carboxyanhydride (NCA), N-chlorosuccinimide (NCS), N-methylmorpholine (NMM), N-methyl-pyrrolidone (NMP), pyridinium chlorochromate (PcM)(PCC), dichloro- ((bis-diphenylphosphino) ferrocenyl) palladium (II) (Pd (dppf) Cl₂) Palladium (II) acetate (Pd (OAc)₂) Tris (dibenzylideneacetone) dipalladium (0) (Pd)₂(dba)₃) Pyridine dichromate(PDC), phenyl (Ph), propyl (Pr), isopropyl (i-Pr), pounds per square inch (psi), pyridine (pyr), 1,2, 3,4, 5-pentaphenyl-1' - (di-t-butylphosphino) ferrocene (Q-Phos), room temperature (RT or RT), sec-butyllithium (sBuLi), t-butyldimethylsilyl, or t-BuMe₂Si (TBDMS), tetra-n-butylammonium fluoride (TBAF), triethylamine (TEA or Et)₃N), 2,2, 6, 6-tetramethylpiperidine 1-oxyl (TEMPO), trimethylsilylethoxymethyl (SEM), trifluoromethanesulfonate or CF₃SO₂- (Tf), trifluoroacetic acid (TFA), 1,1 ' -bis-2, 2,6, 6-tetramethylheptane-2, 6-dione (TMHD), O-benzotriazol-1-yl-N, N, N ', N ' -tetramethylureaTetrafluoroboric acidSalt (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-carboxy anhydride (UNCA). When used for alkyl moieties, conventional Nomenclature including the prefixes positive (n), iso (i-), secondary (sec-), tertiary (tert-), and neo have their customary meaning (J.Rigaudy and D.P.Klesney, Nomenclature in Organic Chemistry, IUPAC1979Pergamon Press, Oxford.).

General conditions

The compounds of the present invention can be prepared by using general synthetic techniques and methods well known to those skilled in the art, starting from commercially available starting materials. The reaction schemes suitable for preparing this compound are outlined below. Further exemplary may be found in the specific embodiments.

Preparation examples

4-bromo-1-methyl-1H-imidazole-2-carboxamide (intermediate-1)

To a solution of 1-methyl-1H-imidazole (2g, 24.4mmol) in 16mL of EDCM was added dropwise 4.4g of trichloroacetyl chloride and the resulting mixture was stirred at 25 ℃ for 6H. The mixture was cooled to 0 ℃ and 3.4mL triethylamine was added dropwise with stirring. The solvent was evaporated and the residue was purified by flash column chromatography (petroleum ether/DCM = 4/1) to give 2,2, 2-trichloro-1- (1-methyl-1H-imidazol-2-yl) ethanone (3.1g, 56% yield).¹H NMR(300MHz，CDCl₃)7.35(s，1H)，7.16(s，1H)，4.06(s，3H)。

To a solution of 2,2, 2-trichloro-1- (1-methyl-1H-imidazol-2-yl) ethanone (1.5g, 6.6mmol) in 26mL anhydrous THF at-10 ℃ was added 2.35g NBS. The resulting mixture was stirred at-10 ℃ for 2h, then at 25 ℃ for 16 h. The solution was evaporated and the residue was purified by silica gel column (DC)M is eluent) to give 1- (-4-bromo-1-methyl-1H-imidazol-2-yl) -2,2, 2-trichloroethanone (1.18g, yield 58%).¹H NMR(300MHz，CDCl₃)7.15(s，1H)，4.05(s，3H)。

To a solution of 1.18g of 1- (-4-bromo-1-methyl-1H-imidazol-2-yl) -2,2, 2-trichloroethanone in 10mL of methanol was added 42mg of sodium methoxide, and the resulting mixture was stirred at 25 ℃ for 20 min. TLC showed the reaction was complete. The solution was evaporated, redissolved in 30mL of DCM, and washed with water (15mL of X2) and brine (15 mL). The solution was dried over anhydrous sodium sulfate and filtered. The residue was purified by silica gel column (DCM/MeOH = 60/1) to give 4-bromo-1-methyl-1H-imidazole-2-carboxylic acid methyl ester (0.575g, yield 68%).¹H NMR(300MHz，CDCl₃)7.02(s, 1H), 4.00(s, 3H), 3.93(s, 3H). LC-MS calculated value C₆H₇BrN₂O₂(M/e) 217.97, found 219 and 221[ M +1 ]]⁺.

To a saturated solution of ammonia in MeOH was added 165mg of 4-bromo-1-methyl-1H-imidazole-2-carboxylic acid methyl ester and the resulting mixture was stirred at 20 ℃ for 16H. The solution was evaporated to give 4-bromo-1-methyl-1H-imidazole-2-carboxamide (154mg, yield 100%). LC-MS calculated value C₅H₆BrN₃O (M/e) 202.97, measured values 204 and 206[ M +1 ]]⁺。

2-bromothiazole-4-carboxamide (intermediate-2)

Methyl 2-bromothiazole-4-carboxylate (50mg) was dissolved in a saturated ammonia solution in methanol (4 ml). The reaction solution was stirred at room temperature overnight. The solvent was removed under reduced pressure to give 2-bromothiazole-4-carboxamide (46mg, yield 100%) as a yellow solid.¹H NMR(300MHz，CDCl₃): 8.11(s, 1H). LC-MS calculated value C₄H₃BrN₂OS (M/e) 205.91, found 207 and 209[ M +1 ]]⁺。

4-bromo-1-methyl-1H-pyrrole-2-carboxamide (intermediate-3)

4-bromo-1-methyl-1H-pyrrole-2-carboxylic acid (122mg, 0.60mmol), HATU (274mg, 0.72mmol) and DIEA (194mg, 1.50mmol) were dissolved in DMF (5 mL). The reaction mixture was stirred at room temperature overnight by continuous bubbling with ammonia gas. The reaction mixture was diluted with 100mL of DCM and washed with water (50 mL). The aqueous layer was extracted with DCM (20 mL. times.2). All organic layers were combined, dried over sodium sulfate, filtered, and concentrated. The residue was purified by silica gel chromatography (petroleum ether/ethyl acetate 6/1) to give 4-bromo-1-methyl-1H-pyrrole-2-carboxamide (56mg, yield 46%).¹H NMR(300MHz，CDCl₃): 6.74(d, J =1.8Hz, 1H), 6.59(d, J =1.8Hz, 1H), 3.92(s, 3H). LC-MS calculated value C₆H₇BrN₂O (M/e) 201.97, measured values 203 and 205[ M +1 ]]⁺。

Acetic acid 2- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -6- (4, 4,5, 5-tetramethyl- [1, 3,2] dioxaborolan-2-yl) -benzyl ester (intermediate-4)

To a solution of 2, 6-dibromotoluene (55.5g, 222.1mmol) and NBS (43.5g, 244.3mmol) in carbon tetrachloride (300mL) was added benzoyl peroxide (5.37g, 22.2 mmol). The reaction mixture was refluxed for 20h, cooled to room temperature, washed with water (300 mL. times.3) and brine (300mL), and dried over anhydrous sodium sulfate. The solution was filtered and evaporated to give 1, 3-dibromo-2-bromomethylbenzene as a yellow powder (71 g). The material was used in the next step without further purification.

To a solution of 1, 3-dibromo-2-bromomethylbenzene (71.3g) in DMF (300mL) was added sodium acetate (91.7g), and the resulting mixture was stirred at 105 ℃ for 6 h. Cooling the mixture withWater (1500mL) was quenched and extracted with ethyl acetate (1000 mL. times.2). The combined organic layers were washed with water (1000mL) and brine (1000mL), dried over anhydrous sodium sulfate, and filtered. The residue was purified by column on silica gel (200-300 mesh, eluting with petroleum ether/ethyl acetate 30/1) to give 2, 6-dibromobenzyl acetate as a yellow oil (59.42g, 89% yield).¹HNMR(300MHz，d6-DMSO)7.57(d，J＝8.0Hz，2H)，7.07(t，J=7.8Hz，1H)，5.41(s，2H)，2.11(s，3H)。

In N₂In the atmosphere, 2, 6-dibromobenzyl acetate (34.88g, 113mmol), 6-tert-butyl-2H-phthalazin-1-one (4.56g, 22.5mmol), Cs₂CO₃(14.72g, 45mmol), CuI (6.44g, 33.8 mmol) and N, N-dimethylethane-1, 2-diamine (2g, 22.7mmol) were dissolved in DMF (92 mL). The reaction mixture was stirred at 150 ℃ for 4h, then cooled to room temperature, diluted with ethyl acetate (500mL) and washed with 500mL of water. The aqueous layer was separated and extracted with ethyl acetate (200 mL. times.2). The combined organic layers were washed with water (300 mL. times.2) and brine (300mL) and dried over anhydrous sodium sulfate. The solution was filtered and evaporated. The residue was purified by silica gel chromatography (petroleum ether/ethyl acetate 4/1) to give 2-bromo-6- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -benzyl acetate (6.2g, 64% yield).¹H NMR(300MHz，CDCl₃)8.39(d, J =8.3Hz, 1H), 8.25(s, 1H), 7.91-7.87(m, 1H), 7.72(s, 2H), 7.41-7.35(m, 2H), 5.17(s, 2H), 1.91(s, 3H), 1.44(s, 9H). LC-MS calculated value C₂₁H₂₁BrN₂O₃(M/e) 428.07, found 429 and 431[ M +1 ]]⁺。

In N₂Acetic acid 2-bromo-6- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -benzyl ester (1.43g, 10.7mmol), bis (pinacolato) diboron (1.03g, 4.05mmol), KOAc (0.99g, 10.1mmol) and Pd (dppf) Cl₂(0.3g, 0.36mmol) in 16.7mL DMSO was stirred at 80 ℃ for 16 h. The mixture was cooled to room temperature, diluted with ethyl acetate (100mL) and washed with 100mL of water. The aqueous layer was extracted with ethyl acetate (50 mL. times.2). The combined organic layers were washed with water (100 mL. times.2) and brine (100mL) and dried over anhydrous sodium sulfate. The solution was filtered and evaporated. By chromatography on silica gelThe residue was purified by the procedure (petroleum ether/ethyl acetate 2/1) to give acetic acid 2- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -6- (4, 4,5, 5-tetramethyl- [1, 3,2]Dioxolanyl-2-yl) -benzyl ester as a pale green solid (1.45g, 91% yield).¹H NMR(300MHz，CDCl₃)8.39(d, J =8.5Hz, 1H), 8.23(s, 1H), 7.96(dd, J =6.4, 2.5Hz, 1H), 7.86(dd, J =8.5, 1.8Hz, 1H), 7.71(d, J =1.7Hz, 2H), 7.52-7.45(m, 2H), 5.30(s, 2H), 1.86(s, 3H), 1.43(s, 9H), 1.33(s, 12H). LC-MS calculated value C₂₇H₃₃BN₂O₅(M/e) 476.25, found 477[ M +1 ]]⁺。

Acetic acid 2- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -6- (4, 4,5, 5-tetramethyl- [1, 3,2] dioxaborolan-2-yl) -benzyl ester (intermediate-5)

In N₂In the atmosphere, 2, 6-dibromobenzyl acetate (24.74g, 80.3mmol), 6-tert-butyl-8-fluoro-2H-phthalazin-1-one (3.54g, 16.1mmol), Cs₂CO₃(10.46g, 32.1mmol), CuI (4.61g, 24.2mmol) and N, N-dimethylethane-1, 2-diamine (1.42g, 16.1mmol) were dissolved in DMF (70 mL). The reaction mixture was stirred at 150 ℃ for 4 h. The resulting mixture was cooled to room temperature, diluted with ethyl acetate (200mL), and then 200mL of water was added. The mixture was separated and the aqueous layer was extracted with ethyl acetate (200 mL. times.2). The combined organic layers were washed with water (200mL) and brine (200mL), and the solution was dried over anhydrous sodium sulfate, filtered and concentrated. The resulting crude product was purified by silica gel chromatography (petroleum ether/ethyl acetate 4/1) to give 2-bromo-6- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -benzyl ester (3.21g, 45%).¹H NMR(300MHz，CDCl₃): 8.17(d, J ═ 2.7Hz, 1H), 7.71(t, J =4.8Hz, 1H), 7.50-7.45(m, 2H), 7.36(s, 1H), 7.34(d, J ═ 0.9Hz, 1H), 5.17(s, 2H), 1.94(s, 3H), 1.42(s, 9H). LC-MS calculated value C₂₁H₂₀BrFN₂O₃(M/e) 446.06, found 447 and 449[ M +1 ]]⁺。

In N₂2-bromo-6- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -benzyl ester (1.51 g, 3.39mmol), diboron dipalmondate (1.03g, 4.06mmol), KOAc (0.998g, 10.7mmol) and Pd (dppf) Cl in an atmosphere₂(0.277g, 0.339mmol) was dissolved in dry DMSO (80 mL). The reaction mixture was stirred at 80 ℃ for 24 h. The reaction mixture was diluted with ethyl acetate (100mL) and washed with water (200 mL. times.2). The aqueous layer was extracted with ethyl acetate (100 mL. times.2). The combined organic layers were washed with water (200 mL. times.2) and brine (200mL) and dried over anhydrous sodium sulfate. The solution was filtered and concentrated. The residue was purified by chromatography (petroleum ether/ethyl acetate 2/1) to give acetic acid 2- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -6- (4, 4,5, 5-tetramethyl- [1, 3, 2-tetramethyl- [1, 4,5 ] methyl]Dioxolane-2-yl) -benzyl ester (1.26g, 75%).¹H NMR(300MHz，CDCl₃): 8.17(d, J =2.6, 2.4Hz, 1H), 7.95(dd, J ═ 6.9, 2.2Hz, 1H), 7.49-7.44(m, 4H), 6.31(brs, 2H), 1.90(s, 3H), 1.42(s, 9H), 1.34(s, 12H). LC-MS calculated value C₂₇H₃₂BFN₂O₅(M/e) 494.24, found 495[ M +1 ]]⁺。

6-tert-butyl-2- (3-chloro-2-hydroxymethyl-phenyl) -8-hydroxymethyl-2H-phthalazin-1-one (intermediate-6)

To a 100mL round bottom flask was added 6-tert-butyl-2- [ 3-chloro-2- (hydroxymethyl) phenyl ] -8-fluorophthalazin-1 (2H) -one (671mg, 1.86mmol, prepared according to US 2010/0222325), sodium cyanide (365mg, 7.44mmol) and DMSO (10mL) to give a light yellow suspension. The mixture was stirred at 80 ℃ for 16 hrs. Sodium cyanide (182mg, 3.72mmol) was added and the mixture was stirred at 80 ℃ for a further 60 hrs. The mixture was cooled to room temperature and extracted with ethyl acetate and water. The organic layer was washed with water and brine, dried over sodium sulfate and filtered. The solvent was evaporated and the residue purified by flash column chromatography (220g silica gel, 0% to 40% acetone in heptane) to give 7-tert-butyl-3- [ 3-chloro-2- (hydroxymethyl) phenyl ] -4-oxo-3, 4-dihydrophthalazine-5-carbonitrile as a yellow solid (390mg, 57% yield).

A250 mL round bottom flask was charged with 7-tert-butyl-3- [ 3-chloro-2- (hydroxymethyl) phenyl ] -4-oxo-3, 4-dihydrophthalazine-5-carbonitrile (390mg, 1.06mmol) and toluene (8 mL). The solution was cooled in an ice bath and a solution of DIBAH in toluene (2.33mL, 2.33mmol) was added. The mixture was stirred in an ice bath for 1hr, then diluted with dichloromethane and quenched with 1M hydrochloric acid. The mixture was stirred for 10 minutes and extracted with dichloromethane. The organic layer was washed with water and brine, dried over sodium sulfate and filtered. The solvent was evaporated and the crude material was purified by flash column chromatography (silica gel, 120g, 0% -35% ethyl acetate in heptane) to give 7-tert-butyl-3- [ 3-chloro-2- (hydroxymethyl) phenyl ] -4-oxo-3, 4-dihydrophthalazine-5-carbaldehyde (147mg, 37.4%) as a light yellow powder.

Subjecting the above 7-tert-butyl-3- [ 3-chloro-2- (hydroxymethyl) phenyl group]-4-oxo-3, 4-dihydrophthalazine-5-carbaldehyde (147mg, 0.396mmol) was combined with dichloroethane (5mL) and methanol (5mL) to give a yellow solution. Sodium borohydride (27mg, 0.714mmol) was added and the mixture was stirred at room temperature for 4 hrs. The mixture was treated with 1N hydrochloric acid and extracted with dichloromethane. The organic layer was washed with brine and dried over sodium sulfate. After evaporation of the solvent, the residue was purified by flash column chromatography (silica gel 40g, 0% -40% ethyl acetate in heptane) to give 6-tert-butyl-2- [ 3-chloro-2- (hydroxymethyl) phenyl]-8- (hydroxymethyl) phthalazin-1 (2H) -one as a light yellow solid (39mg, 26.4%). LC/MS calcd for C₂₀H₂₁ClN₂O₃(M/e) 372.12, found 371.1(M-H, ES-).

Example 1

5- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -furan-2-carboxamide

In a microwave flask, 5-bromofuran-2-carboxamide (200mg, 1.05mmol), acetic acid 2- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -6- (4, 4,5, 5-tetramethyl- [1, 3, 2-fluoro-1-oxo-1H-phthalazin-2-yl) -6]Dioxolane-2-yl-benzyl ester (intermediate-5, 1.04g, 2.11mmol), X-PHOS (50.2mg, 0.105mmol), Pd₂(dba)₃A suspension of (48.2mg, 0.052mmol) and potassium phosphate (894mg, 4.21mmol) in dioxane (7ml) and water (0.7ml) was degassed for 3-5 min. The mixture was placed in a microwave at 125 ℃ for 30 min. Water was added and the mixture was extracted 2 times with ethyl acetate. The organic layer was washed with brine, dried over sodium sulfate and concentrated to dryness. The crude material was purified by flash column chromatography (ethyl acetate in hexane, gradient to 100% ethyl acetate) to give acetic acid 2- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -6- (5-carbamoyl-furan-2-yl) -benzyl ester (78.5mg, 16%).

To a solution of the above acetate (78.5mg, 0.164mmol) in methanol (2mL) was added potassium carbonate (4.5mg, 0.033 mmol). The mixture was stirred under an atmosphere of N2 for 5 h. Dichloromethane was added and the mixture was washed with water. The aqueous phase was extracted with dichloromethane. The combined organic phases were dried over sodium sulfate and evaporated to dryness. Adding ethyl acetate, stirring the suspension for 5min, filtering to obtain solid, and drying to obtain 5- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl]Furan-2-carboxamide (42mg, 59%).¹H NMR(300MHz，DMSO-d₆) ppm1.38(s, 9H)4.33-4.53(m, 2H)4.66-4.92(m, 1H)7.05(d, J ═ 3.78Hz, 1H)7.20(d, J ═ 3.40Hz, 1H)7.29-7.47(m, 2H)7.50-7.63(m, 1H)7.75(dd, J =13.41, 1.70Hz, 1H)7.81-8.03(m, 3H)8.52(d, J ═ 2.64Hz, 1H); LC/MS calcd for C₂₄H₂₂FN₃O₄(M/e) 435.16, found 458[ M + Na [ ]]⁺。

Example 2

4- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -furan-2-carboxamide

4-bromofuran-2-carboxamide (150mg, 0.79mmol), 2- (6-tert-butyl-8-fluoro-1-oxophthalazin-2 (1H) -yl) -6- (4, 4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzyl acetate (intermediate 5, 468mg, 0.95mmol), X-PHOS (37.6mg, 0.079mmol), Pd were given argon in a microwave flask₂(dba)₃A suspension of (36.1mg, 0.040mmol) and potassium phosphate (670mg, 3.16mmol) in dioxane (5mL) and water (0.5mL) was degassed for 5 min. The mixture was placed in a microwave at 125 ℃ for 30 min. Water was added and the mixture was extracted 2 times with ethyl acetate. The organic layer was washed with brine, dried over sodium sulfate and concentrated to dryness. The crude material was purified by flash column chromatography (ethyl acetate in hexane, linear gradient to 100% EtOAc) to give 2- (6-tert-butyl-8-fluoro-1-oxophthalazin-2 (1H) -yl) -6- (5-carbamoylfuran-3-yl) benzyl acetate (273mg, 0.572mmol, 72.4% yield).

To a solution of the above acetate (262.6mg, 0.55mmol) in methanol (5mL) was added potassium carbonate (15.2mg, 0.11 mmol). Adding the mixture to N₂Stirring for 3.5h under atmosphere. Dichloromethane (50mL) was added and the mixture was washed with water, dried over sodium sulfate and concentrated to dryness. The crude material was purified by flash column chromatography (ethyl acetate in hexanes, linear gradient to 100% EtOAc) to give 4- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl]Furan-2-carboxamide (192mg, 80%).¹H NMR (300MHz, chlorine FORM-d) ppm1.36-1.62(m, 9H)4.40(br.s., 2H)7.35(dd, J =6.80, 2.64Hz, 1H)7.44-7.64(m, 5H)8.14(d, J =1.13Hz, 4H)8.29(d, J =2.64Hz, 1H); LC/MS calcd for C₂₄H₂₂FN₃O₄(M/e) 435.16, found 436[ M + H]⁺。

Example 3

4- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -1-methyl-1H-imidazole-2-carboxamide

The same procedure as described in example 1 was used, by using 4-bromo-1-methyl-1H-imidazole-2-carboxamide (intermediate-1) and acetic acid 2- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -6- (4, 4,5, 5-tetramethyl- [1, 3, 2-methyl-)]This compound is prepared from dioxaborolan-2-yl) -benzyl ester (intermediate 4). The desired compound was prepared in 2 steps (33% yield).¹H NMR(300MHz，CDCl₃)8.46(d, J =8.5Hz, 1H), 8.36(s, 1H), 7.95-7.91(m, 2H), 7.77(d, J =1.6Hz, 1H), 7.70(s, 1H), 7.54(t, J =7.9Hz, 1H), 7.36(dd, J =7.9, 1.3Hz, 1H), 4.42(brs, 2H), 4.14(s, 3H), 1.46(s, 9H). LC-MS calculated value C₂₄H₂₅N₅O₃(M/e) 431.20, found 432[ M +1 [)]⁺。

Example 4

2- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -thiazole 4-carboxamide

The same procedure as described in example 1 was used, by using 2-bromothiazole-4-carboxamide (intermediate-2) and acetic acid 2- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -6- (4, 4,5, 5-tetramethyl- [1, 3,2]Dioxolane-2-yl-benzyl ester (intermediate)4) The compound is prepared. The desired compound was prepared in 2 steps (25% yield).¹H NMR(300MHz，DMSO-d₆): 8.56(s, 1H), 8.41(s, 1H), 8.24(d, J =8.4Hz, 1H), 8.05-8.00(m, 2H), 7.82(dd, J =1.8, 7.2Hz, 1H), 7.61-7.58(m, 2H), 4.54-4.45(m, 2H), 1.41(s, 9H). LC-MS calculated value C₂₃H₂₂N₄O₃S (M/e) 434.14, found 435[ M +1 ]]⁺。

Example 5

4- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -1-methyl-1H-pyrrole-2-carboxamide

The same procedure as described in example 1 was used, by using 4-bromo-1-methyl-1H-pyrrole-2-carboxamide (intermediate-3) and acetic acid 2- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -6- (4, 4,5, 5-tetramethyl- [1, 3, 2-methyl-)]This compound is prepared from dioxaborolan-2-yl) -benzyl ester (intermediate 4). The desired compound was prepared in 2 steps (20% yield).¹H NMR(300 MHz，DMSO-d₆) 8.54(s, 1H), 8.25(d, J =8.4Hz, 1H), 8.04(d, J =1.5Hz, 1H), 8.01(dd, J =1.8, 8.4Hz, 1H), 7.48-7.45(m, 2H), 7.28-7.23(m, 2H), 7.08(d, J =1.8Hz, 1H), 4.35(s, 2H), 3.89(s, 3H), 1.41(s, 9H). LC-MS calculated value C₂₅H₂₆N₄O₃(M/e) 430.20, found 431[ M +1 ]]⁺。

Example 6

2- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -thiazole-4-carboxamide

The same procedure as described in example 1 was used, by using 2-bromothiazole-4-carboxamide (intermediate-2) and acetic acid 2- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -6- (4, 4,5, 5-tetramethyl- [1, 3, 2-methyl ] -amide]This compound was prepared as dioxolane-2-yl) -benzyl ester (intermediate 5). The desired compound was prepared in 2 steps (10% yield).¹H NMR(300MHz，DMSO-d₆)8.59(d, J =2.4Hz, 1H), 8.46(s, 1H), 7.94(d, J =1.8Hz, 1H), 7.88-7.79(m, 2H), 7.66-7.63(m, 2H), 4.61-4.52(m, 2H), 1.46(s, 9H). LC-MS calculated value C₂₃H₂₁FN₄O₃S452.13, measurement 453[ M +1 ]]⁺。

Example 7

4- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethylphenyl ] -1-methyl-1H-imidazole-2-carboxamide

The same procedure as described in example 1 was used, by using 4-bromo-1-methyl-1H-imidazole-2-carboxamide (intermediate-1) and acetic acid 2- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -6- (4, 4,5, 5-tetramethyl- [1, 3, 2-dimethyl-)]This compound was prepared as dioxolane-2-yl) -benzyl ester (intermediate 5). The desired compound was prepared in 2 steps (12% yield).¹H NMR(300MHz，DMSO-d₆)8.50(d, J =2.7Hz, 1H), 7.87-7.71(m, 5H), 7.50-7.44(m, 2H), 7.30(dd, J =1.2, 7.8Hz, 1H), 4.38-4.33(m, 2H), 4.00(s, 3H), 1.39(s, 9H). LC-MS calculated value C₂₄H₂₄FN₅O₃(M/e) 449.19, found 450[ M +1]⁺。

Example 8

4- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -1-methyl-1H-pyrrole-2-carboxamide

The same procedure as described in example 1 was used, by using 4-bromo-1-methyl-1H-pyrrole-2-carboxamide (intermediate-3) and acetic acid 2- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -6- (4, 4,5, 5-tetramethyl- [1, 3, 2-dimethyl- [1, 3,2]]This compound was prepared as dioxolane-2-yl) -benzyl ester (intermediate 5). The desired compound was prepared in 2 steps (60% yield).¹H NMR(300MHz，d₆-DMSO)8.49-8.48(m,1H), 7.86(s, 1H), 7.73(d, J =13.2Hz, 1H), 7.44-7.42(m, 3H), 7.25-7.23(m, 2H), 7.06(brs, 2H), 4.56(s, 1H), 4.36(s, 2H), 3.88(s, 3H), 1.37(s, 9H). LC-MS calculated value C₂₅H₂₅FN₄O₃(M/e) 448.19, found 449.1[ M + H [)]⁺。

Example 9

4- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -1H-pyrrole-2-carboxamide

Step 1: in N₂Acetic acid 2-bromo-6- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -benzyl ester (prepared as described in intermediate-4, 300mg, 0.699mmol), 4- (4, 4,5, 5-tetramethyl- [1, 3, 2-d) was added under an atmosphere]Dioxopentaborane-2-yl) -pyrrole-1, 2-dicarboxylic acid 1-tert-butyl 2-methyl ester (270mg, 0.769mmol), Pd (dppf) Cl₂(171mg, 0.21mmol) and K₂CO₃A mixture of (289mg, 2.19mmol) in dioxane (15mL) and water (3mL) was heated at 100 ℃ for 4 h. The mixture was concentrated to dryness.The residue was purified by column chromatography on silica gel (eluting with petroleum ether/ethyl acetate at 2/1) to give 4- [ 2-acetoxymethyl-3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -phenyl ] -4- [ 2-acetoxymethyl ] -3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl ] -phenyl]Pyrrole-1, 2-dicarboxylic acid 1-tert-butyl 2-methyl ester (160mg, yield 40%). LC-MS calculated value C₃₂H₃₅N₃O₇(M/e) 573.25, found 473[ M-Boc +1]⁺。

Step 2: to 4- [ 2-acetoxymethyl-3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -phenyl]To a solution of pyrrole-1, 2-dicarboxylic acid 1-tert-butyl ester 2-methyl ester (136mg, 0.24mmol) in 5mL dioxane and 5mL water was added NaOH (40mg) and the reaction mixture was stirred at room temperature for 20 h. The mixture was concentrated under reduced pressure. The residue was dissolved in 8mL of water and acidified to pH3-4 with 1N HCl. The mixture was extracted with ethyl acetate (5 mL. times.3). With Na₂SO₄The combined organic layers were dried and concentrated under reduced pressure to give 4- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl]-1H-pyrrole-2-carboxylic acid (99mg, yield 100%). LC-MS calculated value C₂₄H₂₃N₃O₄(M/e) 417.17, found 418[ M +1 [)]⁺。

And step 3: HATU (104mg, 0.27mmol) and triethylamine (1mL) were added to 4- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl]-1H-pyrrole-2-carboxylic acid (95mg, 0.23mmol) in dry THF (15mL) and ammonia gas was bubbled through the solution for 5H. The mixture was concentrated under reduced pressure. The residue was purified by preparative TLC (petroleum ether/ethyl acetate = 2: 1) to give 4- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl]-1H-pyrrole-2-carboxamide (25mg, yield 26%) as a white solid.¹H NMR(300MHz，CD₃OD)8.43(s, 1H), 8.28(d, J =8.5Hz, 1H), 7.98-7.92(m, 2H), 7.52-7.39(m, 2H), 7.26-7.19(m, 2H), 7.03(d, J =1.6Hz, 1H), 4.38(s, 2H), 1.38(s, 9H). LC-MS calculated value C₂₄H₂₄N₄O₃(M/e) 416.18, found 417[ M +1 ]]⁺。

Example 10

5- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -1-methyl-1H-pyrrole-2-carboxamide

Step 1: in N₂In the atmosphere, acetic acid 2- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -6- (4, 4,5, 5-tetramethyl- [1, 3,2-]Dioxopentaborane-2-yl) -benzyl ester (described in preparation of intermediate 4, 300mg, 0.63mmol), methyl 5-bromo-1-methyl-1H-pyrrole-2-carboxylate (150mg, 0.69mmol), Pd (dppf) Cl₂(150mg, 0.19mmol) and K₂CO₃A mixture of (261mg, 1.9mmol) in dioxane (15mL) and water (3mL) was heated at 100 ℃ for 3 h. The mixture was concentrated to dryness. The residue was purified by silica gel column chromatography (eluting with petroleum ether/ethyl acetate in a ratio of 8: 1 to 5: 1) to give 5- [ 2-acetoxymethyl-3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -phenyl ] -5- [ 2-acetoxymethyl-3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl)]-methyl 1-methyl-1H-pyrrole-2-carboxylate (237mg, yield 77%) as a yellow liquid. LC-MS calculated C28H29N3O5 (M/e) 487.21, found 997[2M + Na]⁺。

Step 2: reacting 5- [ 2-acetoxymethyl-3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -phenyl]-methyl 1-methyl-1H-pyrrole-2-carboxylate (237mg, 0.486mmol) in saturated NH₃The mixture in MeOH (20mL) was stirred at room temperature for 48 h. The mixture was evaporated to dryness to give 5- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl]-methyl 1-methyl-1H-pyrrole-2-carboxylate (150mg, yield 68%) as a yellow solid. LC-MS calculated value C₂₆H₂₇N₃O₄(M/e) 445.20, found 913[2M + Na]⁺。

And step 3: water (5mL) was added to 5- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl]-1-methyl-1H-pyrrole-2-carboxylic acid methyl ester (150mg, 0.337mmol) in dioxane (5 mL). The mixture was stirred for 5min, and NaOH (40mg) was added. Will be provided withThe reaction mixture was stirred at rt for 16 h. The mixture was concentrated under reduced pressure. The residue was extracted with diethyl ether (5 mL. times.3). The combined organic layers were washed with brine, Na₂SO₄Drying, and concentrating under reduced pressure to obtain 5- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl]-1-methyl-1H-pyrrole-2-carboxylic acid (113mg, yield 78%) as a yellow solid. LC-MS calculated value C₂₅H₂₅N₃O₄(M/e) 431.18, found 885[2M + 23%]⁺。

And 4, step 4: HATU (110mg, 0.288mmol) was added to 5- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl]-1-methyl-1H-pyrrole-2-carboxylic acid (113mg, 0.262mmol) in dry THF (15 mL). Ammonia gas was bubbled through the solution. The mixture was brought to NH at room temperature₃(g) Stirring for 16h under atmosphere. The mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC to give 5- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl]-1-methyl-1H-pyrrole-2-carboxamide (25mg, yield 22%) as a white solid.¹HNMR(300 MHz，CD₃OD)8.42(s, 1H), 8.27(d, J =8.5Hz, 1H), 8.02-7.90(m, 2H), 7.54-7.35(m, 2H), 7.24-7.13(m, 2H), 7.02(d, J =1.9 Hz, 1H), 4.37(s, 2H), 3.87(s, 3H), 1.37(s, 9H). LC-MS calculated value C₂₅H₂₆N₄O₃(M/e) 430.20, found 883[2M + Na]⁺。

Example 11

2- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -4-methyl-oxazole-5-carboxylic acid amide

Step 1: in N₂In the atmosphere, acetic acid 2- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -6- (4, 4,5, 5-tetramethyl- [1, 3,2-]Dioxolane-borane-2-yl) -benzyl ester (preparation described in intermediate-4, 72mg, 0.153mmol), ethyl 2-bromo-4-methyl-oxazole-5-carboxylate (34mg, 0.146mmol), Pd (dppf) Cl₂(36mg, 0.0438mmol) and K₂CO₃(61mg, 0.438mmol) in dioxane/H₂O (5: 1, 6 mL). The reaction mixture was stirred at 100 ℃ for about 1.5 h. The solvent was removed under reduced pressure and the residue was purified by silica gel chromatography (petroleum ether/ethyl acetate = 2: 1) to give 2- [ 2-acetoxymethyl-3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -phenyl ] -2- [ 2-acetoxymethyl-3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl ] -phenyl]-4-methyl-oxazole-5-carboxylic acid ethyl ester.

Step 2: mixing the above 2- [ 2-acetoxymethyl-3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -phenyl]-4-methyl-oxazole-5-carboxylic acid ethyl ester dissolved in 10ml NH₃MeOH. The reaction was stirred at room temperature for 2 days. The solvent was removed under reduced pressure and the residue was purified by silica gel chromatography (petroleum ether/ethyl acetate ═ 1: 1) to give 2- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl]-4-methyl-oxazole-5-carboxamide (47mg, yield 68%, 2 steps) as a white solid.¹HNMR(300MHz，DMSO-d₆): 8.57(s, 1H), 8.24(d, J =8.1Hz, 1H), 8.20(dd, J =2.1, 8.4Hz, 1H), 8.06-8.00(m, 2H), 7.66-7.58(m, 2H), 4.73-4.57(m, 2H), 2.46(s, 3H), 1.41(s, 9H). LC-MS calculated value C₂₄H₂₄N₄O₄(M/e) 432.18, found 433[ M + H]⁺。

Example 12

4- [3- (6-cyclopropyl-8-fluoro-1-oxo-1H-isoquinolin-2-yl) -2-hydroxymethyl-phenyl ] -1-methyl-1H-pyrrole-2-carboxamide

Step 1: 1- (4-bromo-1-methyl-1H-pyrrol-2-yl) -2,2, 2-trifluoroacetone (5.0g, 19.5mmol) is dissolved in ammonium hydroxide (72.5ml, 29.4% aqueous solution) and stirred at room temperature for 45 min. The solution was brought to pH =7 with 2 NHCl. The solid precipitate was collected by filtration. The solid was washed with water and dried at 50 ℃ under reduced pressure. The aqueous layer was extracted 2 times with dichloromethane. The organic phase was concentrated in vacuo to give a solid again. The solids from filtration and extraction were combined to give 4-bromo-1-methyl-1H-pyrrole-2-carboxamide (3.2g, 81%) as a white solid: LC/MS-ESI measurement [ M + H ]]⁺203 and 205.

Step 2: to a 25mL microtube was added bis (pinacolato) diboron (2.5g, 9.85mmol), 4-bromo-1-methyl-1H-pyrrole-2-carboxamide (1.0g, 4.93mmol) and potassium acetate (1.45g, 14.8 mmol). Bis (dibenzylideneacetone) palladium (142mg, 246. mu. mol) was added followed by X-phos (235mg, 493. mu. mol). 1, 4-dioxane (10.0mL) was added to give a black solution. The tube was sealed and the mixture was purged with argon for 10 min. The reaction mixture was heated to 65 ℃ and stirred for 18 h. The crude reaction mixture was poured into 100mLH₂O, extracted with ethyl acetate (4 × 100 mL). The organic phase was washed with brine and Na₂SO₄Drying and vacuum concentrating. The crude material was purified by flash chromatography (silica gel, 150g, 5% -50% ethyl acetate/hexanes gradient). Vacuum concentrating to obtain 1-methyl-4- (4, 4,5, 5-tetramethyl- [1, 3,2]]Dioxolane-2-yl) -1H-pyrrole-2-carboxamide (973mg, 79%) as a pale yellow foam. LC/MS-ESI measurement [ M + H ]]⁺251。

And step 3: to a 10mL microwave vial was added 1-methyl-4- (4, 4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1H-pyrrole-2-carboxamide (100mg, 400. mu. mol, Eq: 1.00), 2- (3-chloro-2- (hydroxymethyl) phenyl) -6-cyclopropyl-8-fluoroisoquinolin-1 (2H) -one (137mg, 400. mu. mol, Eq: 1.00), X-phos (19.1mg, 40.0. mu. mol, Eq: 0.1), Pd₂(dba)₃(18.3mg, 20.0. mu. mol, Eq: 0.05) and tripotassium phosphate (212mg, 1.00mmol, Eq: 2.50), followed by the addition of 1, 4-dioxane (5ml) and water (0.5ml) to give a dark brown suspension. The reaction mixture was stirred while purging with argon for 10 min. Will be provided withThe solution was heated in a microwave at 125 ℃ for 30 min. The reaction mixture was cooled to room temperature. The reaction mixture was diluted with 50mL of dichloromethane and extracted with water. The aqueous phase was concentrated in vacuo. The crude material was purified by flash chromatography (silica gel, 25% -50% [ 60: 10: 1 dichloromethane: methanol: ammonium hydroxide aqueous solution)]Dichloromethane gradient) to give 4- [3- (6-cyclopropyl-8-fluoro-1-oxo-1H-isoquinolin-2-yl) -2-hydroxymethyl-phenyl]-1-methyl-1H-pyrrole-2-carboxamide (25.9mg, 15%) as a white solid: LC/MS-ESI measurement [ M + H ]]⁺432。

Example 13

4- [3- (6-tert-butyl-3-methyl-1-oxo-3, 4-dihydro-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -1-methyl-1H-pyrrole-2-carboxamide

Step 1: in a 100mL round bottom flask, 6-tert-butyl-2- (3-chloro-2- (hydroxymethyl) phenyl) -3-methyl-3, 4-dihydrophthalazin-1 (2H) -one (500mg, 1.39mmol, Eq: 1.00), acetic anhydride (711mg, 657. mu.l, 6.97mmol, Eq: 5), and pyridine (331mg, 338. mu.l, 4.18mmol, Eq: 3) were combined with dichloromethane (10.0mL) to give a colorless solution. The reaction mixture was heated to 45 ℃ and stirred for 8 h. Cooled to room temperature and stirred for 48 h. The crude reaction mixture was concentrated in vacuo to a tan oil. The crude material was purified by flash chromatography (silica gel, 20% -30% ethyl acetate/hexanes gradient) to give 2- (6-tert-butyl-3-methyl-1-oxo-3, 4-dihydrophthalazin-2 (1H) -yl) -6-chlorobenzyl acetate (489mg, 88%):¹h NMR (300MHz, chlorine FORM-d) ppm1.37(s, 9H)2.03(s, 3H)2.62(s, 3H)4.45(s, 2H)5.33(s, 2H)7.23(d, J =1.51Hz, 1H)7.35-7.39(m, 2H)7.43-7.52(m, 2H)8.03(d, J =8.31Hz, 1H).

Step 2: in a 50mL 2-necked flask equipped with an argon balloon, condenser and thermometer, acetic acid 2- (6-tert-butyl-3-methyl-1-oxo-3,4-dihydrophthalazin-2 (1H) -yl-6-chlorobenzyl ester (489mg, 1.22mmol, eq.1.00) and bis (pinacolato) diboron (619mg, 2.44mmol, Eq: 2) combined with 1, 4-dioxane (20.0ml) to give a colorless solution. Stirring until dissolved. The mixture was evacuated. Argon was back-filled 3 times, then potassium acetate (359mg, 3.66mmol, Eq: 3) was added. Bis (dibenzylideneacetone) palladium (35.1mg, 61.0. mu. mol, Eq: 0.05) was added. X-phos (58.1mg, 122. mu. mol, Eq.: 1) was added. The mixture was evacuated and back filled with argon 3 times. The reaction mixture was heated to 65 ℃ and stirred for 18 h. The reaction mixture was cooled to room temperature and poured into 50mL of H₂O, then extracted with ethyl acetate (4 × 50 mL). The organic phases were combined and washed with brine. Decolorizing activated carbon was added to the organic layer. The solution was stirred for 5 min. The solution was filtered through celite and then concentrated in vacuo. The crude material was purified by flash chromatography (silica gel, 25% ethyl acetate/hexanes) to give 2- (6-tert-butyl-3-methyl-1-oxo-3, 4-dihydrophthalazin-2 (1H) -yl) -6- (4, 4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzyl acetate (518mg, 82%):¹h NMR (300MHz, chloroform-d) ppm1.35(d, J =9.44Hz, 21H)1.99(s, 3H)2.62(s, 3H)4.44(br.s., 2H)5.44(s, 2H)7.22(d, J =1.51Hz, 1H)7.36-7.58(m, 3H)7.87(dd, J =7.18, 1.89Hz, 1H)8.04(d, J =8.31Hz, 1H).

And step 3: in a 10ml microwave vial, 4-bromo-1-methyl-1H-pyrrole-2-carboxamide (82.5mg, 406. mu. mol, Eq: 1.00) [ prepared in example 12 step 1 ]]Acetic acid 2- (6-tert-butyl-3-methyl-1-oxo-3, 4-dihydrophthalazin-2 (1H) -yl) -6- (4, 4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzyl ester (200mg, 406. mu. mol, Eq: 1.00), X-phos (19.4mg, 40.6. mu. mol, Eq: 0.10) and tripotassium phosphate (259mg, 1.22mmol, Eq: 3.00) were combined with 1, 4-dioxane (5ml) and water (0.5ml) to give a dark brown suspension. The reaction mixture was stirred while purging with argon for 10 min. The reaction mixture was heated in a microwave at 125 ℃ for 30 min. The reaction mixture was diluted with 50mL of dichloromethane and dried (MgSO)₄). And (4) concentrating in vacuum. The crude material was purified by flash chromatography (silica gel, 20% -100% ethyl acetate/hexanes gradient) to afford acetic acid 2- (6-tert-butyl-3-methyl-1-oxo3, 4-dihydrophthalazin-2 (1H) -yl) -6- (5-carbamoyl-1-methyl-1H-pyrrol-3-yl) benzyl ester (31mg, 16%):¹HNMR (300MHz, chloroform-d) ppm1.37(s, 9H)1.98(s, 3H)2.68(s, 3H)3.99(s, 2H)5.28(s, 2H)6.72(d, J =1.89Hz, 1H)6.84-6.94(m, 1H)7.23 (d, J =1.51Hz, 1H)7.27-7.52(m, 5H)7.61(s, 1H)8.04(d, J =7.93Hz, 1H).

And 4, step 4: to acetic acid 2- (6-tert-butyl-3-methyl-1-oxo-3, 4-dihydrophthalazin-2 (1H) -yl) -6- (5-carbamoyl-1-methyl-1H-pyrrol-3-yl) benzyl ester (31mg, 63.4. mu. mol, Eq: 1.00) in tetrahydrofuran was added NaOH (1.0M (aq.) 1.0mL, 1.00mmol, Eq: 15.8). The reaction mixture was heated at 60 ℃ for 4 h. The mixture was cooled to room temperature. With saturated NaHCO₃(aqueous solution) and dichloromethane dilute the solution. The layers were separated. The aqueous layer was extracted 1 time with dichloromethane. The organic extracts were combined and extracted with MgSO₄And (5) drying. The solution was filtered. Vacuum concentrating to obtain 4- [3- (6-tert-butyl-3-methyl-1-oxo-3, 4-dihydro-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl]-1-methyl-1H-pyrrole-2-carboxamide (28mg, 99%):¹h NMR (300MHz, chlorine FORM-d) ppm1.26-1.40(m, 9H)2.56(s, 3H)3.95(s, 3H)4.53(br.s., 2H)5.23(s, 2H)6.93(d, J =1.89Hz, 1H)7.14(d, J =1.89Hz, 1H)7.24-7.38(m, 3H)7.39-7.53(m, 1H)7.64(dd, J =5.67, 3.40Hz, 1H)8.01(d, J =8.31Hz, 1H). LC/MS-ESI measurement [ M + H ]]⁺447。

Example 14

4- [3- (6-tert-butyl-8-hydroxymethyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -1-methyl-1H-pyrrole-2-carboxamide

To a 15mL tube was added 6-tert-butyl-2- [ 3-chloro-2- (hydroxymethyl) phenyl]-8- (hydroxymethyl) phthalazin-1 (2H) -one (39mg, 0.105mmol), 1-methyl-4- (4, 4,5, 5-tetramethyl-1, 3,2Dioxaborolan-2-yl) -1H-pyrrole-2-carboxamide (26.2mg, 0.105mmol, prepared from intermediate-3 and bis (pinacol) diboron under the same conditions as the boronic ester formation shown in the preparation of intermediate-4), X-Phos (4.99mg, 0.0105mmol) and potassium tris-phosphate (55.5mg, 0.262 mmol). Dioxane (5mL) and water (0.5mL) were added followed by Pd₂(dba)₃(4.79mg, 0.00523 mmol). The mixture was purged with argon and then heated to 125 ℃ for 30 minutes with stirring. Then adding Pd₂(dba)₃(6.0mg, 0.00655mmol) and the mixture stirred at 125 ℃ for a further 30 minutes. The mixture was extracted with dichloromethane and water. The organic layer was washed with brine and dried over sodium sulfate. The solvent was evaporated and the residue was purified by flash column chromatography (12g of silica gel, 0% to 10% methanol in dichloromethane). The desired fraction was further purified by preparative TLC (silica gel, dichloromethane/methanol/ammonium hydroxide 90/10/1) to give 4- [3- (6-tert-butyl-8-hydroxymethyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl]-1-methyl-1H-pyrrole-2-carboxamide as a pale yellow powder (7.6mg, 15.8% yield). LC/MS calcd for C₂₆H₂₈N₄O₄(M/e) 460.21, found 459.0(M-H, ES-). 1H-NMR (300MHz, CDCl)₃)。

Example 15

1- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -3- [4- (morpholine-4-carbonyl) -phenylamino ] -1H-pyrazole-4-carboxamide

Step 1: 3-amino-1H-pyrazole-4-carbonitrile (10g, 92.5mmol) was dissolved in 100mL DMF and the solution was stirred at 0 ℃. Sodium hydride (60% solution in mineral oil, 7.4g, 185mmol) was added in small portions. The mixture was stirred for 30min and (2- (chloromethoxy) ethyl) trimethylsilane (90% pure, 17.1g, 92.5mmol) was added. The mixture was stirred at room temperature for 1hr,then extracted with chloroform and aqueous ammonium chloride. The organic layer was washed with brine, dried over sodium sulfate, filtered and concentrated. The residue was purified by ISCO flash column chromatography (220g silica gel, 5% -40% ethyl acetate in hexane, 25 minutes). Evaporation with a slightly larger R_fThe fraction as the main component was then crystallized from a 7% ethyl acetate in hexane to give a white crystalline pure compound as 1N-trimethylsilylethoxymethyl-5-amino-4-cyanopyrazole (3.75 g). Evaporation with a slightly lower R_fThe fraction as the main component was then crystallized from a 4% ethyl acetate in hexane to give the pure crystalline compound as 1N-trimethylsilylethoxymethyl-3-amino-4-cyanopyrazole (3.50 g). The mother liquor and the fractions containing both components were combined and evaporated to give a mixture (7.4g, 67% overall yield of this step). For 1N-trimethylsilylethoxymethyl-5-amino-4-cyanopyrazole: LC/MS calcd for C₁₀H₁₈N₄OSi (M/e) 238.12, found 237.2(M-H, ES-);¹H NMR(400MHz，DMSO-d₆) ppm0.00(s, 9H), 0.82(t, J =8.0Hz, 2H), 3.53(t, J =8.0Hz, 2H), 5.24(s, 2H)6.80(s, 2H)7.58(s, 1H); for 1N-trimethylsilylethoxymethyl-3-amino-4-cyanopyrazole:¹H NMR(400MHz，DMSO-d₆)ppm0.00(s，9H)，0.83(t，J=8.0Hz，2H)，3.51(t，J=8.0Hz，2H)，5.15(s，2H)，5.65(s，2H)，8.28(s，1H)。

step 2: 5-amino-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazole-4-carbonitrile (476mg, 2.0mmol) from step 1 and (4-bromophenyl) (morpholino) methanone (539mg, 2.0mmol, prepared from 4-bromobenzoyl chloride and morpholine) were dissolved in warm toluene (10mL), cesium carbonate (976mg, 3.0mmol) was added, followed by toluene (4 mL). The mixture was degassed with argon and bis (tri-tert-butylphosphino) palladium (102mg, 0.2mmol) was added. The mixture was sealed and stirred at 120 ℃ for 5 hrs. The mixture was filtered and washed with ethyl acetate (60 mL). The organic layer was extracted with ethyl acetate and water. The organic layer was dried over sodium sulfate, filtered, and concentrated. The residue was passed through using a solution of ethyl acetate (containing 8% methanol) in hexane (5% -50%, 15 min, 5%0g silica gel) to give the desired fraction which was crystallized from diethyl ether and hexane to give a white crystalline material which was 5- [4- (morpholine-4-carbonyl) -phenylamino]-1- (2-trimethylsilyl-ethoxymethyl) -1H-pyrazole-4-carbonitrile (631mg, 74.1% yield). LC/MS calcd for C₂₁H₂₉N₅O₃Si (M/e) 427.20, found 428.0(M + H, ES +);¹H NMR(400MHz，CDCl₃)ppm0.02(s，9H)，0.95(t，J=8.0Hz，2H)，3.61(t，J=8.0Hz，2H)，3.50-3.80(m，8H)，5.44(s，2H)，6.57(br.s.，1H)，7.02(d，J=8.5Hz，2H)，7.42(d，J=8.5Hz，2H)，7.68(s，1H)。

and step 3: 5- (4- (morpholine-4-carbonyl) phenylamino) -1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazole-4-carbonitrile (880mg, 2.06mmol) was dissolved in 40mL ethanol. Hydrochloric acid (2N) was added and the mixture was stirred at room temperature overnight. After 16hrs at room temperature, LC/MS showed 60% of the desired material and 40% unreacted starting material. The mixture was heated to 65 ℃ and stirred for 2 hrs. LC/MS showed complete depletion of the starting material to form the desired product. The mixture was treated with sodium hydroxide solution (4g NaOH in 40mL water) in an ice bath. The resulting mixture was extracted 2 times with ethyl acetate (350 mL). The organic layer was washed with brine and dried over sodium sulfate. TLC showed a clear point. The solvent was evaporated and the residue triturated with ether to give 5- [4- (morpholine-4-carbonyl) -phenylamino]-1H-pyrazole-4-carbonitrile as a white solid (530mg, 86.6% yield). H-NMR was consistent with the expected structure. LC/MS is clear and consistent with the desired molecular weight. LC/MS calcd for C₁₅H₁₅N₅O₂(M/e) 297.12, found 298.0(M + H, ES +).

And 4, step 4: potassium tert-butoxide (68mg, 0.60mmol) was dissolved in 3mL of DMSO and stirred at room temperature for 5 minutes. 5- (4- (morpholine-4-carbonyl) phenylamino) -1H-pyrazole-4-carbonitrile (150mg, 0.505mmol) was added and the mixture was stirred for 5 min. 2-fluoro-6-iodobenzaldehyde (378mg, 1.51mmol) was added and the mixture was stirred at room temperature for 24 hrs. The mixture was extracted with ethyl acetate and water. The organic layer was washed with brine and dried. The solvent was evaporated. The residue is reacted with ethyl acetateThe esters are milled together. The solid was filtered to give 71mg of an off-white solid. Analysis of the white solid by TLC and LC/MS showed a clear material. The filtrate was concentrated, dissolved in dichloromethane and purified by ISCO flash column chromatography using methanol in dichloromethane (0-5%, 15 min, 24g silica gel) to give 58mg as a pale pink solid. Two isolated solids from TLC and LC/MS were shown to react with the desired 1- (2-formyl-3-iodo-phenyl) -3- [4- (morpholine-4-carbonyl) -phenylamino]-1H-pyrazole-4-carbonitrile (48.5% yield) was identical. LC/MS calcd for C₂₂H₁₈N₅O₃I (M/e) 527.05, found 526.0(M-H, ES-);¹H NMR(400MHz，CDCl₃)ppm3.52-3.85(m，8H)，6.60(br.s，1H)，7.33(t，J=8.0Hz，1H)，7.38-7.49(m，4H)，7.52(d，J＝8.1Hz，1H)，8.04(s，1H)，8.08(d，J=8.0Hz，1H)，9.93(br.s，1H)。

and 5: 1- (2-formyl-3-iodophenyl) -3- (4- (morpholine-4-carbonyl) phenylamino) -1H-pyrazole-4-carbonitrile (71mg, 0.135mmol), 6-tert-butylphthalazin-1 (2H) -one (27.2mg, 0.135mmol), cuprous iodide (25.6mg, 0.135mmol) and sodium bicarbonate (22.6mg, 0.269mmol) were combined in 2mL of DMSO. The mixture was thoroughly degassed with argon and then stirred in an oil bath preheated to 100 ℃. After 1hr, the mixture was extracted with dichloromethane and water. The organic layer was dried over sodium sulfate and filtered. The solvent was evaporated and the residue was purified by flash column chromatography (12g silica gel, 0% -5% methanol in dichloromethane) to give 1- (3- (6-tert-butyl-1-oxophthalazin-2 (1H) -yl) -2-formylphenyl) -3- [4- (morpholine-4-carbonyl) phenylamino]-1H-pyrazole-4-carbonitrile as a pale pink solid (34mg, 42% yield). LC/MS calcd for C₃₄H₃₁N₇O₄(M/e) 601.24, found 602.1(M + H, ES +);¹H NMR(400MHz，CDCl₃)ppm1.45(s，9H)，3.68(br.m.，8H)，6.68(br.s.，1H)，7.39(d，J=8.6Hz，2H)，7.46(d，J=8.6Hz，2H)，7.57(dd，J=8.0，1.1Hz，1H)，7.65-7.74(m，1H)，7.74-7.81(m，2H)，7.93(dd，J＝8.6，1.8Hz，1H)，8.17(s，1H)，8.40(d，J=8.6Hz，1H)，8.47(br.s.，1H)，9.90(br.s.，1H)。

step 6: 1- (3- (6-tert-butyl-1-oxophthalazin-2 (1H) -yl) -2-formylphenyl) -3- [4- (morpholine-4-carbonyl) phenylamino]Pyrazole-4-carbonitrile (53mg, 0.0875mmol) was dissolved in 6mL of dichloromethane and 2mL of methanol. A solution of sodium borohydride (8.28mg, 0.219mmol) in water (0.5mL) and methanol (1.0mL) was added dropwise. The mixture was stirred at room temperature for 1 hr. LC/MS showed a clear desired product. The mixture was evaporated to dryness and extracted with dichloromethane and water. The organic layer was dried over sodium sulfate and evaporated to give pure desired 1- [3- (6-tert-butyl-1-oxophthalazin-2 (1H) -yl) -2- (hydroxymethyl) phenyl]-3- [4- (morpholine-4-carbonyl) phenylamino]-1H-pyrazole-4-carbonitrile (52.8mg, 100% yield). LC/MS calcd for C₃₄H₃₃N₇O₄(M/e) 603.26, found 604.1(M + H, ES +);¹HNMR(400MHz，CDCl₃)ppm1.47(s，9H)，3.61-3.77(m，8H)，4.35(br.s.，2H)，6.59(br.s.，1H)，7.43(d，J＝8.6Hz，2H)，7.50(d，J＝8.1Hz，1H)，7.58(d，J=8.6Hz，2H)，7.67(t，J=8.1Hz，1H)，7.80(d，J=1.7Hz，1H)，7.83(dd，J＝8.1，1.3Hz，1H)，7.97(dd，J=8.6，1.7Hz，1H)，8.39(d，J=0.5Hz，1H)，8.48(d，J=8.6Hz，1H)，8.79(s，1H)。

and 7: 1- [3- (6-tert-butyl-1-oxophthalazin-2 (1H) -yl) -2- (hydroxymethyl) phenyl]-3- [4- (morpholine-4-carbonyl) phenylamino]-1H-pyrazole-4-carbonitrile (52mg, 0.0861mmol) was dissolved in 4mL THF and 0.4mL water. Dihydrotris (dimethylphosphinous acid) hydrogen platinate (CAS #173416-05-2, 3mg, 8% eq) was then added. The mixture was refluxed for 1 hr. TLC showed complete consumption of starting material. LC/MS showed clear product formation with correct MW. The mixture was evaporated to dryness, then dissolved in dichloromethane, dried over sodium sulfate and filtered. After evaporation of the solvent, the residue was purified by flash column chromatography (24g silica gel, methanol in dichloromethane, 0% -5%, 16 min) to give 1- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl]-3- [4- (morpholine-4-carbonyl) -phenylamino]-1H-pyrazole-4-carboxamide as a white solid (41mg, 76.6% yield). LC/MS calcd for C₃₄H₃₅N₇O₅(M/e) 621.27, found 622.1(M + H, ES +);¹H NMR(400MHz，DMSO-d6)ppm1.41(s，9H)3.41-3.71(m，8H)，4.37(br.s.，2H)，4.74(br.s.，1H)，7.36(d，J=6.3Hz，3H)，7.49-7.71(m，5H)，7.80(br.s.，1H)，8.04(d，J＝9.1Hz，2H)，8.25(d，J＝6.6Hz，1H)，8.56(br.s.，2H)，9.44(br.s.，1H)。

example 16

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -3- [4- (morpholine-4-carbonyl) -phenylamino ] -1H-pyrazole-4-carboxamide

This compound was prepared using the same method as shown in example 15 (steps 5,6 and 7 of example 15) by using 6-tert-butyl-8-fluoro-2H-phthalazin-1-one (prepared according to US 2010/0222325).

1- (2-formyl-3-iodophenyl) -3- (4- (morpholine-4-carbonyl) phenylamino) -1H-pyrazole-4-carbonitrile (115mg, 0.218mmol), 6-tert-butyl-8-fluorophthalazin-1 (2H) -one (48mg, 0.218mmol), cuprous iodide (41.5mg, 0.218mmol) and sodium bicarbonate (36.6mg, 0.436mmol) were combined in 2mL of DMMSO. The mixture was thoroughly degassed with argon and stirred in an oil bath preheated to 100 ℃ for 1 hr. The mixture was extracted with dichloromethane and water. The organic layer was washed with brine, dried over sodium sulfate and filtered. The solvent was evaporated and the residue was purified by flash column chromatography using methanol in dichloromethane (0% to 5%, 16 min, 24g silica gel) to give 1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazine_-2-yl) -2-formyl-phenyl]-3- [4- (morpholine-4-carbonyl) -phenylamino]-1H-pyrazole-4-carbonitrile as a pale pink solid (54mg, 40% yield). LC/MS calcd for C₃₄H₃₀FN₇O₄(M/e) 619.23, found 620.0(M + H, ES +);¹H NMR(400MHz，CDCl₃)ppm1.43(s，9 H)，3.56-3.76(m，8H)，6.61br.s.，1H)，7.40(d，J＝8.6Hz，2H)，7.46(d，J＝8.6Hz，2H)，7.52(dd，J=12.4，1.8Hz，1H)，7.55-7.59(m，2H)，7.64(d，J=7.8Hz，1H)，7.78(t，J＝8.1Hz，1H)，8.16(s，1H)，8.32(s，1H)，9.97(s，1H)。

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-formyl-phenyl]-3- [4- (morpholine-4-carbonyl) -phenylamino]-1H-pyrazole-4-carbonitrile (52mg, 0.0839mmol) was dissolved in 6mL of dichloromethane and 2mL of methanol. A solution of sodium borohydride (9.52mg, 0.252mmol) in water (0.5mL) and methanol (1.0mL) was added dropwise. The mixture was stirred at room temperature for 1 hr. LC/MS showed a clear desired product. The mixture was evaporated to dryness and extracted with dichloromethane and water. The organic layer was dried over sodium sulfate and evaporated to give pure desired 1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl]-3- [4- (morpholine-4-carbonyl) -phenylamino]-1H-pyrazole-4-carbonitrile (51mg, 98% yield). LC/MS calcd for C₃₄H₃₂FN₇O₄(M/e) 621.25, found 622.1(M + H, ES +);¹HNMR(400MHz，CDCl₃)ppm1.45(s，9H)，3.59-3.77(m，8H)，4.37(br.s.，2H)，6.59(br.s.，1H)，7.43(d，JJ＝8.6Hz，2H)，7.48(dd，J＝7.8，1.3Hz，1H)，7.54-7.61(m，4H)，7.66(t，J＝8.1Hz，1H)，7.81(dd，J＝8.1，1.3Hz，1H)，8.32(d，J=2.5Hz，1H)，8.74(s，1H)。

1- (3- (6-tert-butyl-8-fluoro-1-oxophthalazin-2 (1H) -yl) -2- (hydroxymethyl) phenyl) -3- (4- (morpholine-4-carbonyl) phenylamino) -1H-pyrazole-4-carbonitrile (46mg, 0.074mmol) was dissolved in 4mL THF and 0.5mL water. Dihydrotris (dimethylphosphinic acid) hydrogen platinate (3mg, 0.0069mmol) was then added. The mixture was stirred under reflux. After 1hr, TLC showed complete consumption of starting material. LC/MS showed the formation of a clear desired product. The mixture was evaporated, the residue dissolved in dichloromethane, dried over sodium sulfate, filtered through a micron filter and evaporated. The residue was purified by flash column chromatography using methanol in dichloromethane (1.5% -5% methanol, 10min, 12g silica gel) to give 1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl]-3- [4- (morpholine-4-carbonyl) -phenylamino]-1H-pyrazole-4-carboxamide, is whiteA colored solid (36mg, 76.1% yield). LC/MS calcd for C₃₄H₃₄FN₇O₅(M/e) 639.26, found 640.1(M + H, ES +);¹H NMR(400MHz，DMSO-d6)ppm1.38(s，9H)，3.50(br.，4H)，3.59(br.，4H)，4.38(br.s.，2H)，4.78(t，J=5.2Hz，1H)，7.36(d，J＝8.6Hz，3H)，7.52-7.69(m，5H)， 7.76(d，J=13.1Hz，2H)，7.88(s，1H)，8.54(m，2H)，9.44(s，1H)。

example 17

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -phenyl ] -3- [4- (morpholine-4-carbonyl) -phenylamino ] -1H-pyrazole-4-carboxamide

Step 1: potassium tert-butoxide (120mg, 1.07mmol) was dissolved in 5mL of DMSO and stirred at rt for 5 min. 5- (4- (morpholine-4-carbonyl) phenylamino) -1H-pyrazole-4-carbonitrile (290mg, 0.975mmol) was then added and the mixture was stirred for 5 min. To the solution was added 2-bromo-6-fluorobenzaldehyde (396mg, 1.95mmol), and the mixture was stirred at room temperature overnight. The mixture was extracted with ethyl acetate and water. The organic layer was washed with water and brine, dried over sodium sulfate and evaporated. The residue was triturated with 20mL ethyl acetate and filtered to give the desired compound 70 mg. The filtrate was purified by flash column chromatography to give a second crop of the desired compound (110mg) as 1- (3-bromo-2-formyl-phenyl) -3- [4- (morpholine-4-carbonyl) -phenylamino]-1H-pyrazole-4-carbonitrile (38.4% yield). LC/MS calcd for C₂₂H₁₈BrN₅O₃(M/e) 479.06, found 477.8(M-H, ES-);¹H NMR(300MHz，DMSO-d6)ppm3.50(br.s.，4H)，3.59(d，J=3.8Hz，4H)，7.34(d，J=8.7Hz，2H)，7.53(d，J=8.7Hz，2H)，7.65(t，J＝7.9Hz，1H)，7.79(d，J=7.9Hz，1H)，7.85(d，J＝7.9Hz，1H)，9.08(s，1H)，9.47(s，1H)，10.07(s，1H)。¹NOE analysis by H-NMRThe desired regiochemistry is achieved.

Step 2: 1- (3-bromo-2-formylphenyl) -3- (4- (morpholine-4-carbonyl) phenylamino) -1H-pyrazole-4-carbonitrile (34mg, 0.0708mmol), 6-tert-butyl-8-fluorophthalazin-1 (2H) -one (31.2mg, 0.142mmol), cuprous iodide (27mg, 0.142mmol) and sodium bicarbonate (14.9mg, 0.177 mmol 1) were combined in 1mL of DMSO. The solution was degassed with argon and then heated in a microwave at 120 ℃ for 1 hr. The resulting mixture was extracted with ethyl acetate and ammonium chloride solution. The organic layer was concentrated and purified by flash column chromatography using ethyl acetate (5% methanol in hexanes) in a linear gradient of 5% to 80% for 15 minutes on 12g silica gel to afford the pure desired product. This material was triturated with diethyl ether in hexane and filtered to give 1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -phenyl]-3- [4- (morpholine-4-carbonyl) -phenylamino]-1H-pyrazole-4-carbonitrile (18.2mg, 41.5% yield) as a pale pink solid. LC/MS calcd for C₃₃H₃₀FN₇O₃(M/e) 591.24, found 592.0(M + H, ES +);¹H NMR(400MHz，DMSO-d6)ppm1.38(s，9H)，3.50(br.，4H)，3.59(br.，4H)，7.37(d，J=8.6Hz，2H)，7.59(d，J＝8.3Hz，1H)，7.65-7.71(m，3H)，7.78(d，J=13.1Hz，1H)，7.87-7.93(m，2H)，8.08(s，1H)，8.58(d，J＝2.3Hz，1H)，9.29(s，1H)，9.47(s，1H)。

and step 3: 1- (3- (6-tert-butyl-8-fluoro-1-oxophthalazin-2 (1H) -yl) phenyl) -3- (4- (morpholine-4-carbonyl) phenylamino) -1H-pyrazole-4-carbonitrile (30mg, 0.050mmol) was dissolved in 4mL THF and 0.5mL water. Dihydrotris (dimethylphosphinous acid) hydrogen platinate (2.3mg, 0.0052mmol) was then added and the mixture was stirred under reflux. After 1hr, TLC showed the starting material was consumed. LC/MS showed clear formation of the desired product. The mixture was evaporated, the residue dissolved in dichloromethane, dried over sodium sulfate, filtered through a micron filter and evaporated. The residue was purified by flash column chromatography using methanol in dichloromethane (0% to 5% methanol, 10min, 12g silica gel) to give 1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -phenyl]-3- [4- (morpholine-4-carbonyl) -phenylamino]-1H-pyrazole-4-carboxamide, white in colorSolid (21mg, 68% yield). LC/MS calcd for C₃₃H₃₂FN₇O₄(M/e) 609.25, found 608.0(M-H, ES-);¹H NMR(400MHz，DMSO-d6)ppm1.39(s，9H)，3.51(br.s.，4H)，3.59(br.s.，4H)，7.39(d，J=8.3Hz，3H)，7.56(d，J=7.8Hz，1H)，7.65-7.73(m，3H)，7.75-7.86(m，3H)，7.89(s，1H)，7.98(br.s.，1H)，8.60(d，J=2.0Hz，1H)，9.04(s，1H)，9.42(s，1H)。

example 18

1- (3- (6-tert-butyl-8-fluoro-1-oxophthalazin-2 (1H) -yl) -2- (hydroxymethyl) phenyl) -1H-indole-3-carboxamide

Step 1: to a cooled (ice bath) solution of 1H-indole-3-carbonitrile (1g, 7.03mmol) in dry dimethylformamide (10mL) was added sodium hydride (383mg, 8.44mmol, 60% in oil) in portions over about 4 minutes under a nitrogen atmosphere. The material was stirred for 5 minutes, then the ice bath was removed and the mixture was warmed to ambient temperature. 2-bromo-6-fluorobenzaldehyde (1.43g, 7.03mmol) was added as a powder 1 time. The mass was stirred vigorously for 2 hours. The mixture was placed on a rotary evaporator connected to a mechanical pump and the solvent was stripped. The remaining portion was dissolved in 5% aqueous ammonium chloride (40ml) and ethyl acetate (40ml), which was transferred to a separatory funnel. The organic phase was collected and washed with 50% dilute aqueous salt solution (40 ml). The ethyl acetate phase was collected and the aqueous phase was back-extracted with ethyl acetate (2X35 mL). The organic phases were combined, dried (magnesium sulfate), filtered and stripped. The crude product was adsorbed on silica gel (10g from dichloromethane) and purified by HPLC (dry load; silica gel; 40g) eluting with 100% dichloromethane to give the semi-pure product (990 mg). The material was purified by trituration from hot dichloromethane/hexanes to give 1- (3-bromo-2-formylphenyl) -1H-indole-3-carbonitrile as an orange solid (354mg, 16% yield).¹H NMR (300MHz, chloroform-d)ppm7.02-7.11(m，1H)7.27-7.38(m，2H)7.45(d，J＝7.93Hz，1H)7.61(t，J＝8.12Hz，1H)7.68(s，1H)7.80-7.86(m，1H)7.90(dd，J=7.93，1.13Hz，1H)10.05(s，1H)。

Step 2: to a solution of 36mg, 0.16mmol) of 6-tert-butyl-8-fluorophthalazin-1 (2H) -one (prepared according to US 2010/0222325) and 1- (3-bromo-2-formylphenyl) -1H-indole-3-carbonitrile (59mg, 0.18mmol) in dry dimethylsulfoxide (1.8mL) was added sodium bicarbonate (31mg, 0.36mmol) under an argon atmosphere. Cuprous iodide (35mg, 0.18mmol) was then added and the mixture was heated to 110 ℃ for 2 h. The reaction was cooled to ambient temperature and dissolved in water (40ml) and dichloromethane (40 ml). The material was passed through a pad of celite, rinsing well with dichloromethane. The filtrate was transferred to a separatory funnel and the dichloromethane phase was collected. Washed with 50% dilute aqueous salt solution (40 ml). The organic phase was collected and the aqueous phase was back-extracted with dichloromethane (2 × 35 ml). The dichloromethane phases were combined and dried (MgSO)₄) Filtering and steam stripping. The same reaction was then repeated on a larger scale using 1- (3-bromo-2-formylphenyl) -1H-indole-3-carbonitrile (290mg, 0.89mmol) and the above-mentioned reagents in similar amounts, under the same methods and work-up conditions. The crude products from both reactions were combined and purified by HPLC (silica gel, 100% CH)₂Cl₂-1％MeOH／CH₂Cl₂Elution) to give a semi-pure product. The material was further purified by preparative thin layer chromatography (3 plates with 0.5% MeOH/CH)₂Cl₂Elute, then with 0.75%, then 1% MeOH/CH₂Cl₂Re-elution). The product band was collected to give 1- (3- (6-tert-butyl-8-fluoro-1-oxophthalazin-2 (1H) -yl) -2-formylphenyl) -1H-indole-3-carbonitrile as a yellow foamy solid. (384mg, 77% yield). LC/MS calcd for C₂₈H₂₁FN₄O₂(M/e) 464.49, found 465.0(M + H, ES +):¹h NMR (300MHz, chloroform-d) ppm1.42(s, 9H)7.24-7.32(m, 1H)7.34-7.41(m, 2H)7.46-7.60(m, 3H)7.75(d, J =7.55Hz, 1H)7.81(s, 1H)7.83-7.87(m, 1H)7.90(t, J =7.96Hz, 1H)8.25(d, J =2.64Hz, 1H)9.56(s, 1H).

And step 3: round bottom flaskComprising 1- (3- (6-tert-butyl-8-fluoro-1-oxophthalazin-2 (1H) -yl) -2-formylphenyl) -1H-indole-3-carbonitrile (12mg, 0.03mmol) dissolved in 10% water/tetrahydrofuran (1.5 ml). Followed by addition of hydrogen (dimethyl-phosphinous acid-kp) [ hydrogen bis- (dimethyl-phosphinous acid-kp)]Platinum (II) catalyst (2mg, 0.005mmol) and the mixture was heated to reflux (oil bath). After 1 hour, the mixture was cooled to ambient temperature and the volatiles were stripped (rotary evaporator) to give the crude product. The reaction was repeated using a 61mg scale (0.24mmol) (as described above-but using 10% H2O/ethanol as solvent) and work-up was carried out as described. The combined crude material from 2 reactions was purified by preparative thin layer chromatography (3 plates with 5% MeOH/CH)₂Cl₂Eluted, then eluted with 5% MeOH/CH₂Cl₂Re-deployed). Collecting the product band to obtain 1- (3- (6-tert-butyl-8-fluoro-1-oxophthalazine)_-2(1H) -yl) -2-formylphenyl) -1H-indole-3-carboxamide as a pale yellow solid (51 mg). LC/MS calcd for C₂₈H₂₃FN₄O₃(M/e) 482.52, found 483.0(M + H, ES +):¹HNMR (300MHz, chloroform-d) ppm1.40(s, 9H)6.03(br.s, 2H)7.20-7.36(m, 3H)7.44-7.59(m, 3H)7.66(d, J =7.93Hz, 1H)7.81(t, J ═ 7.60Hz, 1H)7.91(s, 1H)8.19(m, 1H)8.23(d, J =2.64Hz, 1H)9.52(s, 1H).

And 4, step 4: to a cooled (ice bath) flask containing 1- (3- (6-tert-butyl-8-fluoro-1-oxophthalazin-2 (1H) -yl) -2-formylphenyl) -1H-indole-3-carboxamide (51mg0.11mmol) in methanol/dichloromethane (1.35ml 2.9: 1) was added a solution of sodium borohydride (20mg, 0.53mmol) in water (0.35ml) slowly dropwise. The mixture was stirred for 10 minutes and then dissolved in dichloromethane (20ml) and water (20 ml). The contents were poured into a separatory funnel and stirred. The organic phase was collected and washed with 50% dilute aqueous salt solution (20 ml). The dichloromethane layer was collected and the aqueous phase was back-extracted with dichloromethane (2X20 mL). The combined organic phases were dried over magnesium sulfate, filtered and stripped. The crude material was purified by filtration through a short silica gel column eluting with 7.5% methanol in dichloromethane. The desired fraction was collected and the material was then crystallized from a hot dichloromethane/hexane solution to give a white crystalline product, 1- (3- (6-tert-butyl-8-fluoro-1-oxophthalazin-2 (1H) -yl) -2- (hydroxymethyl) phenyl) -1H-indole-3-carboxamide (37 mg). LC/MS calcd for C₂₈H₂₅FN₄O₃(M/e) 484.54, found 485.0(M + H, ES +):¹h NMR (300MHz, chloroform-d) ppm1.44(s, 9H)4.03-4.37(m, 2H)5.49-6.00(br.s, 2H) 7.18-7.38(m, 3H)7.49-7.71(m, 5H)8.13-8.28(m, 2H)8.32(d, J =2.64Hz, 1H).

Example 19

1- (3- (6-tert-butyl-1-oxophthalazin-2 (1H) -yl) -2- (hydroxymethyl) -phenyl) -1H-indole-3-carboxamide

Step 1: 1- (3- (6-tert-butyl-1-oxophthalazin-2 (1H) -yl) -2-formylphenyl) -1H-indole-3-carbonitrile was prepared in analogy to the procedure described in example 18 by reacting 1- (3-bromo-2-formylphenyl) -1H-indole-3-carbonitrile (222mg, 40% pure, 0.27mmol, synthesized as described in step 1 of example 18 above) with 6-tert-butylphthalazin-1 (2H) -one (138mg, 0.68 mmol). Similar workup and purification gave the desired product as a pale yellowish white solid (82mg, 66% yield). LC/MS calcd for C₂₈H₂₂N₄O₂(M/e) 446.51, found 447.0(M + H, ES +):¹h NMR (300MHz, chloroform-d) ppm1.44(s, 9H)7.23-7.40(m, 3H)7.55(dd, J =7.93, 1.13Hz, 2H)7.72-7.94(m, 6H)8.32(s, 1H)8.39(d, J ═ 8.31Hz, 1H)9.59(s, 1H).

Step 2: 1- (3- (6-tert-butyl-1-oxophthalazin-2 (1H) -yl) -2-formylphenyl) -1H-indole-3-carbonitrile (82mg, 0.18mmol) was hydrolyzed by nitrile and hydrogen (dimethylphosphinic acid-kp) [ Hydrobis- (dimethylphosphinic acid-kp) was used as a catalyst in a similar manner to that described in example 18]Preparation of 1- (3- (6-tert-butyl-1-oxophthalazin-2 (1H) -yl) -2-formylphenyl) -1H-indole-3-carboxamide over platinum (II) catalyst (5mg, 0.064mmol). Similar workup and purification gave the desired product as a light yellow glassy solid (54mg, 63% yield). LC/MS calcd for C₂₈H₂₄N₄O₃(M/e) 464.5, found 465.0(M + H, ES +):¹h NMR (300MHz, chloroform-d) ppm1.44(s, 9H)7.21-7.37(m, 3H)7.48-7.59(m, 2H)7.67-7.76(m, 2H)7.85(t, J =7.93, 1.00Hz, 1H)7.90(s, 1H)8.14-8.20(m, 1H)8.30(s, 1H)8.37(d, J =8.31Hz, 1H)9.56(s, 1H).

And step 3: 1- (3- (6-tert-butyl-1-oxophthalazin-2 (1H) -yl) -2- (hydroxymethyl) -phenyl) -1H-indole-3-carboxamide was prepared by reducing 1- (3- (6-tert-butyl-1-oxophthalazin-2 (1H) -yl) -2-formylphenyl) -1H-indole-3-carbonitrile (54mg, 0.12mmol) in analogy to the procedure described in example 18, by using sodium borohydride (22mg, 0.58 mmol). Similar workup and purification gave the desired product as a white crystalline solid (36mg, 64% yield). LC/MS calcd for C₂₈H₂₆N₄O₃(M/e) 466.54, found 467.0(M + H, ES +):¹h NMR (300MHz, chloroform-d) ppm1.46(s, 9H)4.07(b r.d, J =11.70Hz, 1H)4.30(br.d, J =11.70Hz, 1H)5.60-5.97(m, 2H)7.20-7.38(m, 3H)7.52-7.71(m, 3H)7.80(d, J ═ 1.89Hz, 1H)7.96(dd, J =8.31, 1.89Hz, 1H)8.17-8.29(m, 2H)8.39(s, 1H)8.46(d, J =8.31Hz, 1H).

Example 20

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -1H-pyrrolo [2, 3-b ] pyridine-3-carboxamide

Step 1:in a 100mL pear-shaped flask, 1H-pyrrolo [2, 3-b ] is added]Pyridine-3-carbonitrile (150mg, 1.05mmol, Eq: 1.00), copper (II) acetate (381mg, 2.1mmol, Eq: 2), pyridine (166mg, 170. mu.l, 2.1mmol, Eq: 2), and 6-tert-butyl-8-fluoro-2- (1-hydroxy-1, 3-dihydro-benzo [ c ] -)][1，2]Oxetaborolan-4-yl) -2H-phthalazin-1-one (706mg, 1.15mmol, Eq: 1.1) was combined with dichloromethane (10ml) to give a torquise suspension. The reaction mixture was purged with nitrogen. The reaction mixture was heated to 80 ℃ and stirred for 16 hr. With saturated NH₄The reaction was diluted with Cl (50mL) and extracted with EtOAc (3X50 mL). The mixture was separated on ISCO with 30-50% EtOAc in hexane affording 1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl]-1H-pyrrolo [2, 3-b]Pyridine-3-carbonitrile (122mg, 25%) as an off-white foam.¹H NMR(DMSO-d₆)：8.64(dd，J=7.9，1.6Hz，1H)，8.60(d，J=2.5Hz，1H)，8.45(s，1H)，8.36(dd，J=4.8，1.8Hz，1H)，7.94(d，J=1.8Hz，1H)，7.82(dd，J=13.2，1.6Hz，1H)，7.66-7.78(m，3H)，7.63(dd，J=7.3，2.3Hz，1H)，7.36(dd，J=8.0，4.8Hz，1H)，7.18(br.s.，1H)，4.72(t，J=5.4Hz，1H)，4.11-4.34(m，2H)，1.44(s，9H)。MS m／e468.5(M+H⁺)。

Step 2: stirring 1- (3- (6-tert-butyl-8-fluoro-1-oxophthalazin-2 (1H) -yl) -2- (hydroxymethyl) phenyl) -1H-pyrrolo [2, 3-b]A solution of pyridine-3-carbonitrile (610mg, 783. mu. mol, Eq: 1.00), acetaldoxime (139mg, 143. mu.l, 2.35mmol, Eq: 3) and indium (III) chloride (8.66mg, 39.1. mu. mol, Eq: 0.05) in toluene (25.2ml) was heated to 110 ℃ and stirred for 3 h. The reaction mixture was poured into 15mL EtOAc and saturated NH₄Cl (1 × 25 mL). With MgSO₄The organic layer was dried and concentrated in vacuo. After removal of the solvent and separation of the residue by flash chromatography (silica gel, 12g, 80% -100% EtOAc in hexanes), 66mg of the desired product was obtained, with impurities. The material was repurified by preparative reverse phase HPLC with TFA. With NaHCO₃(1x25mL) neutralize the desired product. With MgSO₄The organic layer was dried, concentrated in vacuo and extracted with ethyl acetate/methanol (9: 1) to give 1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazine-2-yl) -2-hydroxymethyl-phenyl]-1H-pyrrolo [2, 3-b]Pyridine-3-carboxamide (43mgs, 12%) as a white solid.¹H NMR(DMSO-d₆)：8.64(dd，J=7.9，1.6Hz，1H)，8.60(d，J=2.5Hz，1H)，8.45(s，1H)，8.36(dd，J=4.8，1.8Hz，1H)，7.94(d，J=1.8Hz，1H)，7.82(dd，J=13.2，1.6Hz，1H)，7.66-7.78(m，3H)，7.63(dd，J=7.3，2.3Hz，1H)，7.36(dd，J=8.0，4.8Hz，1H)，7.18(br.s.，1H)，4.72(t，J=5.4Hz，1H)，4.11-4.34(m，2H)，1.44(s，9H)。MS m／e486.6(M+H⁺)。

Example 21

Step 1: in a 100mL round bottom flask, 1-methyl-4- (4, 4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1H-pyrrole-2-carboxamide (61.0mg, 244. mu. mol, Eq: 1.10), 2- (6-tert-butyl-8-fluoro-1-oxophthalazin-2 (1H) -yl) -4-iodonicotinaldehyde (100mg, 222. mu. mol, Eq: 1.00) and [1, 1' -bis (diphenylphosphino) ferrocene]Palladium (II) dichloride (16.2mg, 22.2. mu. mol, Eq: 0.1) was combined with dioxane (667. mu.l) to give a red solution. A solution of potassium carbonate (61.3mg, 443. mu. mol, Eq: 2) in water (66.7. mu.l) was added and the resulting suspension was heated to 70 ℃ and stirred for 32 h. The reaction was diluted with ethyl acetate (10mL), washed with brine, and Na₂SO₄Drying and vacuum concentrating. The crude material was purified by flash chromatography (silica gel, 12g, 10% -15% EtOAc in hexane to afford 4- [2- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -3-formyl-pyridin-4-yl]-1-methyl-1H-pyrrole-2-carboxamide (28mg, 28.2%) as an off-white solid.¹H NMR(DMSO-d₆)：10.06(s，1H)，8.72(d，J=5.3Hz，1H)，8.53(d，J=2.5Hz，1H)，7.91(d，J=1.8Hz，1H)，7.79(dd，J=13.3，1.8Hz，1H)，7.73(d，J=5.3Hz，1H)，7.69-7.76(m，1H)，7.50(d，J=1.8Hz，1H)，7.17(d，J=2.0Hz，1H)，7.12(br.s.，1H)，3.93(s，3H)，1.39(s，9H)；MS m／e448.5(M+H⁺)。

Step 2: in a 25mL pear-shaped flask, 4- (2- (6-tert-butyl-8-fluoro-1-oxophthalazin-2 (1H) -yl) -3-formylpyridin-4-yl) -1-methyl-1H-pyrrole-2-carboxamide (28mg, 62.6. mu. mol, Eq: 1.00) was reacted with CH₂Cl₂(3ml) and MeOH (1ml) were combined to give a colorless solution. Sodium borohydride (4.73mg, 125. mu. mol, Eq: 2.00) was added. The reaction mixture was stirred for 1 h. The reaction mixture was poured into EtOAc (25mL) with saturated NH₄Cl (3 × 10 mL). With MgSO₄The organic layer was dried, concentrated in vacuo and then lyophilized to give 4- [2- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -3-hydroxymethyl-pyridin-4-yl]-1-methyl-1H-pyrrole-2-carboxamide (14mg, 50%) as a white solid.¹H NMR(DMSO-d₆)ppm8.52(d，J=2.8Hz，1H)，8.46(d，J=5.0Hz，1H)，7.90(d，J=1.8Hz，1H)，7.77(dd，J=13.3，1.8Hz，1H)，7.55-7.72(m，1H)，7.52(d，J=5.0Hz，1H)，7.49(d，J=1.8Hz，1H)，7.23(d，J=2.0Hz，1H)，7.04(br.s.，1H)，4.88(br.s.，1H)，4.29-4.57(m，2H)，3.91(s，3H)，1.39(s，9H)；MS m／e450.5(M+H⁺)。

Example 22

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -3- [4- (1-hydroxy-1-methyl-ethyl) -phenylamino ] -1H-pyrazole-4-carboxamide

Step 1: 5-amino-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazole-4-carbonitrile (500mg, 2.1mmol), 2- (4-bromophenyl) propan-2-ol (519mg, 2.41mmol), and cesium carbonate (1.03g, 3.15mmol) from example 15, step 1 were dissolved in anhydrous toluene (14 ml). The mixture was degassed with argon and bis (tri-tert-butylphosphino) palladium (107mg, 0.21mmol) was added. The mixture was degassed with argonAnd then stirred at 120C for 4.5 hours under an argon atmosphere. Further, 2- (4-bromophenyl) propan-2-ol (50mg) was added to the solution, and the mixture was heated for 2 hours or more. The material was cooled to ambient temperature and stirred overnight. The crude material was filtered through a pad of celite, rinsing well with ethyl acetate (60 ml). The organic layer was shaken well with water (60ml) argon in a separatory funnel and collected. The aqueous phase was back-extracted with ethyl acetate (2 × 40 ml). The combined organic phases were dried over magnesium sulfate, filtered and stripped. The residue was purified by Analogix flash column chromatography using ethyl acetate in hexane (10% -45% gradient, 23g silica gel) to give 5- (4- (2-hydroxyprop-2-yl) phenylamino) -1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazole-4-carbonitrile as a red-brown viscous oil (482mg, 62% yield). LC/MS calcd for C₁₉H₂₈N₄O₂Si (M/e) 372.55, found 371(M-H, ES-).

Step 2: 5- (4- (2-hydroxypropan-2-yl) phenylamino) -1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazole-4-carbonitrile (480mg, 1.13mmol) was dissolved in a tetrabutylammonium fluoride solution (1M, 16.8ml), and the flask was sealed. The mixture was placed in an oil bath heated to 110 ℃ and stirred for 28 hours. The mixture was cooled to ambient temperature. Water (60ml) and ether (60ml) were added, the material was shaken in a separatory funnel, and the organic phase was collected. The aqueous phase was back-extracted with diethyl ether (2 × 50 ml). The combined organic phases were dried over magnesium sulfate, filtered and stripped. The crude residue was purified by Analogix flash column chromatography using ethyl acetate in hexane (30% -90% gradient, 40g silica gel) to give 3- (4- (2-hydroxyprop-2-yl) phenylamino) -1H-pyrazole-4-carbonitrile as an off-white powder (128mg, 47% yield). LC/MS calcd for C₁₃H₁₄N₄O (M/e) 242.28, found 241(M-H, ES-).

And step 3: 3- (4- (2-hydroxypropan-2-yl) phenylamino) -1H-pyrazole-4-carbonitrile is converted into 1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-formyl-phenyl ] -3- [4- (1-hydroxy-1-methyl-ethyl) -phenylamino ] -1H-pyrazole-4-carbonitrile by an analogous scheme to that described in example 15.

And 4, step 4: 1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-formyl-phenyl]-3- [4- (1-hydroxy-1-methyl-ethyl) -phenylamino]-1H-pyrazole-4-carbonitrile (78mg, 0.14mmol) and dihydrotris (dimethylphosphinic acid) hydrogen platinate (7.1mg, 0.12eq) were dissolved in tetrahydrofuran (3.1ml) and water (0.31 ml). The mass was stirred and heated to reflux (oil bath) for 1 hour. The mixture was cooled to ambient temperature and the volatiles were stripped (rotary evaporator). The crude material was purified by preparative thin layer chromatography (2 plates, eluting first with 7% methanol in dichloromethane and then developed again with 6% methanol in dichloromethane). Collecting the product band to obtain the desired 1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-formyl-phenyl]-3- [4- (1-hydroxy-1-methyl-ethyl) -phenylamino]-1H-pyrazole-4-carboxamide as a pale yellow powder (41 mg). LC/MS calcd for C₃₂H_31FN₆O₄(M/e) 582.62, found 581.0(M-H, ES-).

And 5: 1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-formyl-phenyl]-3- [4- (1-hydroxy-1-methyl-ethyl) -phenylamino]-1H-pyrazole-4-carboxamide (34mg, 0.06mmol) dissolved in 1: 1 in methanol and dichloromethane (15 ml). To this was added by dropwise addition a solution of sodium borohydride (11mg, 0.29mmol) dissolved in water (0.25 ml). After stirring for 10min, the volatiles were stripped (rotary evaporator) and the residue was dissolved in dichloromethane (30ml) and water (30ml) and shaken in a separatory funnel. The organic phase was collected and the aqueous phase was back-extracted with dichloromethane (2 × 25 ml). The organic extracts were combined, dried over magnesium sulfate, filtered and stripped. The crude material was purified by silica gel chromatography (1g, eluting with 7.5% methanol in dichloromethane) to give the desired product 1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl]-3- [4- (1-hydroxy-1-methyl-ethyl) -phenylamino]-1H-pyrazole-4-carboxamide as an off-white powder (32 mg). LC/MS calcd for C₃₂H₃₃FN₆O₄(M/e) 584.66, found 583(M-H, ES-).

Example 23

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -3- (5-chloro-pyridin-2-ylamino) -1H-pyrazole-4-carboxamide

Step 1: the procedure was carried out analogously to that described in WO 2005/5414A 2. Thus, 3-amino-4-cyanopyrazole (11g, 102mmol) was dissolved in diiodomethane (150ml, 1.87mol) and the flask was cooled to-10 ℃. Isoamyl nitrite (92.4ml, 688mmol) was added dropwise over 40 minutes with efficient stirring. After the addition was complete, the mixture was stirred for 5 minutes. The addition flask was then removed. The mass was heated (oil bath) at 100 ℃ for 2 hours, replacing with a high efficiency condenser. The flask was cooled to ambient temperature and the solvent was removed (rotary evaporator, final mechanical pump). The residue was dissolved in ethyl acetate (120ml) and transferred to a separatory funnel. 5% aqueous sodium metabisulphite (120ml) was added and the biphasic mixture was shaken. The organic phase was collected and shaken with a 1N hydrochloric acid solution (aqueous solution, 120ml) and then with water (120 ml). The organic phase was collected and the aqueous phase was back-extracted with ethyl acetate (2 × 100 ml). The organic extracts were combined, dried over magnesium sulfate, filtered and stripped. The crude product was adsorbed on silica gel (about 25g from dichloromethane) and purified (dry loading) by Analogix flash column chromatography using ethyl acetate in hexane (23% -50% gradient, 80g silica gel) to give a light yellow powder (20 g). The material was further purified by thermal crystallization from hot ethyl acetate/hexanes to give 3-iodo-1H-pyrazole-4-carbonitrile (11.3g) as a light brown solid, which was collected by filtration (rinsing well with 5% ethyl acetate/hexanes). A second crop of material (3.76g) was again obtained from the mother liquor. LC/MS calcd for C₄H₂IN₃(M/e) 218.98, found 218(M-H, ES-).

Step 2: 3-iodo-1H-pyrazole-4-carbonitrile (3.62g, 16.5mmol) was dissolved in dry tetrahydrofuran (67ml) under a nitrogen atmosphere. Sodium hydride (992mg, 24.8mmol, 60% solution in oil) was added 1 time and the mixture was placed inHeat (50 ℃) the ultrasonic bath for 50 minutes. To this mixture was added 2-fluoro-6-iodobenzaldehyde (5.37g, 21.5mmol) and the mixture was put in an oil bath heated to 60-65 ℃. After 2 hours, 2-fluoro-6-iodobenzaldehyde (350mg, 1.4mmol) was added and the material was stirred for more than 1 hour. The flask was cooled to ambient temperature and nearly 90% of the solvent was stripped (rotary evaporator). Diethyl ether (30ml) and water (50ml) were added and the mixture was stirred vigorously for 30 minutes. The precipitated product was collected by filtration, washed thoroughly with ether and water, and dried in a vacuum oven to give a light brown powder (4.78 g). The solid product was dissolved in 2% methanol in dichloromethane (about 60ml, heated to dissolve) and transferred to a separatory funnel. Water (60ml) was added, the material was shaken and the organic phase collected. Drying over magnesium sulfate, filtration and stripping gave the desired 1- (2-formyl-3-iodo-phenyl) -3-iodo-1H-pyrazole-4-carbonitrile as a light yellow powder (3.973 g). LC/MS calcd for C₁₁H₅I₂N₃O (M/e) 448.99, found 450(M + H, ES +).

And step 3: to an oven dried flask was added 1- (2-formyl-3-iodo-phenyl) -3-iodo-1H-pyrazole-4-carbonitrile (1.703g, 3.79mmol), 6-tert-butyl-8-fluorophthalazin-1 (2H) -one [ prepared according to US 2010/0222325](919mg, 4.17mmol) and sodium bicarbonate (637mg, 7.59mmol) were dissolved in dry dimethylsulfoxide (30 ml). The mixture was degassed with argon in an ultrasonic bath. Cuprous iodide (722mg, 3.79mmol) was added and the material was again degassed thoroughly. The mixture was heated to 60 ℃ for 2.5 hours under sonication, and kept at ambient temperature overnight. Additional cuprous iodide (360mg) was added and the mass was heated under sonication at 60C for 4 hours. The flask was cooled to ambient temperature and dichloromethane (40ml) and water (40ml) were added with vigorous stirring. After 5 minutes, the material was filtered through a pad of celite, rinsing well with 1% methanol in dichloromethane. The filtrate was transferred to a separatory funnel and the organic phase was collected. This was shaken with 50% dilute saline solution (60ml, some crumbs). The dichloromethane phase was collected and the aqueous phase was back-extracted with dichloromethane (note: some debris was observed; it was helpful to use larger volumes of organic and aqueous solutions in the back-extraction). The combined organic phases were dried over magnesium sulfate, filtered and stripped. The residue was dissolved in dichloromethane and purified by Analogix flash column chromatography eluting first with 100% dichloromethane (5 min hold) and then switched to a gradient of 1% to 3% methanol in dichloromethane (25g silica gel) to give the desired 1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-formyl-phenyl]3-iodo-1H-pyrazole-4-carbonitrile as a light brown powder (1.2 g). LC/MS calcd for C₂₃H₁₇FIN₅O₂(M/e) 541.32, found 542(M + H, ES +).

And 4, step 4: 1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-formyl-phenyl]-3-iodo-1H-pyrazole-4-carbonitrile (1.2g, 2.22mmol) was dissolved in a solution of dichloromethane (40ml) and methanol (80 ml). To this was added dropwise a solution of sodium borohydride (21mg, 0.53mmol) dissolved in water (0.35 ml). After stirring for 10min, the volatiles were stripped (rotary evaporator) and the residue was dissolved in dichloromethane (60ml) and water (50ml) and shaken in a separatory funnel. The organic phase was collected and the aqueous phase was back-extracted with dichloromethane (2 × 50 ml). The organic extracts were combined, dried over magnesium sulfate, filtered and stripped. The crude product was purified by Analogix flash column chromatography (40g column, first eluting with 100% -dichloromethane [ hold for 10 min)]And then switched to a solution of 1% methanol in dichloromethane to give the desired product 1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl]3-iodo-1H-pyrazole-4-carbonitrile as a light orange oil which solidified on standing (1.029 g). LC/MS calcd for C₂₃H_wFIN₅O₂(M/e) 543.33, found 544(M + H, ES +).

And 5: adding 1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl to a small round bottom flask]-3-iodo-1H-pyrazole-4-carbonitrile (212mg, 0.39mmol), 5-chloropyridin-2-amine (65mg, 0.51mmol), XANTPHOS (56mg, 0.098mmol) and cesium carbonate (381mg, 1.17 mmol). Dry dioxane (5.7ml) was added and the mixture was degassed thoroughly with argon. Adding Pd₂(dba)₃(46mg, 0.051mmol), the material was degassed again with argon. The flask was placed in an oil bath heated to 95 ℃ for 2.5 hours. The flask was cooled to ambient temperature and ethyl acetate (30ml) and water (30ml) were addedml). The contents were shaken in a separatory funnel and the organic phase was collected. The aqueous phase was back-extracted with ethyl acetate (2 × 30ml), the organic phases were combined, dried over magnesium sulfate, filtered and stripped. The crude material was purified by preparative thin layer chromatography (2 plates, eluting with 4% methanol in dichloromethane). Collecting the product band to obtain the desired 1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl]-3- (5-chloro-pyridin-2-ylamino) -1H-pyrazole-4-carbonitrile as a pale yellow powder (111 mg). LC/MS calcd for C₂₈H₂₃ClFN₇O₂(M/e) 543.98, found 544(M + H, ES +).

Step 6: 1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl]-3- (5-chloro-pyridin-2-ylamino) -1H-pyrazole-4-carbonitrile (111mg, 0.204mmol) and dihydrotris (dimethylphosphinic acid) hydrogen platinate salt (10.5mg, 0.025mmol) were dissolved in tetrahydrofuran (3.8ml) and water (0.38 ml). The mass was stirred and heated to reflux (oil bath) for 1 hour. The mixture was cooled to ambient temperature and the volatiles were stripped (rotary evaporator). The crude residue was purified by preparative thin layer chromatography (2 plates, eluting first with 7% methanol in dichloromethane and then developed again with 7% methanol in dichloromethane). Collecting the product band to obtain the desired 1- [3- (6-tert-butyl-8-fluoro-1-oxo-1-H-phthalazin-2-yl) -2-hydroxymethyl-phenyl]-3- (5-chloro-pyridin-2-ylamino) -1H-pyrazole-4-carboxamide as an off-white powder (70 mg). LC/MS calcd for C₂₈H₂₅ClFN₇O₃(M/e) 561.99, found 562(M + H, ES +).

Example 24

3- [5- (2-azetidin-3-yl-1, 1-dimethyl-ethoxy) -pyridin-2-ylamino ] -1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -1H-pyrazole-4-carboxamide

Step 1: preparation of 2- (6-Chloropyridin-3-yloxy) -2-methylpropionaldehyde [ according to US 2012/40949A 1 ] under a nitrogen atmosphere](28g, 140mmol, Eq: 1.00) was dissolved in dry dichloromethane (252ml) and cooled to-10 deg.C (dry ice/acetonitrile cooling bath). Acetic acid (10.4ml, 182mmol) and sodium triacetoxyborohydride (41.6g, 196mmol) were added. Azetidine (17ml, 252mmol) was next added via dropping funnel over 6 minutes with efficient stirring. After the addition was complete, the mixture was stirred for 5 minutes, then the cooling bath was removed and the material was warmed to ambient temperature. After 1 hour, saturated aqueous sodium bicarbonate (200ml) and dichloromethane (80ml) were added. Transferring the biphasic material to a separating funnel, stirring and collecting an organic phase. This was shaken with 5% sodium bicarbonate solution (200ml) and then with 50% dilute saline solution (200 ml). The organic phase was collected and the aqueous phase was back-extracted with dichloromethane (2100 ml). The combined organic phases were dried over magnesium sulfate, filtered and stripped. The crude remaining material was purified by column chromatography (silica gel, 40g) eluting with 4% methanol in dichloromethane to give the desired 5- (2-azetidin-1-yl-1, 1-dimethyl-ethoxy) -2-chloro-pyridine (29.66g) as a gold brown, flowable oil. LC/MS calcd for C₁₂H₁₇ClN₂O (M/e) 240.73, found 241(M + H, ES +).

Step 2: to a solution of degassed 5- (2-azetidin-1-yl-1, 1-dimethyl-ethoxy) -2-chloro-pyridine (21.2g, 88.1mmol) in anhydrous tetrahydrofuran (314ml) were added 2- (dicyclohexylphosphino) biphenyl (6.17g, 17.6mmol) and tris (dibenzylideneacetone) dipalladium (0) (8.06g, 8.81 mmol). A1M solution of lithium bis (trimethylsilyl) amide in THF (264ml, 264mmol) was then added over 5 minutes via an addition funnel. The reaction mixture was stirred at 75 ℃ overnight under an argon atmosphere. The reaction mixture was poured into a saturated aqueous ammonium chloride solution (400ml), which was subjected to extraction with ethyl acetate (350 ml). The organic phase was collected and washed with 50% dilute brine (350 ml). The organic phase was collected and the aqueous phase was back-extracted with ethyl acetate (2X200 ml). The combined organic phases were dried over magnesium sulfate, filtered and stripped. The residue was purified by Analogix flash column chromatography (80g column) using 0% to 12% methanol in dichloromethaneLiquid elution afforded the desired 5- (2-azetidin-1-yl-1, 1-dimethyl-ethoxy) -pyridin-2-ylamine (10.59g) as a dark brown semi-viscous oil (and less pure fractions which could be repurified under similar conditions to give 4.01g more product). LC/MS calcd for C₁₂H₁₉N₃O (M/e) 221.3, found 222(M + H, ES +).

And 3, step 3 and step 4: the 5- (2-azetidin-1-yl-1, 1-dimethyl-ethoxy) -pyridin-2-ylamine prepared in step 2 above was converted to the desired product using a similar protocol as described in example 16-C, steps 5 and 6 to give the crude product. This material was purified by preparative thin layer chromatography (2 plates eluting first with 14% methanol in dichloromethane and then developed more than 2 times with 12% methanol in dichloromethane) to afford the desired 3- [5- (2-azetidin-1-yl-1, 1-dimethyl-ethoxy) -pyridin-2-ylamino- ] -a]-1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl]-1H-pyrazole-4-carboxamide as a light brown powder (27 mg). LC/MS calcd for C₃₅H₃₉FN₈O₄(M/e) 654.73, found 655(M + H, ES +).

Table I describes additional analogs prepared using methods similar to those described in the above examples.

Table I

Example 39

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -3- (5-cyano-pyridin-2-ylamino) -1H-pyrazole-4-carboxamide

Step 1: 1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -3- (5-chloro-pyridin-2-ylamino) -1H-pyrazole-4-carboxamide (prepared in example 23 above) (31mg, 55.2. mu. mol), zinc cyanide (51.8mg, 441. mu. mol) and 2-dicyclohexylphosphino-2 ', 6' -dimethoxybiphenyl (6mg, 14.6. mu. mol) were dissolved in 1.5mL of dry dimethylformamide in a small volume microwave tube and degassed thoroughly with argon. Tris (dibenzylideneacetone) dipalladium (0) (7mg, 7.6. mu. mol) was added and degassed. The mixture was heated in a microwave reactor at 150 ℃ for 60 minutes. The solvent was stripped (rotary evaporator/pump) and the residue was dissolved in ethyl acetate (25mL) and water (25mL) and shaken in a separatory funnel. The organic phase was collected and the aqueous phase was back-extracted with ethyl acetate (2 × 20 ml). The combined organic phases were dried over magnesium sulfate, filtered and stripped. The crude product was purified by preparative thin layer chromatography (1 plate, eluting with 9.5% methanol in dichloromethane). The product band was collected to give the semi-pure desired product (21mg, 91% pure). For greater purity, the material was applied to 1 more preparative TLC plates and eluted with 7% methanol in dichloromethane. The plate was re-expanded with a 7%, then 8.5% and finally 9% solution of methanol in dichloromethane, at which point the more polar impurities were separated. The less polar product band was collected to give the desired 1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -3- (5-cyano-pyridin-2-ylamino) -1H-pyrazole-4-carboxamide as a yellow-white solid (17mg, 56%). LC/MS calculated C29H25FN8O3 (M/e) 552.57, found 553(M + H, ES +).

Example 40

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -1H-pyrazole-3-carboxamide

Step 1: 1H-pyrazole-3-carbonitrile (850mg, 9.13mmol) was dissolved in 15mL dry DMSO, and potassium tert-butoxide (1.08g, 9.13mmol, Eq: 1.00) was added. The mixture was stirred at room temperature for 20 minutes, and 2-bromo-6-fluorobenzaldehyde (3.71g, 18.3mmol, Eq: 2) was added. The mixture was stirred at room temperature overnight and then extracted with ethyl acetate and water. The organic layer was dried and concentrated. The crude material was purified by flash chromatography (silica gel, 40g, 20% -40% EtOAc in hexanes) to give 1- (3-bromo-2-formylphenyl) -1H-pyrazole-3-carbonitrile (1.83g, 73%).

Step 2: in a 25mL vessel, 1- (3-bromo-2-formylphenyl) -1H-pyrazole-3-carbonitrile (50mg, 181. mu. mol, Eq: 1.00), 6-tert-butyl-8-fluorophthalazin-1 (2H) -one (79.8mg, 362. mu. mol, Eq: 2), and copper (I) iodide (69.0mg, 362. mu. mol, Eq: 2.00) were combined with DMSO (2.00mL) to give a yellow suspension. Sodium bicarbonate (38.0mg, 453. mu. mol, Eq: 2.5) was added thereto. The mixture was heated in a microwave at 120 ℃ for 1 hr. The reaction mixture was poured into 25mL of saturated NH4Cl and extracted with EtOAc (3 × 25 mL). With MgSO₄The organic layer was dried and concentrated in vacuo. The organic layer was concentrated and purified by ISCO flash column chromatography using ethyl acetate (5% methanol in hexanes) with a linear gradient of 5% to 80% over 15 minutes, 12g silica gel to afford the pure desired product, which was triturated with diethyl ether in hexanes and filtered to afford 1- (3- (6-tert-butyl-8-fluoro-1-oxophthalazin-2 (1H) -yl) -2-formylphenyl) -1H-pyrazole-3-carbonitrile (19mg, 25%).

And step 3: in a 25mL vessel, 1- (3- (6-tert-butyl-8-fluoro-1-oxophthalazin-2 (1H) -yl) -2-formylphenyl) -1H-pyrazole-3-carbonitrile (18mg, 43.3. mu. mol, Eq: 1.00), sodium borohydride (6.56mg, 173. mu. mol, Eq: 4) and CH₂Cl₂(2mL) and MeOH (1mL) were combined to give a white suspension. Stirring the mixtureStirring for 1 hr. The reaction mixture was poured into 25mL of saturated NH₄Cl, extracted with EtOAc (3 × 25 mL). With MgSO₄The organic layer was dried and concentrated in vacuo. The organic layer was concentrated to give 1- (3- (6-tert-butyl-8-fluoro-1-oxophthalazin-2 (1H) -yl) -2- (hydroxymethyl) phenyl) -1H-pyrazole-3-carbonitrile (16mg, 99%) as a white product.

And 4, step 4: in a 10mL round bottom flask, 1- (3- (6-tert-butyl-8-fluoro-1-oxophthalazin-2 (1H) -yl) -2- (hydroxymethyl) phenyl) -1H-pyrazole-3-carbonitrile (16mg, 38.3. mu. mol, Eq: 1.00) and [ hydrobis (dimethylphosphinous acid-kP) ] platinum (II) (1mg, 2.33. mu. mol, Eq: 0.0608) were combined with ethanol (821. mu.l) and water (410. mu.l) to give a colorless solution. The reaction mixture was heated to 80 ℃ and stirred for 2 h. The reaction mixture was diluted with DCM. The reaction mixture was filtered through a glass fiber filter paper. The crude material was purified by preparative HPLC to give 1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -1H-pyrazole-3-carboxamide (11mg, 66%, [ M + H ] +436) as a lyophilised white solid.

EXAMPLE 41

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -1H-pyrazole-4-carboxamide

Step 1: 1H-pyrazole-4-carbonitrile (935mg, 10.0mmol, Eq: 1.00) was dissolved in 15mL dry DMSO, and potassium tert-butoxide (1.19g, 10.0mmol, Eq: 1.00) was added. The mixture was stirred at room temperature for 20 minutes, and 2-bromo-6-fluorobenzaldehyde (4.08g, 20.1mmol, Eq: 2) was added. The mixture was stirred at room temperature overnight and then extracted with ethyl acetate and water. The organic layer was dried and concentrated. The crude material was purified by flash chromatography (silica gel, 40g, 20% -40% EtOAc in hexanes) to give 1- (3-bromo-2-formylphenyl) -1H-pyrazole-4-carbonitrile (1.53g, 55%).

Step 2: in thatIn a 25mL vessel, 1- (3-bromo-2-formylphenyl) -1H-pyrazole-4-carbonitrile (100mg, 362. mu. mol, Eq: 1.00), 6-tert-butyl-8-fluorophthalazin-1 (2H) -one (160mg, 724. mu. mol, Eq: 2), and copper (I) iodide (138mg, 724. mu. mol, Eq: 2.00) were combined with DMSO (2.00mL) to give a yellow suspension. Sodium bicarbonate (76.1mg, 906. mu. mol, Eq: 2.5) was added thereto. The mixture was heated at 120C for 1hr in a microwave. The reaction mixture was poured into 25mL of saturated NH4Cl and extracted with EtOAc (3 × 25 mL). With MgSO₄The organic layer was dried and concentrated in vacuo. The organic layer was concentrated and purified by ISCO flash column chromatography using ethyl acetate (5% methanol in hexanes) with a linear gradient of 5% to 80% over 15 minutes, 24g silica gel to give the pure desired product, trituration with diethyl ether in hexanes, filtration to give 1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-formyl-phenyl ] -phenyl]-1H-pyrazole-4-carbonitrile (110mg, 73%).

And step 3: the aldehyde was reduced to the alcohol as described in example 22.

And 4, step 4: in a 25mL round bottom flask, 1- (3- (6-tert-butyl-8-fluoro-1-oxophthalazin-2 (1H) -yl) -2- (hydroxymethyl) phenyl) -1H-pyrazole-4-carbonitrile (89mg, 213. mu. mol, Eq: 1.00) and hydrogen (dimethylphosphoric acid-kP) (hydrido (dimethylphosphinothus acid)) (4.58mg, 10.7. mu. mol, Eq: 0.05) were combined with ethanol (1mL) and water (1.00mL) to give a colorless solution. The reaction mixture was heated to 85 ℃ and stirred for 45 min. The mixture was brought to room temperature and the solvent was removed in vacuo. Purification by reverse phase HPLC gave 1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -1H-pyrazole-4-carboxamide (51mg, 56%, [ M + H ] + 436).

Example 42

7- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -7H-pyrrolo [2, 3-d ] pyrimidine-5-carboxamide

Step 1: in a 250mL round bottom flask, 7H-pyrrolo [2, 3-d ] pyrimidine (4.34g, 36.4mmol, Eq: 1.00) and N-iodosuccinimide (8.61g, 38.3mmol, Eq: 1.05) were combined with acetonitrile (60mL) to give a light brown suspension. The reaction mixture was stirred for 3 h. The reaction mixture was poured into 100mLH2O and extracted with EtOAc (3 × 50 mL). The organic layer was dried over MgSO4 and concentrated in vacuo. The crude material was triturated with ether (2 × 25mL) to give 5-iodo-7H-pyrrolo [2, 3-d ] pyrimidine (7.58g, 85%) as an orange solid.

Step 2: in a 200mL round-bottom flask, add 5-iodo-7H-pyrrolo [2, 3-d ]]Pyrimidine (2.5g, 10.2mmol, Eq: 1.00), triethylamine (1.14g, 1.56ml, 11.2mmol, Eq: 1.1) and DMAP (74.8mg, 612. mu. mol, Eq: 0.06) with CH₂Cl₂(50.0ml) were combined to give an orange suspension. tosyl-Cl (2.00g, 10.5mmol, Eq: 1.03) was added 1 time to the reaction mixture, and the reaction mixture was stirred for an additional 3 hrs. The reaction mixture was poured into 25mL of H₂O, extracted with DCM (3 × 25 mL). With MgSO₄The organic layer was dried and concentrated in vacuo to give 2gm of a red oil. The crude material was purified by flash chromatography (silica gel, 80g, 25% -70% EtOAc in hexane) to afford 5-iodo-7-tosyl-7H-pyrrolo [2, 3-d ] -a]Pyrimidine (1g, 24.6%) as a yellow solid.

And step 3: in a 20mL round-bottom flask, 5-iodo-7-tosyl-7H-pyrrolo [2, 3-d ] was added]Pyrimidine (3.32g, 8.32mmol, Eq: 1.00), copper (I) cyanide (2.98g, 33.3mmol, Eq: 4), Pd₂(dba)₃(305mg, 333. mu. mol, Eq: 0.04) and DPPF (738mg, 1.33mmol, Eq: 0.16) were combined with dioxane (48.1ml) to give a yellow suspension. The reaction was purged with argon, the mixture was heated to 80 ℃ and stirred for 4 h. LCMS showed reaction completion at 4 h. The crude reaction mixture was concentrated in vacuo. The crude material was triturated with ethanol (2 × 15mL) to give 1g of an off-white solid.

And 4, step 4: in a 15mL round-bottom flask, 7-tosyl-7H-pyrrolo [2, 3-d ] is added]Pyrimidine-5-carbonitrile (1g, 3.35mmol, Eq:1.00) and TBAF (13.4ml, 13.4mmol, Eq: 4.00) was combined with tetrahydrofuran to give a yellowish white solution. The reaction mixture was stirred for 3 h. The reaction mixture was poured into 20mL of saturated NH₄Cl, extracted with EtOAc (3 × 25 mL). With MgSO₄The organic layer was dried and concentrated in vacuo. The crude material was triturated with ether (1X20mL) to give 7H-pyrrolo [2, 3-d]Pyrimidine-5-carbonitrile (312mg, 65%).

And 5: in a 25mL round-bottom flask, add 7H-pyrrolo [2, 3-d]Pyrimidine-5-carbonitrile (40.9mg, 284. mu. mol, Eq: 1.00), 6-tert-butyl-8-fluoro-2- (1-hydroxy-1, 3-dihydrobenzo [ c ]][1，2]Oxacyclopentylboran-4-yl) phthalazin-1 (2H) -one (100mg, 284 μmol, Eq: 1.00), copper acetate (34.8mg) and pyridine (44.9mg, 45.9 μ l, 568 μmol, Eq: 2) combine with dichloroethane to give a dark blue suspension. The reaction mixture was purged with nitrogen. The reaction mixture was heated to 80 ℃ and stirred for 16 hr. With saturated NH₄The reaction was diluted with Cl (50mL) and extracted with EtOAc (3X50 mL). The mixture was separated by flash column using 30-50% EtOAc in hexane to give 7- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl]-7H-pyrrolo [2, 3-d]Pyrimidine-5-carbonitrile (42mg, 31%) as a lyophilized white powder.

Step 6: in a 10mL round bottom flask, 7- (3- (6-tert-butyl-8-fluoro-1-oxophthalazin-2 (1H) -yl) -2- (hydroxymethyl) phenyl) -7H-pyrrolo [2, 3-d ] pyrimidine-5-carbonitrile (39mg, 83.2. mu. mol, Eq: 1.00) and [ hydrobis (dimethylphosphinous acid-kP) ] platinum (II) (1mg, 2.33. mu. mol, Eq: 0.0280) were combined with ethanol (2mL) and water (1mL) to give a colorless solution. The reaction mixture was heated to 80 ℃ and stirred for 2 h. The reaction mixture was diluted with DCM. The reaction mixture was filtered through a glass fiber filter paper. Purification by HPLC afforded 7- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -7H-pyrrolo [2, 3-d ] pyrimidine-5-carboxamide (33mg, 66%, [ M + H ] + 487).

Example 43

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -6- (4-methyl-piperazin-1-yl) -1H-pyrrolo [2, 3-b ] pyridine-3-carboxamide

Step 1: in a 25mL round-bottom flask, add 6-bromo-1H-pyrrolo [2, 3-b ]]Pyridine (250mg, 1.27mmol, Eq: 1.00), TIPS-OTf (972mg, 860. mu.l, 3.17mmol, Eq: 2.5) and DIEA (492mg, 665. mu.l, 3.81mmol, Eq: 3) were combined with dioxane (6.25ml) to give a light brown solution. The reaction mixture was heated to 55 ℃ and stirred for 16 h. The reaction mixture was poured into 20mL EtOAc and saturated NaHCO₃(3 × 10 mL). With MgSO₄The organic layer was dried and concentrated in vacuo. The crude material was purified by flash chromatography (silica gel, 12g, 5% -10% EtOAc in hexane) to give 6-bromo-1- (triisopropylsilyl) -1H-pyrrolo [2, 3-b ] -c]Pyridine (380mg, 85%) was a colorless oil.

Step 2: in a 25mL round bottom flask, 6-bromo-1- (triisopropylsilyl) -1H-pyrrolo [2, 3-b ]]Pyridine (520mg, 1.47mmol, Eq: 1.00), palladium (II) acetate (165mg, 736. mu. mol, Eq: 0.5) and tert-butylphosphine (149mg, 182. mu.l, 736. mu. mol, Eq: 0.5) were combined with toluene to give a yellow solution. 1-methylpiperazine (442mg, 491. mu.l, 4.41mmol, Eq: 3) and sodium tert-butoxide (424mg, 4.41mmol, Eq: 3) were added. The reaction mixture was heated to 80 ℃ and stirred for 1 h. The reaction mixture was poured into 20ml of etoac and extracted with saturated NaCl (3 × 20 mL). With MgSO₄The organic layer was dried and concentrated in vacuo. The crude material was purified by flash chromatography (silica gel, 12g, 2% -5% MeOH in DCM) to give 6- (4-methylpiperazin-1-yl) -1- (triisopropylsilyl) -1H-pyrrolo [2, 3-b%]Pyridine (326mg, 60%) as a yellow oil solidified upon standing.

And step 3: in a 25mL round-bottom flask, 6- (4-methylpiperazin-1-yl) -1- (triisopropylsilyl) -1H-pyrrolo [2, 3-b ] pyridine (320mg, 859. mu. mol, Eq: 1.00) was combined with DMF (11.8mL) to give a colorless solution. The reaction mixture was cooled to-20 ℃ and stirred for 5 min. Chlorosulfonyl isocyanate (365mg, 224. mu.l, 2.58mmol, Eq: 3) in acetonitrile (11.8ml) was added dropwise and the resulting cooled reaction system was stirred at-20 ℃ for 3 hrs. The reaction mixture was poured into 25ml of letoac and extracted with saturated NaCl (3 × 20 mL). The crude material was purified by flash chromatography (silica gel, 12g, 5% -10% MeOH in DCM) to give 6- (4-methylpiperazin-1-yl) -1H-pyrrolo [2, 3-b ] pyridine-3-carbonitrile (113mg, 55%).

And 4, step 4: in a 25mL round-bottom flask, 6- (4-methylpiperazin-1-yl) -1H-pyrrolo [2, 3-b]Pyridine-3-carbonitrile (110mg, 456. mu. mol), 6-tert-butyl-8-fluoro-2- (1-hydroxy-1, 3-dihydrobenzo [ c ]][1，2]Oxetaborolan-4-yl) phthalazin-1 (2H) -one (177mg, 501. mu. mol, Eq: 1.10) and copper acetate (112mg) were combined with 1, 2-dichloroethane (3.03ml) to give a blue suspension. Pyridine (72.1mg, 73.7. mu.l, 912. mu. mol, Eq: 2) was added. The reaction mixture was heated to 45 ℃ and stirred for 2 d. The reaction mixture was poured into 20ml of LEtOAc and saturated NH was used₄Cl (3 × 20 mL). With MgSO₄The organic layer was dried and concentrated in vacuo. The crude material was purified by flash chromatography (silica gel, 40g, 5% -10% MeOH in DCM) to give 1- (3- (6-tert-butyl-8-fluoro-1-oxophthalazin-2 (1H) -yl) -2- (hydroxymethyl) phenyl) -6- (4-methylpiperazin-1-yl) -1H-pyrrolo [2, 3-b]Pyridine-3-carbonitrile (43mg, 17%) as an off-white solid.

And 5: in a 25mL round bottom flask, 1- (3- (6-tert-butyl-8-fluoro-1-oxophthalazin-2 (1H) -yl) -2- (hydroxymethyl) phenyl) -6- (4-methylpiperazin-1-yl) -1H-pyrrolo [2, 3-b ] pyridine-3-carbonitrile (43mg, 76.0. mu. mol) and [ hydrobis (dimethylphosphino-kP) ] platinum (II) (2.00mg, 4.66. mu. mol, Eq: 0.0613) were combined with ethanol (1.00mL) and water (1.00mL) to give a colorless solution. The reaction mixture was heated to 45 ℃ and stirred for 1 h. The crude reaction mixture was concentrated in vacuo. The mixture was diluted with acetonitrile and water and filtered. The resulting filtrate was lyophilized to give 1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -6- (4-methyl-piperazin-1-yl) -1H-pyrrolo [2, 3-b ] pyridine-3-carboxamide (35mg, 97%, [ M + H ] + 584).

Example 44

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -6-morpholin-4-yl-1H-pyrrolo [2, 3-b ] pyridine-3-carboxamide

Step 1: in a 25mL round bottom flask, 6-bromo-1- (triisopropylsilyl) -1H-pyrrolo [2, 3-b ]]Pyridine (800mg, 2.26mmol, Eq: 1.00), palladium (II) acetate (254mg, 1.13mmol, Eq: 0.5) and tert-butylphosphine (229mg, 279. mu.l, 1.13mmol, Eq: 0.5) were combined with toluene to give a yellow solution. Morpholine (789mg, 789. mu.l, 9.06mmol, Eq: 4) and sodium tert-butoxide (653mg, 6.79mmol, Eq: 3) were added. The reaction mixture was heated to 80 ℃ and stirred for 1 h. The reaction mixture was poured into 20mL of saturated NaCl and extracted with EtOAc (3 × 20 mL). With MgSO₄The organic layer was dried and concentrated in vacuo. The crude material was purified by flash chromatography (silica gel, 80g, 10% -15% EtOAc in hexane) to give 4- (1- (triisopropylsilyl) -1H-pyrrolo [2, 3-b ] -c]Pyridin-6-yl) morpholine (703mg, 86%) as a light brown oil.

Step 2: in a 25mL round-bottom flask, 4- (1- (triisopropylsilyl) -1H-pyrrolo [2, 3-b)]Pyridin-6-yl) morpholine (803mg, 2.23mmol) was combined with DMF (1.00ml) to give a colorless solution. The reaction mixture was cooled to-20C and stirred for 5 min. Chlorosulfonyl isocyanate (474mg, 291. mu.l, 3.35mmol, Eq: 1.5) in acetonitrile (1ml) was added dropwise and the resulting cooled reaction system was stirred at-20 ℃ for 3 h. The reaction mixture was poured into saturated NaCl (25mL) and extracted with EtOAc (3 × 20 mL). With MgSO₄The organic layer was dried and concentrated in vacuo to give 390mg of oil. In a 25mL round bottom flask, the crude cyano compound and TBAF (1.02mL, 1.02mmol, Eq: 1) were combined with THF (2.00mL) to give a white suspension. The reaction mixture was stirred for 1 h. LC-MS showed reaction completion at t =1 h. The reaction mixture was poured over20mL of DCM were charged with saturated NaHCO₃(2X25 mL). With MgSO₄The organic layer was dried and concentrated in vacuo. The crude material was purified by flash chromatography (silica gel, 24g, 50% EtOAc in hexane) to give 6-morpholino-1H-pyrrolo [2, 3-b ] -c]Pyridine-3-carbonitrile (180mg, 35%) as a white solid.

And step 3: in a 25mL round-bottom flask, 6-morpholino-1H-pyrrolo [2, 3-b ] was placed]Pyridine-3-carbonitrile (65mg, 285. mu. mol, Eq: 1.00), 6-tert-butyl-8-fluoro-2- (1-hydroxy-1, 3-dihydrobenzo [ c ]][1，2]Oxetaborolan-4-yl) phthalazin-1 (2H) -one (150mg, 427. mu. mol, Eq: 1.5) and copper acetate (69.8mg) were combined with 1, 2-dichloroethane (3ml) to give a blue suspension. Pyridine (45.1mg, 46.1. mu.l, 570. mu. mol, Eq: 2) was added. The reaction mixture was heated to 45 ℃ and stirred for 2 d. The reaction mixture was poured into 20mL of saturated NH₄Cl, extracted with EtOAc (3 × 20 mL). With MgSO₄The organic layer was dried and concentrated in vacuo. The crude material was purified by flash chromatography (silica gel, 40g, 5% -10% MeOH in DCM, then silica gel, 12g, 30% -45% EtOAc in hexane) to give 1- (3- (6-tert-butyl-8-fluoro-1-oxophthalazin-2 (1H) -yl) -2- (hydroxymethyl) phenyl) -6-morpholino-1H-pyrrolo [2, 3-b%]Pyridine-3-carbonitrile (157mg, 50%) as a colorless oil, which was a foam upon drying.

And 4, step 4: in a 25mL round bottom flask, 1- (3- (6-tert-butyl-8-fluoro-1-oxophthalazin-2 (1H) -yl) -2- (hydroxymethyl) phenyl) -6-morpholino-1H-pyrrolo [2, 3-b ] pyridine-3-carbonitrile (75mg, 136. mu. mol, Eq: 1.00) and [ hydro bis (dimethylphosphinous acid-kP) ] platinum (II) (2mg, 4.66. mu. mol, Eq: 0.0343) were combined with ethanol (1mL) and water (1.00mL) to give a colorless solution. The reaction mixture was heated to 45 ℃, stirred for 1h, and then concentrated in vacuo. The mixture was diluted with acetonitrile and water and filtered. The resulting filtrate was lyophilized to give 1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -6-morpholin-4-yl-1H-pyrrolo [2, 3-b ] pyridine-3-carboxamide (70mg, 90%, [ M + H ] + 571).

Example 45

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -6- (6-ethoxy-pyridin-3-yl) -1H-pyrrolo [2, 3-b ] pyridine-3-carboxamide

Step 1: in a 100mL round-bottom flask, 1-acetyl-6-bromo-1H-pyrrolo [2, 3-b ] is added]Pyridine-3-carbonitrile (250mg, 947. mu. mol, Eq: 1.00), triethylamine (575mg, 792. mu.l, 5.68mmol, Eq: 6), and X-PHOS (181mg, 379. mu. mol, Eq: 0.40) were combined with dioxane (25ml) to give a colorless solution. Adding [1, 1' -bis (diphenylphosphino) ferrocene]Dichloropalladium (II) (173mg, 237. mu. mol, Eq: 0.25) and 2-ethoxy-5-pyridineboronic acid (205mg, 1.23mmol, Eq: 1.3), and the resulting mixture was degassed under a nitrogen atmosphere for 5 minutes. The reaction mixture was heated to 100 ℃ and O/N h was stirred. The reaction mixture was poured into 50mL of H₂O, extracted with EtOAc (3 × 50 mL). With MgSO₄The organic layer was dried and concentrated in vacuo. The crude material was purified by flash chromatography (silica gel, 40g, 20% -30% EtOAc in hexane) to give 6- (6-ethoxypyridin-3-yl) -1H-pyrrolo [2, 3-b ] -a]Pyridine-3-carbonitrile (167mg, 67%).

Step 2: in a 25mL round-bottom flask, 6- (6-ethoxypyridin-3-yl) -1H-pyrrolo [2, 3-b ] was placed]Pyridine-3-carbonitrile (110mg, 416. mu. mol, Eq: 1.00), 6-tert-butyl-8-fluoro-2- (1-hydroxy-1, 3-dihydrobenzo [ c ]][1，2]Oxetaborolan-4-yl) phthalazin-1 (2H) -one (161mg, 458. mu. mol, Eq: 1.10) and copper acetate (102mg) were combined with 1, 2-dichloroethane (3ml) to give a blue suspension. Pyridine (65.8mg, 67.3. mu.l, 832. mu. mol, Eq: 2) was added. The reaction mixture was heated to 45 ℃ and stirred for 2 d. The reaction mixture was poured into 20mL of saturated NH₄Cl, extracted with EtOAc (3 × 20 mL). With MgSO₄The organic layer was dried and concentrated in vacuo. The crude material was purified by flash chromatography (silica gel, 40g, 5% -10% MeOH in DCM) followed by SFC to give 1- (3- (6-tert-butyl-8-fluoro-1-oxophthalazin-2 (1H) -yl) -2- (hydroxy) phthalazin-2 (1H) -yl)Ylmethyl) phenyl) -6- (6-ethoxypyridin-3-yl) -1H-pyrrolo [2, 3-b]Pyridine-3-carbonitrile (36mg, 15%).

And step 3: in a 25mL round bottom flask, 1- (3- (6-tert-butyl-8-fluoro-1-oxophthalazin-2 (1H) -yl) -2- (hydroxymethyl) phenyl) -6- (6-ethoxypyridin-3-yl) -1H-pyrrolo [2, 3-b]Pyridine-3-carbonitrile (36mg, 61.2. mu. mol) and [ Hydrobis (dimethylphosphinic acid-kP)]Platinum (II) (263. mu.g, 0.612. mu. mo)_lAnd Eq: 0.01) was combined with ethanol (480. mu.l) and water (480. mu.l) to give a colorless solution. The reaction mixture was heated to 45 ℃ and stirred for 1 h. The crude reaction mixture was concentrated in vacuo. The mixture was diluted with acetonitrile and water and filtered. Lyophilizing the resulting filtrate to obtain 1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl]-6- (6-ethoxy-pyridin-3-yl) -1H-pyrrolo [2, 3-b]Pyridine-3-carboxamide (31mg, 84%, [ M + H ]]+607)。

Example 46

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -6- (2-fluoro-phenyl) -1H-pyrrolo [2, 3-b ] pyridine-3-carboxamide

Step 1: in a 100mL round-bottom flask, 1-acetyl-6-bromo-1H-pyrrolo [2, 3-b ] is added]Pyridine-3-carbonitrile (250mg, 947. mu. mol, Eq: 1.00), triethylamine (575mg, 792. mu.l, 5.68mmol, Eq: 6), and X-PHOS (181mg, 379. mu. mol, Eq: 0.40) were combined with dioxane (25.0ml) to give a colorless solution. Adding [1, 1' -bis (diphenylphosphino) ferrocene]Dichloropalladium (II) (173mg, 237. mu. mol, Eq: 0.25) and 2-fluorophenylboronic acid (265mg, 1.89mmol), and the resulting mixture was degassed under a nitrogen atmosphere for 5 minutes. The reaction mixture was heated to 100 ℃ and microwave irradiated for 1 hr. The reaction mixture was poured into 50mL EtOAc and washed with H₂And O washing. With MgSO₄The organic layer was dried and concentrated in vacuo. Purification of the crude material by flash chromatography(silica gel, 40g, 20% -30% EtOAc in hexane) to give 6- (2-fluorophenyl) -1H-pyrrolo [2, 3-b ] -salt]Pyridine-3-carbonitrile (220mg, 98%).

Step 2: in a 25mL round-bottom flask, 6- (2-fluorophenyl) -1H-pyrrolo [2, 3-b ] is added]Pyridine-3-carbonitrile (200mg, 843. mu. mol), 6-tert-butyl-8-fluoro-2- (1-hydroxy-1, 3-dihydrobenzo [ c ]][1，2]Oxacyclopentylboran-4-yl) phthalazin-1 (2H) -one (297mg, 843 μmol, Eq: 1.00) and copper acetate (207mg,) were combined with 1, 2-dichloroethane (10ml) to give a blue suspension. Pyridine (133mg, 136. mu.l, 1.69mmol, Eq: 2) was added. The reaction mixture was heated to 45 ℃ and stirred for 2 d. The reaction mixture was poured into 20mL of saturated NH₄C) Extracted with EtOAc (3 × 20 mL). With MgSO₄The organic layer was dried and concentrated in vacuo. The crude material was purified by flash chromatography (silica gel, 40g, 50% -60% EtOAc in hexane) followed by SFC to give 1- (3- (6-tert-butyl-8-fluoro-1-oxophthalazin-2 (1H) -yl) -2- (hydroxymethyl) phenyl) -6- (2-fluorophenyl) -1H-pyrrolo [2, 3-b]Pyridine-3-carbonitrile (163mg, 13%).

And step 3: in a 25mL round bottom flask, 1- (3- (6-tert-butyl-8-fluoro-1-oxophthalazin-2 (1H) -yl) -2- (hydroxymethyl) phenyl) -6- (2-fluorophenyl) -1H-pyrrolo [2, 3-b ] pyridine-3-carbonitrile (65mg, 116. mu. mol) and [ hydrobis (dimethylphosphinous acid-kP) ] platinum (II) (497. mu.g, 1.16. mu. mol, Eq: 0.01) were combined with ethanol (853. mu.l) and water (853. mu.l) to give a colorless solution. The reaction mixture was heated to 45 ℃ and stirred for 1 h. The mixture was concentrated in vacuo, then diluted with acetonitrile and water and filtered. The resulting filtrate was lyophilized to give 1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -6- (2-fluoro-phenyl) -1H-pyrrolo [2, 3-b ] pyridine-3-carboxamide (61mg, 91%, [ M + H ] + 580).

Example 47

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -6- (2-chloro-phenyl) -1H-pyrrolo [2, 3-b ] pyridine-3-carboxamide

Step 1: in a 100mL round-bottom flask, 1-acetyl-6-bromo-1H-pyrrolo [2, 3-b ] is added]Pyridine-3-carbonitrile (250mg, 947. mu. mol, Eq: 1.00), triethylamine (575mg, 792. mu.l, 5.68mmol, Eq: 6), and X-PHOS (181mg, 379. mu. mol, Eq: 0.40) were combined with dioxane (25.0ml) to give a colorless solution. Adding [1, 1' -bis (diphenylphosphino) ferrocene]Dichloropalladium (II) (173mg, 237. mu. mol, Eq: 0.25) and 2-chlorophenylboronic acid (296mg, 1.89mmol), and the resulting mixture was degassed under a nitrogen atmosphere for 5 minutes. The reaction mixture was heated to 100 ℃ and stirred overnight. The reaction mixture was poured into 50mL of H₂O, extracted with EtOAc (3 × 50 mL). With MgSO₄The organic layer was dried and concentrated in vacuo. The crude material was purified by flash chromatography (silica gel, 40g, 20% -30% EtOAc in hexane) to give 6- (2-chlorophenyl) -1H-pyrrolo [2, 3-b ] -c]Pyridine-3-carbonitrile (142mg, 59%).

Step 2: in a 25mL round-bottom flask, 6- (2-chlorophenyl) -1H-pyrrolo [2, 3-b ] was placed]Pyridine-3-carbonitrile (125mg, 493. mu. mol), 6-tert-butyl-8-fluoro-2- (1-hydroxy-1, 3-dihydrobenzo [ c ]][1，2]Oxetaborolan-4-yl) phthalazin-1 (2H) -one (174mg, 493. mu. mol, Eq: 1.00) and copper acetate (121mg) were combined with 1, 2-dichloroethane (6.25ml) to give a blue suspension. Pyridine (78.0mg, 79.7. mu.l, 985. mu. mol, Eq: 2) was added. The reaction mixture was heated to 45 ℃ and stirred for 2 d. The reaction mixture was poured into saturated NH₄Cl (20mL), extracted with EtOAc (3 × 20 mL). With MgSO₄The organic layer was dried and concentrated in vacuo. The crude material was purified by flash chromatography (silica gel, 40g, 5% -10% MeOH in DCM) followed by SFC to give 1- (3- (6-tert-butyl-8-fluoro-1-oxophthalazin-2 (1H) -yl) -2- (hydroxymethyl) phenyl) -6- (2-chlorophenyl) -1H-pyrrolo [2, 3-b]Pyridine-3-carbonitrile (4.5mg, 2%).

And step 3: in a 25mL round bottom flask, 1- (3- (6-tert-butyl-8-fluoro-1-oxophthalazin-2 (1H) -yl) -2- (hydroxymethyl) phenyl) -6- (2-chlorophenyl) -1H-pyrrolo [2, 3-b ] pyridine-3-carbonitrile (4mg, 6.92. mu. mol) and [ hydrobis (dimethylphosphinous acid-kP) ] platinum (II) (29.7. mu.g, 0.0692. mu. mol, Eq: 0.01) were combined with ethanol (853. mu.l) and water (853. mu.l) to give a colorless solution. The reaction mixture was heated to 45 ℃, stirred for 1h, and then concentrated in vacuo. The mixture was diluted with acetonitrile and water and filtered. The resulting filtrate was lyophilized to give 1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -6- (2-chloro-phenyl) -1H-pyrrolo [2, 3-b ] pyridine-3-carboxamide (3.9mg, 95%, [ M + H ] + 596).

Example 48

6-bromo-1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -1H-pyrrolo [2, 3-b ] pyridine-3-carboxamide

Step 1: in a 25mL round-bottom flask, add 6-bromo-1H-pyrrolo [2, 3-b ]]Pyridine-3-carbonitrile (75.0mg, 338. mu. mol, Eq: 1.19), 6-tert-butyl-8-fluoro-2- (1-hydroxy-1, 3-dihydrobenzo [ c ]][1，2]Oxacyclopentylboran-4-yl) phthalazin-1 (2H) -one (100mg, 284 μmol, Eq: 1.00) and acetone (52.2mg) were combined with 1, 2-dichloroethane (3.00ml) to give a blue suspension. Pyridine (44.9mg, 45.9. mu.l, 568. mu. mol, Eq: 2) was added. The reaction mixture was heated to 45 ℃ and stirred for 2 d. The mixture was poured into 20mL of saturated NH₄Cl, extracted with EtOAc (3 × 20 mL). With MgSO₄The organic layer was dried and concentrated in vacuo. The crude material was purified by flash chromatography (silica gel, 40g, 25% -45% EtOAc in hexane) to give 6-bromo-1- (3- (6-tert-butyl-8-fluoro-1-oxophthalazin-2 (1H) -yl) -2- (hydroxymethyl) phenyl) -1H-pyrrolo [2, 3-b]Pyridine-3-carbonitrile (16mg, 10.3%).

Step 2: in a 25mL round bottom flask, 6-bromo-1- (3- (6-tert-butyl-8-fluoro-1-oxophthalazin-2 (1H) -yl) -2- (hydroxymethyl) phenyl) -1H-pyrrolo [2, 3-b ] pyridine-3-carbonitrile (14mg, 25.6. mu. mol) and [ hydrobis (dimethylphosphinic acid-kP) ] platinum (II) (110. mu.g, 0.256. mu. mol, Eq: 0.01) were combined with ethanol (2.99mL) and water (2.99mL) to give a colorless solution. The reaction mixture was heated to 45 ℃ and stirred for 1 h. The crude reaction mixture was concentrated in vacuo. Purification by HPLC afforded 6-bromo-1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -1H-pyrrolo [2, 3-b ] pyridine-3-carboxamide (11mg, 77%, [ M + H ] + 565).

Example 49

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -6- (1, 2-dihydroxy-ethyl) -1H-pyrrolo [2, 3-b ] pyridine-3-carboxamide

Step 1: in a 25mL round bottom flask, 1-acetyl-6-bromo-1H-pyrrolo [2, 3-b ] is added]Pyridine-3-carbonitrile (372mg, 1.41mmol, Eq: 1.00), 2, 6-di-tert-butyl-4-methylphenol (5mg, 22.7. mu. mol, Eq: 0.0161), and tributyl (vinyl) tin (536mg, 494. mu.l, 1.69mmol, Eq: 1.20) were combined with toluene (8ml) to give a light yellow solution. The mixture was degassed by bubbling nitrogen therethrough for 5 minutes, tetrakis (triphenylphosphine) palladium (0) (130mg, 113. mu. mol, Eq: 0.08) was added, the reaction system was degassed with nitrogen, and the reaction mixture was heated to 80 ℃ and stirred overnight. The mixture was poured into 15mL of saturated NaHCO₃Above, extract with EtOAc (3 × 25 mL). With MgSO₄The organic layer was dried and concentrated in vacuo. The crude material was purified by flash chromatography (silica gel, 40g, 10% -20% EtOAc in hexanes) to afford two products: 1-acetyl-6-vinyl-1H-pyrrolo [2, 3-b]Pyridine-3-carbonitrile (160mg) and 6-vinyl-1H-pyrrolo [2, 3-b ]]Pyridine-3-carbonitrile (138 mg).

Step 2: in a 25mL round-bottom flask, 6-vinyl-1H-pyrrolo [2, 3-b ] was added]Pyridine-3-carbonitrile (138mg, 816. mu. mol, Eq: 1.00), 6-tert-butyl-8-fluoro-2- (1-hydroxy-1, 3-dihydrobenzo [ c ]][1，2]Oxetaborolan-4-yl) phthalazin-1 (2H) -one (287mg, 816. mu. mol, Eq: 1.00) and acetone (200mg) were combined with 1, 2-dichloroethane (3.58ml) to give a blue suspension. Pyridine (129mg, 132. mu.l, 1.63mmol, Eq: 2) was added. The reaction mixture was heated to 45 ℃ and stirred for 2 d. The reaction mixture was poured into 20mL of saturated NH₄Cl, extracted with saturated EtOAc (3 × 20 mL). With MgSO₄The organic layer was dried and concentrated in vacuo. The crude material was purified by flash chromatography (silica gel, 40g, 5% -10% MeOH in DCM) followed by HPLC to give 1- (3- (6-tert-butyl-8-fluoro-1-oxophthalazin-2 (1H) -yl) -2- (hydroxymethyl) phenyl) -6-vinyl-1H-pyrrolo [2, 3-b]Pyridine-3-carbonitrile (51mg, 13%).

And step 3: in a 10mL pear-shaped flask, 1- (3- (6-tert-butyl-8-fluoro-1-oxophthalazin-2 (1H) -yl) -2- (hydroxymethyl) phenyl) -6-vinyl-1H-pyrrolo [2, 3-b ] pyridine-3-carbonitrile (50mg, 101. mu. mol, Eq: 1.00), 4-methylmorpholine N-oxide (17.8mg, 152. mu. mol, Eq: 1.50) and osmium tetroxide (41.2mg, 50.9. mu.l, 4.05. mu. mol, Eq: 0.04) were combined with acetone (2mL) to give a colorless solution. The reaction mixture was stirred for 2 d. The reaction was quenched with sodium sulfite, extracted with ethyl acetate and evaporated. 4-methylmorpholine N-oxide (17.8mg, 152. mu. mol, Eq: 1.50), osmium tetroxide (41.2mg, 50.9. mu.l, 4.05. mu. mol, Eq: 0.04) and acetone (3ml) were further added to give a brown solution, and the reaction was continued for 1 day. The reaction was quenched with sodium sulfite, extracted with ethyl acetate and evaporated. Crude 1- (3- (6-tert-butyl-8-fluoro-1-oxophthalazin-2 (1H) -yl) -2- (hydroxymethyl) phenyl) -6- (1, 2-dihydroxyethyl) -1H-pyrrolo [2, 3-b ] pyridine-3-carbonitrile (15mg) was used in the next step.

And 4, step 4: in a 25mL round bottom flask, 1- (3- (6-tert-butyl-8-fluoro-1-oxophthalazin-2 (1H) -yl) -2- (hydroxymethyl) phenyl) -6- (1, 2-dihydroxyethyl) -1H-pyrrolo [2, 3-b ] pyridine-3-carbonitrile (14mg, 26.5. mu. mol, Eq: 1.00) and [ hydro bis (dimethylphosphinous acid-kP) ] platinum (II) (373. mu.g, 0.870. mu. mol, Eq: 0.0328) were combined with ethanol (200. mu.l) and water (200. mu.l) to give a colorless solution. The reaction mixture was heated to 45 ℃ and stirred for 1 h. The crude reaction mixture was concentrated in vacuo. The mixture was diluted with acetonitrile and water and filtered. The resulting filtrate was lyophilized to give 1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -6- (1, 2-dihydroxy-ethyl) -1H-pyrrolo [2, 3-b ] pyridine-3-carboxamide (11.2mg, 77%, [ M + H ] + 546).

Example 50

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -6- (1, 1-dioxo-1 λ 6 x-thiomorpholin-4-yl) -1H-pyrrolo [2, 3-b ] pyridine-3-carboxamide

Step 1: in a 25mL round bottom flask, 6-bromo-1- (triisopropylsilyl) -1H-pyrrolo [2, 3-b ]]Pyridine (420mg, 1.19mmol, Eq: 1.00) and thiomorpholine 1, 1-dioxide (482mg, 3.57mmol) were combined with toluene (3ml) to give a yellow solution. Bis (tri-tert-butylphosphine) palladium (0) (60.7mg, 119. mu. mol) and sodium tert-butoxide (400mg, 4.16mmol, Eq: 3.5) were added. The reaction mixture was heated to 80 ℃ and stirred for 1 h. The reaction mixture was poured into 20mL of saturated NaCl and extracted with EtOAc (3 × 20 mL). With MgSO₄The organic layer was dried and concentrated in vacuo. The crude material was purified by flash chromatography (silica gel, 40g, 20% -40% EtOAc in hexanes) to give 6- (1, 1-dioxo-1 λ 6-thiomorpholin-4-yl) -1-triisopropylsilyl-1H-pyrrolo [2, 3-b ] -a]Pyridine (327mg, 68%) as a yellow oil solidified to a yellow-white solid on standing.

Step 2: in a 25mL round bottom flask, 6- (1, 1-dioxo-1. lamda. 6. lamda. -thiomorpholin-4-yl) -1-triisopropylsilyl-1H-pyrrolo [2, 3-b ]]Pyridine (322mg, 790. mu. mol, Eq: 1.00) was combined with DMF (11.9ml) to give a colorless solution. The reaction mixture was cooled to-20 ℃ and stirred for 5 min. Chlorosulfonyl isocyanate (335mg, 206. mu.l, 2.37mmol, Eq: 3) in acetonitrile (11.9ml) was added dropwise and the resulting cooled reaction system was stirred at-20 ℃ for 3 hrs. The reaction mixturePour into 25mL EtOAc and wash with saturated NaCl (3X20 mL). After evaporation, the crude material was purified by flash column chromatography (silica gel, 12g, 5% -10% MeOH in DCM) to give 6- (1, 1-dioxo_-1 lambda 6 thiomorpholin-4-yl) -1H-pyrrolo [2, 3-b]Pyridine-3-carbonitrile (122mg, 56%).

And step 3: in a 25mL round bottom flask, 6- (1, 1-dioxo-1 λ 6-thiomorpholin-4-yl) -1H-pyrrolo [2, 3-b ] was placed]Pyridine-3-carbonitrile (122mg, 442. mu. mol), 6-tert-butyl-8-fluoro-2- (1-hydroxy-1, 3-dihydrobenzo [ c ]][1，2]Oxetaborolan-4-yl) phthalazin-1 (2H) -one (155mg, 442. mu. mol, Eq: 1.00) and copper acetate (108mg) were combined with 1, 2-dichloroethane (3.89ml) to give a blue suspension. Pyridine (69.8mg, 71.4. mu.l, 883. mu. mol, Eq: 2) was added. The reaction mixture was heated to 45 ℃ and stirred for 2 d. The reaction mixture was poured into 20mL of saturated NH₄Cl, extracted with EtOAc (3 × 20 mL). With MgSO₄The organic layer was dried and concentrated in vacuo. The crude material was purified by flash chromatography (silica gel, 40g, 5% -10% MeOH in DCM) followed by HPLC to give 1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl]-6- (1, 1-dioxo-1 λ 6-thiomorpholin-4-yl) -1H-pyrrolo [2, 3-b]Pyridine-3-carbonitrile (22mg, 8%).

And 4, step 4: in a 25mL round bottom flask, 1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -6- (1, 1-dioxo-1 λ 6-thiomorpholin-4-yl) -1H-pyrrolo [2, 3-b ] pyridine-3-carbonitrile (22mg, 36.6 μmol) and [ hydro bis (dimethylphosphinous acid-kP) ] platinum (II) (1.1mg, 2.56 μmol, Eq: 0.07) were combined with ethanol (200 μ l) and water (200 μ l) to give a colorless solution. The reaction mixture was heated to 45 ℃ and stirred for 1 h. The crude reaction mixture was concentrated in vacuo, then diluted with acetonitrile and water, filtered and purified by HPLC to give 1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -6- (1, 1-dioxo-1 λ x 6 x-thiomorpholin-4-yl) -1H-pyrrolo [2, 3-b ] pyridine-3-carboxamide (15.5mg, 68%, [ M + H ] + 619).

Example 51

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -6- (2-dimethylamino-ethylamino) -1H-pyrrolo [2, 3-b ] pyridine-3-carboxamide

Step 1: in a 25mL round bottom flask, 6-bromo-1- (triisopropylsilyl) -1H-pyrrolo [2, 3-b ]]Pyridine (713mg, 2.02mmol, Eq: 1.00), N-dimethylethylenediamine (1.07g, 1.32ml, 12.1mmol, Eq: 6), and bis (tri-tert-butylphosphine) palladium (0) (206mg, 404. mu. mol, Eq: 0.2) were combined with toluene (2ml) to give a yellow solution. Sodium tert-butoxide (582mg, 6.05mmol, Eq: 3) was added. The reaction mixture was heated to 80 ℃, stirred for 1h, then poured into 20mL of saturated NaCl and extracted with EtOAc (3 × 20 mL). With MgSO₄The organic layer was dried and concentrated in vacuo. The crude material was purified by flash chromatography (silica gel, 12g, 2% -5% MeOH in DCM) to give N1, N1-dimethyl-N2- (1- (triisopropylsilyl) -1H-pyrrolo [2, 3-b ] -c]Pyridin-6-yl) ethane-1, 2-diamine (405mg, 56%) was a yellow oil that solidified upon standing.

Step 2: in a 25mL round bottom flask, N1, N1-dimethyl-N2- (1- (triisopropylsilyl) -1H-pyrrolo [2, 3-b ] pyridin-6-yl) ethane-1, 2-diamine (405mg, 1.12mmol) was combined with DMF (14.9mL) to give a colorless solution. The reaction mixture was cooled to-20 ℃ and stirred for 5 min. Chlorosulfonyl isocyanate (477mg, 293. mu.l, 3.37mmol, Eq: 3) in acetonitrile (14.9ml) was added dropwise and the resulting cooled reaction system was stirred at-20 ℃ for 3 hrs. The reaction mixture was poured into 25mL of saturated NaCl and extracted with EtOAc (3 × 20 mL). After evaporation, the crude material was purified by flash chromatography (silica gel, 12g, 5% -10% MeOH in DCM) to give 6- (2- (dimethylamino) ethylamino) -1H-pyrrolo [2, 3-b ] pyridine-3-carbonitrile (211mg, 82%).

And step 3: in a 25mL round bottom flask, 6- (2- (dimethylamino) ethylamino) -1H-pyrrolo [2, 3-b]Pyridine-3-carbonitrile (211mg, 920. mu. mol), 6-tert-butyl-8-fluoro-2- (1-hydroxy-1, 3-dihydrobenzo [ c ]][1，2]Oxetaborolan-4-yl) phthalazin-1 (2H) -one (324mg, 920. mu. mol, Eq: 1.00) and copper acetate (226mg) were combined with 1, 2-dichloroethane (6.73ml) to give a blue suspension. Pyridine (146mg, 149. mu.l, 1.84mmol, Eq: 2) was added. The reaction mixture was heated to 45 ℃ and stirred for 2 d. The mixture was poured into 20mL of saturated NH₄Cl, extracted with EtOAc (3 × 20 mL). With MgSO₄The organic layer was dried and concentrated in vacuo. The crude material was purified by flash chromatography (silica gel, 40g, 5% -10% MeOH in DCM) followed by HPLC to give 1- (3- (6-tert-butyl-8-fluoro-1-oxophthalazin-2 (1H) -yl) -2- (hydroxymethyl) phenyl) -6- (2- (dimethylamino) ethylamino) -1H-pyrrolo [2, 3-b]Pyridine-3-carbonitrile (18mg, 3.5%).

And 4, step 4: in a 25mL round bottom flask, 1- (3- (6-tert-butyl-8-fluoro-1-oxophthalazin-2 (1H) -yl) -2- (hydroxymethyl) phenyl) -6- (2- (dimethylamino) ethylamino) -1H-pyrrolo [2, 3-b ] pyridine-3-carbonitrile (19mg, 34.3. mu. mol, Eq: 1.00) and [ hydro-bis (dimethylphosphinous acid-kP) ] platinum (II) (1.47mg, 3.43. mu. mol, Eq: 0.1) were combined with ethanol (271. mu.l) and water (271. mu.l) to give a colorless solution. The reaction mixture was heated to 45 ℃ and stirred for 1 h. The reaction mixture was concentrated in vacuo, then diluted with acetonitrile and water and filtered. The resulting filtrate was lyophilized to give 1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -6- (2-dimethylamino-ethylamino) -1H-pyrrolo [2, 3-b ] pyridine-3-carboxamide (14.2mg, 71%, [ M + H ] + 572).

Example 52

Step 1: in a 10mL pear-shaped flask, 6-vinyl-1H-pyrrolo [2, 3-b ] pyridine-3-carbonitrile (274mg, 1.62mmol), 4-methylmorpholine N-oxide (17.8mg, 152. mu. mol, Eq: 1.50), and osmium tetroxide (3.29g, 4.07mL, 324. mu. mol, Eq: 0.2) were combined with acetone (3mL) to give a colorless solution. The reaction mixture was maintained at room temperature and stirred for 2 d. The reaction was quenched with sodium sulfite, extracted with ethyl acetate and evaporated. 4-methylmorpholine N-oxide (17.8mg, 152. mu. mol, Eq: 1.50) and osmium tetroxide (41.2mg, 50.9. mu.l, 4.05. mu. mol, Eq: 0.04) and acetone (3ml) were then added to give a brown solution, and the reaction was allowed to continue for 1 day. The reaction was quenched with sodium sulfite, extracted with ethyl acetate and evaporated. Crude 6-formyl-1H-pyrrolo [2, 3-b ] pyridine-3-carbonitrile was taken for the next step.

And 2, step 3: after standard reductive amination conditions, 6- ((dimethylamino) methyl) -1H-pyrrolo [2, 3-b]Pyridine-3-carbonitrile (18mg, 89.9. mu. mol), 6-tert-butyl-8-fluoro-2- (1-hydroxy-1, 3-dihydrobenzo [ c ]][1，2]Oxacyclopentylboran-4-yl) phthalazin-1 (2H) -one (38.0mg, 108 μmol, Eq: 1.20) and Ketone acetate (22.0mg) with 1, 2-dichloroethane (574)_μl) were combined to give a blue suspension. Pyridine (14.2mg, 14.5. mu.l, 180. mu. mol, Eq: 2) was added. The reaction mixture was heated to 45 ℃ and stirred for 2 d. The reaction mixture was poured into 20mL of EtOAc and extracted with saturated NH4Cl (3 × 20 mL). The organic layer was dried over MgSO4 and concentrated in vacuo. Taking crude 1- (3- (6-tert-butyl-8-fluoro-1-oxophthalazin-2 (1H) -yl) -2- (hydroxymethyl) phenyl) -6- ((dimethylamino) methyl) -1H-pyrrolo [2, 3-b ] without purification]Pyridine-3-carbonitrile (8mg) was used in the next step.

And 4, step 4: in a 25mL round bottom flask, 1- (3- (6-tert-butyl-8-fluoro-1-oxophthalazin-2 (1H) -yl) -2- (hydroxymethyl) phenyl) -6- ((dimethylamino) methyl) -1H-pyrrolo [2, 3-b ] pyridine-3-carbonitrile (8mg, 15.3. mu. mol, Eq: 1.00) and [ hydrobis (dimethylphosphinous acid-kP) ] palladium (II) (655. mu.g, 1.53. mu. mol, Eq: 0.1) were combined with ethanol (1mL) and water (1mL) to give a colorless solution. The reaction mixture was heated to 45 ℃ and stirred for 1 h. The reaction mixture was concentrated in vacuo. LCMS of the crude product showed the desired product. The reaction mixture was diluted with acetonitrile and water, filtered, purified by HPLC, then lyophilized to give 1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -6-dimethylaminomethyl-1H-pyrrolo [2, 3-b ] pyridine-3-carboxamide (2.5mg, 30%, [ M + H ] + 543).

Example 53

3- (4-acetyl-phenylamino) -1- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -1H-pyrazole-4-carbonitrile

Step 1: to dry acetonitrile (10mL) and dry tetrahydrofuran (30mL) was added 5-amino-1H-pyrazole-4-carbonitrile (1.0g, 9.25mmol) followed by triethylamine (1.08mL, 1.74 mmol). The resulting suspension was stirred at room temperature for 5 minutes and then cooled to 0 ℃. A solution of (bromomethane trityl) triphenyl (1.89g, 5.84mmol) in dry tetrahydrofuran (10mL) was added dropwise through an addition funnel fitted with a nitrogen inlet at such a rate that the temperature was no higher than 5 ℃. The addition funnel was then rinsed with dry tetrahydrofuran (5mL) and the reaction mixture was stirred at 0 deg.C for 10 minutes, then warmed to room temperature and stirred for 2 hours. The volatiles were removed in vacuo and the residue was dissolved in EtOAc (150mL), washed 1 time with water (100mL), 1 time with saturated aqueous sodium chloride, and Na₂SO₄Drying, filtering and vacuum concentrating. The crude product was loaded onto a 80g silica gel column using dichloromethane, and the column was eluted with a gradient of 10-30% EtOAc in hexane. The product-containing fractions were combined to give 3- (trityl-amino) -1H-pyrazole-4-carbonitrile (1.3g, 41%) as a yellow-white semi-crystalline solid.

Step 2: to degassed DMF (1.5mL) was added 5- (tritylamino) -1H-pyrazole-4-carbonitrile (1.36g, 3.89mmol), 2-bromo-6- (6-tert-butyl-1-oxophthalazin-2 (1H) -yl) benzaldehyde (1.0g, 2.6mmol), copper (I) iodide (494mg, 2.6mmol) and potassium carbonate (717mg, 5.19 mmol). The reaction was inertized 5 times by alternating vacuum and nitrogen purges, then heated to 100 ℃ (external) 8 hr. TLC showed trace amounts of starting material the next morning. The reaction was diluted with EtOAc (50mL) and passed through a pad of Celite. The pad was rinsed with EtOAc (50mL) and concentrated in vacuo. The crude material was bound to a silica gel column, purified using a 40g silica gel column, eluting the column with 20-40% EtOAc/hexane, isocratically maintained for 5 minutes, followed by a 40-100% EtOAc/hexane gradient for 20 minutes. The product-containing fractions were combined and concentrated to give 1- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-formyl-phenyl ] -3- (trityl-amino) -1H-pyrazole-4-carbonitrile (632mg, 37%).

And step 3: to 1- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-formyl-phenyl at 0 deg.C]To a solution of-3- (trityl-amino) -1H-pyrazole-4-carbonitrile (632mg, 0.965mmol) in dichloromethane (20mL) and methanol (10mL) was added sodium borohydride (37mg, 0.965 mmol). The resulting cloudy reaction mixture was stirred at 0 ℃ for 5 minutes and then at room temperature for 10 minutes. The reaction was then judged complete by TLC. The volatiles were removed in vacuo and the crude product was combined with silica gel, purified using 40g silica gel column, and purified using 20g silica gel_-Gradient elution column 50% EtOAc/hexanes to give 1- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl]-3- (trityl-amino) -1H-pyrazole-4-carbonitrile (482mg, 76%).

And 4, step 4: 1- (3- (6-tert-butyl-1-oxophthalazin-2 (1H) -yl) -2- (hydroxymethyl) phenyl) -3- (tritylamino) -1H-pyrazole-4-carbonitrile (382mg0.582mmol) was dissolved in Et2O (30mL) and cooled to 0 ℃. MeOH (2mL) was added, followed by saturated ethereal HCl (2mL, prepared by bubbling HCl gas into 100mL diethyl ether). The reaction mixture was stirred at 0 ℃ for 30 minutes, after which the reaction was judged to be complete by TLC. Volatiles were removed in vacuo and the residue was partitioned with saturated NaHCO₃The aqueous solution (100mL) was stirred vigorously with EtOAc (100 mL). The aqueous layer was separated and extracted with EtOAc (50 mL). With Na₂SO₄The combined organic layers were dried, filtered, and concentrated in vacuo. The oily residue was combined with silica gel and the crude product was purified on a 25g silica gel column, eluting the column with a 20-70% EtOAc/hexanes gradient to give 3-amino-1- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl]-1H-pyrazole-4-carbonitrile (178mg, 74%) as a white powdery solid.

And 5: 3-amino-1- (3- (6-tert-butyl) was introduced into a 5mL microwave reaction flaskButyl-1-oxophthalazin-2 (1H) -yl) -2- (hydroxymethyl) phenyl) -1H-pyrazole-4-carbonitrile (50mg, 0.121mmol), 1- (4-bromophenyl) ethanone (29mg, 0.145mmol), Pd₂(dba)₃(7.7mg, 0.008mmol), 2- (dicyclohexylphosphino) -3, 6-dimethoxy-2 '-4' -6 '-tri-isopropyl-1, 1' -biphenyl (Brett-Phos) (9.1mg, 0.017mmol), cesium carbonate (59mg, 0.181mmol) and suspended in t-butanol (1.5 mL). The reaction flask was inerted 3 times by alternating vacuum and Ar gas purges, then stirred at room temperature for 5 minutes, resulting in a heterogeneous mixture that changed color from purple to red-orange. The reaction was heated to 100 ℃ (external) during which the color of the reaction mixture became orange after 2 minutes of heating. After 1.5hr, the reaction was judged complete by TLC. The mixture was diluted with EtOAc, filtered through a pad of celite, and combined with silica gel. By 12_gPurifying the crude product with a silica gel column, eluting the column with a 30% to 100% EtOAc/hexanes gradient to provide 3- (4-acetyl-phenylamino) -1- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl]-1H-pyrazole-4-carbonitrile (40mg, 62%) as a white powder.

Step 6: the procedure was carried out in the same manner as in step 4 of example 22. Thus, 3- (4-acetyl-phenylamino) -1- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -1H-pyrazole-4-carbonitrile (114mg, 0.214mmol) gave crude formamide, which was purified on silica gel using a 12g Redisep silica gel column eluting with a 30% to 100% (20% MeOH/EtOAc)/hexane gradient to give 3- (4-acetyl-phenylamino) -1- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -1H-pyrazole-4-carboxamide (99mg, 84%, [ M + H ] +551) as a yellowish white powder.

Example 54

1- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -3- (pyridin-2-ylamino) -1H-pyrazole-4-carboxamide

Step 1: into a 5mL microwave reaction flask, 3-amino-1- (3- (6-tert-butyl-1-oxophthalazin-2 (1H) -yl) -2- (hydroxymethyl) phenyl) -1H-pyrazole-4-carbonitrile (25mg, 0.060mmol), 2-bromopyridine (11mg, 0.072mmol), Pd₂(dba)₃(3.9mg, 0.004mmol), 2- (dicyclohexylphosphino) -3, 6-dimethoxy-2 '-4' -6 '-tri-isopropyl-1, 1' -biphenyl (Brett-Phos) (4.5mg, 0.008mmol), cesium carbonate (29.5mg, 0.091mmol) and suspended in t-butanol (0.609 mL). The reaction flask was inerted 3 times by alternating vacuum and Ar gas purges, then stirred at room temperature for 5 minutes, resulting in a heterogeneous mixture that changed color from purple to red-orange. The reaction was heated to 100 ℃ (external) during which the color of the reaction mixture became orange after 2 minutes of heating. After 2hr, the reaction was judged complete by TLC. The mixture was diluted with EtOAc, filtered through a pad of celite, and combined with silica gel. The crude product was purified on a 12g RediSep silica gel column eluting with a 30% to 100% EtOAc/hexanes gradient to give 1- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl]-3- (pyridin-2-ylamino) -1H-pyrazole-4-carbonitrile (15mg, 51%) as a white powder.

Step 2: the procedure was carried out in the same manner as in step 4 of example 22. Thus, 1- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -3- (pyridin-2-ylamino) -1H-pyrazole-4-carbonitrile (15mg, 0.0305mmol) gave crude formamide, which was purified on silica gel using a 12g Redisep silica gel column eluting with a 30% to 100% (20% MeOH/EtOAc)/hexane gradient to give 1- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -3- (pyridin-2-ylamino) -1H-pyrazole-4-carboxamide (10mg, 67%, [ M + H ] +510) as a yellowish white powder containing 5-10% of amide by-product of pyridine hydrolysis.

Example 55

1- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -3- (5-dimethylaminomethyl-pyridin-2-ylamino) -1H-pyrazole-4-carboxamide

Step 1: into a 5mL microwave reaction flask, 3-amino-1- (3- (6-tert-butyl-1-oxophthalazin-2 (1H) -yl) -2- (hydroxymethyl) phenyl) -1H-pyrazole-4-carbonitrile (25mg, 0.06mmol), 1- (6-chloropyridin-3-yl) -N, N-dimethylmethylamine (13mg, 0.075mmol), and Pd₂(dba)₃(4mg, 0.004mmol), 2- (dicyclohexylphosphino) -3, 6-dimethoxy-2 '-4' -6 '-tri-isopropyl-1, 1' -biphenyl (Brett-Phos) (4.5mg, 0.008mmol), cesium carbonate (30mg, 0.091mmol) and suspended in t-butanol (0.609 mL). The reaction flask was inerted 3 times by alternating vacuum and Ar gas purges, then stirred at room temperature for 5 minutes, resulting in a heterogeneous mixture that changed color from purple to red-orange. The reaction was heated to 100 ℃ (external) during which the color of the reaction mixture became orange after 2 minutes of heating. After 4hr, the reaction was judged complete by TLC. The mixture was diluted with EtOAc, filtered through a pad of celite, and combined with silica gel. The crude product was purified on a 12g RediSep silica gel column eluting with a 30% to 100% EtOAc/hexanes gradient to give 1- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl]-3- (5-dimethylaminomethyl-pyridin-2-ylamino) -1H-pyrazole-4-carbonitrile (18mg, 54%) as a white powder.

Step 2. the same procedure as in step 4 of example 22 was followed. Thus, 1- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -3- (5-dimethylaminomethyl-pyridin-2-ylamino) -1H-pyrazole-4-carbonitrile (18mg, 0.0328mmol) gave crude formamide, which was purified on silica gel using a 12g RediseP silica gel column eluting with a 30% to 100% (20% MeOH/EtOAc) hexane gradient to give 1- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -3- (5-dimethylaminomethyl-pyridin-2-ylamino) -1H-pyrazole-4-carboxamide (10mg, 54%, [ M + H ] +567) as a yellowish white powder.

Example 56

1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -phenyl ] -1H-pyrazole-4-carboxamide

Step 1: 1- (3-bromo-2-formylphenyl) -1H-pyrazole-4-carbonitrile (100mg, 362. mu. mol) obtained in example 41 above was converted into 1- (3- (6-tert-butyl-8-fluoro-1-oxophthalazin-2 (1H) -yl) phenyl) -1H-pyrazole-4-carbonitrile (88mg, 63%) according to a similar manner to example 21, step 2.

Step 2: in a 25mL round bottom flask, 1- (3- (6-tert-butyl-8-fluoro-1-oxophthalazin-2 (1H) -yl) phenyl) -1H-pyrazole-4-carbonitrile (87mg, 225. mu. mol, Eq: 1.00) and hydrogen (dimethylphosphoric acid-kP) (4.82mg, 11.2. mu. mol, Eq: 0.05) were combined with ethanol (1mL) and water (1.00mL) to give a colorless solution. The reaction mixture was heated to 85 ℃ and stirred for 45 min. After the desired reaction time (inversion check my LCMS and TLC), the reaction was brought to room temperature and the solvent was removed in vacuo. Then filtered through a HPLC Teflon filter and lyophilized overnight by reverse phase HPLC to give 1- [3- (6-tert-butyl-8-fluoro-1-oxo-1H-phthalazin-2-yl) -phenyl ] -1H-pyrazole-4-carboxamide (49mg, 54%, [ M + H ] + 406).

Biological examples

Tyrosine kinase (Btk) inhibition assay

The assay is carried out by capturing radioactivity by filtration³³p phosphorylation product. Btk, biotinylated SH₂The interaction of the peptide substrate (Src homology) with ATP leads to phosphorylation of the peptide substrate. The biotinylated product is bound streptavidin agarose beads. All bound, radiolabeled products were detected by scintillation counter.

The plates assayed were 96-well polypropylene (Greiner) and 96-well 1.2 μm hydrophilic PVDF filter plates (Millipore). The concentrations reported here are finalAnd (3) measuring the concentration: 10-100. mu.M compound, 5-10nM Btk enzyme (His-tagged, full-length), 30. mu.M peptide substrate (biotin-Aca-AAAEEIYGEI-NH) in DMSO (Burdick and Jackson)₂) 100. mu.M ATP (Sigma), 8mM imidazole (Sigma, pH7.2), 8mM glycerol-2-phosphate (Sigma), 200. mu.M EGTA (Roche Diagnostics), 1mM MnCl₂(Sigma), 20mM MgCl₂(Sigma), 0.1 mg/ml BSA (Sigma), 2mM DTT (Sigma), 1. mu. Ci³³P ATP (Amersham), 20% streptavidin agarose beads (Amersham), 50mM EDTA (Gibco), 2M NaCl w/1% phosphoric acid (Gibco), microscint-20 (platinum Elmer).

IC was calculated from 10 data points per compound using data generated from standard 96-well plate assay templates₅₀And (d) measuring the value. One control compound and seven unknown inhibitors were tested on each plate, twice per plate. Typically, compounds are diluted in half log (half log) starting at 100 μ M and ending at 3 nM. The control compound was staurosporine. The background was counted in the absence of peptide substrate. In the presence of peptide substrate, the total activity was determined. The following method was used to determine Btk inhibition.

1) Sample preparation: test compounds were added in half log increments in assay buffer (imidazole, glycerol-2-phosphate, EGTA, MnCl)₂，MgCl₂BSA).

2) Bead preparation

a.) bead rinsing by centrifugation at 500g

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

3) The reaction mixture without substrate (assay buffer, DTT, ATP,³³p ATP) and a mixture with substrate (assay buffer, DTT, ATP,³³p ATP, peptide substrate) for 15 minutes.

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

5) To Btk and compound, 30 μ L of the reaction mixture containing no or substrate was added.

6) 50 μ L of the total assay mixture was incubated at 30 ℃ for 30 minutes.

7) The reaction was stopped by transferring 40. mu.L of assay to 150. mu.L of bead slurry in the filter plate.

8) After 30 minutes, the filter plate was washed with the following steps:

a.3x250μL NaCl

3 x250. mu. LNaCl containing 1% phosphoric acid

c.1x250μL H₂O

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

10) Add 50. mu.L microscint-20 and count on scintillation counter³³p cpm。

Percent activity was calculated from raw data expressed as cpm

Percent activity = (sample-bgg)/(total activity-bgg) x100

Calculation of IC from percent Activity Using a one-site dose-response sigmoidal model₅₀

y=A+((B-A)／(1+((x／C)^D))))

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

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

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

human Whole blood: (HWB) were obtained from healthy volunteers, satisfying the following limits: the preparation is not taken for 24hr, and is not suitable for smokers. Blood was collected by venipuncture into Vacutainer tubes anticoagulated with sodium heparin. Test compounds were diluted 10-fold to the desired initial drug concentration (20x) in PBS, followed by three-fold serial dilutions in 10% DMSO in PBS, resulting in a 9-point dose response curve. Mu.l of each compound dilution was added in duplicate to 2ml of 96 well V-bottomed plates (Analytical salts and Services, # 59623-23); to control and non-stimulated wells 5.5 μ l of 10% DMSO in PBS was added. HWB (100. mu.l) was added to each well and plates were mixed at 37C, 5% CO₂100% humidity incubation for 30 minutes. To each well (except for non-irritating wells) sheep F (ab') 2 anti-human IgM (Southern Biotech, #2022-14) (10 μ Ι of 500 μ g/ml solution, 50 μ g/ml final concentration) was added with stirring and the plates were incubated for an additional 20 hours.

At the end of the 20 hour incubation, the samples were mixed with fluorescent probe-labeled antibodies (15. mu.l PE mouse anti-human CD20, BD Pharmingen, #555623, and/or 20. mu.l APC mouse anti-human CD69, BD Pharmingen #555533) at 37C, 5% CO₂100% humidity incubation for 30 minutes. Including induced control, unstained and single stain for compensation of adjustments and initial voltage settings. The sample was then lysed with 1ml of 1X Pharmingen Lyse Buffer (BD Pharmingen #555899), and the plate was centrifuged at 1800rpm for 5 minutes. The supernatant was removed by aspiration, the remaining pellet was re-lysed with another 1ml of 1X Pharmingen Lyse Buffer, and the plate was centrifuged (spun down) as before. The supernatant was aspirated and the remaining pellet was washed in FACs buffer (PBS + 1% FBS). After the final spin, the supernatant was removed and the pellet was resuspended in 180. mu.l of FACs buffer. Samples were transferred to 96-well plates suitable for running on the HTS96 well system of BD LSR II flow cytometer.

Data were acquired using excitation and emission wavelengths appropriate to the fluorophore used and percentage positive Cell values were obtained using Cell Quest Software. The results were initially analyzed using FACS analysis software (Flow Jo). The IC50 of the test compound was defined as the concentration that reduced the percentage of CD69 positive cells by 50%, which CD69 positive cells were also CD20 positive after stimulation with anti-IgM (average of 8 control wells after subtraction of the average of 8 wells without a stimulating background). The IC50 value was calculated using XLfit software version 3, equation 201.

Data for representative compounds of this assay are listed in table II below.

Table II.

B-inhibition of cell activation-B cell FLlPR assay in Ramos cells

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

The B cell FLIPR assay is a cell-based functional method to determine the effect of potential inhibitors on intracellular calcium increase caused by stimulation with anti-IgM antibodies. Ramos cells (human Burkitt's lymphoma cell line. ATCC-No. CRL-1596) were cultured in growth medium (described below). One day prior to assay, Ramos cells were resuspended in fresh growth medium (same as above) and at 0.5x10⁶The concentration of/mL was placed in a tissue culture flask. On the day of assay, cells were counted and counted at 1 × 10⁶Perml concentration into tissue culture flasks supplemented with 1. mu. MFLUO_-3AM (TefLabs Cat No. 0116, prepared in anhydrous DMSO and 10% block polyetheracid (Pluronic acid)) in growth medium and at 37 deg.C (4% CO)₂) Incubate for 1 hour. To get rid ofCells were harvested by centrifugation (5 min, 1000rpm) except for extracellular dye and at 1 × 10⁶cells/mL were resuspended in FLIPR buffer (described below) followed by 1 × 10⁵Cells/well were distributed in 96-well poly-D-lysine coated black/clear plates (BD catalog No. 356692). Test compounds were added at various concentrations ranging from 100 μ M to 0.03 μ M (7 concentrations, detailed below) and incubated with the cells for 30 minutes at room temperature. Ramos cells Ca were stimulated by addition of 10. mu.g/mL anti-IgM (Southern Biotech, Cat. No. 2020-01)²⁺Signals were conducted and measured on a FLIPR (Molecular Devices, using a CCD camera with an argon laser excited at 480nM to capture images of 96-well plates).

Medium/buffer:

growth medium: RPMI1640 medium containing L-glutamine (Invitrogen, Cat. No. 61870-010), 10% fetal bovine serum (FBS, Summit Biotechnology Cat. No. FP-100-05); 1mM sodium pyruvate (Invitrogen Cat No. 11360-070).

FLIPR buffer: HBSS (Invitrogen, catalog No. 141175-079), 2mMCaCl₂(Sigma, Cat. No. C-4901), HEPES (Invitrogen, Cat. No. 15630-080), 2.5mM probenecid (Sigma, Cat. No. P-8761), 0.1% BSA (Sigma, Cat. No. A-7906), 11mM glucose (Sigma, Cat. No. G-7528).

Compound dilution details:

to obtain the highest final assay concentration of 100. mu.M, 24. mu.L of a 10mM stock solution of the compound (prepared in DMSO) was added directly to 576. mu.L of FLIPR buffer. Test compounds were diluted in FLIPR buffer (using Biomek2000 auto pipettor) to give the following dilution scheme: solvent, 1.00x10^-4M，1.00x10^-5，3.16x10^-6，1.00x10^-6，3.16x10^-7，1.00x10^-7，3.16x10^-8。

Determination and analysis：

The most usedLarge-minimum statistics (using Molecular Devices FLIPR controls and statistical output software, subtracting the resting baseline from the peak resulting from addition of the stimulating antibody) report intracellular increases in calcium. IC determination using non-linear curve fitting (GraphPad Prism software)₅₀。

Mouse collagen-induced arthritis (mCIA)

On day 0, mice were injected with an emulsion of type II collagen in Complete Freund's Adjuvant (CFA), injected intradermally (i.d.) at several sites at the base of the tail or on the back. Following collagen immunization, animals will develop arthritis between about 21 and 35 days. The onset of arthritis was synchronized (enhanced) by systemic administration of collagen in incomplete Freund's adjuvant (IFA; i.d.) on day 21. Animals were examined daily after day 20 for any onset of mild arthritis (score 1 or 2; described with reference to the scores below), which was an enhanced signal. After boost, mice are scored and the candidate therapeutic agent is dosed for a predetermined time (typically 2-3 weeks) and dosing frequency, once daily (QD) or twice daily (BID).

Rat aminoprotein _- Induced arthritis (rCIA)

On day 0, rats were injected with an emulsion of type II bovine collagen in Incomplete Freund's Adjuvant (IFA), injected intradermally (i.d.) at several sites on the back. A booster injection of collagen emulsion was provided at an alternate site on the base or back of the tail on about day 7 (i.d.). Arthritis is usually observed 12-14 days after the initial collagen injection. From day 14 onwards, the progression of arthritis in the animals can be evaluated as described below (evaluation of arthritis). Animals are dosed with the candidate therapeutic agent in a prophylactic manner, starting at the time of the second challenge, and at a predetermined time (typically 2-3 weeks) and dosing frequency, once daily (QD) or twice daily (BID).

Evaluation of arthritis：

In both models, the inflammatory development of the paw and limb joints was quantified using a scoring system comprising the evaluation of 4 paws according to the criteria described below:

and (3) scoring: 1= swelling and/or redness of the paw or one toe.

2= two or more joints swelling.

3= total swelling of the paw, involving more than two joints.

4= severe arthritis of the entire paw and toe.

The evaluation was performed as follows: baseline measurements were taken on day 0, starting again at the first sign or swelling, up to three times per week until the end of the experiment. The arthritis index was obtained for each mouse by summing the four single paw scores, with a maximum score of 16 for each animal.

Rat in vivo asthma model

Male Brown Norway (Brown-Norway) rats were sensitized i.p. once a week for three weeks (days 0, 7 and 14) with 100 μ gOA (ovalbumin) in 0.2ml alum. On day 21 (one week after the last sensitization), rats were dosed subcutaneously with a q.d. dose of solvent or compound formulation, with OA aerosol challenge (1% OA for 45 minutes) after 0.5 hours and terminated 4 or 24 hours after challenge. At sacrifice, serum and plasma were collected from all animals for serological studies and PK, respectively. The tracheal tube was inserted and the lungs were lavaged 3X with PBS. BAL fluid was analyzed for total and differential white blood cell counts. The total white blood cell count in cell aliquots (20-100. mu.l) was determined by a Coulter Counter. For differential white blood cell counts, 50-200. mu.l of the sample was centrifuged in Cytospin and the slides stained with Diff-Quik. The proportion of monocytes, eosinophils, neutrophils and lymphocytes was counted under a light microscope using standard morphological criteria and expressed as a percentage. Representative inhibitors of Btk show: total leukocyte counts in BAL of OA sensitized and challenged rats were decreased compared to control levels.

The foregoing invention has been described in some detail by way of illustration and example for purposes of illustration and understanding. It will be apparent to those skilled in the art that changes and modifications may be made within the scope of the appended claims. Accordingly, it is to be understood that the above description is intended to be illustrative, and not restrictive. The scope of the invention should, therefore, be determined not with reference to the above description, but instead should be determined with reference to the 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 herein incorporated by reference in their entirety for all purposes to the same extent as if each patent, patent application, or publication were individually indicated to be so incorporated by reference.

Claims

1. A compound of the formula I,

wherein:

is a double bond;

a is pyrazolyl substituted with one or more A';

a' is-NHR;

r is-R¹、-R¹-R²-R³、-R¹-R³；

R¹Is aryl, heteroaryl, or heteroaryl fused with heterocycloalkyl, each of which is optionally substituted with one or more R^1’Or R^1”Substitution;

R^1’each independently is halogen, cyano;

R^1”each independently is C_1-6Alkyl radical, C_1-6Alkoxy, each of which is optionally substituted by one or more R^1”’Substitution;

R^1”’each independently is hydroxy, halogen, or heterocycloalkyl;

R²is-C (═ O), or-S (═ O)₂；

R³Is H or R⁴；

R⁴Is C_1-6Alkyl radical, C_1-6Haloalkyl, C_1-6Alkoxy radical, C_1-6Alkylamino, heterocycloalkyl, (C)_1-6Alkyl) heterocycloalkyl, each of which is optionally substituted by one or more C_1-6Alkyl, halogen, di (C)_1-6Alkyl) amino, hydroxy C_1-6Alkyl substitution of which two are C_1-6The alkyl groups may together form a ring;

q is CH;

x is N;

Y⁰is H;

Y¹is Y^1b；

Y^1bIs tert-butyl or isopropyl;

Y²is H;

Y³is H or F; and is

Y⁴Is H or C_1-6A hydroxyalkyl group;

wherein heteroaryl means a monocyclic or bicyclic group of 5 to 12 ring atoms having at least one aromatic ring, each ring containing 4 to 8 atoms, to which is bonded one or more N, O or S heteroatoms, the remaining ring atoms being carbon, wherein the point of attachment of the heteroaryl is on the aromatic ring; heterocyclic ringsAlkyl represents a monovalent saturated cyclic group consisting of 1 to 2 rings of 3 to 8 atoms per ring to which one or more atoms selected from N, O or S (O)_0-2A ring heteroatom of (a);

or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1, wherein Y is⁰Is H, Y²Is H, and Y⁴Is hydroxymethyl.

3. The compound of claim 1, wherein Y is¹Is a tert-butyl group.

4. A compound according to any one of claims 1 to 3, wherein Y is³Is F.

5. A compound selected from:

1- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -3- (pyridin-2-ylamino) -1H-pyrazole-4-carboxamide; and

1- [3- (6-tert-butyl-1-oxo-1H-phthalazin-2-yl) -2-hydroxymethyl-phenyl ] -3- (5-dimethylaminomethyl-pyridin-2-ylamino) -1H-pyrazole-4-carboxamide.

6. A pharmaceutical composition comprising a compound according to any one of claims 1 to 5 together with at least one pharmaceutically acceptable carrier, excipient or diluent.

7. Use of a compound according to any one of claims 1 to 5 in the manufacture of a medicament for the treatment of an inflammatory and/or autoimmune disorder.