WO2023155886A1

WO2023155886A1 - Pyrazolopyridine compounds as tam inhibitors

Info

Publication number: WO2023155886A1
Application number: PCT/CN2023/076794
Authority: WO
Inventors: Bin Jiang; Ming Zhang
Original assignee: Shanghai Antengene Corporation Limited; Antengene Discovery Limited
Priority date: 2022-02-17
Filing date: 2023-02-17
Publication date: 2023-08-24

Abstract

The present disclosure relates to pyrazolopyridine compounds useful as inhibitors of TAM kinases, in particular as intibitors of AXL and MER kinases, as well as pharmaceutical compositions comprising these compounds and methods of treatment by administration of these compounds or the pharmaceutical compositions.

Description

PYRAZOLOPYRIDINE COMPOUNDS AS TAM INHIBITORS

FIELD OF THE DISCLOSURE

The present disclosure generally relates to pyrazolopyridine compounds useful as inhibitors of TAM kinases, in particular as intibitors of AXL and MER kinases, as well as pharmaceutical compositions comprising these compounds and methods of treatment by administration of these compounds or the pharmaceutical compositions.

BACKGROUND OF THE DISCLOSURE

Receptor tyrosine kinases (RTK) are cell-surface transmembrane receptors that contain regulated kinase activity within their cytoplasmic domain and play an important role in signal transduction in both normal and malignant cells. Overexpression or ectopic expression of the TAM receptors has been detected in a wide array of human cancers.

AXL and MER are two members of the TAM (TYRO3-AXL-MER) family of receptor tyrosine kinases, which, when activated, can regulate tumor cell survival, proliferation, migration and invasion, angiogenesis, and tumor-host interactions. An increasing body of evidence strongly suggests that these receptors play major roles in resistance to targeted therapies and conventional cytotoxic agents. In addition, it has been shown that both AXL and MER are critical regulators of innate immunity, phagocytosis, and immune-suppressive activity. Therefore, targeting both AXL and MER kinases may not only impact the growth, survival and malignant progression of neoplastic cells directly, but also has the potential to restore and enhance host immunity against cancers.

Accordingly, there is a need for compounds for inhibition of TAM kinases, in particular AXL and MER kinases.

SUMMARY OF THE DISCLOSURE

The present disclosure provides compounds or pharmaceutically acceptable salts thereof, which are capable of inhibiting TAM kinases, in particular AXL and MER kinases. Methods for use of such compounds for treatment of various diseases or conditions, such as cancer, are also provided.

In one aspect, the present disclosure provides a compound having Formula (II) :

or a pharmaceutical acceptable salt thereof,

wherein

Ring A is phenyl or 6-membered heteroaryl comprising 1 or 2 nitrogen atoms;

Ring B is phenyl or 6-membered heteroaryl comprising 1 or 2 nitrogen atoms, each of which is optionally substituted with one or two R²;

Ring C is cycloalkyl, heterocyclyl, aryl or heteroaryl, each of which is optionally substituted with one or more R^a;

W is CH, N, O, or S;

R¹ is cycloalkyl, heterocyclyl, aryl or heteroaryl, each of which is optionally substituted with one or more R^b;

R² is halogen, hydroxyl, cyano, amino or alkyl;

R³ is null, hydrogen, oxo, alkyl or haloalkyl;

R⁴ is NH₂ or haloalkyl;

each R^a is independently selected from the group consisting of hydrogen, halogen, hydroxyl, cyano, amino, alkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, and –C (=O) R^c; or

two adjacent R^a together with the atoms they attached form a cycloalkyl or heterocyclyl;

each R^b is independently selected from oxo, hydroxyl, alkyl, alkoxyl, haloalkyl, haloalkoxyl, cycloalkyl, heterocyclyl, aryl, heteroaryl or –C (O) NH-R^c, wherein the cycloalkyl, heterocyclyl, aryl, and heteroaryl are optionally substituted with one or more groups independently selected from halogen, hydroxyl, cyano, amino or alkyl; and

R^c is alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein the cycloalkyl, heterocyclyl, aryl, and heteroaryl are optionally substituted with one or more groups independently selected from halogen, hydroxyl, cyano, amino or alkyl.

In a further aspect, the present disclosure provides a compound selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.

In another aspect, the present disclosure provides a pharmaceutical composition comprising the compound of the present disclosure or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

In a further aspect, the present disclosure provides a method for inhibiting a TAM kinase, comprising contacting the TAM kinase with the compound of the present disclosure or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of the present disclosure.

In a further aspect, the present disclosure provides a method for inhibiting AXL and MER kinases, comprising contacting the AXL and MER kinases with the compound of the present disclosure or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of the present disclosure.

In a further aspect, the present disclosure provides a method for treating a disease or disorder mediated by TAM in a subject in need thereof, comprising administering an effective amount of the compound of the present disclosure or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of the present disclosure to the subject.

DETAILED DESCRIPTION OF THE DISCLOSURE

Reference will now be made in detail to certain embodiments of the present disclosure, examples of which are illustrated in the accompanying structures and formulas. While the present disclosure will be described in conjunction with the enumerated embodiments, it will be understood that they are not intended to limit the present disclosure to those embodiments. On the contrary, the present disclosure is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present disclosure as defined by the claims. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present disclosure. The present disclosure is in no way limited to the methods and materials described. In the event that one or more of the incorporated references and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, the present disclosure controls. All references, patents, patent applications cited in the present disclosure are hereby incorporated by reference in their entireties.

It is appreciated that certain features of the present disclosure, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the present disclosure, which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable sub-combination. It must be noted that, as used in the specification and the appended claims, the singular forms “a, ” “an, ” and “the” include plural forms of the same unless the context clearly dictates otherwise. Thus, for example, reference to “acompound” includes a plurality of compounds.

Definitions

Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75^th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, 2^nd Edition, University Science Books, Sausalito, 2006; Smith and March March’s Advanced Organic Chemistry, 6^th Edition, John Wiley &Sons, Inc., New York, 2007; Larock, Comprehensive Organic Transformations, 3^rd Edition, VCH Publishers, Inc., New York, 2018; Carruthers, Some Modern Methods of Organic Synthesis, 4^th Edition, Cambridge University Press, Cambridge, 2004; the entire contents of each of which are incorporated herein by reference.

At various places in the present disclosure, linking substituents are described. It is specifically intended that each linking substituent includes both the forward and backward forms of the linking substituent. For example, -NR (CR’R”) -includes both -NR (CR’R”) -and - (CR’R”) NR-. Where the structure clearly requires a linking group, the Markush variables listed for that group are understood to be linking groups. For example, if the structure requires a linking group and the Markush group definition for that variable lists “alkyl” , then it is understood that the “alkyl” represents a linking alkylene group.

When a bond to a substituent is shown to cross a bond connecting two atoms in a ring, then such substituent may be bonded to any atom in the ring. When a substituent is listed without indicating the atom via which such substituent is bonded to the rest of the compound of a given formula, then such substituent may be bonded via any atom in such formula. Combinations of substituents and/or variables are permissible, but only if such combinations result in stable compounds.

When any variable (e.g., Rⁱ) occurs more than one time in any constituent or formula for a compound, its definition at each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is shown to be substituted with 0-2 Rⁱ moieties, then the group may optionally be substituted with up to two Rⁱ moieties and Rⁱ at each occurrence is selected independently from the definition of Rⁱ. Also, combinations of substituents and/or variables are permissible, but only if such combinations result in stable compounds.

As used herein, the term “C_i-j” indicates a range of the carbon atoms numbers, wherein i and j are integers and the range of the carbon atoms numbers includes the endpoints (i.e. i and j) and each integer point in between, and wherein j is greater than i. For examples, C_1-6 indicates a range of one to six carbon atoms, including one carbon atom, two carbon atoms, three carbon atoms, four carbon atoms, five carbon atoms and six carbon atoms. In some embodiments, the term “C_1-12” indicates 1 to 12, particularly 1 to 10, particularly 1 to 8, particularly 1 to 6, particularly 1 to 5, particularly 1 to 4, particularly 1 to 3 or particularly 1 to 2 carbon atoms.

As used herein, the term “alkyl” , whether as part of another term or used independently, refers to a saturated linear or branched-chain hydrocarbon radical, which may be optionally substituted independently with one or more substituents described below. The term “C_i-j alkyl” refers to an alkyl having i to j carbon atoms. In some embodiments, alkyl groups contain 1 to 10 carbon atoms. In some embodiments, alkyl groups contain 1 to 9 carbon atoms. In some embodiments, alkyl groups contain 1 to 8 carbon atoms, 1 to 7 carbon atoms, 1 to 6 carbon atoms, 1 to 5 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms. Examples of “C_1-10 alkyl” include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl. Examples of “C_1-6 alkyl” are methyl, ethyl, propyl, isopropyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2, 3-dimethyl-2-butyl, 3, 3-dimethyl-2-butyl, and the like.

As used herein, the term “alkoxyl” , whether as part of another term or used independently, refers to an alkyl group, as previously defined, attached to the parent molecule through an oxygen atom. The term “C_i-j alkoxy” means that the alkyl moiety of the alkoxy group has i to j carbon atoms. In some embodiments, alkoxy groups contain 1 to 10 carbon atoms. In some embodiments, alkoxy groups contain 1 to 9 carbon atoms. In some embodiments, alkoxy groups contain 1 to 8 carbon atoms, 1 to 7 carbon atoms, 1 to 6 carbon atoms, 1 to 5 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms. Examples of “C_1-6 alkoxyl” include, but are not limited to, methoxy, ethoxy, propoxy (e.g. n-propoxy and isopropoxy) , t-butoxy, neopentoxy, n-hexoxy, and the like.

As used herein, the term “amino” refers to –NH₂ group. Amino groups may also be substituted with one or more groups such as alkyl, aryl, carbonyl or other amino groups.

As used herein, the term “aryl” , whether as part of another term or used independently, refers to monocyclic and polycyclic ring systems having a total of 5 to 20 ring members, wherein at least one ring in the system is aromatic and wherein each ring in the system contains 3 to 12 ring members. Examples of “aryl” include, but are not limited to, phenyl, biphenyl, naphthyl, anthracyl and the like, which may bear one or more substituents. Also included within the scope of the term “aryl” , as it is used herein, is a group in which an aromatic ring is fused to one or more additional rings. In the case of polycyclic ring system, only one of the rings needs to be aromatic (e.g., 2, 3-dihydroindole) , although all of the rings may be aromatic (e.g., quinoline) . The second ring can also be fused or bridged. Examples of polycyclic aryl include, but are not limited to, benzofuranyl, indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, and the like. Aryl groups can be substituted at one or more ring positions with substituents as described above.

As used herein, the term “cycloalkyl” , whether as part of another term or used independently, refer to a monovalent non-aromatic, saturated or partially unsaturated monocyclic and polycyclic ring system, in which all the ring atoms are carbon and which contains at least three ring forming carbon atoms. In some embodiments, the cycloalkyl may contain 3 to 12 ring forming carbon atoms, 3 to 10 ring forming carbon atoms, 3 to 9 ring forming carbon atoms, 3 to 8 ring forming carbon atoms, 3 to 7 ring forming carbon atoms, 3 to 6 ring forming carbon atoms, 3 to 5 ring forming carbon atoms, 4 to 12 ring forming carbon atoms, 4 to 10 ring forming carbon atoms, 4 to 9 ring forming carbon atoms, 4 to 8 ring forming carbon atoms, 4 to 7 ring forming carbon atoms, 4 to 6 ring forming carbon atoms, 4 to 5 ring forming carbon atoms. Cycloalkyl groups may be saturated or partially unsaturated. Cycloalkyl groups may be substituted. In some embodiments, the cycloalkyl group may be a saturated cyclic alkyl group. In some embodiments, the cycloalkyl group may be a partially unsaturated cyclic alkyl group that contains at least one double bond or triple bond in its ring system. In some embodiments, the cycloalkyl group may be monocyclic or polycyclic. Examples of monocyclic cycloalkyl group include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl and cyclododecyl. Examples of polycyclic cycloalkyl group include, but are not limited to, adamantyl, norbornyl, fluorenyl, spiro-pentadienyl, spiro [3.6] -decanyl, bicyclo [1, 1, 1] pentenyl, bicyclo [2, 2, 1] heptenyl, and the like.

As used herein, the term “cyano” refers to –CN.

As used herein, the term “halogen” refers to an atom selected from fluorine (or fluoro) , chlorine (or chloro) , bromine (or bromo) and iodine (or iodo) .

As used herein, the term “haloalkyl” refers to alkyl as defined herein substituted with one or more halogen atoms.

As used herein, the term “haloalkoxyl” refers to alkoxyl as defined herein substituted with one or more halogen atoms.

As used herein, the term “heteroatom” refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen (including N-oxides) .

As used herein, the term “heteroaryl” , whether as part of another term or used independently, refers to an aryl group having, in addition to carbon atoms, one or more heteroatoms. The heteroaryl group can be monocyclic. Examples of monocyclic heteroaryl include, but are not limited to, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, benzofuranyl and pteridinyl. The heteroaryl group also includes polycyclic groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring. Examples of polycyclic heteroaryl include, but are not limited to, indolyl, isoindolyl, benzothienyl, benzofuranyl, benzo [1, 3] dioxolyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, dihydroquinolinyl, dihydroisoquinolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.

As used herein, the term “heterocyclyl” refers to a saturated or partially unsaturated carbocyclyl group in which one or more ring atoms are heteroatoms independently selected from oxygen, sulfur, nitrogen, phosphorus, and the like, the remaining ring atoms being carbon, wherein one or more ring atoms may be optionally substituted independently with one or more substituents. In some embodiments, the heterocyclyl is a saturated heterocyclyl. In some embodiments, the heterocyclyl is a partially unsaturated heterocyclyl having one or more double bonds in its ring system. In some embodiments, the heterocyclyl may contains any oxidized form of carbon, nitrogen or sulfur, and any quaternized form of a basic nitrogen. “Heterocyclyl” also includes radicals wherein the heterocyclyl radicals are fused with a saturated, partially unsaturated, or fully unsaturated (i.e., aromatic) carbocyclic or heterocyclic ring. The heterocyclyl radical may be carbon linked or nitrogen linked where such is possible. In some embodiments, the heterocycle is carbon linked. In some embodiments, the heterocycle is nitrogen linked. For example, a group derived from pyrrole may be pyrrol-1-yl (nitrogen linked) or pyrrol-3-yl (carbon linked) . Further, a group derived from imidazole may be imidazol-1-yl (nitrogen linked) or imidazol-3-yl (carbon linked) .

In some embodiments, the term “3-to 12-membered heterocyclyl” refers to a 3-to 12-membered saturated or partially unsaturated monocyclic or polycyclic heterocyclic ring system having 1 to 3 heteroatoms independently selected from nitrogen, oxygen, or sulfur. The fused, spiro and bridged ring systems are also included within the scope of this definition. Examples of monocyclic heterocyclyl include, but are not limited to oxetanyl, 1, 1-dioxothietanylpyrrolidyl, tetrahydrofuryl, tetrahydrothienyl, pyrrolyl, furanyl, thienyl, pyrazolyl, imidazolyl, triazolyl, oxazolyl, thiazolyl, piperidyl, piperazinyl, piperidinyl, morpholinyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, pyridonyl, pyrimidonyl, pyrazinonyl, pyrimidonyl, pyridazonyl, pyrrolidinyl, triazinonyl, and the like. Examples of fused heterocyclyl include, but are not limited to, phenyl fused ring or pyridinyl fused ring, such as quinolinyl, isoquinolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, quinoxalinyl, quinolizinyl, quinazolinyl, azaindolizinyl, pteridinyl, chromenyl, isochromenyl, indolyl, isoindolyl, indolizinyl, indazolyl, purinyl, benzofuranyl, isobenzofuranyl, benzimidazolyl, benzothienyl, benzothiazolyl, carbazolyl, phenazinyl, phenothiazinyl, phenanthridinyl, imidazo [1, 2-a] pyridinyl, [1, 2, 4] triazolo [4, 3-a] pyridinyl, [1, 2, 3] triazolo [4, 3-a] pyridinyl groups, and the like. Examples of spiro heterocyclyl include, but are not limited to, spiropyranyl, spirooxazinyl, diazaspiro [3.3] heptanyl and the like. Examples of bridged heterocyclyl include, but are not limited to, morphanyl, hexamethylenetetraminyl, 3-aza-bicyclo [3.1.0] hexane, 8-aza-bicyclo [3.2.1] octane, 1-aza-bicyclo [2.2.2] octane, 1, 4-diazabicyclo [2.2.2] octane (DABCO) , and the like.

As used herein, the term “hydroxyl” or “hydroxy” refers to –OH.

As used herein, the term “oxo” refers to =O substituent.

As used herein, the term “partially unsaturated” refers to a radical that includes at least one double or triple bond. The term “partially unsaturated” is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aromatic (i.e., fully unsaturated) moieties.

As used herein, the term “substituted” , whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and that the substitution results in a stable or chemically feasible compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. It will be understood by those skilled in the art that substituents can themselves be substituted, if appropriate. Unless specifically stated as “unsubstituted” , references to chemical moieties herein are understood to include substituted variants. For example, reference to an “aryl” group or moiety implicitly includes both substituted and unsubstituted variants.

Compounds

The present disclosure provides a novel compound of Formula (I) and pharmaceutically acceptable salts thereof, synthetic methods for making the compound, pharmaceutical compositions and various uses of the disclosed compounds.

In one aspect, the present disclosure provides a compound having Formula (II) :

or a pharmaceutical acceptable salt thereof,

wherein

Ring A is phenyl or 6-membered heteroaryl comprising 1 or 2 nitrogen atoms;

W is CH, N, O, or S;

R² is halogen, hydroxyl, cyano, amino or alkyl;

R³ is null, hydrogen, oxo, alkyl or haloalkyl;

R⁴ is NH₂ or haloalkyl;

each R^a is independently selected from the group consisting of hydrogen, oxo, halogen, hydroxyl, cyano, amino, alkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, and –C (=O) R^c; or

In some embodiments, Ring A is phenyl.

In some embodiments, Ring A is a 6-membered heteroaryl comprising 1 nitrogen atom.

In certain embodiments, is selected from the group consisting of :

In some embodiments, W is N.

In some embodiments, W is O or S.

In some embodiments, W is CH.

In some embodiments, Ring B is phenyl.

In some embodiments, Ring B is 6-membered heteroaryl comprising 1 or 2 nitrogen atoms. In certain embodiments, Ring B is pyridinyl.

In some embodiments, R¹ is cycloalkyl optionally substituted with one or more R^b. In certain embodiments, R¹ is C_3-8 cycloalkyl, C_3-7 cycloalkyl, C_3-6 cycloalkyl, or C_3-5 cycloalkyl, each optionally substituted with one or more R^b. In certain embodiments, R¹ is cyclopropyl optionally substituted with one or more R^b.

In some embodiments, R¹ is heterocyclyl optionally substituted with one or more R^b. In certain embodiments, R¹ is C_3-8 heterocyclyl, C_3-7 heterocyclyl, C_3-6 heterocyclyl, or C_3-5 heterocyclyl, each optionally substituted with one or more R^b.

In some embodiments, each R^b is independently selected from oxo, hydroxyl, alkyl, alkoxyl, haloalkyl, haloalkoxyl, aryl, heteroaryl or –C (O) NH-R^c, wherein the aryl and heteroaryl are optionally substituted with one or more groups independently selected from halogen, hydroxyl, or cyano.

In some embodiments, R¹ is cycloalkyl optionally substituted with one or more R^b, R^b is –C (O) NH-R^c, and R^c is aryl or heteroaryl, wherein the aryl and heteroaryl are optionally substituted with one or more groups independently selected from halogen, hydroxyl, cyano, amino or alkyl.

In some embodiments, R¹ is heterocyclyl optionally substituted with one or more R^b, each R^b is independently selected from oxo, alkyl, alkoxyl, aryl, or heteroaryl, wherein the aryl and heteroaryl are optionally substituted with one or more groups independently selected from halogen, hydroxyl, cyano, amino or alkyl.

In some embodiments, R¹ is selected from the group consisting of:

In some embodiments, R² is halogen. In certain embodiments, R² is fluoro or chloro. In certain embodiments, R² is fluoro.

In some embodiments, R² is alkyl. In certain embodiments, R² is methyl.

In some embodiments, R² is haloalkyl. In certain embodiments, R² is trifluoromethyl.

In some embodiments, Ring C is heterocyclyl or aryl, each of which is optionally substituted with one or more R^a.

In some embodiments, Ring C is 6-membered heterocyclyl optionally substituted with one or more R^a.

In certain embodiments, Ring C is piperidinyl optionally substituted with one or more R^a. In certain embodiments, Ring C is 4-piperidinyl optionally substituted with one or more R^a.

In certain embodiments, Ring C is piperazinyl optionally substituted with one or more R^a. In certain embodiments, Ring C is 4-piperazinyl optionally substituted with one or more R^a.

In certain embodiments, Ring C is 7-to 9-membered heterocyclyl optionally substituted with one or more R^a.

In certain embodiments, Ring C is octahydroindolizinyl, octahydropyrrolo [1, 2-a] pyrazinyl, or 2, 6-diazaspiro [3.3] heptanyl, each optionally substituted with one or more R^a.

In certain embodiments, each R^a is independently selected from the group consisting of hydrogen, oxo, halogen, hydroxyl, cyano, amino, alkyl, and –C (=O) R^c. In certain embodiments, R^c is alkyl, C_3-8 cycloalkyl, 3-8 membered heterocyclyl or 5-6 membered heteroaryl.

In certain embodiments, each R^a is independently selected from the group consisting of hydrogen, halogen, hydroxyl, cyano, amino, alkyl, and –C (=O) R^c. In certain embodiments, R^c is alkyl, C_3-8 cycloalkyl, 3-8 membered heterocyclyl or 5-6 membered heteroaryl.

In embodiments, each R^a is independently selected from the group consisting of hydrogen, oxo, alkyl, and –C (=O) R^c.

In some embodiments, Ring C is 5-6 membered aryl optionally substituted with one or more R^a.

In certain embodiments, Ring C is phenyl optionally substituted with one or more R^a.

In certain embodiments, each R^a is independently selected from the group consisting of halogen, cyano, alkyl, C_3-8 cycloalkyl and 5-6 membered heteroaryl.

In certain embodiments, two adjacent R^a together with the atom they attached form C_5-8 cycloalkyl or 5-8 membered heterocyclyl.

In certain embodiments, Ring C is selected from the group consisting of:

In some embodiment, R³ is null.

In some embodiments, R³ is hydrogen.

In some embodiments, R³ is oxo.

In some embodiments, R³ is alkyl. In certain embodiments, R³ is C_1-6 alkyl, C_1-5 alkyl, C_1-4 alkyl or C_1-3 alkyl. In certain embodiments, R³ is methyl or ethyl.

In some embodiments, R³ is haloalkyl. In certain embodiments, R³ is C_1-6 haloalkyl, C_1-5 haloalkyl, C_1-4 haloalkyl or C_1-3 haloalkyl. In certain embodiments, R³ is difluoromethyl, trifluoroethyl or difluoroethyl.

In some embodiments, R⁴ is NH₂.

In some embodiments, R⁴ is haloalkyl. In certain embodiments, R⁴ is difluoromethyl.

In one aspect, the present disclosure provides a compound selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.

Exemplary compounds of the present disclosure are set forth in Table 1 below.

Table 1

The compound of the present disclosure may exist in a number of different forms or derivatives, including but not limited to prodrugs, soft drugs, active metabolic derivatives (active metabolites) , and their pharmaceutically acceptable salts, all within the scope of the present disclosure.

As used herein, the term “prodrugs” refers to compounds or pharmaceutically acceptable salts thereof which, when metabolized under physiological conditions or when converted by solvolysis, yield the desired active compound. Prodrugs include, without limitation, esters, amides, carbamates, carbonates, ureides, solvates, or hydrates of the active compound. Typically, the prodrug is inactive, or less active than the active compound, but may provide one or more advantageous handling, administration, and/or metabolic properties. For example, some prodrugs are esters of the active compound; during metabolysis, the ester group is cleaved to yield the active drug. Also, some prodrugs are activated enzymatically to yield the active compound, or a compound which, upon further chemical reaction, yields the active compound. Prodrugs may proceed from prodrug form to active form in a single step or may have one or more intermediate forms which may themselves have activity or may be inactive. Preparation and use of prodrugs is discussed in T. Higuchi and V. Stella, “Pro-drugs as Novel Delivery Systems” , Vol. 14 of the A.C.S. Symposium Series, in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987; in Prodrugs: Challenges and Rewards, ed. V. Stella, R. Borchardt, M. Hageman, R. Oliyai, H. Maag, J. Tilley,Springer-Verlag New York, 2007, all of which are hereby incorporated by reference in their entirety.

As used herein, the term “soft drug” refers to compounds that exert a pharmacological effect but break down to inactive metabolites degradants so that the activity is of limited time. See, for example, “Soft drugs: Principles and methods for the design of safe drugs” , Nicholas Bodor, Medicinal Research Reviews, Vol. 4, No. 4, 449-469, 1984, which is hereby incorporated by reference in its entirety.

As used herein, the term “metabolite” , e.g., active metabolite overlaps with prodrug as described above. Thus, such metabolites are pharmacologically active compounds or compounds that further metabolize to pharmacologically active compounds that are derivatives resulting from metabolic process in the body of a subject. For example, such metabolites may result from oxidation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, enzymatic cleavage, and the like, of the administered compound or salt or prodrug. Of these, active metabolites are such pharmacologically active derivative compounds. For prodrugs, the prodrug compound is generally inactive or of lower activity than the metabolic product. For active metabolites, the parent compound may be either an active compound or may be an inactive prodrug.

Prodrugs and active metabolites may be identified using routine techniques know in the art. See, e.g., Bertolini et al, 1997, J Med Chem 40: 2011-2016; Shan et al., J Pharm Sci 86: 756-757; Bagshawe, 1995, DrugDev Res 34: 220-230; Wermuth, supra.

As used herein, the term “pharmaceutically acceptable” indicates that the substance or composition is compatible chemically and/or toxicologically, with the other ingredients comprising a formulation, and/or the subjects being treated therewith.

As used herein, the term “pharmaceutically acceptable salt” , unless otherwise indicated, includes salts that retain the biological effectiveness of the free acids and bases of the specified compound and that are not biologically or otherwise undesirable. Contemplated pharmaceutically acceptable salt forms include, but are not limited to, mono, bis, tris, tetrakis, and so on. Pharmaceutically acceptable salts are non-toxic in the amounts and concentrations at which they are administered. The preparation of such salts can facilitate the pharmacological use by altering the physical characteristics of a compound without preventing it from exerting its physiological effect. Useful alterations in physical properties include lowering the melting point to facilitate transmucosal administration and increasing the solubility to facilitate administering higher concentrations of the drug.

Pharmaceutically acceptable salts include acid addition salts such as those containing sulfate, chloride, hydrochloride, fumarate, maleate, phosphate, sulfamate, acetate, citrate, lactate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate and quinate. Pharmaceutically acceptable salts can be obtained from acids such as hydrochloric acid, maleic acid, sulfuric acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, fumaric acid, and quinic acid.

Pharmaceutically acceptable salts also include basic addition salts such as those containing benzathine, chloroprocaine, choline, diethanolamine, ethanolamine, t-butylamine, ethylenediamine, meglumine, procaine, aluminum, calcium, lithium, magnesium, potassium, sodium, ammonium, alkylamine, and zinc, when acidic functional groups, such as carboxylic acid or phenol are present. For example, see Remington's Pharmaceutical Sciences, 19^thed., Mack Publishing Co., Easton, PA, Vol. 2, p. 1457, 1995; “Handbook of Pharmaceutical Salts: Properties, Selection, and Use” by Stahl and Wermuth, Wiley-VCH, Weinheim, Germany, 2002. Such salts can be prepared using the appropriate corresponding bases.

Pharmaceutically acceptable salts can be prepared by standard techniques. For example, the free-base form of a compound can be dissolved in a suitable solvent, such as an aqueous or aqueous-alcohol solution containing the appropriate acid and then isolated by evaporating the solution. Thus, if the particular compound is a base, the desired pharmaceutically acceptable salt may be prepared by any suitable method available in the art, for example, treatment of the free base with an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, or with an organic acid, such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, a pyranosidyl acid, such as glucuronic acid or galacturonic acid, an alpha-hydroxy acid, such as citric acid or tartaric acid, an amino acid, such as aspartic acid or glutamic acid, an aromatic acid, such as benzoic acid or cinnamic acid, a sulfonic acid, such as p-toluenesulfonic acid or ethanesulfonic acid, or the like.

Similarly, if the particular compound is an acid, the desired pharmaceutically acceptable salt may be prepared by any suitable method, for example, treatment of the free acid with an inorganic or organic base, such as an amine (primary, secondary or tertiary) , an alkali metal hydroxide or alkaline earth metal hydroxide, or the like. Illustrative examples of suitable salts include organic salts derived from amino acids, such as L-glycine, L-lysine, and L-arginine, ammonia, primary, secondary, and tertiary amines, and cyclic amines, such as hydroxyethylpyrrolidine, piperidine, morpholine or piperazine, and inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum and lithium.

It is also to be understood that the compound of present disclosure can exist in unsolvated forms, solvated forms (e.g., hydrated forms) , and solid forms (e.g., crystal or polymorphic forms) , and the present disclosure is intended to encompass all such forms.

As used herein, the term “solvate” or “solvated form” refers to solvent addition forms that contain either stoichiometric or non-stoichiometric amounts of solvent. Some compounds have a tendency to trap a fixed molar ratio of solvent molecules in the crystalline solid state, thus forming a solvate. If the solvent is water the solvate formed is a hydrate; and if the solvent is alcohol, the solvate formed is an alcoholate. Hydrates are formed by the combination of one or more molecules of water with one molecule of the substance in which the water retains its molecular state as H₂O. Examples of solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.

As used herein, the terms “crystal form” , “crystalline form” , “polymorphic forms” and “polymorphs” can be used interchangeably, and mean crystal structures in which a compound (or a salt or solvate thereof) can crystallize in different crystal packing arrangements, all of which have the same elemental composition. Different crystal forms usually have different X-ray diffraction patterns, infrared spectral, melting points, density hardness, crystal shape, optical and electrical properties, stability and solubility. Recrystallization solvent, rate of crystallization, storage temperature, and other factors may cause one crystal form to dominate. Crystal polymorphs of the compounds can be prepared by crystallization under different conditions.

The compound of the present disclosure may also exist in different tautomeric forms, and all such forms are embraced within the scope of the present disclosure. The term “tautomer” or “tautomeric form” refers to structural isomers of different energies which are interconvertible via a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via migration of a proton, such as keto-enol, amide-imidic acid, lactam-lactim, imine-enamine isomerizations and annular forms where a proton can occupy two or more positions of a heterocyclic system (for example, 1H-and 3H-imidazole, 1H-, 2H-and 4H-1, 2, 4-triazole, 1H-and 2H-isoindole, and 1H-and 2H-pyrazole) . Valence tautomers include interconversions by reorganization of some of the bonding electrons. Tautomers can be in equilibrium or sterically locked into one form by appropriate substitution. Compounds of the present disclosure identified by name or structure as one particular tautomeric form are intended to include other tautomeric forms unless otherwise specified.

The present disclosure is also intended to include all isotopes of atoms in the compounds. Isotopes of an atom include atoms having the same atomic number but different mass numbers. For example, unless otherwise specified, hydrogen, carbon, nitrogen, oxygen, phosphorous, sulfur, fluorine, chlorine, bromide or iodine in the compounds of present disclosure are meant to also include their isotopes, such as but not limited to ¹H, ²H, ³H, ¹¹C, ¹²C, ¹³C, ¹⁴C, ¹⁴N, ¹⁵N, ¹⁶O, ¹⁷O, ¹⁸O, ³¹P, ³²P, ³²S, ³³S, ³⁴S, ³⁶S, ¹⁷F, ¹⁸F, ¹⁹F, ³⁵Cl, ³⁷Cl, ⁷⁹Br, ⁸¹Br, ¹²⁴I, ¹²⁷I and ¹³¹I. In some embodiments, hydrogen includes protium, deuterium and tritium. In some embodiments, carbon includes ¹²C and ¹³C.

The term “compound” as used herein is meant to include all forms or derivatives of the structure depicted. The compound herein identified by name or structure as one particular tautomeric form are intended to include other tautomeric forms unless otherwise specified.

Synthesis of Compounds

Synthesis of the compound provided herein, including pharmaceutically acceptable salts thereof, is illustrated in the synthetic schemes in the example. The compound provided herein can be prepared using any known organic synthesis techniques and can be synthesized according to any of numerous possible synthetic routes, and thus these schemes are illustrative only and are not meant to limit other possible methods that can be used to prepare the compounds provided herein. Additionally, the steps in the schemes are for better illustration and can be changed as appropriate. The embodiments of the compound in examples were synthesized for the purposes of research and potentially submission to regulatory agencies.

The reactions for preparing the compound of the present disclosure can be carried out in suitable solvents, which can be readily selected by one skilled in the art of organic synthesis. Suitable solvents can be substantially non-reactive with the starting materials (reactants) , the intermediates, or products at the temperatures at which the reactions are carried out, e.g. temperatures that can range from the solvent’s freezing temperature to the solvent's boiling temperature. A given reaction can be carried out in one solvent or a mixture of more than one solvent. Depending on the particular reaction step, suitable solvents for a particular reaction step can be selected by one skilled in the art.

Preparation of the compound of the present disclosure can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups, can be readily determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 3rd Ed., Wiley &Sons, Inc., New York (1999) , in P. Kocienski, Protecting Groups, Georg Thieme Verlag, 2003, and in Peter G. M. Wuts, Greene's Protective Groups in Organic Synthesis, 5^th Edition, Wiley, 2014, all of which are incorporated herein by reference in its entirety.

Reactions can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g. ¹H or ¹³C) , infrared spectroscopy, spectrophotometry (e.g. UV-visible) , mass spectrometry, or by chromatographic methods such as high performance liquid chromatography (HPLC) , liquid chromatography-mass spectroscopy (LCMS) , or thin layer chromatography (TLC) . Compounds can be purified by one skilled in the art by a variety of methods, including high performance liquid chromatography (HPLC) ( “Preparative LC-MS Purification: Improved Compound Specific Method Optimization” Karl F. Blom, Brian Glass, Richard Sparks, Andrew P. Combs J. Combi. Chem. 2004, 6 (6) , 874-883, which is incorporated herein by reference in its entirety) , and normal phase silica chromatography.

The known starting materials of the present disclosure can be synthesized by using or according to the known methods in the art, or can be purchased from commercial suppliers. Unless otherwise noted, analytical grade solvents and commercially available reagents were used without further purification.

Unless otherwise specified, the reactions of the present disclosure were all done under a positive pressure of nitrogen or argon or with a drying tube in anhydrous solvents, and the reaction flasks were typically fitted with rubber septa for the introduction of substrates and reagents via syringe. Glassware was oven dried and/or heat dried.

For illustrative purposes, the Examples section below shows synthetic route for preparing the compound of the present disclosure as well as key intermediates. Those skilled in the art will appreciate that other synthetic routes may be used to synthesize the inventive compounds. Although specific starting materials and reagents are depicted, other starting materials and reagents can be easily substituted to provide a variety of derivatives and/or reaction conditions. In addition, many of the compounds prepared by the methods described below can be further modified in light of this disclosure using conventional chemistry well known to those skilled in the art.

Pharmaceutical Compositions

In a further aspect, there is provided pharmaceutical compositions comprising the compound of the present disclosure, or a pharmaceutically acceptable salt thereof.

In another aspect, there is provided pharmaceutical composition comprising the compound of the present disclosure, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutical acceptable excipient.

As used herein, the term “pharmaceutical composition” refers to a formulation containing the molecule or compound of the present disclosure in a form suitable for administration to a subject.

As used herein, the term “pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes excipient that is acceptable for veterinary use as well as human pharmaceutical use. A “pharmaceutically acceptable excipient” as used herein includes both one and more than one such excipient. The term “pharmaceutically acceptable excipient” also encompasses “pharmaceutically acceptable carrier” and “pharmaceutically acceptable diluent” .

The particular excipient used will depend upon the means and purpose for which the compounds of the present disclosure is being applied. Solvents are generally selected based on solvents recognized by persons skilled in the art as safe to be administered to a mammal including humans. In general, safe solvents are non-toxic aqueous solvents such as water and other non-toxic solvents that are soluble or miscible in water. Suitable aqueous solvents include water, ethanol, propylene glycol, polyethylene glycols (e.g., PEG 400, PEG 300) , etc. and mixtures thereof.

In some embodiments, suitable excipients may include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol) ; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes) ; and/or non-ionic surfactants such as TWEEN^TM, PLURONICS^TM or polyethylene glycol (PEG) .

In some embodiments, suitable excipients may include one or more stabilizing agents, surfactants, wetting agents, lubricating agents, emulsifiers, suspending agents, preservatives, antioxidants, opaquing agents, glidants, processing aids, colorants, sweeteners, perfuming agents, flavoring agents and other known additives to provide an elegant presentation of the drug (i.e., a compound of the present disclosure or pharmaceutical composition thereof) or aid in the manufacturing of the pharmaceutical product (i.e., medicament) . The active pharmaceutical ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) . A “liposome” is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug (such as the compounds disclosed herein and, optionally, a chemotherapeutic agent) to a mammal including humans. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.

The pharmaceutical compositions provided herein can be in any form that allows for the composition to be administered to a subject, including, but not limited to a human, and formulated to be compatible with an intended route of administration.

A variety of routes are contemplated for the pharmaceutical compositions provided herein, and accordingly the pharmaceutical composition provided herein may be supplied in bulk or in unit dosage form depending on the intended administration route. For example, for oral, buccal, and sublingual administration, powders, suspensions, granules, tablets, pills, capsules, gelcaps, and caplets may be acceptable as solid dosage forms, and emulsions, syrups, elixirs, suspensions, and solutions may be acceptable as liquid dosage forms. For injection administration, emulsions and suspensions may be acceptable as liquid dosage forms, and a powder suitable for reconstitution with an appropriate solution as solid dosage forms. For inhalation administration, solutions, sprays, dry powders, and aerosols may be acceptable dosage form. For topical (including buccal and sublingual) or transdermal administration, powders, sprays, ointments, pastes, creams, lotions, gels, solutions, and patches may be acceptable dosage form. For vaginal administration, pessaries, tampons, creams, gels, pastes, foams and spray may be acceptable dosage form.

The quantity of active ingredient in a unit dosage form of composition is a therapeutically effective amount and is varied according to the particular treatment involved. As used herein, the term “therapeutically effective amount” refers to an amount of a molecule, compound, or composition comprising the molecule or compound to treat, ameliorate, or prevent an identified disease or condition, or to exhibit a detectable therapeutic or inhibitory effect. The effect can be detected by any assay method known in the art. The precise effective amount for a subject will depend upon the subject’s body weight, size, and health; the nature and extent of the condition; the rate of administration; the therapeutic or combination of therapeutics selected for administration; and the discretion of the prescribing physician. Therapeutically effective amounts for a given situation can be determined by routine experimentation that is within the skill and judgment of the clinician.

In some embodiments, the pharmaceutical compositions of the present disclosure may be in a form of formulation for oral administration.

In certain embodiments, the pharmaceutical compositions of the present disclosure may be in the form of tablet formulations. Suitable pharmaceutically- acceptable excipients for a tablet formulation include, for example, inert diluents such as lactose, sodium carbonate, calcium phosphate or calcium carbonate, granulating and disintegrating agents such as corn starch or algenic acid; binding agents such as starch; lubricating agents such as magnesium stearate, stearic acid or talc; preservative agents such as ethyl or propyl p-hydroxybenzoate, and anti-oxidants, such as ascorbic acid. Tablet formulations may be uncoated or coated either to modify their disintegration and the subsequent absorption of the active ingredient within the gastrointestinal tract, or to improve their stability and/or appearance, in either case using conventional coating agents and procedures well known in the art.

In certain embodiments, the pharmaceutical compositions of the present disclosure may be in a form of hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules in which the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin, or olive oil.

In certain embodiments, the pharmaceutical compositions of the present disclosure may be in the form of aqueous suspensions, which generally contain the active ingredient in finely powdered form together with one or more suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents such as lecithin or condensation products of an alkylene oxide with fatty acids (for example polyoxethylene stearate) , or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives (such as ethyl or propyl p-hydroxybenzoate, anti-oxidants (such as ascorbic acid) , coloring agents, flavoring agents, and/or sweetening agents (such as sucrose, saccharine or aspartame) .

In certain embodiments, the pharmaceutical compositions of the present disclosure may be in the form of oily suspensions, which generally contain suspended active ingredient in a vegetable oil (such as arachis oil, olive oil, sesame oil or coconut oil) or in a mineral oil (such as liquid paraffin) . The oily suspensions may also contain a thickening agent such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set out above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

In certain embodiments, the pharmaceutical compositions of the present disclosure may be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, such as olive oil or arachis oil, or a mineral oil, such as for example liquid paraffin or a mixture of any of these. Suitable emulsifying agents may be, for example, naturally-occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as soya bean, lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides (for example sorbitan monooleate) and condensation products of the said partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening, flavoring and preservative agents.

In certain embodiments, the pharmaceutical compositions provided herein may be in the form of syrups and elixirs, which may contain sweetening agents such as glycerol, propylene glycol, sorbitol, aspartame or sucrose, a demulcent, a preservative, a flavoring and/or coloring agent.

In some embodiments, the pharmaceutical compositions of the present disclosure may be in a form of formulation for injection administration.

In certain embodiments, the pharmaceutical compositions of the present disclosure may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents, which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1, 3-butanediol or prepared as a lyophilized powder. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils may conventionally be employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid may likewise be used in the preparation of injectables.

In some embodiments, the pharmaceutical compositions of the present disclosure may be in a form of formulation for inhalation administration.

In certain embodiments, the pharmaceutical compositions of the present disclosure may be in the form of aqueous and nonaqueous (e.g., in a fluorocarbon propellant) aerosols containing any appropriate solvents and optionally other compounds such as, but not limited to, stabilizers, antimicrobial agents, antioxidants, pH modifiers, surfactants, bioavailability modifiers and combinations of these. The carriers and stabilizers vary with the requirements of the particular compound, but typically include nonionic surfactants (Tweens, Pluronics, or polyethylene glycol) , innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols.

In some embodiments, the pharmaceutical compositions of the present disclosure may be in a form of formulation for topical or transdermal administration.

In certain embodiments, the pharmaceutical compositions provided herein may be in the form of creams, ointments, gels and aqueous or oily solutions or suspensions, which may generally be obtained by formulating an active ingredient with a conventional, topically acceptable excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

In certain embodiments, the pharmaceutical compositions provided herein may be formulated in the form of transdermal skin patches that are well known to those of ordinary skill in the art.

Besides those representative dosage forms described above, pharmaceutically acceptable excipients and carriers are generally known to those skilled in the art and are thus included in the present disclosure. Such excipients and carriers are described, for example, in “Remingtons Pharmaceutical Sciences” Mack Pub. Co., New Jersey (1991) , in “Remington: The Science and Practice of Pharmacy” , Ed. University of the Sciences in Philadelphia, 21^st Edition, LWW (2005) , which are incorporated herein by reference.

In some embodiments, the pharmaceutical compositions of the present disclosure can be formulated as a single dosage form. The amount of the compounds provided herein in the single dosage form will vary depending on the subject treated and particular mode of administration.

In some embodiments, the pharmaceutical compositions of the present disclosure can be formulated so that a dosage of between 0.001-1000 mg/kg body weight/day, for example, 0.01-800 mg/kg body weight/day, 0.01-700 mg/kg body weight/day, 0.01-600 mg/kg body weight/day, 0.01-500 mg/kg body weight/day, 0.01-400 mg/kg body weight/day, 0.01-300 mg/kg body weight/day, 0.1-200 mg/kg body weight/day, 0.1-150 mg/kg body weight/day, 0.1-100 mg/kg body weight/day, 0.5-100 mg/kg body weight/day, 0.5-80 mg/kg body weight/day, 0.5-60 mg/kg body weight/day, 0.5-50 mg/kg body weight/day, 1-50 mg/kg body weight/day, 1-45 mg/kg body weight/day, 1-40 mg/kg body weight/day, 1-35 mg/kg body weight/day, 1-30 mg/kg body weight/day, 1-25 mg/kg body weight/day of the compounds provided herein, or a pharmaceutically acceptable salt thereof, can be administered. In some instances, dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing any harmful side effect, provided that such larger doses are first divided into several small doses for administration throughout the day. For further information on routes of administration and dosage regimes, see Chapter 25.3 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board) , Pergamon Press 1990, which is specifically incorporated herein by reference.

In some embodiments, the pharmaceutical compositions of the present disclosure can be formulated as short-acting, fast-releasing, long-acting, and sustained-releasing. Accordingly, the pharmaceutical formulations of the present disclosure may also be formulated for controlled release or for slow release.

In a further aspect, there is also provided veterinary compositions comprising one or more molecules or compounds of the present disclosure or pharmaceutically acceptable salts thereof and a veterinary carrier. Veterinary carriers are materials useful for the purpose of administering the composition and may be solid, liquid or gaseous materials which are otherwise inert or acceptable in the veterinary art and are compatible with the active ingredient. These veterinary compositions may be administered parenterally, orally or by any other desired route.

The pharmaceutical compositions or veterinary compositions may be packaged in a variety of ways depending upon the method used for administering the drug. For example, an article for distribution can include a container having deposited therein the compositions in an appropriate form. Suitable containers are well known to those skilled in the art and include materials such as bottles (plastic and glass) , sachets, ampoules, plastic bags, metal cylinders, and the like. The container may also include a tamper-proof assemblage to prevent indiscreet access to the contents of the package. In addition, the container has deposited thereon a label that describes the contents of the container. The label may also include appropriate warnings. The compositions may also be packaged in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water, for injection immediately prior to use. Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules and tablets of the kind previously described.

Method of Use

The compound of Formula (I) or pharmaceutically acceptable salts thereof are capable of inhibiting the activity of a TAM kinase. For example, the compound of the disclosure can be used to inhibit activity of a TAM kinase in a cell or in a subject in need of inhibition of the kinases by administering an inhibiting amount of the compound of the disclosure to the cell or subject. The inhibitory properties of the compound may be demonstrated using the test procedures set out herein.

As used herein, the term “cell” refers to a cell that is in vitro, ex vivo or in vivo. In some embodiments, an ex vivo cell can be part of a tissue sample excised from an organism such as a mammal. In some embodiments, an in vitro cell can be a cell in a cell culture. In some embodiments, an in vivo cell is a cell living in an organism such as a mammal.

As used herein, a “subject” refers to a human and a non-human animal. Examples of a non-human animal include all vertebrates, e.g., mammals, such as non-human primates (particularly higher primates) , dog, rodent (e.g., mouse or rat) , guinea pig, cat, and non-mammals, such as birds, amphibians, reptiles, etc. In a preferred embodiment, the subject is a human. In another embodiment, the subject is an experimental animal or animal suitable as a disease model.

In some embodiments, the compound of the disclosure or pharmaceutically acceptable salts thereof are selective for the TAM kinases over one or more of other kinases. In some embodiments, the selectivity is 2-fold or more, 3-fold or more, 5-fold or more, 10-fold or more, 25-fold or more, 50-fold or more, or 100-fold or more.

In some embodiments, the compound of the disclosure or pharmaceutically acceptable salts thereof can inhibit one or more of AXL, MER and TYRO3. In some embodiments, the compound of the disclosure or pharmaceutically acceptable salts thereof are selective for one TAM kinase over another.

As used herein, the term “selective” means that the compound binds to or inhibits a TAM kinase with greater affinity or potency, compared to a reference enzyme, such as another TAM kinase or kinases other than TAM kinases.

For example, the compound of the disclosure or pharmaceutically acceptable salts thereof can be selective for AXL over MER and TYRO3, selective for MER over AXL and TYRO3, or selective for AXL and MER over TYRO3. In some embodiments, the compound of the disclosure or pharmaceutically acceptable salts thereof inhibit all of the TAM family members (e.g., AXL, MER and TYRO3) . In some embodiments, the compound of the disclosure or pharmaceutically acceptable salts thereof can be selective for AXL and MER over TYRO3 and other kinases.

Accordingly, in one aspect, the present disclosure provides a method for inhibiting a TAM kinase, which comprises contacting the TAM kinase with the compound provided herein, or a pharmaceutically acceptable salt thereof.

In a further aspect, the present disclosure provides a method for inhibiting AXL and MER kinases, which comprises contacting the AXL and MER kinases with the compound provided herein, or a pharmaceutically acceptable salt thereof.

As used herein, the term “contacting” refers to the bringing together of indicated moieties in an in vitro system or an in vivo system. For example, “contacting” the TAM kinase with the compound provided herein or a pharmaceutically acceptable salt thereof includes the administration of the compound provided herein, or a pharmaceutically acceptable salt thereof to a subject having the TAM kinase, as well as, for example, introducing the compound provided herein or a pharmaceutically acceptable salt thereof into a sample containing a cellular or purified preparation containing the TAM kinase.

Since the compound of the present disclosure or pharmaceutically acceptable salts thereof has an inhibitory activity against TAM kinases, they are useful in the treatment (therapeutic or prophylactic) of conditions or diseases associated with TAM kinases.

As used herein, the term “therapy” or “therapeutic” is intended to have its normal meaning of dealing with a disease in order to entirely or partially relieve one, some or all of its symptoms, or to correct or compensate for the underlying pathology, thereby achieving beneficial or desired clinical results. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total) , whether detectable or undetectable. “Therapy” can also mean prolonging survival as compared to expected survival if not receiving it. Those in need of therapy include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

As used herein, the term “prophylaxis” or “prophylactic” is intended to have its normal meaning and includes primary prophylaxis to prevent the development of the disease and secondary prophylaxis whereby the disease has already developed and the patient is temporarily or permanently protected against exacerbation or worsening of the disease or the development of new symptoms associated with the disease.

In some embodiments, the conditions or diseases associated with TAM kinases include proliferative disorders such as cancers, kidney diseases, immune system diseases, circulatory system diseases and viral diseases.

In some embodiments, the compound provided herein or pharmaceutically acceptable salts thereof are useful for the treatment of cancer, for example but not limited to leukemia (e.g., acute myeloid leukemia, chronic myeloid leukemia, acute lymphatic leukemia) , melanoma, glioma, lymphoma (e.g., chronic lymphocytic lymphoma, B-cell lymphoma, cutaneous T-cell lymphoma, Hodgkin’s or non-Hodgkin’s lymphoma, hairy cell lymphoma, chronic myelogenic lymphoma, acute lymphoblastic lymphoma, AIDS-related lymphomas, and Burkitt’s lymphoma) , bladder cancer, breast cancer, cervical cancer, colorectal cancer, small intestine cancer, large intestine cancer, colon cancer, rectal cancer, cancer of the anus, endometrial cancer, gastric cancer, head and neck cancer (e.g., cancers of the larynx, hypopharynx, nasopharynx, oropharynx, lips, and mouth) , kidney cancer, liver cancer (e.g., hepatocellular carcinoma, cholangiocellular carcinoma) , lung cancer (e.g., adenocarcinoma, small cell lung cancer and non-small cell lung carcinomas, parvicellular and non-parvicellular carcinoma, bronchial carcinoma, bronchial adenoma, pleuropulmonary blastoma) , ovarian cancer, prostate cancer, testicular cancer, uterine cancer, esophageal cancer, gall bladder cancer, pancreatic cancer (e.g. exocrine pancreatic carcinoma) , stomach cancer, thyroid cancer, parathyroid cancer, skin cancer (e.g., squamous cell carcinoma, Kaposi sarcoma, Merkel cell skin cancer) , and brain cancer (e.g., astrocytoma, medulloblastoma, ependymoma, neuro-ectodermal tumors, pineal tumors) .

In some embodiments, the compound provided herein or pharmaceutically acceptable salts thereof are useful for the treatment of kidney diseases, for example but not limited to glomerular nephritis, chronic nephritis, IgA nephritis, sequential (secondary) nephritis, nephrosis nephritis, acute renal failure, chronic renal failure, diabetic nephropathy, gouty nephropathy, interstitial nephritis, and nephro-pyelitis.

In some embodiments, the compound provided herein or pharmaceutically acceptable salts thereof are useful for the treatment of immune system disease, for example but not limited to psoriasis and rheumatoid arthritis.

In some embodiments, the compound provided herein or pharmaceutically acceptable salts thereof are useful for the treatment of circulatory system diseases, for example but not limited to atherosclerosis and thrombosis.

In some embodiments, the compound provided herein or pharmaceutically acceptable salts thereof are useful for the treatment of viral diseases such as viral infections. Examples of viruses causing infections include but are not limited to human immunodeficiency virus, papillomavirus, influenza virus, hepatitis A, B, C or D viruses, adenovirus, poxvirus, herpes viruses (e.g., N7N, HSV-1, HAV-6, HSVII, and CMV, Epstein Barr virus) , human cytomegalovirus, severe acute respiratory syndrome virus, respiratory syncytial virus, ebola virus, Marburg virus, measles virus, flaviviruses (e.g., West Nile, dengue, tick-home encephalitis, yellow fever, Zika) , echovirus, rhinovirus, coxsackie virus, comovirus, mumpsvirus, rotavirus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, molluscum virus, poliovirus, rabies virus, JC virus and arboviral encephalitis virus.

The compound provided herein or pharmaceutically acceptable salts thereof can be used as a metastasis suppressing agent to cancer cell.

Combination Use

The compound provided herein or pharmaceutically acceptable salts thereof may be used in combination with one or more additional pharmaceutical agents or therapies in order to supplement and/or enhance the preventive and/or therapeutic effect of the compound, to improve the kinetics, improvement of absorption, and reduction of the dose of the compound; and/or to eliminate the side effects of the compound.

The compound provided herein or pharmaceutically acceptable salts thereof can be administered simultaneously (as a single preparation or separate preparation) or sequentially to the additional pharmaceutical agents or therapies. In the sequential administration, the compound provided herein or pharmaceutically acceptable salts thereof may be administered before the additional pharmaceutical agents or therapies. Alternatively, the additional pharmaceutical agents or therapies may be administered before the compound provided herein or pharmaceutically acceptable salts. The method for the administration of these pharmaceutical agents or therapies may be the same as each other or different from each other.

Examples of pharmaceutical agents or therapies that can be used in combination with the compound provided herein or pharmaceutically acceptable salts thereof for the treatment of cancer include, but are not limited to chemotherapeutic agents, targeted cancer therapies, immunotherapy or radiation therapy, for example, alkylating agents (e.g., nitrogen mustard N-oxide hydrochloride, cyclophosphamide, ifosfa-mide, melphalan, thiotepa, carboquone, busulfan, nimustine hydrochloride, dacarbazine, ranimustine, carmustine, chlorambucil, bendamustine, and mechlorethamine) , antimetabolites (e.g., folic acid antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors) , anticancer antibiotics (e.g., actinomycin D, mitomycin C, daunorubicin hydrochloride, doxorubicin hydrochloride, aclarubicin hydrochloride, neocarzinostatin, pirarubicin hydrochloride, epirubicin (hydrochloride) , idarubicin hydrochloride, chromomycin A3, bleomycin (hydrochloride) , peplomycin sulfate, therarubicin, zinostatin stimalamer, gemtuzumab ozogamicin) , plant alkaloids, hormones, platinum compounds, anti-CD20 antibodies (e.g., rituximab, ibritumomab, ibritumomab tiuxetan, and ocreli-zumab. ) , anti-CD52 antibodies (e.g., alemtuzumab) , anti-PD-1 antibodies (e.g., nivolumab, pembrolizumab) , an adoptive cell transfer, an angiogenesis inhibitor, Bacillus Calmette-Guerin therapy, biochemotherapy, a cancer vaccine, a chimeric antigen receptor (CAR) T-cell therapy, a cytokine therapy, gene therapy, an immune checkpoint modulator, an immunoconjugate, a radioconjugate, an oncolytic virus therapy, a targeted drug therapy, G-CSF formulations (e.g., pegfilgrastim, filgrastim, lenograstim, and nartograstim) , acute promyelocytic leukemia differentiation-inducing agents, topoisomerase inhibitors (e.g., topotecan, teniposide, irinotecan, and sobuzoxane) , aromatase inhibitors (e.g., exemestane) , kinase inhibitors or antibodies (against, for example, EGFR, Her2, VEGFR, c-Met, PI3, JAK, Ret, IGFR1, PDGFR, FGFR1, FGFR2, FGFR3, FGFR4, TrkA, TrkB, TrkC, ROS, c-Kit, Fit-3, CSF1R, Bcr-Abl or EML4-Alk) , and other anticancer agents (e.g., BET inhibitors, HDAC inhibitors, Bcl2 inhibitors, beta catenin pathway inhibitors, notch pathway inhibitors, hedgehog pathway inhibitors) .

Examples of pharmaceutical agents or therapies that can be used in combination with the compound provided herein or pharmaceutically acceptable salts thereof for the treatment of kidney diseases include but are not limited to steroids, immunosuppressants (e.g., azathioprine, ascomycin, everolimus, salazosulfapyridine, cyclosporine, cyclophosphamide, sirolimus, tacrolimus, bucillamine, methotrexate, and leflunomide) , angiotensin II antagonistic drugs (e.g., losartan, candesartan, valsartan, irbesartan, olm-esartan, telmisartan) , angiotensin-converting enzyme inhibitors (e.g., alacepril, imidapril hydrochloride, quinapril hydrochloride, temocapril hydrochloride, delapril hydrochloride, benazepril hydrochloride, captopril, trandolapril, perindopril erbumine, enalapril maleate, lisinopril) , antiplatelet drugs (e.g., dipyridamole, and dilazep hydrochloride hydrate) , and anticoagulant drugs (e.g., dipyridamole, dilazep hydrochloride hydrate, apixaban, dabigatran, edoxaban, fondaparinex, heparin, rivaroxaban, and warfarin) .

Examples of pharmaceutical agents or therapies that can be used in combination with the compound provided herein or pharmaceutically acceptable salts thereof for the treatment of immune system diseases include but are not limited to immunosuppressants, steroid, diseasemodifying anti-rheumatic drugs, prostaglandins, prostaglandin synthase inhibitors, phosphodiesterase inhibitors, metalloprotease inhibitors, anti-cytokine protein formulations such as anti-TNF-α formulations, anti-IL-1 formulations, and anti-IL-6 formulation, cytokine inhibitors, and nonsteroidal anti-inflammatory agents.

Examples of pharmaceutical agents or therapies that can be used in combination with the compound provided herein or pharmaceutically acceptable salts thereof for the treatment of circulatory system diseases include but are not limited to antiplatelet drugs, angiotensin II antagonistic drugs, angiotensin-converting enzyme inhibitors, HMG-CoA reductase inhibitors, and thiazolidine derivatives.

In some embodiments, the compound provided herein or pharmaceutically acceptable salts thereof are used in combination with radiation therapy or surgeries. Radiation is commonly delivered internally (implantation of radioactive material near cancer site) or externally from a machine that employs photon (x-ray or gamma-ray) or particle radiation. Where the combination therapy further comprises radiation treatment, the radiation treatment may be conducted at any suitable time so long as a beneficial effect from the co-action of the combination of the therapeutic agents and radiation treatment is achieved.

Labelled Compounds and Assay Methods

In another aspect, the present disclosure relates to labeled compounds provided herein (radio-labeled, fluorescent-labeled, and the like. ) that would be useful not only in imaging techniques but also in assays, both in vitro and in vivo, for localizing and quantitating TAM kinases in tissue samples, including human, and for identifying TAM kinase ligands by inhibition binding of a labeled compound. Accordingly, the present disclosure includes TAM kinase assays that contain such labeled compounds.

The present disclosure further includes isotopically-labeled compounds provided herein. An “isotopically” or “radio-labeled” compound is a compound provided herein where one or more atoms are replaced or substituted by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature (i.e., naturally occurring) . The radionuclide that is incorporated in the instant radio-labeled compounds will depend on the specific application of that radio-labeled compound. For example, for in vitro TAM kinases labeling and competition assays, compounds that incorporate ³H, ¹⁴C, ⁸²Br, ¹²⁵I, ¹³¹I, or ³⁵S will generally be most useful. For radio-imaging applications ¹¹C, ¹⁸F, ¹²⁵I, ¹²³I, ¹²⁴I, ¹³¹I, ⁷⁵Br, ⁷⁶Br or ⁷⁷Br will generally be most useful. Synthetic methods for incorporating radio-isotopes into organic compounds are applicable to the compouns provided herein and are well known in the art.

A radio-labeled compound provided herein can be used in a screening assay to identify/evaluate compounds. In general terms, a newly synthesized or identified compound (i.e., test compound) can be evaluated for its ability to reduce binding of the radio-labeled compound of the application to the TAM kinases. Accordingly, the ability of a test compound to compete with the radio-labeled compound for binding to the TAM kinases directly correlates to its binding affinity.

EXAMPLES

For the purpose of illustration, the following examples are included. However, it is to be understood that these examples do not limit the present disclosure and are only meant to suggest a method of practicing the present disclosure. Persons skilled in the art will recognize that the chemical reactions described may be readily adapted to prepare a number of other compounds of the present disclosure, and alternative methods for preparing the compounds of the present disclosure are deemed to be within the scope of the present disclosure. For example, the synthesis of non-exemplified compounds according to the present disclosure may be successfully performed by modifications apparent to those skilled in the art, e.g., by appropriately protecting interfering groups, by utilizing other suitable reagents and building blocks known in the art other than those described, and/or by making routine modifications of reaction conditions. Alternatively, other reactions disclosed herein or known in the art will be recognized as having applicability for preparing other compounds of the present disclosure.

Example 1

N- (4- (3-amino-7- (1-isobutyrylpiperidin-4-yl) - [1, 2, 4] triazolo [4, 3-a] pyridin-5-yl) phenyl) -1-isopropyl-2, 4-dioxo-3- (pyridin-2-yl) -1, 2, 3, 4-tetrahydropyrimidine-5-carboxamide (Compound 17)

Step 1. 4-bromo-2-chloro-6-hydrazineylpyridine

To a solution of compound 17-1 (12.8 g, 56.5 mmol, 1.00 eq) in dioxane (130 mL) was added N₂H₄·H₂O (57.7 g, 979 mmol, 56.0 mL, 85.0%purity, 17.4 eq) . The mixture was stirred at 50℃ for 12 hrs. LC-MS showed there was desired mass detected. TLC indicated there was new spot. Water (60.0 mL) was added into the mixture. The mixture was extracted with EtOAc (150 mL *3) . The combined organic layers were washed with brine (100 mL *2) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (basic condition; column: Welch Ultimate XB -CN 250 *70 *10 um; mobile phase: [Hexane -EtOH (0.1%NH₃·H₂O) ] ; B%: 1%-40%, 15 min) . Compound 17-2 (7.00 g, 28.9 mmol, 51.2%yield, 91.9%purity) was obtained as a pink solid. H NMR (400 MHz, DMSO-d6) : δ 8.21 (s, 1H) , 6.87 (s, 1H) , 6.77 (d, J =1.6 Hz, 1H) , 4.33 (s, 2H) . LC-MS (M+H) ⁺: 223.9.

Step 2. ethyl (7-bromo-5-chloro- [1, 2, 4] triazolo [4, 3-a] pyridin-3-yl) carbamate

To a solution of compound 17-2 (2.00 g, 8.26 mmol, 91.9%purity, 1.00 eq) in 2-methyltetrahydrofuran (30.0 mL) was added compound 17-3 (1.30 g, 9.92 mmol, 1.17 mL, 1.20 eq) , then the mixture was stirred at 20 ℃ for 1 min. TLC showed the starting material was consumed completely. The mixture was concentrated under reduced pressure. Then the mixture was dissolved in DCM (20.0 mL) . And the mixture was added compound 17-4 (1.84 g, 7.19 mmol, 0.87 eq) and TEA (919 mg, 9.09 mmol, 1.27 mL, 1.10 eq) , then stirred at 20 ℃ for 1 min. TLC indicated there were new spots. The mixture was concentrated under reduced pressure. The residue was purified by column chromatography (SiO₂, PE: EtOAc = 1: 0 to 100: 51) . Compound 17-5 (1.27 g, 3.96 mmol, 47.9%yield, 99.5%purity) was obtained as a yellow solid.

H NMR (400 MHz, CDCl₃) : δ 7.87 (s, 1H) , 7.59 (s, 1H) , 7.00 (d, J = 0.8 Hz, 1H) , 4.28 -4.23 (m, 2H) , 1.30 -1.27 (m, 3H) . LC-MS (M+H) ⁺: 320.9.

Step 3. 1- (4- (3-amino-5-chloro- [1, 2, 4] triazolo [4, 3-a] pyridin-7-yl) -3, 6-dihydropyridin-1 (2H) -yl) -2-methylpropan-1-one

To a solution of compound 17-5 (900 mg, 2.81 mmol, 99.5%purity, 1.00 eq) , compound 17-6 (744 mg, 2.66 mmol, 0.95 eq) in dioxane (7.50 mL) was added Na₂CO₃ (297 mg, 2.81 mmol, 1.00 eq) in H₂O (1.50 mL) and Pd (PPh₃) ₂Cl₂ (196 mg, 280 μmol, 0.10 eq) . The mixture was taken up into a microwave tube. The sealed tube was heated at 120 ℃ for 4 hrs under microwave. LC-MS showed there was desired mass detected. TLC showed there were new spots. The reaction mixture was filtered and the filter was concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC. Compound 17-7 (410 mg, 1.04 mmol, 37.1%yield, 81.3%purity) was obtained as a yellow solid.

H NMR (400 MHz, CDCl₃) : δ 7.31 -7.26 (m, 1H) , 6.78 -6.72 (m, 1H) , 6.29 -6.20 (m, 1H) , 5.14 (s, 2H) , 4.30 -4.25 (m, 2H) , 3.84 -3.74 (m, 2H) , 2.90 -2.81 (m, 1H) , 2.56 -2.50 (m, 2H) , 1.19 -1.15 (m, 6H) .

LC-MS (M+H) ⁺: 320.1.

Step 4. 1- (4- (3-amino-5- (4-aminophenyl) - [1, 2, 4] triazolo [4, 3-a] pyridin-7-yl) -3, 6-dihydropyridin-1 (2H) -yl) -2-methylpropan-1-one

To a solution of compound 17-7 (310 mg, 788 μmol, 81.3%purity, 1.00 eq) and compound 17-8 (345 mg, 1.58 mmol, 2.00 eq) in dioxane (5.00 mL) was added K₃PO₄ (501 mg, 2.36 mmol, 3.00 eq) in H₂O (1.00 mL) and XPHOS-PD-G2 (93.0 mg, 118 μmol, 0.15 eq) . The mixture was degassed and purged with N₂ for 3 times, then stirred at 100 ℃ for 12 hrs. LC-MS showed there was desired mass detected. TLC indicated there were new spots. The solution was filtered and the filter was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, DCM: MeOH = 100: 0 to 100: 3) . Compound 17-9 (193 mg, 500 μmol, 63.4%yield, 97.6%purity) was obtained as a yellow solid.

H NMR (400 MHz, CDCl₃) : δ 7.50 -7.48 (m, 1H) , 7.39 -7.27 (m, 2H) , 6.82 -6.77 (m, 2H) , 6.61 -6.58 (m, 1H) , 6.32 -6.23 (m, 1H) , 4.42 -4.40 (m, 2H) , 4.31 -4.25 (m, 2H) , 3.88 -3.87 (m, 1H) , 3.79 -3.76 (m, 1H) , 2.94 -2.91 (m, 1H) , 2.64 - 2.57 (m, 2H) , 1.22 -1.17 (m, 6H) .

LC-MS (M+H) ⁺: 377.1.

Step 5. 1- (4- (3-amino-5- (4-aminophenyl) imidazo [1, 2-a] pyridin-7-yl) piperidin-1-yl) -2-methylpropan-1-one

To a solution of compound 17-9 (193 mg, 500 μmol, 97.6%purity, 1.00 eq) in MeOH (3.00 mL) was added Pd/C (20.0 mg, 10.0%purity) , degassed and purged with H₂ (15 psi) for 3 times, then stirred at 20 ℃ for 4 hrs. LC-MS showed there was desired mass detected and the starting material was consumed (～95.0 %) . TLC indicated there were new spots. TLC indicated the starting material was consumed completely (KMnO₄) . The mixture was filtered and filter was concentrated under reduced pressure to give a residue. Compound 17-10 (120 mg, 317 μmol, 63.3%yield) was obtained as a light yellow solid.

H NMR (400 MHz, CDCl₃) : δ 7.28 (s, 1H) , 7.26 -7.24 (m, 2H) , 6.80 -6.78 (m, 2H) , 6.32 (d, J = 1.2 Hz, 1H) , 4.85 (d, J = 12.0 Hz, 1H) , 4.26 (s, 2H) , 4.11 (d, J =12.8 Hz, 1H) , 3.19 -3.13 (m, 1H) , 2.89 -2.82 (m, 1H) , 2.78 -2.71 (m, 1H) , 2.67 -2.61 (m, 1H) , 2.02 -1.95 (m, 2H) , 1.65 -1.59 (m, 2H) , 1.18 -1.15 (m, 6H) .

Step 6. N- (4- (3-amino-7- (1-isobutyrylpiperidin-4-yl) - [1, 2, 4] triazolo [4, 3-a] pyridin-5-yl) phenyl) -1-isopropyl-2, 4-dioxo-3- (pyridin-2-yl) -1, 2, 3, 4-tetrahydropyrimidine-5-carboxamide

To a solution of compound 17-10 (50.0 mg, 132 μmol, 1.00 eq) and compound 17-11 (36.3 mg, 132 μmol, 1.00 eq) in pyridine (3.00 mL) was added EDCI (101 mg, 528 μmol, 4.00 eq) . Then the mixture was stirred at 20 ℃ for 1 hr. LC-MS showed there was desired mass detected. The mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC. Compound 17 (25.0 mg, 38.2 μmol, 28.9%yield, 97.2%purity) was obtained as confirmed by H NMR and LC-MS.

H NMR (400 MHz, CDCl₃) : δ 10.98 (s, 1H) , 8.75 -8.71 (m, 2H) , 8.02 -7.97 (m, 1H) , 7.82 -7.80 (m, 2H) , 7.53 -7.50 (m, 1H) , 7.45 -7.38 (m, 3H) , 7.28 -7.27 (m, 1H) , 6.36 (s, 1H) , 5.01 -4.97 (m, 1H) , 4.85 -4.82 (m, 1H) , 4.09 (s, 3H) , 3.18 -3.12 (m, 1H) , 2.87 -2.81 (m, 1H) , 2.78 -2.72 (m, 1H) , 2.66 -2.60 (m, 1H) , 2.01 -1.95 (m, 2H) , 1.64 -1.58 (m, 2H) , 1.52 -1.51 (m, 6H) , 1.14 -1.13 (m, 6H) .

LC-MS (M+H) ⁺: 636.4.

HPLC: purity: 97.2% (220 nm) .

Example 2

N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1-methyl-1H-indazol-4-yl) phenyl) -1-isopropyl-2, 4-dioxo-3- (pyridin-2-yl) -1, 2, 3, 4-tetrahydropyrimidine-5-carboxamide (Compound 18)

Step 1. 2-amino-4-bromo-6-fluorobenzonitrile

Two reactions were carried out in parallel. To a solution of compound 18-1 (12.5 g, 57.3 mmol, 1.00 eq) in i-PrOH (20.0 mL) was added NH₃·H₂O (43.1 g, 344 mmol, 47.3 mL, 28.0%purity, 6.00 eq) . The mixture was stirred at 80 ℃ for 36 hrs. LC-MS showed no desired mass was detected. TLC indicated the starting material was consumed completely and there was new spot. Two reactions were combined for work-up. The reaction mixture was poured into water (200 mL) . A white solid was filtered and washed with water. The solid was dissolved with EtOAc (200 mL) . The organic layer was dried over Na₂SO₄, then the solvent was evaporated under reduced pressure. Compound 18-2 (24.2 g, 112 mmol, 98.1%yield) was obtained as a white solid.

H NMR (400 MHz, CDCl₃) : δ 6.73 (s, 1H) , 6.68 (d, J = 8.4 Hz, 1H) , 4.62 (s, 2H) .

Step 2. 4-bromo-2-fluoro-6-iodobenzonitrile

To a solution of compound 18-2 (4.00 g, 18.6 mmol, 1.00 eq) in H₂O (40.0 mL) and HCl (12.0 M, 40.0 mL, 25.8 eq) was added NaNO₂ (1.93 g, 27.9 mmol, 1.50 eq) in H₂O (10.0 mL) at 0 ℃. Then the mixture was added MeCN (30.0 mL) stirred at 0 ℃ for 10 mins. And the mixture was added KI (7.72 g, 46.5 mmol, 2.50 eq) in H₂O (10.0 mL) at 0 ℃. The mixture was stirred at 20 ℃ for 6 hrs. LC-MS showed no desired mass detected. TLC showed the starting material was consumed completely. TLC indicated there were new spots. The reaction mixture was extracted with EtOAc (100 mL *3) . The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, PE: EtOAc = 100: 0 to 100: 3) . Compound 18-3 (3.50 g, 10.7 mmol, 57.7%yield) was obtained as a white solid.

H NMR (400 MHz, CDCl₃) δ 7.92 (d, J = 1.2 Hz, 1H) , 7.46 -7.42 (m, 1H) .

Step 3. 6-bromo-4-iodo-1H-indazol-3-amine

To a solution of compound 18-3 (3.00 g, 9.21 mmol, 1.00 eq) in butan-1-ol (30.0 mL) was added N₂H₄·H₂O (1.04 g, 20.34 mmol, 1.00 mL, 98.0%purity, 2.21 eq) . Then the mixture was stirred at 110 ℃ for 6 hrs. LC-MS showed there was desired mass detected. The mixture was filtered. And the solid was washed by MeOH (20.0 mL *3) and dried under reduced pressure to give a residue. Compound 18-4 (2.60 g, 7.42 mmol, 80.6%yield, 96.4%purity) was obtained as a white solid. F NMR showed there was not F in the structure of desired mass.

H NMR: (400 MHz, DMSO-d6) δ 11.91 (s, 1H) , 7.50 (s, 1H) , 7.46 (s, 1H) , 5.12 (s, 2H) . LC-M (M+H) ⁺: 337.9.

Step 4. 2- (6-bromo-4-iodo-1H-indazol-3-yl) isoindoline-1, 3-dione

To a solution of compound 18-4 (2.00 g, 5.71 mmol, 96.4%purity, 1.00 eq) in DMF (20.0 mL) was added compound 18-5 (1.27 g, 8.56 mmol, 1.50 eq) . Then the mixture was stirred at 140 ℃ for 4 hrs. LC-MS showed there was desired mass detected. The mixture can be purified directly. The mixture was purified by prep-HPLC. Compound 18-6 (1.62 g, 3.16 mmol, 55.2%yield, 91.1%purity) was obtained as a off-white solid.

H NMR (400 MHz, DMSO-d6) δ 13.97 (s, 1H) , 8.11 -8.10 (m, 2H) , 8.00 -7.99 (m, 2H) , 7.97 -7.96 (m, 1H) , 7.80 (s, 1H) .

LC-MS: (M+H) ⁺: 467.9.

HPLC: purity: 97.0% (220 nm) .

Step 5. 2- (6-bromo-4-iodo-1-methyl-1H-indazol-3-yl) isoindoline-1, 3-dione

To a solution of compound 18-6 (500 mg, 974 μmol, 91.1%purity, 1.00 eq) in DMF (5.00 mL) was added K₂CO₃ (134 mg, 974 μmol, 1.00 eq) and MeI (138 mg, 976 μmol, 60.7 uL, 1.00 eq) at 0 ℃. Then the mixture was stirred at 20 ℃ for 3 hrs under N₂ atmosphere. LC-MS showed there was desired mass detected. The mixture was poured into water (20.0 mL) and filtered. Then the solid was washed by water (10.0 mL *3) . Compound 18-7 (600 mg, crude) was obtained as a white solid.

Step 6. 6-bromo-4-iodo-1-methyl-1H-indazol-3-amine

To a solution of compound 18-7 (600 mg, 1.24 mmol, 1.00 eq) in MeOH (5.00 mL) and DCM (5.00 mL) was added N₂H₄·H₂O (1.50 g, 29.3 mmol, 1.46 mL, 98.0%purity, 23.5 eq) . The mixture was stirred at 20 ℃ for 2 hrs. TLC indicated the starting material was consumed completely. LC-MS showed there was desired mass detected. The mixture was washed by water (10.0 mL) , the water was extracted with EtOAc (5.00 mL *3) . The combined organic layers was dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, PE: EtOAc = 100: 20 to 100: 40) . Compound 18-8 (300 mg, 841 μmol, 67.6%yield, 98.7%purity) was obtained as a white solid.

H NMR (400 MHz, CDCl₃) : δ 7.59 -7.58 (m, 1H) , 7.42 (s, 1H) , 4.56 (s, 2H) , 3.86 (s, 3H) . LC-MS (M+H) ⁺: 351.9.

Step 7. 4- (4-aminophenyl) -6-bromo-1-methyl-1H-indazol-3-amine

To a solution of compound 18-8 (185 mg, 519 μmol, 98.7%purity, 1.00 eq) and compound 18-9 (102 mg, 467 μmol, 0.90 eq) in THF (6.00 mL) was added K₂CO₃ (150 mg, 1.09 mmol, 2.10 eq) in H₂O (1.50 mL) and Pd (PPh₃) ₄ (59.9 mg, 51.9 μmol, 0.10 eq) . The mixture was degassed and purged with N₂ for 3 times, then stirred at 80 ℃ for 8 hrs. LC-MS showed there was desired mass detected. TLC indicated there were new spots. The mixture was extracted by EtOAc (10.0 mL*3) . The combined organic layers was dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC. Compound 18-10 (110 mg, 326 μmol, 62.9%yield, 94.1%purity) was obtained as a off-white solid.

H NMR (400 MHz, CDCl₃) δ 7.32 -7.31 (m, 2H) , 7.28 -7.27 (m, 1H) , 6.98 (s, 1H) , 6.80 -6.78 (m, 2H) , 3.82 (s, 3H) .

LC-MS (M+H) ⁺: 318.9.

Step 8. 1- (4- (3-amino-4- (4-aminophenyl) -1-methyl-1H-indazol-6-yl) -3, 6-dihydropyridin-1 (2H) -yl) -2-methylpropan-1-one

To a solution of compound 18-10 (110 mg, 326 μmol, 94.1%purity, 1.00 eq) and compound 18-11 (182 mg, 652 μmol, 2.00 eq) in dioxane (12.0 mL) was added K₂CO₃ (90.2 mg, 652 μmol, 2.00 eq) in H₂O (3.00 mL) and Pd (PPh₃) ₄ (37.7 mg, 32.6 μmol, 0.10 eq) under N₂ atmosphere. Then the mixture was stirred at 90 ℃ for 12 hrs. LC-MS showed there was desired mass detected. TLC indicated there were new spots. The mixture was poured into water (10.0 mL) and extracted with EtOAc (10.0 mL *3) . The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC. Compound 18-12 (141 mg, crude) was obtained as a yellow solid.

H NMR (400 MHz, CDCl₃) δ 7.33 -7.31 (m, 2H) , 7.06 (s, 1H) , 6.93 -6.92 (m, 1H) , 6.81 -6.79 (m, 2H) , 6.22 -6.15 (m, 1H) , 4.27 -4.23 (m, 2H) , 3.87 -3.85 (m, 1H) , 3.82 (s, 3H) , 3.79 -3.74 (m, 1H) , 2.92 -2.87 (m, 1H) , 2.69 -2.64 (m, 2H) , 1.16 -1.13 (m, 6H) .

Step 9. 1- (4- (3-amino-4- (4-aminophenyl) -1-methyl-1H-indazol-6-yl) piperidin-1-yl) -2-methylpropan-1-one

To a solution of compound 18-12 (141 mg, 362 μmol, 1.00 eq) in MeOH (3.00 mL) was added Pd/C (14.0 mg, 10%purity) , degassed and purged with H₂ (15 psi) for 3 times, then stirred at 20 ℃ for 2 hrs. Then the mixture was stirred at 20 ℃for 4 hrs. LC-MS indicated the starting material was consumed completely and there was desired mass detected. The mixture was filtered and filter was concentrated under reduced pressure to give a residue. Compound 18-13 (65.0 mg, 162 μmol, 44.9%yield, 98.0%purity) was obtained as a white solid.

H NMR (400 MHz, CDCl₃) : δ 7.33 -7.31 (m, 2H) , 6.93 (s, 1H) , 6.81 -6.78 (m, 2H) , 6.72 (s, 1H) , 4.88 -4.85 (m, 1H) , 4.11 -4.08 (m, 1H) , 3.84 (s, 3H) , 3.20 - 3.17 (m, 1H) , 2.90 -2.85 (m, 2H) , 2.66 -2.64 (m, 1H) , 2.03 -2.00 (m, 2H) , 1.73 - 1.71 (m, 2H) , 1.17 -1.12 (m, 6H) .

LC-MS (M+H) ⁺: 392.3

Step 10. N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1-methyl-1H-indazol-4-yl) phenyl) -1-isopropyl-2, 4-dioxo-3- (pyridin-2-yl) -1, 2, 3, 4-tetrahydropyrimidine-5-carboxamide

To a solution of compound 18-13 (25.5 mg, 63.8 μmol, 98.0%purity, 1.00 eq) and compound 18-14 (16.7 mg, 60.6 μmol, 0.95 eq) in pyridine (2.00 mL) was added EDCI (48.9 mg, 255 μmol, 4.00 eq) . Then the mixture was stirred at 20 ℃for 2 hrs. LC-MS showed there was desired mass detected. The mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC. Compound 18 (8.00 mg, 12.0 μmol, 18.8%yield, 97.6%purity) was obtained as confirmed by H NMR and LC-MS.

H NMR: (400 MHz, CDCl₃) δ 10.83 (s, 1H) , 8.76 -8.75 (m, 1H) , 8.72 (s, 1H) , 7.99 -7.98 (m, 1H) , 7.77 -7.75 (m, 2H) , 7.52 -7.48 (m, 3H) , 7.41 -7.39 (m, 1H) , 6.97 (s, 1H) , 6.75 (s, 1H) , 5.02 -4.95 (m, 1H) , 4.88 -4.85 (m, 1H) , 4.12 -4.09 (m, 1H) , 3.85 (s, 3H) , 3.78 (s, 2H) , 3.20 -3.17 (m, 1H) , 2.89 -2.83 (m, 2H) , 2.66 - 2.64 (m, 1H) , 2.05 -2.01 (m, 2H) , 1.73 -1.70 (m, 2H) , 1.52 -1.50 (m, 6H) , 1.19 -1.17 (m, 6H) .

LC-MS (M+H) ⁺: 649.4.

HPLC: purity: 97.6% (220 nm) .

Example 3

N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) - [1, 2, 3] triazolo [1, 5-a] pyridin-4-yl) phenyl) -1-isopropyl-2, 4-dioxo-3- (pyridin-2-yl) -1, 2, 3, 4-tetrahydropyrimidine-5-carboxamide (Compound 23)

Step 1. diethyl 2- (5-bromo-3-chloropyridin-2-yl) malonate

To a solution of compound 23-1 (10.0 g, 47.5 mmol, 1.00 eq) , compound 23-2 (15.2 g, 95.0 mmol, 14.3 mL, 2.00 eq) in DMSO (100 mL) was added Cs₂CO₃ (30.9 g, 95.0 mmol, 2.00 eq) , the mixture was stirred at 100 ℃ for 2 hrs. LC-MS showed the desired mass was detected. The mixture was filtered and diluted with water (300 mL) , extracted with EtOAc (100 mL *3) , the combined organic layer was washed with brine (200 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to get the desired product compound 23-3 (16.0 g, 45.6 mmol, 96.0%yield) as yellow oil.

H NMR: (400 MHz, CDCl₃) δ 8.55 (d, J = 2.0 Hz, 1H) , 7.89 (d, J = 2.0 Hz, 1H) , 5.14 (s, 1H) , 4.31 -4.26 (m, 4H) , 1.28 (t, J = 7.2 Hz, 6H) .

Step 2. ethyl 2- (5-bromo-3-chloropyridin-2-yl) acetate

To a solution of compound 23-3 (8.00 g, 22.8 mmol, 1.00 eq) in DMSO (160 mL) was added LiCl (1.93 g, 45.6 mmol, 2.00 eq) , H₂O (411 mg, 22.8 mmol, 411 μL, 1.00 eq) , the mixture was stirred at 100 ℃ for 4 hrs. The mixture was heated to 120 ℃ for 10 hrs. LC-MS showed the desired mass detected. The reaction mixture was diluted with water (500 mL) , extracted with EtOAc (150 mL *3) , the combined organic layer was washed with brine (300 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to get the desired product ethyl compound 23-4 (6.00 g, 21.5 mmol, 94.4%yield) as yellow oil.

H NMR: (400 MHz, CDCl₃) δ 8.52 (d, J = 2.0 Hz, 1H) , 7.87 (d, J = 2.0 Hz, 1H) , 4.21 -4.19 (m, 2H) , 3.96 (s, 2H) , 1.28 -1.26 (m, 3H) .

Step 3. ethyl 6-bromo-4-chloro- [1, 2, 3] triazolo [1, 5-a] pyridine-3-carboxylate

To a solution of compound 23-4 (5.00 g, 17.9 mmol, 1.00 eq) in ACN (50.0 mL) was added DBU (9.57 g, 62.8 mmol, 9.47 mL, 3.50 eq) at 0 ℃ under N₂ atmosphere, then compound 23-5 (6.47 g, 26.9 mmol, 1.50 eq) was added in portions at 0 ℃, the mixture was stirred at 0 ℃ for 4 hrs. LC-MS showed the desired mass detected. The reaction mixture was diluted with water (50.0 mL) , extracted with EtOAc (30.0 mL *3) , the combined organic layer was washed with brine (70.0 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column (SiO₂, PE: EtOAc = 1: 0 to 10: 1, Plate 1, PE: EtOAc = 5: 1, R_f = 0.3) to get the desired product compound 23-6 (1.30 g, 4.27 mmol, 23.7%yield) as light yellow solid.

Step 4. ethyl 4-chloro-6- (1-isobutyryl-1, 2, 3, 6-tetrahydropyridin-4-yl) - [1, 2, 3] triazolo [1, 5-a] pyridine-3-carboxylate

To a solution of compound 23-6 (700 mg, 2.30 mmol, 1.00 eq) , compound 23-7 (674 mg, 2.41 mmol, 1.05 eq) in dioxane (10.0 mL) , H₂O (2.00 mL) was added Pd (PPh₃) ₄ (265mg, 229 μmol, 0.100 eq) , Na₂CO₃ (487 mg, 4.60 mmol, 2.00 eq) under N₂ atmosphere, the mixture was heated at 90 ℃ for 2 hrs. LC-MS showed the desired mass detected. The residue was filtered and concentrated under reduced pressure to give a residue. The residue was purified by column (SiO₂, PE: EtOAc = 100: 1 to 1: 1, Plate 1, PE: EtOAc = 1: 1) to get the desired product compound 23-8 (750 mg, 1.99 mmol, 86.5%yield) as yellow solid.

H NMR: (400 MHz, CDCl₃) δ 8.69 -8.66 (m, 1H) , 7.66 (s, 1H) , 6.39 -6.30 (m, 1H) , 4.54 -4.49 (m, 2H) , 4.33 -4.28 (m, 2H) , 3.89 -3.79 (m, 2H) , 2.90 -2.80 (m, 1H) , 2.61 -2.55 (m, 2H) , 1.48 (t, J = 7.2 Hz, 3H) , 1.19 -1.16 (m, 6H) .

Step 5. ethyl 6- (1-isobutyryl-1, 2, 3, 6-tetrahydropyridin-4-yl) -4- (4-nitrophenyl) - [1, 2, 3] triazolo [1, 5-a] pyridine-3-carboxylate

To a solution of compound 23-8 (740 mg, 1.96 mmol, 1.00 eq) , compound 23-9 (393 mg, 2.36 mmol, 1.20 eq) in dioxane (10.0 mL) , H₂O (2.00 mL) was added Na₂CO₃ (624 mg, 5.89 mmol, 3.00 eq) , Pd (PPh₃) ₄ (227 mg, 196 μmol, 0.100 eq) at N₂ atmosphere, the mixture was stirred at 80 ℃ for 2 hrs. LC-MS showed the desired mass detected. The mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by column (SiO₂, PE: EtOAc = 10: 1 to 0: 1, Plate 1, PE: EtOAc = 0: 1, R_f = 0.4) to get the desired product compound 23-10 (650 mg, 1.40 mmol, 71.4%yield) as yellow solid.

H NMR: (400 MHz, CDCl₃) δ 8.85 -8.82 (m, 1H) , 8.34 (d, J = 8.8 Hz, 2H) , 7.58 -7.55 (m, 3H) , 6.43 -6.34 (m, 1H) , 4.34 -4.29 (m, 2H) , 4.20 -4.15 (m, 2H) , 3.91 -3.82 (m, 2H) , 2.92 -2.82 (m, 1H) , 2.68 -2.62 (m, 2H) , 1.28 -1.16 (m, 9H) .

Step 6. ethyl 4- (4-aminophenyl) -6- (1-isobutyrylpiperidin-4-yl) - [1, 2, 3] triazolo [1, 5-a] pyridine-3-carboxylate

To a solution of compound 23-10 (190 mg, 410 μmol, 1.00 eq) in DCM (2.00 mL) , THF (2.00 mL) was added Pd/C (100 mg, 10%purity) , then degassed and purged with H₂ three times, the mixture was stirred at 20 ℃ for 5 hrs (15 psi) . LC-MS showed the desired mass detected. The mixture was filtered and concentrated to get the desired product compound 23-11 (150 mg, 344 μmol, 84.0%yield) as white solid.

Step 7. ethyl 6- (1-isobutyrylpiperidin-4-yl) -4- (4- (1-isopropyl-2, 4-dioxo-3- (pyridin-2-yl) -1, 2, 3, 4-tetrahydropyrimidine-5-carboxamido) phenyl) - [1, 2, 3] triazolo [1, 5-a] pyridine-3-carboxylate

To a solution of compound 23-11 (150 mg, 344 μmol, 1.00 eq) , compound 23-12 (114 mg, 413 μmol, 1.20 eq) in DMF (2.00 mL) was added DIEA (222 mg, 1.72 mmol, 299 uL, 5.00 eq) , HATU (262 mg, 688 μmol, 2.00 eq) , the mixture was stirred at 20 ℃ for 5 hrs. LC-MS showed the desired mass detected. The reaction mixture was diluted with sat. aq. NaHCO₃ (3.00 mL) and water (3.00 mL) , extracted with DCM (10.0 mL *3) , the combined organic layer was washed with water (20.0 mL *2) , brine (20.0 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by Prep-TLC (Plate 1, DCM: MeOH = 10: 1, R_f = 0.6) to get the desired product compound 23-13 (170 mg, 245 μmol, 71.2%yield) as white solid.

H NMR: (400 MHz, CDCl₃) δ 10.88 (s, 1H) , 8.76 -8.74 (m, 1H) , 8.71 (s, 1H) , 8.63 (brs, 1H) , 8.01 -7.87 (m, 1H) , 7.76 -7.74 (m, 2H) , 7.53 -7.49 (m, 1H) , 7.43 -7.32 (m, 4H) , 5.02 -4.95 (m, 1H) , 4.93 -4.87 (m, 1H) , 4.17 -4.12 (m, 1H) , 4.10 -4.05 (m, 2H) , 3.20 (t, J = 12.4 Hz, 1H) , 2.67 (t, J = 12.4 Hz, 1H) , 2.10 -2.03 (m, 2H) , 1.74 -1.67 (m, 2H) , 1.51 (d, J = 6.8 Hz, 6H) , 1.18 -1.14 (m, 6H) , 1.06 (t, J =7.2 Hz, 3H) .

Step 8. 6- (1-isobutyrylpiperidin-4-yl) -4- (4- (1-isopropyl-2, 4-dioxo-3- (pyridin-2-yl) -1, 2, 3, 4-tetrahydropyrimidine-5-carboxamido) phenyl) - [1, 2, 3] triazolo [1, 5-a] pyridine-3-carboxylic acid

To a solution of compound 23-13 (70.0 mg, 101 μmol, 1.00 eq) in toluene (2.50 mL) was added (Bu₃Sn) ₂O (120mg, 202 μmol, 103 μL, 2.00 eq) , the mixture was stirred at 110℃ for 24 hrs. LC-MS showed the desired mass detected. The reaction mixture was diluted with sat. aq. KF (3.00 mL) and water (3.00 mL) , extracted with DCM (5.00 mL *3) , the combined organic layer was washed with brine (10.0 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by Prep-TLC (SiO₂, Plate 1, DCM: MeOH = 10: 1, R_f = 0.3) to get the desired product compound 23-14 (60.0 mg, 90.2 μmol, 89.3%yield) as white solid.

Step 9. N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) - [1, 2, 3] triazolo [1, 5-a] pyridin-4-yl) phenyl) -1-isopropyl-2, 4-dioxo-3- (pyridin-2-yl) -1, 2, 3, 4-tetrahydropyrimidine-5-carboxamide

To a solution of compound 23-14 (40.0 mg, 60.1 μmol, 1.00 eq) in toluene (1.00 mL) was added DPPA (24.8 mg, 90.2 μmol, 19.5 μL, 1.50 eq) , TEA (18.2 mg, 180 μmol, 25.1 μL, 3.00 eq) , the mixture was stirred at 20 ℃ for 12 hrs, then heated to 35 ℃ for 6 hrs. LC-MS showed the desired mass detected. The mixture was concentrated to remove solvent. The residue was purified by Prep-TLC (SiO₂, Plate 1, DCM: MeOH = 10: 1, R_f = 0.65) . The residue was purified by Prep-HPLC (column: Waters Xbridge 150 *25 mm *5 um; mobile phase: [water (NH₄HCO₃) -ACN] ; B%: 32%-62%, 9 min) to get the desired product compound 23 (2.10 mg, 3.10 μmol, 5.15%yield, 93.8%purity) as confirmed by H NMR, LC-MS and HPLC.

H NMR: (400 MHz, CDCl₃) δ 10.90 (s, 1H) , 8.76 (d, J = 1.2 Hz, 1H) , 8.71 (s, 1H) , 8.27 (s, 1H) , 8.01 -7.97 (m, 1H) , 7.81 -7.79 (m, 2H) , 7.53 -7.49 (m, 1H) , 7.48 -7.45 (m, 2H) , 7.41 -7.38 (m, 1H) , 6.73 (s, 1H) , 5.02 -4.95 (m, 1H) , 4.88 -4.84 (m, 1H) , 4.12 (d, J = 11.2 Hz, 1H) , 3.70 (s, 2H) , 3.17 (t, J =13.6 Hz, 1H) , 2.88 -2.77 (m, 2H) , 2.68 -2.62 (m, 1H) , 2.07 -1.99 (m, 2H) , 1.70 -1.65 (m, 2H) , 1.51 (d, J = 6.8 Hz, 6H) , 1.16 (brs, 6H) .

LC-MS (M+H) ⁺: 636.5

HPLC: 93.8%purity (220 nm)

Example 4

N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1H-pyrazolo [3, 4-b] pyridin-4-yl) phenyl) -1-isopropyl-2, 4-dioxo-3- (pyridin-2-yl) -1, 2, 3, 4-tetrahydropyrimidine-5-carboxamide (Compound 24)

Step 1. 2-hydrazineyl-4-iodonicotinonitrile

To a solution of compound 24-1 (25.0 g, 94.5 mmol, 1.00 eq) in MeOH (100 mL) was added N₂H₄ ^. H₂O (48.1 g, 817 mmol, 46.7 mL, 85.0%purity, 8.65 eq) . The mixture was stirred at 20 ℃ for 0.5 hr. LC-MS showed desired mass detected. The reaction mixture was filtered. The cake was washed with MeOH (100 mL) , then concentrated to give a residue. Compound 24-2 and compound 24-2A (mixture, 15.0 g, crude) was obtained as a yellow solid.

Step 2. 4-iodo-1H-pyrazolo [3, 4-b] pyridin-3-amine

To a solution of compound 24-2 and compound 24-2A (mixture, 15.0 g, crude) in DCM (100 mL) and anisole (10.0 mL) was added TFA (15.0 g, 131 mmol, 9.74 mL, 3.17 eq) . The mixture was stirred at 25 ℃ for 0.5 hr. LC-MS showed desired mass detected. The reaction mixture was concentrated to give a residue. The residue was adjusted pH to 8 by NH₃·H₂O, then filtered. The cake was concentrated under reduced pressure to give a residue.

The residue was purified by prep-HPLC (FA condition; column: Phenomenex luna C18 (250 *70 mm, 10 um) ; mobile phase: [water (FA) -ACN] ; B%: 0%-30%, 21 min) . Compound 24-3 (4.80 g, 18.4 mmol, 44.4%yield) was obtained as a yellow solid. The structure was confirmed by H NMR. Compound 24-3A (7.00 g, 41.5 mmol, 100%yield) was obtained as a yellow solid. The structure was confirmed by H NMR.

H NMR of Compound 24-3: (400 MHz, DMSO-d6) δ 12.39 (brs, 1H) , 7.92 (d, J = 4.8 Hz, 1H) , 7.43 (d, J = 4.8 Hz, 1H) , 5.16 (s, 2H)

H NMR of Compound 24-3A: (400 MHz, DMSO-d6) δ 12.31 (brs, 1H) , 7.91 (d, J = 6.0 Hz, 1H) , 7.22 (d, J = 6.0 Hz, 1H) , 5.52 (brs, 2H) .

Step 3. 2- (4-iodo-1H-pyrazolo [3, 4-b] pyridin-3-yl) isoindoline-1, 3-dione

A mixture of compound 24-3 (2.00 g, 7.69 mmol, 1.00 eq) and compound 24-4 (1.71 g, 11.5 mmol, 1.50 eq) in HOAc (20.0 mL) was stirred at 120 ℃ for 12 hrs. LC-MS showed desired mass detected. The reaction mixture was filtered. The cake was washed with H₂O 40.0 mL, then concentrated to give a crude product. Compound 24-5 (1.90 g, 4.87 mmol, 63.3%yield) was obtained as a white solid. The structure was confirmed by H NMR.

H NMR: (400 MHz, DMSO-d6) δ 14.46 (s, 1H) , 8.25 (d, J = 4.8 Hz, 1H) , 8.13 -8.11 (m, 2H) , 8.02 -7.99 (m, 2H) , 7.79 (d, J = 4.8 Hz, 1H) .

Step 4. 3- (1, 3-dioxoisoindolin-2-yl) -4-iodo-1H-pyrazolo [3, 4-b] pyridine 7-oxide

To a solution of compound 24-5 (3.30 g, 8.46 mmol, 1.00 eq) in HOAc (30.0 mL) was added m-CPBA (6.87 g, 33.8 mmol, 85.0%purity, 4 eq) at 0 ℃. The mixture was stirred at 85 ℃ for 12 hrs. LC-MS showed desired mass detected. The reaction mixture was concentrated to give a residue. The residue was adjusted pH to 9 by sat. aq NaHCO₃ and extracted with DCM 200 mL (100 mL *2) . The combined organic layers were washed with sat. aq NaHCO₃ 200 mL (100 mL *2) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether: EtOAc = 100: 1 to 0: 1) . (Plate 1: DCM: MeOH = 10: 1) . Compound 24-6 (2.00 g, 4.92 mmol, 58.2%yield) was obtained as a yellow solid.

Step 5. 2- (6-chloro-4-iodo-1H-pyrazolo [3, 4-b] pyridin-3-yl) isoindoline-1, 3-dione

To a solution of compound 24-6 (2.00 g, 3.67 mmol, 74.6%purity, 1.00 eq) in ACN (10.0 mL) was added POCl₃ (3.30 g, 21.5 mmol, 2.00 mL, 5.86 eq) at 0 ℃. The mixture was stirred at 25 ℃ for 1 hr. LC-MS showed desired mass detected. The reaction mixture was filtered, then concentrated to give a crude product. Compound 24-7 (1.00 g, 2.36 mmol, 64.1%yield) was obtained as a white solid.

Step 6. 6-chloro-4-iodo-1H-pyrazolo [3, 4-b] pyridin-3-amine

To a solution of compound 24-7 (1.00 g, 2.36 mmol, 1.00 eq) in MeOH (10.0 mL) was added NH₂NH₂ ^. H₂O (4.86 g, 82.5 mmol, 4.72 mL, 85.0%purity, 35.0 eq) at 15 ℃. The mixture was stirred at 15 ℃ for 1 hr. LC-MS showed desired mass detected. The reaction mixture was filtered. The filtrate was concentrated to give a residue. Then diluted with H₂O 50.0 mL and extracted with DCM 200 mL (100 mL *2) . The combined organic layers were washed with brine 200 mL, dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. Compound 24-8 (0.270 g, 916 μmol, 38.9%yield) was obtained as a yellow solid. The structure was confirmed by H NMR.

H NMR: (400 MHz, DMSO-d6) δ 7.51 (s, 1H) , 5.27 (s, 2H) .

Step 7. 6-chloro-4- (4-nitrophenyl) -1H-pyrazolo [3, 4-b] pyridin-3-amine

To a solution of compound 24-8 (120 mg, 407 μmol, 1.00 eq) and compound 24-9 (71.4 mg, 427 μmol, 1.05 eq) in dioxane (2.00 mL) and H₂O (1.00 mL) was added Pd (PPh₃) ₄ (47.0 mg, 40.7 μmol, 0.100 eq) and K₂CO₃ (168 mg, 1.22 mmol, 3.00 eq) . The mixture was stirred at 90 ℃ for 2 hrs. LC-MS showed desired mass detected. The reaction mixture was diluted with H₂O 10.0 mL and extracted with EtOAc 40.0 mL (20.0 mL *2) . The combined organic layers were washed with brine 60.0 mL (20.0 mL *3) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiO₂, Petroleum ether: EtOAc = 1: 2) . (Plate 1: Petroleum ether: EtOAc = 1: 2) . Compound 24-10 (60.0 mg, 207 μmol, 50.8%yield) was obtained as a yellow solid.

Step 8. 1- (4- (3-amino-4- (4-nitrophenyl) -1H-pyrazolo [3, 4-b] pyridin-6-yl) -3, 6-dihydropyridin-1 (2H) -yl) -2-methylpropan-1-one

To a solution of compound 24-10 (60.0 mg, 207 μmol, 1.00 eq) and compound 24-11 (86.7 mg, 310 μmol, 1.50 eq) in dioxane (2.00 mL) and H₂O (1.00 mL) was added Xphos Pd G4 (17.8 mg, 20.7 μmol, 0.100 eq) and K₃PO₄ (87.9 mg, 414 μmol, 2.00 eq) . The mixture was stirred at 90 ℃ for 3 hrs. LC-MS showed desired mass detected. The reaction mixture was diluted with H₂O 30.0 mL and extracted with EtOAc 100 mL (50.0 mL *2) . The combined organic layers were washed with brine 100 mL (50.0 mL *2) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiO₂, DCM: MeOH = 10: 1) . (Plate 1: DCM: MeOH = 10: 1) . Compound 24-12 (60.0 mg, 147 μmol, 71.2%yield) was obtained as a yellow solid.
Step 9. 1- (4- (3-amino-4- (4-aminophenyl) -1H-pyrazolo [3, 4-b] pyridin-6-
yl) piperidin-1-yl) -2-methylpropan-1-one

To a solution of compound 24-12 (60.0 mg, 147 μmol, 1.00 eq) in THF (6.00 mL) was added Pd/C (30.0 mg, 10.0%purity) under N₂. The suspension was degassed under vacuum and purged with H₂ several times. The mixture was stirred under H₂ (15 psi) at 15 ℃ for 2 hrs. LC-MS showed desired mass detected. The reaction mixture was filtered and the cake was washed with 100 mL DCM, then concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiO₂, DCM: MeOH = 10: 1) . (Plate 1: DCM: MeOH = 10: 1) . Compound 24-13 (40.0 mg, 105 μmol, 71.5%yield) was obtained as a white solid.

Step 10. N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1H-pyrazolo [3, 4-b] pyridin-4-yl) phenyl) -1-isopropyl-2, 4-dioxo-3- (pyridin-2-yl) -1, 2, 3, 4-tetrahydropyrimidine-5-carboxamide

To a solution of compound 24-13 (30.0 mg, 79.2 μmol, 1.00 eq) and compound 24-14 (21.8 mg, 79.2 μmol, 1.00 eq) in Py (2.00 mL) was added EDCI (30.3 mg, 158 μmol, 2.00 eq) . The mixture was stirred at 15 ℃ for 1 hr. LC-MS showed desired mass detected. The reaction mixture was added 30.0 mL H₂O and extracted with EtOAc 100 mL (50.0 mL *2) . The combined organic layers were washed with sat. aq NaHCO₃ 60.0 mL, dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiO₂, DCM: MeOH = 10: 1) . (Plate 1: DCM: MeOH = 10: 1) . The crude product was purified by prep-HPLC (FA condition; column: Phenomenex C18 75 *30 mm *3 um;mobile phase: [water (FA) -ACN] ; B%: 32%-62%, 7 mins) . Compound 24 (5.37 mg, 8.08 μmol, 10.1%yield, and 95.6%purity) was obtained as confirmed by H NMR, LC-MS, and HPLC.

H NMR (400 MHz, CDCl₃) : δ 10.92 (s, 1H) , 8.77 -8.72 (m, 2H) , 8.02 -7.98 (m, 1H) , 7.83 (d, J = 8.4 Hz, 2H) , 7.57 -7.50 (m, 4H) , 7.40 (d, J = 7.6 Hz, 1H) , 6.84 (s, 1H) , 5.03 -4.96 (m, 1H) , 4.85 -4.82 (m, 1H) , 4.15 -4.08 (m, 1H) , 4.03 -3.95 (m, 2H) , 3.21 -3.02 (m, 2H) , 2.89 -2.66 (m, 2H) , 2.11 -2.01 (m, 2H) , 1.92 -1.80 (m, 2H) , 1.51 (d, J = 6.8 Hz, 6H) , 1.18 -1.14 (m, 6H) .

LC-MS (M+H) ⁺: 636.5

HPLC: Purity: 95.6% (220 nm)

Example 5

N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1H-pyrazolo [4, 3-c] pyridin-4-yl) phenyl) -1-isopropyl-2, 4-dioxo-3- (pyridin-2-yl) -1, 2, 3, 4-tetrahydropyrimidine-5-carboxamide (Compound 25)

Step 1. tert-butyl 4- (4, 4-dicyano-3, 3-bis (methylthio) butanoyl) piperidine-1-carboxylate

To a solution of compound 25-1 (10.0 g, 58.7 mmol, 1.00 eq) and compound 25-2 (26.7 g, 117 mmol, 2.00 eq) in DMSO (100 mL) was added KOH (9.89 g, 176 mmol, 3.00 eq) . The mixture was stirred at 25 ℃ for 8 hrs. LC-MS showed desired mass detected. The reaction mixture was poured into 100 mL ice water, and acidified with 1M HCl to pH to 6 and extracted with EtOAc 400 mL (100 mL *3) . The combined organic layers were washed with brine 600 mL (200 mL *3) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether: EtOAc = 100: 1 to 0: 1) . (Plate 1: Petroleum ether: EtOAc = 1: 1) . Compound 25-3 (5.88 g, 14.7 mmol, 25.1%yield) was obtained as a yellow solid. The structure was confirmed by H NMR.

H NMR: (400 MHz, DMSO-d6) δ 7.52 -7.29 (m, 2H) , 5.76 -5.74 (m, 1H) , 4.05 -3.97 (m, 3H) , 3.30 (s, 3H) , 2.70 -2.67 (m, 2H) , 2.34 -2.19 (m, 7H) , 1.64 -1.50 (m, 2H) , 1.39 (s, 9H) .

Step 2. tert-butyl 4- (5-cyano-4- (methylthio) -6-oxo-1, 6-dihydropyridin-2-yl) piperidine-1-carboxylate

To a solution of compound 25-3 (5.88 g, 14.7 mmol, 1.00 eq) in DMSO (50.0 mL) was added KOH (2.49 g, 44.3 mmol, 3.00 eq) . The mixture was stirred at 25 ℃for 12 hrs. LC-MS showed desired mass detected. The reaction mixture was poured into 100 mL ice water, and acidified with 1M HCl to pH to 6 and extracted with EtOAc 150 mL (75.0 mL *2) . The combined organic layers were washed with brine 300 mL (60.0 mL *5) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (FA condition; column: Phenomenex luna C18 (250 *70 mm, 10 um) ; mobile phase: [water (FA) -ACN] ; B%: 35%-65%, 20 mins) . Compound 25-4 (1.00 g, 2.86 mmol, 19.3%yield) was obtained as a white solid. The structure was confirmed by H NMR.

H NMR: (400 MHz, DMSO-d6) δ 6.21 (s, 1H) , 4.14 -4.06 (m, 2H) , 2.72 -2.61 (m, 6H) , 1.78 -1.75 (m, 2H) , 1.65 -1.59 (m, 2H) , 1.38 (s, 9H) .

Step 3. 4- (methylthio) -2-oxo-6- (piperidin-4-yl) -1, 2-dihydropyridine-3-carbonitrile

To a solution of compound 25-4 (1.00 g, 2.86 mmol, 1.00 eq) in DCM (1.00 mL) was added HCl/dioxane (4 M, 25.0 mL, 34.9 eq) . The mixture was stirred at 15 ℃ for 0.5 hr. LC-MS showed desired mass detected. The reaction mixture was concentrated to give a residue. Compound 25-5 (0.700 g, 2.45 mmol, 85.5%yield, HCl) was obtained as a yellow solid.

Step 4. 6- (1-isobutyrylpiperidin-4-yl) -4- (methylthio) -2-oxo-1, 2-dihydropyridine-3-carbonitrile

To a solution of compound 25-5 (0.700 g, 2.45 mmol, 1.00 eq, HCl) and TEA (743 mg, 7.35 mmol, 1.02 mL, 3.00 eq) in THF (10.0 mL) was added compound 25-6 (260 mg, 2.45 mmol, 255 μL, 1.00 eq) . The mixture was stirred at 15 ℃ for 0.5 hr. LiOH^. H₂O (1.00 g) in 2.00 mL H₂O was added to the reaction mixture and stirred at 15 ℃ for 0.5 hr. LC-MS (showed desired mass detected. The reaction mixture was added 10.0 mL H₂O and extracted with DCM 40.0 mL (20.0 mL *2) . The combined organic layers were washed with sat. aq NaHCO₃ 40.0 mL , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiO₂, DCM: MeOH = 10: 1) . (Plate 1: DCM: MeOH = 10: 1) . Compound 25-7 (550 mg, 1.72 mmol, 70.3%yield) was obtained as a white solid. The structure was confirmed by H NMR.

H NMR: (400 MHz, DMSO-d6) δ 12.21 (s, 1H) , 6.22 (s, 1H) , 4.58 -4.55 (m, 1H) , 4.09 -4.05 (m, 1H) , 3.04 -2.67 (m, 4H) , 2.61 (s, 3H) , 1.82 -1.68 (m, 4H) , 1.03 -0.98 (m, 6H) .

Step 5. 3-amino-6- (1-isobutyrylpiperidin-4-yl) -1, 5-dihydro-4H-pyrazolo [4, 3-c] pyridin-4-one

Compound 25-7 (550 mg, 1.72 mmol, 1.00 eq) , and N₂H₄ ^. H₂O (2.69 g, 52.6 mmol, 2.61 mL, 98.0%purity, 30.5 eq) were taken up into a microwave tube in i- PrOH (4.00 mL) . The sealed tube was heated at 120 ℃ for 60 mins under microwave. TLC indicated compound 25-7 was consumed completely and one new spot formed. The reaction was clean according to TLC. (Plate 1: DCM: MeOH =10: 1) . The reaction mixture was concentrated to give a residue. Compound 25-8 (400 mg, 1.32 mmol, 76.5%yield) was obtained as a white solid. The structure was confirmed by H NMR.

H NMR: (400 MHz, DMSO-d6) δ 5.95 (s, 1H) , 5.19 (s, 2H) , 4.53 (d, J =11.6 Hz, 1H) , 4.04 (d, J = 13.2 Hz, 1H) , 3.05 -3.02 (m, 1H) , 2.99 -2.84 (m, 1H) , 2.67 -2.61 (m, 2H) , 1.89 -1.81 (m, 2H) , 1.55 -1.38 (m, 2H) , 1.02 -0.97 (m, 6H) .

Step 6. 1- (4- (3-amino-4-bromo-1H-pyrazolo [4, 3-c] pyridin-6-yl) piperidin-1-yl) -2-methylpropan-1-one

POBr₃ (2.84 g, 9.89 mmol, 1.01 mL, 10.0 eq) was melted at 60 ℃. Compound 25-8 (300 mg, 988 μmol, 1.00 eq) was added to the reaction mixture and stirred at 75 ℃ for 1 hr. LC-MS showed desired mass detected. The reaction mixture was poured into 100 mL ice sat. aq NaHCO₃, and then extracted with EtOAc 200 mL (50.0 mL *4) . The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiO₂, DCM: MeOH = 7: 1) . (Plate 1: DCM: MeOH = 7: 1) . Compound 25-9 (80.0 mg, 218 μmol, 22.0%yield) was obtained as a white solid.

Step 7. N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1H-pyrazolo [4, 3-c] pyridin-4-yl) phenyl) -1-isopropyl-2, 4-dioxo-3- (pyridin-2-yl) -1, 2, 3, 4-tetrahydropyrimidine-5-carboxamide

Compound 25-9 (30.0 mg, 81.9 μmol, 1.00 eq) , compound 25-10 (117 mg, 245 μmol, 3.00 eq) and DIEA (31.7 mg, 245 μmol, 42.8 μL, 3.00 eq) Pd (t-Bu₃P) ₂ (4.19 mg, 8.19 μmol, 0.100 eq) were taken up into a microwave tube in H₂O (0.150 mL) , dioxane (1.50 mL) . The sealed tube was heated at 130 ℃ for 60 mins under microwave. LC-MS showed desired mass detected. The reaction mixture was concentrated to give a residue. The residue was purified by prep-TLC (SiO₂, DCM: MeOH = 10: 1) . (Plate 1: DCM: MeOH = 10: 1) . Compound 25 was obtained as confirmed by H NMR, LC-MS, and HPLC.

H NMR (400 MHz, CDCl₃) δ 10.92 (s, 1H) , 8.74 (d, J = 4.0 Hz, 1H) , 8.70 (s, 1H) , 8.01 -7.97 (m, 1H) , 7.81 (d, J = 8.4 Hz, 2H) , 7.21 (d, J = 8.4 Hz, 2H) , 7.53 - 7.40 (m, 3H) , 7.04 (s, 1H) , 5.01 -4.98 (m, 1H) , 4.80 -4.77 (m, 1H) , 4.21 -4.09 (m, 3H) , 3.24 -3.17 (m, 1H) , 2.93 -2.82 (m, 1H) , 2.23 -2.09 (m, 2H) , 1.72 -1.65 (m, 2H) , 1.54 -1.45 (m, 8H) , 1.14 (t, J = 6.8 Hz, 6H) .

Example 6

N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) benzo [d] isoxazol-4-yl) phenyl) -1-isopropyl-2, 4-dioxo-3- (pyridin-2-yl) -1, 2, 3, 4-tetrahydropyrimidine-5-carboxamide (Compound 27)

Step 1. 4'-amino-5-bromo-3-fluoro- [1, 1'-biphenyl] -2-carbonitrile

To a solution of compound 27-1 (1.00 g, 3.07 mmol, 1.00 eq) and compound 27-2 (470 mg, 2.15 mmol, 0.700 eq) in THF (12.0 mL) was added Na₂CO₃ (975 mg, 9.21 mmol, 3.00 eq) in H₂O (3.00 mL) and Pd (dppf) Cl₂ (449 mg, 613 μmol, 0.200 eq) . The mixture was degassed and purged with N₂ for 3 times, then stirred at 60 ℃for 3 hrs. LC-MS showed the desired mass was detected. The reaction mixture was quenched by water (20.0 mL) , and then extracted with EtOAc (30.0 mL) . The organic layers were washed by water (10.0 mL *3) and brine (10.0 mL *3) , and the combined aqueous layers were extracted with EtOAc (30.0 mL *3) . The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue (PE: EtOAc = 3: 1, R_f (P1) = 0.500) was purified by column chromatography (SiO₂, PE: EtOAc = 100: 0 to 100: 30) . Then the residue was triturated with EtOAc: MeOH = 10: 1 (100 mL) at 25 ℃ for 30 min. The mixture was filtered and concentrated under reduced pressure to give a residue. Compound 27-3 (330 mg, 742 μmol, 24.2%yield) was obtained as a brown solid, confirmed by LC-MS.

LC-MS: (M+H) ⁺: 291.0

Step 2. 4'-amino-3-fluoro-5- (1-isobutyryl-1, 2, 3, 6-tetrahydropyridin-4-yl) - [1, 1'-biphenyl] -2-carbonitrile

To a solution of compound 27-3 (320 mg, 720 μmol, 65.5%purity, 1.00 eq) and compound 27-4 (301 mg, 1.08 mmol, 1.50 eq) in THF (12.0 mL) was added K₂CO₃ (298 mg, 2.16 mmol, 3.00 eq) in H₂O (3.00 mL) and Pd (PPh₃) ₄ (166 mg, 144 μmol, 0.200 eq) . The mixture was stirred at 75 ℃ for 12 hrs under N₂ atmosphere. LC-MS showed the desired mass was detected. The reaction mixture was quenched by water (20.0 mL) , and then extracted with EtOAc (30.0 mL) . The organic layers were washed by water (10.0 mL *3) and brine (10.0 mL *3) , and the combined aqueous layers were extracted with EtOAc (30.0 mL *3) . The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (DCM: MeOH = 10: 1, R_f (P1) = 0.600) . Crude 27-5 (550 mg) was obtained as a brown solid.

Step 3. 4'-amino-3-fluoro-5- (1-isobutyrylpiperidin-4-yl) - [1, 1'-biphenyl] -2-carbonitrile

To a solution of compound 27-5 (550 mg, 1.51 mmol, 1.00 eq) in THF (80.0 mL) was added Pd/C (8.00 g, 10.0%purity) , degassed and purged with H₂ (15 Psi) for 3 times, then stirred at 20 ℃ for 2 hrs. LC-MS showed the desired mass was detected. The mixture was filtered and filtrate was concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (EtOAc: MeOH = 10: 1, R_f (P1) =0.800) and further purified by prep-HPLC (neutral condition; column: Welch Ultimate XB-SiOH 250 *70.0 *10.0 um; mobile phase: [Hexane-EtOH] ; B%: 15.0%-55.0%, 15 min) . Compound 27-6 (120 mg, 322 μmol, 21.2%yield) was obtained as a yellow solid, confirmed by H NMR , F NMR and LC-MS.

H NMR: (400 MHz, CDCl₃) δ 7.40 -7.38 (m, 2H) , 7.10 (s, 1H) , 6.97 -6.95 (m, 1H) , 6.81 -6.79 (m, 2H) , 4.86 (d, J = 14.8 Hz, 1H) , 4.10 (d, J = 12.8 Hz, 1H) , 3.19 -3.12 (m, 1H) , 2.88 -2.81 (m, 2H) , 2.66 -2.60 (m, 1H) , 1.98 -1.92 (m, 2H) , 1.67 -1.61 (m, 2H) , 1.20 -1.11 (m, 6H) .

LC-MS: (M+H) ⁺: 366.3

Step 4. 4'-amino-5- (1-isobutyrylpiperidin-4-yl) -3- ( (propan-2-ylideneamino) oxy) - [1, 1'-biphenyl] -2-carbonitrile

To a solution of compound 27-7 (23.5 mg, 322 μmol, 26.1 μL, 2.00 eq) in DMF (2.00 mL) was added t-BuOK (36.1 mg, 322 μmol, 2.00 eq) . The mixture was stirred at 20 ℃ for 1 hr. Then compound 27-6 (60.0 mg, 161 μmol, 98.1%purity, 1.00 eq) was added to the mixture. And the mixture was stirred at 20 ℃ for 6 hrs. LC-MS showed the desired mass was detected. The reaction mixture was quenched by water (10.0 mL) , and then extracted with EtOAc (10.0 mL *5) . The combined organic layers were washed by water (10.0 mL *4) and brine (10.0 mL *4) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. Crude 27-8 (80.0 mg) was obtained as a brown solid.

Step 5. 1- (4- (3-amino-4- (4-aminophenyl) benzo [d] isoxazol-6-yl) piperidin-1-yl) -2-methylpropan-1-one

To a solution of compound 27-8 (80.0 mg, 191 μmol, 1.00 eq) in MeOH (4.00 mL) and DCM (2.00 mL) was added HCl (6.00 M, 4.00 mL, 125 eq) . Then the mixture was stirred at 50 ℃ for 10 hrs. LC-MS showed the desired mass was detected. The reaction mixture was concentrated to give a residue. The residue was adjusted pH to 7 by sat. NaHCO₃. Then the mixture was extracted with EtOAc (30.0 mL *3) . The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (DCM: MeOH = 10: 1, R_f (P1) = 0.600) . Compound 27-9 (40.0 mg, 104 μmol, 54.9%yield) was obtained as an off-white solid, confirmed by LC-MS.

LC-MS: (M+H) ⁺: 379.4

Step 6. N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) benzo [d] isoxazol-4-yl) phenyl) -1-isopropyl-2, 4-dioxo-3- (pyridin-2-yl) -1, 2, 3, 4-tetrahydropyrimidine-5-carboxamide (Compound 27)

To a solution of compound 27-9 (30.0 mg, 78.7 μmol, 99.3%purity, 1.00 eq) and compound 27-10 (21.6 mg, 78.7 μmol, 1.00 eq) in pyridine (3.00 mL) was added EDCI (60.3 mg, 314 μmol, 4.00 eq) . The mixture was stirred at 20 ℃ for 2 hrs. LC-MS showed the desired mass was detected. The reaction mixture was quenched by water (10.0 mL) , and then extracted with DCM (30.0 mL *3) . The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (DCM: MeOH = 10: 1, R_f (P1) = 0.500) . Compound 27 (34.5 mg, 53.0 μmol, 67.2%yield, 97.6%purity) was obtained as confirmed by H NMR, 2D NMR and LC-MS.

H NMR: (400 MHz, DMSO-d₆) δ 10.88 (s, 1H) , 8.72 (s, 1H) , 8.64 -8.63 (m, 1H) , 8.08 -8.05 (m, 1H) , 7.83 -7.81 (m, 2H) , 7.58 -7.55 (m, 2H) , 7.51 -7.49 (m, 2H) , 7.37 (s, 1H) , 7.07 (s, 1H) , 5.19 -5.17 (m, 2H) , 4.79 -4.75 (m, 1H) , 4.59 (d, J =11.2 Hz, 1H) , 4.07 (d, J = 13.6 Hz, 1H) , 3.15 -3.09 (m, 1H) , 2.99 -2.86 (m, 2H) , 2.61 -2.55 (m, 1H) , 1.92 -1.84 (m, 2H) , 1.69 -1.51 (m, 2H) , 1.45 -1.43 (m, 6H) , 1.03 - 0.99 (m, 6H) .

LC-MS: (M+H) ⁺: 636.4

HPLC purity: 97.6% (220 nm)

Example 7

N- (4- (3-amino-1-ethyl-6- (1-isobutyrylpiperidin-4-yl) -1H-indazol-4-yl) phenyl) -1-isopropyl-2, 4-dioxo-3- (pyridin-2-yl) -1, 2, 3, 4-tetrahydropyrimidine-5-carboxamide (Compound 30)

Step 1. 2- (6-bromo-1-ethyl-4-iodo-1H-indazol-3-yl) isoindoline-1, 3-dione

To a solution of compound 30-1 (500 mg, 974 μmol, 91.2%purity, 1.00 eq) in DMF (5.00 mL) was added Cs₂CO₃ (317 mg, 974 μmol, 1.00 eq) and EtI (197 mg, 1.27 mmol, 101 μL, 1.30 eq) at 0 ℃. Then the mixture was stirred at 20 ℃ for 0.5 hr. Then EtI (30.4 mg, 195 μmol, 15.6 μL, 0.200 eq) was added to the mixture. And the mixture was stirred at 20 ℃ for 0.5 hr. LC-MS showed desired mass was detected. The mixture was poured into water (10.0 mL) and extracted with DCM: MeOH = 10: 1 (10.0 mL *3) . And the combined organic layers were washed with brine (10.0 mL * 3) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. Compound 30-2 (513 mg, crude) was obtained as a white solid.

LC-MS: (M+H) ⁺: 497.9

Step 2. 6-bromo-1-ethyl-4-iodo-1H-indazol-3-amine

To a solution of compound 30-2 (513 mg, 1.03 mmol, 1.00 eq) in MeOH (5.00 mL) and DCM (5.00 mL) was added N₂H₄ ^. H₂O (920 mg, 18.0 mmol, 893 μL, 98.0%purity, 17.4 eq) . The mixture was stirred at 20 ℃ for 2 hrs. LC-MS showed desired mass was detected. TLC (Petroleum ether: EtOAc = 1: 1, R_f (P1) = 0.20) indicated the starting material was consumed completely. The mixture was washed by water (10.0 mL) , the water was extracted with DCM: MeOH = 10: 1 (5.00 mL *3) . The combined orgaic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue (Petroleum ether: EtOAc = 1: 1, R_f (P1) = 0.20) was purified by column chromatography (SiO₂, PE: EtOAc = 100: 20 to 100: 40) . Compound 30-3 (276 mg, 720 μmol, 69.6%yield, 95.5%purity) was obtained as a white solid, confirmed by H NMR, 2D NMR and LC-MS.

H NMR: (400 MHz, DMSO-d₆) δ 7.79 (s, 1H) , 7.45 (s, 1H) , 5.21 (s, 2H) , 4.16 -4.10 (m, 2H) , 1.25 (t, J = 7.2 Hz, 3H) .

LC-MS: (M+H) ⁺: 367.9

Step 3. 4- (4-aminophenyl) -6-bromo-1-ethyl-1H-indazol-3-amine

A mixture of compound 30-3 (0.250 g, 683 μmol, 1.00 eq) , compound 30-4 (149 mg, 683 μmol, 1.00 eq) , Pd (PPh₃) ₄ (78.9 mg, 68.3 μmol, 0.100 eq) , Na₂CO₃ (361 mg, 3.42 mmol, 5.00 eq) , EtOH (2.00 mL) and H₂O (2.00 mL) in toluene (2.00 mL) was degassed and purged with N₂ for 3 times, and then the mixture was stirred at 90 ℃ for 3 hrs under N₂ atmosphere. LC-MS showed ～ 31.1%of desired mass was detected. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiO₂, DCM: MeOH = 10: 1) . Compound 30-5 (175 mg, 528 μmol, 77.35%yield) was obtained as a yellow solid.

LC-MS: (M+H) ⁺: 331.0

Step 4. 1- (4- (3-amino-4- (4-aminophenyl) -1-ethyl-1H-indazol-6-yl) -3, 6-dihydropyridin-1 (2H) -yl) -2-methylpropan-1-one

A mixture of compound 30-5 (163 mg, 492 μmol, 1.00 eq) , compound 30-6 (206 mg, 738 μmol, 1.50 eq) , Pd (PPh₃) ₄ (56.8 mg, 49.2 μmol, 0.100 eq) , K₂CO₃ (204 mg, 1.48 mmol, 3.00 eq) and H₂O (0.750 mL) in dioxane (3.00 mL) was degassed and purged with N₂ for 3 times, and then the mixture was stirred at 90 ℃ for 4 hrs under N₂ atmosphere. LC-MS showed ～ 51.5%of desired mass was detected. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiO₂, DCM: MeOH = 10: 1) . Compound 30-7 (166 mg, 408 μmol, 82.92%yield, 99.2%purity) was obtained as a yellow solid and confirmed by LC-MS.

LC-MS: (M+H) ⁺: 404.2

Step 5. 1- (4- (3-amino-4- (4-aminophenyl) -1-ethyl-1H-indazol-6-yl) piperidin-1-yl) -2-methylpropan-1-one

To a solution of compound 30-7 (156 mg, 386 μmol, 1.00 eq) in MeOH (3.00 mL) was added Pd/C (10%, 150 mg) under N₂ atmosphere. The suspension was degassed and purged with H₂ for 3 times. The mixture was stirred under H₂ (15 Psi) at 25 ℃ for 2 hrs. LC-MS showed compound 30-7 was consumed completely and one main peak with desired mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. Compound 30-8 (140 mg, crude) was obtained as a yellow solid.

LC-MS: (M+H) ⁺: 406.2

Step 6. N- (4- (3-amino-1-ethyl-6- (1-isobutyrylpiperidin-4-yl) -1H-indazol-4-yl) phenyl) -1-isopropyl-2, 4-dioxo-3- (pyridin-2-yl) -1, 2, 3, 4-tetrahydropyrimidine-5-carboxamide (Compound 30)

A mixture of compound 30-8 (70.0 mg, 172 μmol, 1.00 eq) , compound 30-9 (52.2 mg, 189 μmol, 1.10 eq) , EDCI (99.2 mg, 517 μmol, 3.00 eq) in Py (2.00 mL) was degassed and purged with N₂ for 3 times, and then the mixture was stirred at 25 ℃ for 2 hrs under N₂ atmosphere. LC-MS showed ～ 54.9%of desired mass was detected. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (neutral condition; column: Waters Xbridge 150 *25 mm *5 um; mobile phase: [water (NH₄HCO₃) -ACN] ; B%: 42%-72%, 8 min) . Compound 30 (46.5 mg, 67.6 μmol, 39.21%yield, 96.3%purity) was obtained as confirmed by H NMR, LC-MS and HPLC.

H NMR: (400 MHz, CDCl₃) δ 10.8 (s, 1H) , 8.77 -8.66 (m, 2H) , 7.96 (td, J₁ = 8.0 Hz, J₂ = 2.0 Hz, 1H) , 7.74 (d, J = 8.0 Hz, 2H) , 7.51 -7.43 (m, 3H) , 7.38 (d, J =8.0 Hz, 1H) , 6.98 (s, 1H) , 6.73 (s, 1H) , 5.02 -4.91 (m, 1H) , 4.84 (d, J = 12.0 Hz, 1H) , 4.19 (q, J = 8.0 Hz, 2H) , 4.08 (d, J = 12.0 Hz, 1H) , 3.79 (s, 2H) , 3.15 (t, J = 12.0 Hz, 1H) , 2.95 -2.76 (m, 2H) , 2.63 (t, J = 12.0 Hz, 1H) , 2.07 -1.91 (m, 2H) , 1.80 -1.59 (m, 2H) , 1.48 (d, J = 8.0 Hz, 6H) , 1.41 (d, J = 8.0 Hz, 3H) , 1.14 (q, J = 8.0 Hz, 6H) .

LC-MS: (M+H) ⁺: 663.4

HPLC Purity: 96.3% (220 nm) .

Example 8

N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1H-indazol-4-yl) phenyl) -1- (4-fluorophenyl) -4, 5-dimethyl-2-oxo-1, 2-dihydropyridine-3-carboxamide (Compound 35)

Step 1. 5-bromo-4-methyl-2-oxo-1, 2-dihydropyridine-3-carbonitrile

To a solution of compound 35-1 (6.00 g, 44.7 mmol, 1.00 eq) in DMF (50.0 mL) was added NBS (8.76 g, 49.2 mmol, 1.10 eq) at 0 ℃ under N₂. The mixture was stirred at 40 ℃ for 2 hrs. LC-MS showed desired mass detected. The reaction mixture was poured into 100 mL ice water and there's solid precipitation. Then filtered and the cake was washed with PE 100 mL, concentrated to give a crude product. Compound 35-2 (6.00 g, 28.1 mmol, 62.9%yield) was obtained as a yellow solid. The structure was confirmed by H NMR.

H NMR: (400 MHz, DMSO-d6) δ 12.75 (s, 1H) , 8.06 (s, 1H) , 2.42 (s, 3H) .

Step 2. 5-bromo-4-methyl-2-oxo-1, 2-dihydropyridine-3-carboxylic acid

A mixture of compound 35-2 (2.00 g, 9.39 mmol, 1.00 eq) in 20.0 mL H₂SO₄ (50.0%purity) and stirred at 120 ℃ for 4 hrs. LC-MS showed desired mass detected. The reaction mixture was added 20.0 mL H₂O and filtered. The cake was concentrated under reduced pressure to give a residue. Compound 35-3 (0.800 g, 3.45 mmol, 36.7%yield) was obtained as a yellow solid.

Step 3. methyl 5-bromo-4-methyl-2-oxo-1, 2-dihydropyridine-3-carboxylate

A mixture of compound 35-3 (0.800 g, 3.45 mmol, 1.00 eq) and H₂SO₄ (1.84 g, 18.7 mmol, 1.00 mL, 5.44 eq) in MeOH (10.0 mL) was stirred at 80 ℃ for 12 hrs. LC-MS showed desired mass detected. The reaction mixture was diluted with H₂O 20.0 mL, and added sat. aq Na₂CO₃ to adjusted pH to 8. The mixture was extracted with DCM 100 mL (50.0 mL *2) . The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. Compound 35-4 (0.650 g, 2.64 mmol, 76.6%yield) was obtained as a yellow solid. The structure was confirmed by H NMR.

H NMR: (400 MHz, DMSO-d6) δ 12.19 (s, 1H) , 7.82 (s, 1H) , 3.78 (s, 3H) , 2.14 (s, 3H) .

Step 4. methyl 5-bromo-1- (4-fluorophenyl) -4-methyl-2-oxo-1, 2-dihydropyridine-3-carboxylate

To a solution of compound 35-4 (0.650 g, 2.64 mmol, 1.00 eq) and compound 35-5 (997 mg, 7.13 mmol, 2.70 eq) in DCM (10.0 mL) was added Cu (OAc) ₂ (959 mg, 5.28 mmol, 2.00 eq) and Py (835 mg, 10.5 mmol, 852 μL, 4.00 eq) . The mixture was stirred at 25 ℃ for 12 hrs. LC-MS showed desired mass detected. The reaction mixture was filtered and the filtrate was concentrated to give a crude product. The residue was purified by column chromatography (SiO₂, Petroleum ether: EtOAc = 100: 1 to 1: 1) . (Petroleum ether: EtOAc = 2: 1) . Compound 35-6 (0.800 g, 2.35 mmol, 89.0%yield) was obtained as a white solid. The structure was confirmed by H NMR.

H NMR: (400 MHz, CDCl₃) δ 7.56 (s, 1H) , 7.37 -7.34 (m, 2H) , 7.19 -7.15 (m, 2H) , 3.93 (s, 3H) , 2.33 (s, 3H) .

Step 5. methyl 1- (4-fluorophenyl) -4, 5-dimethyl-2-oxo-1, 2-dihydropyridine-3-carboxylate

To a solution of compound 35-6 (0.100 g, 293 μmol, 1.00 eq) and compound 35-7 (175 mg, 2.94 mmol, 10.0 eq) in H₂O (0.500 mL) and dioxane (1.50 mL) was added K₂CO₃ (121 mg, 881 μmol, 3.00 eq) and Pd (dppf) Cl₂ ^. CH₂Cl₂ (24.0 mg, 29.4 μmol, 0.100 eq) . The mixture was stirred at 120 ℃ for 1 hr. TLC indicated compound 35-6 was consumed completely and many new spots formed. The reaction was messy according to TLC. (Petroleum ether: EtOAc = 1: 1) . The reaction mixture was diluted with H₂O 10.0 mL and extracted with EtOAc 40.0 mL (20.0 mL *2) . The combined organic layers were washed with brine 60.0 mL (20.0 mL *3) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiO₂, Petroleum ether: Ethyl acetate = 1: 1) . Compound 35-8 (85.0 mg, 213 μmol, 72.4%yield, 69%purity) was obtained as a white solid. The structure was confirmed by LC-MS.

Step 6. 1- (4-fluorophenyl) -4, 5-dimethyl-2-oxo-1, 2-dihydropyridine-3-carboxylic acid

To a solution of compound 35-8 (85.0 mg, 213 μmol, 69%purity, 1.00 eq) in EtOH (4.00 mL) was added NaOH (2.00 M, 3.00 mL, 28.1 eq) . The mixture was stirred at 90 ℃ for 2 hrs. LC-MS showed desired mass detected. The reaction mixture was diluted with H₂O 10.0 mL, and added 1 M HCl to adjusted pH to 2. The mixture was extracted with EtOAc 100 mL (50.0 mL *2) . The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (FA condition; column: Phenomenex C18 75.0 *30.0 mm *3.00 um; mobile phase: [water (FA) - ACN] ; B%: 22.0%-52.0%, 7 min) . 35-9 (50.0 mg, 191 μmol, 89.8%yield) was obtained as a white solid. The structure was confirmed by H NMR.

H NMR: (400 MHz, CDCl₃) δ 15.22 (s, 1H) , 7.39 -7.36 (m, 3H) , 7.26 -7.22 (m, 2H) , 2.83 (s, 3H) , 2.21 (s, 3H) .

Step 7. N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1H-indazol-4-yl) phenyl) -1- (4-fluorophenyl) -4, 5-dimethyl-2-oxo-1, 2-dihydropyridine-3-carboxamide

To a solution of compound 35-10 (50.0 mg, 122 μmol, 92.7%purity, 1.00 eq) and compound 35-9 (28.8 mg, 110 μmol, 0.900 eq) in pyridine (3.00 mL) was added EDCI (94.1 mg, 491 μmol, 4.00 eq) . The mixture was stirred at 20 ℃ for 2 hrs. LC-MS showed the desired mass was detected. The reaction mixture was quenched by water (5.00 mL) , and then extracted with DCM: MeOH = 10: 1 (10.0 mL *3) . The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (HCl condition; column: 3_Phenomenex Luna C18 75 *30.0 mm *3.00 um; mobile phase: [water (HCl) -ACN] ; B%: 26.0%-46.0%, 8 min) . Compound 35 (8.00 mg, 12.7 μmol, 10.3%yield, 98.8%purity) was obtained as confirmed by H NMR and LC-MS.

H NMR: (400 MHz, DMSO-d₆) δ 11.60 (s, 1H) , 10.54 (s, 1H) , 7.82 -7.81 (m, 2H) , 7.59 (s, 1H) , 7.49 -7.45 (m, 4H) , 7.43 -7.37 (m, 2H) , 7.06 (s, 1H) , 6.71 (s, 1H) , 4.59 (d, J = 14.4 Hz, 1H) , 4.27 (d, J = 10.0 Hz, 2H) , 4.08 (d, J = 14.8 Hz, 1H) , 3.17 -3.08 (m, 1H) , 2.94 -2.86 (m, 3H) , 2.20 (s, 3H) , 2.08 (s, 3H) , 1.93 -1.85 (m, 2H) , 1.62 -1.47 (m, 2H) , 1.04 -0.99 (m, 6H) .

LC-MS: (M+H) ⁺: 621.4

HPLC: purity: 98.8% (220 nm)

Example 9

N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1H-indazol-4-yl) phenyl) -5- (4-fluorophenyl) -4-hydroxynicotinamide (Compound 36)

Step 1. 5- (4-fluorophenyl) -4-hydroxynicotinic acid

To a solution of compound 36-1 (1.00 g, 4.59 mmol, 1.00 eq) in dioxane (10.0 mL) and H₂O (2.00 mL) was added Pd (PPh₃) ₄ (530 mg, 458 μmol, 0.100 eq) and Na₂CO₃ (972 mg, 9.17 mmol, 2.00 eq) and compound 36-2 (963 mg, 6.88 mmol, 1.50 eq) at 20 ℃. The reaction mixture was stirred at 90 ℃ for 12 hrs. LC-MS showed compound 36-1 was consumed completely. Several new peaks were shown on LC-MS and desired m/z (MS=234.0 RT=0.688) was detected. The reaction mixture was quenched by 1.00 M HCl until pH is around 2, then extracted with EtOAc 300 mL (100 mL *3) . The combined organic layers were dried over anhydrous Na₂SO₄, then filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (FA condition, column: Phenomenex luna C18 150 *25.0 mm *10.0 um; mobile phase: [water (FA) -ACN] ; B%:12.0%-42.0%, 10 min) . Compound 36-3 (0.150 g, 643 μmol, 14.0%yield, 100%purity) was obtained as confirmed by LC-MS and H NMR.

H NMR: (400 MHz, DMSO-d₆) δ 8.61 (s, 1H) , 8.25 (s, 1H) , 7.74 -7.71 (m, 2H) , 7.30 -7.26 (m, 2H) .

LC-MS: (M+H) ⁺: 234.2

Step 2. N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1H-indazol-4-yl) phenyl) -5- (4-fluorophenyl) -4-hydroxynicotinamide

To a solution of compound 36-4 (80.0 mg, 188 μmol, 88.9%purity, 1.00 eq) , compound 36-3 (43.9 mg, 188 μmol, 100%purity, 1.00 eq) and HATU (107 mg, 282 μmol, 1.50 eq) in DMF (5.00 mL) was added DIEA (73.1 mg, 565 μmol, 98.5 μL, 3.00 eq) . The mixture was stirred at 20 ℃ for 3 hrs. LC-MS showed desired mass was detected. The reaction mixture was quenched by water (10.0 mL) , and then extracted with EtOAc (10.0 mL *3) . The combined organic layers were washed with water (5.00 *3mL) , dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (DCM: MeOH = 10: 1, R_f (P1) = 0.200) and was further purified by prep-HPLC (HCl condition, column: 3_Phenomenex Luna C18 75.0 *30.0 mm *3.00 um; mobile phase: [water (HCl) -ACN] ; B%: 28.0%-48.0%, 8 min) . Compound 36 (20.0 mg, 33.2 μmol, 17.6%yield, 98.4%purity) was obtained as confirmed by H NMR, LC-MS and HPLC.

H NMR: (400 MHz, DMSO-d₆) δ 13.15 (s, 1H) , 11.61 (s, 1H) , 8.63 (s, 1H) , 8.09 (s, 1H) , 7.84 -7.82 (m, 2H) , 7.74 -7.70 (m, 2H) , 7.48 -7.45 (m, 2H) , 7.29 -7.27 (m, 2H) , 7.07 (s, 1H) , 6.73 (s, 1H) , 4.58 (d, J = 11.6 Hz, 1H) , 4.30 (s, 2H) , 4.07 (d, J = 12.8 Hz, 1H) , 3.16 -3.13 (m, 1H) , 2.92 -2.87 (m, 2H) , 2.57 -2.50 (m, 1H) , 1.89 -1.85 (m, 2H) , 1.62 -1.47 (m, 2H) , 1.04 -0.99 (m, 6H) .

LC-MS: (M+H) ⁺: 593.3

HPLC: purity: 98.9% (220 nm)

Example 10

N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1H-indazol-4-yl) phenyl) -5- (4-fluorophenyl) -4-hydroxy-6-methylnicotinamide (Compound 37)

Step 1. 5-bromo-6-methyl-4-oxo-1, 4-dihydropyridine-3-carboxylic acid

To a solution of compound 37-1 (5.00 g, 32.6 mmol, 1.00 eq) in AcOH (50.0 mL) and H₂O (10.0 mL) was added Br₂ (6.26 g, 39.1 mmol, 2.02 mL, 1.20 eq) dropwise at 20 ℃. The reaction mixture was stirred at 60 ℃ for 5 hrs. LC-MS showed the starting material was consumed completely. The reaction mixture was concentrated under reduced pressure to remove AcOH, then diluted with MeOH (30.0 mL) , and then filtered and filter cake was washed with MeOH (10.0 mL) . The filter cake was concentrated under reduced pressure to give a residue. Compound 37-2 (5.12 g, 22.0 mmol, 67.5%yield) was obtained as white solid, confirmed by H NMR.

H NMR: (400 MHz, DMSO-d₆) δ 15.77 (s, 1H) , 13.43 (s, 1H) , 8.55 (s, 1H) , 2.53 (s, 3H) .

Step 2. 5- (4-fluorophenyl) -4-hydroxy-6-methylnicotinic acid

To a solution of compound 37-2 (2.00 g, 8.62 mmol, 1.00 eq) in dioxane (20.0 mL) and H₂O (4.00 mL) was added Na₂CO₃ (1.83 g, 17.2 mmol, 2.00 eq) and Pd(PPh₃) ₄ (996 mg, 861 μmol, 0.100 eq) and compound 37-3 (2.41 g, 17.2 mmol, 2.00 eq) at 20 ℃. The reaction mixture was stirred at 90 ℃ for 12 hrs. LC-MS showed ～5%of the starting material was remained. Several new peaks were shown on LC-MS and desired m/z (MS=248.1 RT=0.700) was detected. The reaction mixture was quenched by 1 M HCl until pH was around 2, then filtered and the filter cake was dissolved in MeOH (10.0 mL) and DCM (90.0 mL) , then dried over anhydrous Na₂SO₄, and then filtered and concentrated under reduced pressure to give a residue. Compound 37-4 (0.700 g, 2.69 mmol, 31.2%yield, 95.0%purity) was obtained as confirmed by H NMR and LC-MS.

H NMR: (400 MHz, DMSO-d₆) δ 13.11 (s, 1H) , 8.57 (s, 1H) , 7.35 -7.25 (m, 4H) , 2.22 (s, 3H) .

LC-MS: (M+H) ⁺: 248.1

Step 3. N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1H-indazol-4-yl) phenyl) -5- (4-fluorophenyl) -4-hydroxy-6-methylnicotinamide

To a solution of compound 37-5 (78.0 mg, 200 μmol, 97.0%purity, 1.00 eq) and compound 37-4 (46.9 mg, 180 μmol, 95.0%purity, 0.900 eq) in pyridine (5.00 mL) was added EDCI (153 mg, 801 μmol, 4.00 eq) . The mixture was stirred at 40 ℃ for 2 hrs. LC-MS showed desired mass was detected. The reaction mixture was quenched by water (5.00 mL) , and then extracted with DCM: MeOH = 10: 1 (10.0 mL *3) . The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (DCM: MeOH = 10: 1, R_f (P1) = 0.200) and was further purified by prep-HPLC (HCl condition; column: 3_Phenomenex Luna C18 75.0 *30.0 mm *3.00 um; mobile phase: [water (HCl) -ACN] ; B%: 28.0%-48.0%, 8 min) . Compound 37 (18.0 mg, 29.5 μmol, 14.7%yield, 99.5%purity) was obtained as confirmed by H NMR and LC-MS.

H NMR: (400 MHz, DMSO-d₆) δ 13.16 (s, 1H) , 11.60 (s, 1H) , 8.54 (s, 1H) , 7.80 -7.78 (m, 2H) , 7.45 -7.43 (m, 2H) , 7.33 -7.06 (m, 4H) , 7.06 (s, 1H) , 6.72 (s, 1H) , 4.58 (d, J = 12.0 Hz, 1H) , 4.27 (d, J = 10.0 Hz, 1H) , 4.07 (d, J = 12.0 Hz, 1H) , 3.28 -3.25 (m, 1H) , 3.16 -3.09 (m, 1H) , 2.94 -2.86 (m, 2H) , 2.70 -2.62 (m, 1H) , 2.17 (s, 3H) , 1.89 -1.85 (m, 2H) , 1.64 -1.47 (m, 2H) , 1.03 -0.99 (m, 6H) .

LC-MS: (M+H) ⁺: 607.4

HPLC: purity: 99.5% (220 nm)

Example 11

N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1H-indazol-4-yl) phenyl) -5- (4-fluorophenyl) -4-hydroxy-2, 6-dimethylnicotinamide (Compound 38)

Step 1. ethyl 4-hydroxy-2, 6-dimethylnicotinate

A mixture of compound 38-1 (19.8 g, 139 mmol, 18.3 mL, 1.80 eq) and compound 38-2 (10.0 g, 77.4 mmol, 1.00 eq) was stirred at 130℃ for 5 hrs. TLC indicated compound 38-2 was consumed completely and many new spots formed. The reaction was messy according to TLC (Petroleum ether: Ethyl acetate = 3: 1) . The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was triturated with MTBE (50.0 mL) at 25 ℃ for 20 mins. Compound 38-3 (4.80 g, 24.5 mmol, 31.7%yield, 100%purity) was obtained as a white solid. The structure was confirmed by LC-MS and H NMR.

H NMR: (400 MHz, DMSO-d₆) δ 11.29 (s, 1H) , 5.89 (s, 1H) , 4.20 -4.14 (m, 2H) , 2.16 (s, 6H) , 1.22 (t, J = 6.80 Hz, 3H) .

LC-MS: (M-H) ^-: 194.1

Step 2. ethyl 5-bromo-4-hydroxy-2, 6-dimethylnicotinate

To a solution of compound 38-3 (2.00 g, 10.2 mmol, 100%purity, 1.00 eq) in CCl₄ (20.0 mL) was added NBS (3.65 g, 20.4 mmol, 2.00 eq) . The mixture was stirred at 70℃ for 5 hrs. LC-MS showed desired mass was detected. The reaction mixture was filtered and the cake was washed DCM (100 mL) . The filtrate was concentrated to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether: Ethyl acetate = 100: 1 to 0: 1) . (DCM: MeOH = 10: 1) . Compound 38-4 (2.50 g, 9.12 mmol, 89.0%yield) was obtained as a white solid. The structure was confirmed by H NMR.

H NMR: (400 MHz, DMSO-d₆) δ 11.80 (s, 1H) , 4.22 -4.20 (m, 2H) , 2.39 (s, 3H) , 2.20 (s, 3H) , 1.24 (t, J = 7.20 Hz, 3H) .

Step 3. 5- (4-fluorophenyl) -4-hydroxy-2, 6-dimethylnicotinic acid

To a solution of compound 38-4 (1.00 g, 3.65 mmol, 1.00 eq) and compound 38-5 (612 mg, 4.38 mmol, 1.20 eq) in dioxane (10.0 mL) and H₂O (2.00 mL) was added Pd (PPh₃) ₄ (421 mg, 364 μmol, 0.100 eq) and K₂CO₃ (1.01 g, 7.30 mmol, 2.00 eq) . The mixture was stirred at 100℃ for 12 hrs. TLC indicated the starting material was consumed completely and many new spots formed. The reaction was messy according to TLC (DCM : MeOH = 10: 1) . The reaction mixture was diluted with H₂O (80.0 mL) and extracted with EtOAc (100 mL *3) . The combined organic layers were washed with brine (200 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The crude product was triturated with MeOH: MTBE = 1: 10 (20.0 mL) at 15℃ for 60 min. Compound 38-6 (0.550 g, 2.02 mmol, 55.4%yield, 96.1%purity) was obtained as a gray solid. The structure was confirmed by H NMR and LC-MS.

H NMR: (400 MHz, DMSO-d₆) δ 12.73 (s, 1H) , 7.32 -7.26 (m, 4H) , 2.79 (s, 3H) , 2.21 (s, 3H) .

LC-MS: (M+H) ⁺: 261.9

Step 4. N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1H-indazol-4-yl) phenyl) -5- (4-fluorophenyl) -4-hydroxy-2, 6-dimethylnicotinamide

To a solution of compound 38-7 (60.0 mg, 138 μmol, 87.3%purity, 1.00 eq) , compound 38-6 (37.7 mg, 138 μmol, 96.1%purity, 1.00 eq) and HATU (79.2 mg, 208 μmol, 1.50 eq) in DMF (3.00 mL) was added DIEA (53.8 mg, 416 μmol, 72.5 μL, 3.00 eq) . The mixture was stirred at 20 ℃ for 2 hrs. LC-MS showed the desired mass was detected. The reaction mixture was quenched by addition water (30.0 mL) , and then extracted with EtOAc (30.0 mL *5) . The combined organic layers were washed with water (60.0 mL *5) and brine (60.0 mL *3) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (plate1, DCM: MeOH = 8: 1, R_f (P1) = 0.40) and was further purified by prep-HPLC (column: 3_Phenomenex Luna C18 75 *30 mm *3 um; mobile phase: [water (HCl) -ACN] ; B%: 28%-48%, 8 min) . Compound 38 (15.0 mg, 23.3 μmol, 16.8%yield, 96.5%purity) was obtained as confirmed by H NMR and LC-MS.

H NMR: (400 MHz, DMSO-d₆) δ 14.23 (s, 1H) , 11.59 (s, 1H) , 7.77 -7.75 (m, 2H) , 7.39 -7.37 (m, 2H) , 7.26 -7.04 (m, 4H) , 6.87 (s, 1H) , 6.70 (s, 1H) , 4.58 (d, J = 11.6 Hz, 1H) , 4.30 (s, 2H) , 4.06 (d, J = 11.6 Hz, 1H) , 3.16 -3.09 (m, 2H) , 2.95 - 2.85 (m, 2H) , 2.71 (s, 3H) , 2.06 (s, 3H) , 1.92 -1.85 (m, 2H) , 1.64 -1.45 (m, 2H) , 1.07 -0.99 (m, 6H) .

LC-MS: (M+H) ⁺: 621.4

HPLC: purity: 98.0% (220 nm)

Example 12

N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1H-indazol-4-yl) phenyl) -1-ethyl-4- (2, 2, 2-trifluoroethoxy) -1H-pyrazole-3-carboxamide (Compound 39)

Step 1. ethyl (Z) -4-chloro-2- (2- (4-methoxyphenyl) hydrazineylidene) -3-oxobutanoate

To a solution of HCl (3.00 M, 45.0 mL, 3.33 eq) was added compound 39-2 (5.00 g, 40.6 mmol, 1.00 eq) slowly at 10 ℃. Then the mixture was cooled to 0 ℃, and a solution of NaNO₂ (2.94 g, 42.6 mmol, 1.05 eq) in H₂O (10.0 mL) was added, the mixture turned to a yellow solution (solution A) . To a mixture of compound 39-1 (7.35 g, 44.6 mmol, 1.10 eq) and NaOAc (16.6 g, 203 mmol, 5.00 eq) in EtOH (10.0 mL) and H₂O (10.0 mL) was added solution A at 0 -5 ℃. Then the mixture was stirred at 15 ℃ for 1 hr. TLC indicated compound 39-2 was consumed completely and one new spot formed. The reaction was clean according to TLC. (Petroleum ether : Ethyl acetate = 1: 1, R_f (P1) = 0.300) . The mixture was filtrated, and the filter cake was washed with water (200 mL) and dried over high vacuum to afford the product as a yellow solid. Compound 39-3 (10.0 g, 33.4 mmol, 82.4%yield) was obtained as a yellow solid. The structure was confirmed by H NMR.

H NMR: (400 MHz, DMSO-d₆) δ 12.34 (s, 1H) , 7.52 -7.50 (m, 2H) , 7.00 -6.98 (m, 2H) , 4.93 (s, 2H) , 4.33 -4.28 (m, 2H) , 3.76 (s, 3H) , 1.30 (t, J = 2.80 Hz, 3H) .

Step 2. ethyl 4-hydroxy-1- (4-methoxyphenyl) -1H-pyrazole-3-carboxylate

To a solution of compound 39-3 (5.00 g, 16.7 mmol, 1.00 eq) in EtOH (50.0 mL) was added KOAc (3.29 g, 33.4 mmol, 2.00 eq) . The mixture was stirred at 78℃ for 12 hrs. LC-MS showed desired mass was detected. The reaction mixture was concentrated under reduced pressure to remove EtOH. The residue was diluted with H₂O (100 mL) and extracted with EtOAc (100 mL *2) . The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. Compound 39-4 (4.00 g, 15.2 mmol, 91.1%yield) was obtained as a yellow solid.

Step 3. ethyl 1- (4-methoxyphenyl) -4- (2, 2, 2-trifluoroethoxy) -1H-pyrazole-3-carboxylate

To a solution of compound 39-4 (4.00 g, 15.2 mmol, 1.00 eq) in DMF (20.0 mL) was added Cs₂CO₃ (9.94 g, 30.5 mmol, 2.00 eq) and compound 39-5 (4.25 g, 18.3 mmol, 1.20 eq) . The mixture was stirred at 20℃ for 10 hrs. LC-MS showed the starting material was consumed and the desired mass was found. The mixture was duilted with water (40.0 mL) , extracted with METB (20.0 mL*3) . The combined organic layers were washed with brine (25.0 mL) and dried over Na₂SO₄, filtered and concentrated in vacuo. Compound 39-6 (4.90 g, 14.2 mmol, 93.3%yield) was obtained as a brown solid, which was confirmed by H NMR.

H NMR: (400 MHz, DMSO-d₆) δ 8.56 (s, 1H) , 7.74 -7.72 (m, 2H) , 7.11 -7.09 (m, 2H) , 4.77 -4.70 (m, 2H) , 4.34 -4.27 (m, 2H) , 3.81 (s, 3H) , 1.30 (t, J = 7.20 Hz, 3H) .

Step 4. ethyl 4- (2, 2, 2-trifluoroethoxy) -1H-pyrazole-3-carboxylate

To a solution of compound 39-6 (4.90 g, 14.2 mmol, 1.00 eq) in IPA (30.0 mL) was added a solution of CAN (27.3 g, 49.8 mmol, 24.8 mL, 3.50 eq) in H₂O (30.0 mL) at 0℃. The mixture was stirred at 20℃ for 10 hrs. LC-MS showed the desired mass was found. The reaction mixture was extracted with EtOAc (30.0 mL*2) . The combined organic layers was washed with saturated aqueous NaHCO₃ (30.0 mL *2) , brine (50.0 mL) , dried over Na₂SO₄, filtered and the filtrate was concentrated in vacuo. The crude product was purified by reversed-phase HPLC (0.100%HCl condition) . Compound 39-7 (1.30 g, 5.46 mmol, 38.3%yield) was obtained as a yellow solid.

Step 5. ethyl 1-ethyl-4- (2, 2, 2-trifluoroethoxy) -1H-pyrazole-3-carboxylate

To a solution of compound 39-7 (1.30 g, 5.46 mmol, 1.00 eq) in dioxane (20.0 mL) was added Cs₂CO₃ (3.56 g, 10.9 mmol, 2.00 eq) and compound 39-8 (1.28 g, 8.19 mmol, 654 μL, 1.50 eq) . The mixture was stirred at 20℃ for 10 hrs. LC-MS showed the starting material was consumed and the desired mass was found. The mixture was filtered and concentrated under reduced pressure to give a residue. Compound 39-9 (1.55 g, crude) was obtained as a yellow solid.

Step 6. 1-ethyl-4- (2, 2, 2-trifluoroethoxy) -1H-pyrazole-3-carboxylic acid

To a solution of compound 39-9 (700 mg, 2.63 mmol, 1.00 eq) in MeOH (6.00 mL) was added NaOH (210 mg, 5.26 mmol, 2.00 eq) in H₂O (6.00 mL) at 0℃. The mixture was stirred at 20 ℃ for 12 hrs. LC-MS showed the desired mass was detected. TLC (PE: EtOAc = 1: 1, R_f (R1) = 0.300) indicated the starting material was consumed completely. The mixture was adjusted to pH = 3 with HCl (1M) at 0 ℃, and extracted with DCM (50.0 mL *3) . The combined organic layers were dried over Na₂SO₄, and the solvent was evaporated under reduced pressure. Compound 39-10 (550 mg, 2.31 mmol, 87.8%yield) was obtained as a white solid, confirmed by H NMR.

H NMR: (400 MHz, DMSO-d₆) δ 12.57 (s, 1H) , 7.83 (s, 1H) , 4.64 -4.57 (m, 2H) , 4.12 -4.07 (m, 2H) , 1.36 (t, J = 7.20 Hz, 3H) .

Step 7. N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1H-indazol-4-yl) phenyl) -1-ethyl-4- (2, 2, 2-trifluoroethoxy) -1H-pyrazole-3-carboxamide

To a solution of compound 39-11 (50.0 mg, 122 μmol, 92.7%purity, 1.00 eq) and compound 39-10 (26.3 mg, 110 μmol, 0.900 eq) in pyridine (3.00 mL) was added EDCI (94.1 mg, 491 μmol, 4.00 eq) . The mixture was stirred at 20 ℃ for 2 hrs. LC-MS showed there was desired mass detected. The reaction mixture was diluted with DCM (60.0 mL) and washed with water (20.0 mL *3) . The organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: 3_Phenomenex Luna C18 75.0 *30.0 mm *3.00 um; mobile phase: [water (HCl) -ACN] ; B%: 28.0%- 48.0%, 8 min) . Compound 39 (15.0 mg, 24.9 μmol, 20.2%yield, 99.3%purity) was obtained as confirmed by H NMR and LC-MS.

H NMR: (400 MHz, DMSO-d₆) δ 11.61 (s, 1H) , 9.97 (s, 1H) , 7.93 -7.90 (m, 3H) , 7.45 -7.43 (m, 2H) , 7.07 (s, 1H) , 6.72 (s, 1H) , 4.75 -4.68 (m, 2H) , 4.59 (d, J =13.6 Hz, 1H) , 4.30 (s, 2H) , 4.21 -4.15 (m, 2H) , 4.07 (d, J = 13.2 Hz, 1H) , 3.16 -3.10 (m, 1H) , 2.94 -2.87 (m, 2H) , 2.63 -2.60 (m, 1H) , 1.93 -1.85 (m, 2H) , 1.62 -1.50 (m, 2H) , 1.45 -1.41 (m, 3H) , 1.04 -0.99 (m, 6H) .

LC-MS: (M+H) ⁺: 598.4

HPLC: purity: 99.3% (220 nm)

Example 13

N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1H-indazol-4-yl) phenyl) -1- (4-fluorophenyl) -4- (2, 2, 2-trifluoroethoxy) -1H-pyrazole-3-carboxamide (Compound 40)

Step 1. ethyl (Z) -4-chloro-2- (2- (4-fluorophenyl) hydrazineylidene) -3-oxobutanoate

To HCl (3.00 M, 45.0 mL, 3.00 eq) was added compound 40-2 (5.00 g, 45.0 mmol, 4.31 mL, 1.00 eq) at 0 ℃, then to the reaction mixture was added NaNO₂ (3.26 g, 47.2 mmol, 1.05 eq) in H₂O (10.0 mL) at 0 ℃. The reaction mixture was stirred at 0 ℃ for 1 hr. After first monitoring, to compound 40-1 (8.15 g, 49.5 mmol, 1.10 eq) and NaOAc (18.4 g, 224 mmol, 5.00 eq) in EtOH (50.0 mL) and H₂O (50.0 mL) was added the previous reaction mixture dropwise at 0 ℃. The reaction mixture was stirred at 20 ℃ for 5 hrs. LC-MS showed compound 40-2 was consumed completely. One main peak was shown on LC-MS with desired m/z (MS=287, RT=0.891) . The reaction mixture was filtered and the filter cake was washed by H₂O (50.0 mL *2) , then concentrated under reduced pressure to give a residue. Compound 40-3 (12.0 g, crude) was obtained as a red solid.

Step 2. ethyl 1- (4-fluorophenyl) -4-hydroxy-1H-pyrazole-3-carboxylate

To a solution of compound 40-3 (12.0 g, 41.8 mmol, 1.00 eq) in EtOH (100 mL) was added KOAc (8.22 g, 83.7 mmol, 2.00 eq) at 20 ℃. The reaction mixture was stirred at 80 ℃ for 2 hrs. LC-MS showed compound 40-3 was consumed completely. One main peak was shown on LC-MS with desired m/z (MS=251, RT=0.788) . The reaction mixture was concentrated under reduced pressure to remove EtOH, then diluted with H₂O (100 mL) , and then extracted with EtOAc (100 mL *3) . The combined organic layers were dried over anhydrous Na₂SO₄, then filtered and concentrated under reduced pressure to give a residue. Compound 40-4 (10.4 g, crude) was obtained as a red solid.

Step 3. ethyl 1- (4-fluorophenyl) -4- (2, 2, 2-trifluoroethoxy) -1H-pyrazole-3-carboxylate

To a solution of compound 40-4 (2.00 g, 7.99 mmol, 1.00 eq) in DMF (20.0 mL) was added NaH (639 mg, 15.9 mmol, 60.0%purity, 2.00 eq) at 20 ℃. The reaction mixture was stirred at 20 ℃ for 30 min. To the reaction mixture was added compound 40-5 (3.71 g, 15.9 mmol, 2.00 eq) at 20 ℃. The reaction mixture was stirred at 60 ℃ for 1 hr. LC-MS showed compound 40-4 was consumed completely. One main peak was shown on LC-MS with desired m/z (MS=333, RT=0.895) . The reaction mixture was diluted with H₂O (60.0 mL) , then extracted with EtOAc (60 mL *3) . The combined organic layers were washed by H₂O (60.0 mL) and brine (60.0 mL) , then dried over anhydrous Na₂SO₄, and then filtered and concentrated under reduced pressure to give a residue. Compound 40-6 (4.00 g, crude) was obtained as a brown solid.

Step 4. 1- (4-fluorophenyl) -4- (2, 2, 2-trifluoroethoxy) -1H-pyrazole-3-carboxylic acid

To a solution of compound 40-6 (3.50 g, 10.5 mmol, 1.00 eq) in MeOH (20.0 mL) was added NaOH (842 mg, 21.0 mmol, 2.00 eq) in H₂O (20.0 mL) at 20 ℃. The reaction mixture was stirred at 20 ℃ for 12 hrs. LC-MS showed compound 40-6 was consumed completely. One main peak was shown on LC-MS with desired m/z (MS=305 RT=0.810) . HPLC showed corresponding peaks to guide the purification. The reaction mixture was quenched by 1 M HCl until pH is around 2, then concentrated under reduced pressure to remove MeOH. The aqueous phase was extracted with EtOAc (100 mL *3) . The combined organic layers were dried over anhydrous Na₂SO₄, then filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (FA condition, column: Phenomenex luna C18 (250 *70 mm, 10 um) ; mobile phase: [water (FA) -ACN] ; B%: 30%-60%, 21 min) . Compound 40-7 (1.50 g, 4.93 mmol, 46.8%yield, 100%purity) was obtained as a white solid, confirmed by H NMR and LC-MS.

HPLC: purity: 87.6% (220 nm)

H NMR: (400 MHz, DMSO-d₆) δ 13.00 (s, 1H) , 8.61 (s, 1H) , 7.87 -7.83 (m, 2H) , 7.43 -7.38 (m, 2H) , 4.76 -4.70 (m, 2H) .

LC-MS: (M+H) ⁺: 305.0

Step 5. N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1H-indazol-4-yl) phenyl) -1- (4-fluorophenyl) -4- (2, 2, 2-trifluoroethoxy) -1H-pyrazole-3-carboxamide

To a solution of compound 40-8 (50.0 mg, 128 μmol, 97.0%purity, 1.00 eq) in DMF (3.00 mL) was added compound 40-7 (37.1 mg, 122 μmol, 100%purity, 0.950 eq) , HATU (73.2 mg, 192 μmol, 1.50 eq) and DIEA (49.8 mg, 385 μmol, 67.1 μL, 3.00 eq) . The mixture was stirred at 20 ℃ for 2 hrs. LC-MS showed the desired mass was detected. The reaction mixture was quenched by addition water (30.0 mL) , and then extracted with EtOAc (30.0 mL *5) . The combined organic layers were washed with water (60.0 mL *5) and brine (60.0 mL *3) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (DCM: MeOH = 10: 1, R_f (P1) = 0.40) and further purified by prep-HPLC (column: 3_Phenomenex Luna C18 75 *30 mm *3 um; mobile phase: [water (HCl) -ACN] ; B%: 36%-56%, 8 min) . Compound 40 (11.0 mg, 15.7 μmol, 12.2%yield, 94.8%purity) was obtained as confirmed by H NMR and LC-MS.

H NMR: (400 MHz, MeOD) δ 8.34 (s, 1H) , 7.93 -7.85 (m, 4H) , 7.52 -7.49 (m, 2H) , 7.29 -7.25 (m, 2H) , 7.16 (s, 1H) , 6.81 (s, 1H) , 4.74 -4.68 (m, 3H) , 4.20 (d, J = 12.8 Hz, 1H) , 3.27 -3.24 (m, 1H) , 3.02 -2.96 (m, 2H) , 2.73 -2.72 (m, 1H) , 2.04 -1.96 (m, 2H) , 1.69 -1.64 (m, 2H) , 1.15 -1.10 (m, 6H) .

LC-MS: (M+H) ⁺: 664.4

HPLC: purity: 94.8% (220 nm)

Example 14

2- ( (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1H-indazol-4-yl) phenyl) carbamoyl) -6- (4-fluorophenyl) pyridine 1-oxide (Compound 42)

Step 1. 6- (4-fluorophenyl) picolinic acid

To a solution of compound 42-1 (1.00 g, 4.95 mmol, 1.20 eq) and compound 42-2 (577 mg, 4.13 mmol, 1.00 eq) in EtOH (10.0 mL) and toluene (10.0 mL) was added Cs₂CO₃ (2.69 g, 8.25 mmol, 2.00 eq) and Pd (PPh₃) ₄ (95.3 mg, 82.5 μmol, 0.0200 eq) at 20 ℃. The reaction mixture was stirred at 90 ℃ for 12 hrs. LC-MS showed compound 42-2 was consumed completely. The reaction mixture was quenched by 1.00 M HCl until pH is around 3, then filtered and concentrated under reduced pressure to remove EtOH and toluene. The residue was filtered and filter cake was concentrated under reduced pressure to give a residue. Compound 42-3 (1.04 g, crude) was obtained as a red solid, confirmed by H NMR.

H NMR: (400 MHz, DMSO-d₆) δ 13.23 (m, 1H) , 8.27 -8.25 (m, 2H) , 8.24 -8.23 (m, 1H) , 8.06 -8.05 (m, 1H) , 8.00 -7.98 (m, 1H) , 7.37 -7.32 (m, 2H) .

Step 2. methyl 6- (4-fluorophenyl) picolinate

To a solution of compound 42-3 (850 mg, 3.91 mmol, 1.00 eq) in MeOH (10.0 mL) was added SOCl₂ (931 mg, 7.83 mmol, 567 μL, 2.00 eq) at 20 ℃. The reaction mixture was stirred at 60 ℃ for 3 hrs. LC-MS showed the starting material was consumed completely. The reaction mixture was quenched by sat. aq. NaHCO₃ until pH is around 8, then extracted with EtOAc 30.0 mL (10.0 mL *3) . The combined organic layers were dried over anhydrous Na₂SO₄, then filtered and concentrated under reduced pressure to give a residue. Compound 42-4 (1.00 g, crude) was obtained as light yellow oil.

Step 3. 2- (4-fluorophenyl) -6- (methoxycarbonyl) pyridine 1-oxide

To a solution of compound 42-4 (300 mg, 1.30 mmol, 1.00 eq) in DCM (10.0 mL) was added m-CPBA (1.12 g, 5.19 mmol, 80.0%purity, 4.00 eq) at 0℃. The mixture was heated to 40℃ for 12 hrs. LC-MS showed the ～30.0%of desired mass detected. The reaction mixture was diluted with sat. aq. NaHCO₃ (20.0 mL) and water (20.0 mL) , extracted with DCM (30.0 mL *3) , the combined organic layer was washed with brine (50.0 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (PE: EtOAc = 1: 1, R_f (P1) = 0.1) . Compound 42-5 (80.0 mg, 323 μmol, 24.9%yield, 100%purity) was obtained as a white solid.

LC-MS: (M+H) ⁺: 248.1

Step 4. 2-carboxy-6- (4-fluorophenyl) pyridine 1-oxide

To a solution of compound 42-5 (160 mg, 647 μmol, 1.00 eq) in MeOH (3.00 mL) was added NaOH (1.00 M, 1.50 mL, 2.32 eq) at 0℃, the mixture was stirred at 20 ℃ for 3 hrs. LC-MS showed the desired mass detected. The residue was concentrated to remove solvent, then dissolved in water (10.0 mL) , adjusted the pH =3 by 1.00M HCl at 0℃, extracted with DCM (10.0 mL *3) , the combined organic layer was washed with brine (20.0 mL) , dried over Na₂SO₄, filtered and concentrated to get a residue. 42-6 (130 mg, 557 μmol, 86.1%yield) was obtained as a light yellow solid.

H NMR: (400 MHz, CDCl₃) δ 8.35 -8.33 (m, 1H) , 8.04 -8.03 (m, 1H) , 7.95 -7.90 (m, 1H) , 7.89 -7.87 (m, 2H) , 7.43 -7.39 (m, 2H) .

Step 5. 2- ( (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1H-indazol-4-yl) phenyl) carbamoyl) -6- (4-fluorophenyl) pyridine 1-oxide

To a solution of compound 42-7 (50.0 mg, 122 μmol, 92.7%purity, 1.00 eq) , 42-6 (25.7 mg, 110 μmol, 0.900 eq) and HATU (70.0 mg, 184 μmol, 1.50 eq) in DMF (4.00 mL) was added DIEA (47.6 mg, 368 μmol, 64.1 μL, 3.00 eq) . The mixture was stirred at 20 ℃ for 2 hrs. LC-MS showed the desired mass was detected. The reaction mixture was quenched by addition water (30.0 mL) , and then extracted with EtOAc (10.0 mL *5) . The combined organic layers were washed with water (10.0 mL *5) and brine (10.0 mL *3) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (DCM: MeOH = 10: 1, R_f (P1) = 0.400) and further purified by prep-HPLC (FA condition; column: Phenomenex Synergi C18 150.0 *25.0 mm *10.0 um; mobile phase: [water (FA) -ACN] ; B%: 41.0%-71.0%, 10 min) . Compound 42 (23.0 mg, 38.2 μmol, 31.1%yield, 98.5%purity) was obtained as confirmed by H NMR, LC-MS and HPLC.

H NMR: (400 MHz, DMSO-d₆) δ 13.51 (s, 1H) , 11.68 (s, 1H) , 8.35 -8.33 (m, 1H) , 7.91 -7.87 (m, 5H) , 7.76 -7.71 (m, 1H) , 7.52 -7.50 (m, 2H) , 7.41 -7.36 (m, 2H) , 7.09 (s, 1H) , 6.74 (s, 1H) , 4.58 (d, J = 13.6 Hz, 1H) , 4.29 (s, 2H) , 4.07 (d, J = 13.2 Hz, 1H) , 3.17 -3.10 (m, 1H) , 2.94 -2.86 (m, 2H) , 2.83 -2.60 (m, 1H) , 1.93 - 1.85 (m, 2H) , 1.62 -1.45 (m, 2H) , 1.07 -0.99 (m, 6H) .

LC-MS: (M+H) ⁺: 593.2

HPLC: purity: 98.5% (220 nm)

Example 15

N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1H-indazol-4-yl) phenyl) -5- (4-fluorophenyl) -1-methyl-4-oxo-1, 4-dihydropyridazine-3-carboxamide (Compound 43)

Step 1. 2- (4-fluorophenyl) acetyl chloride

To a solution of compound 43-1 (10.0 g, 64.8 mmol, 1.00 eq) in DCM (100 mL) was added SOCl₂ (9.26 g, 77.8 mmol, 5.65 mL, 1.20 eq) . The mixture was stirred at 20℃ for 10 hrs. TLC (PE: EtOAc = 3: 1) showed the starting material was consumed and there was a mainly new spot formed. The mixture was concentrated in vacuo. Crude 43-2 (11.0 g, crude) was obtained as yellow oil.

Step 2. ethyl 2-diazo-4- (4-fluorophenyl) -3-oxobutanoate

To a solution of compound 43-2 (11.0 g, 63.7 mmol, 1.00 eq) in EtOAc (100 mL) was added compound 43-3 (14.5 g, 127 mmol, 13.3 mL, 2.00 eq) at 0℃. The mixture was stirred at 20℃ for 10 hrs. LC-MS showed the desired mass was found. The mixture was diluted with sat. K₂CO₃ (10.0 mL) , and stirred for 20 min. The mixture was separated and the water phase was extracted with EtOAc (10.0 mL *3) . The combined organic layer was washed with brine (10.0 mL *2) , dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was purified by column chromatography (SiO₂, PE: EtOAc = 100: 0 to 100: 1) (PE: EtOAc = 5: 1, R_f (P1) =0.400) . Compound 43-4 (4.70 g, 18.7 mmol, 29.4%yield) was obtained as a yellow solid, confirmed by H NMR.

H NMR: (400 MHz, CDCl₃) δ 7.31 -7.27 (m, 2H) , 7.05 -7.01 (m, 2H) , 4.38 -4.32 (m, 2H) , 4.16 (s, 2H) , 1.39 -1.35 (m, 3H) .

Step 3. ethyl (E) -4- (4-fluorophenyl) -2-hydrazineylidene-3-oxobutanoate

To a solution of compound 43-4 (3.00 g, 11.9 mmol, 1.00 eq) in MTBE (15.0 mL) was added n-Bu₃P (2.91 g, 14.3 mmol, 3.55 mL, 1.20 eq) at 0℃. The mixture was stirred at 20℃ for 10 hrs. LC-MS showed the desired mass was found. H NMR showed there was desired product. EtOAc (10.0 mL) and water (20.0 mL) were added, and the layers were separated. The organic phase was washed with saturated brine (20.0 mL) , dried over Na₂SO₄, filtered and concentrated in vacuo. Compound 43-5 (3.00 g, 11.8 mmol, 99.2%yield) was obtained as yellow oil.

H NMR: (400 MHz, DMSO-d₆) δ 7.23 -7.19 (m, 2H) , 7.11 -7.07 (m, 2H) , 4.09 -4.04 (m, 2H) , 3.92 (s, 2H) , 3.32 (s, 2H) , 1.17 -1.15 (m, 3H) .

Step 4. tert-butyl (E) -2- (1-ethoxy-4- (4-fluorophenyl) -1, 3-dioxobutan-2-ylidene) hydrazine-1-carboxylate

To a solution of compound 43-5 (1.00 g, 3.96 mmol, 1.00 eq) in THF (10.0 mL) was added DMAP (145 mg, 1.19 mmol, 0.300 eq) , TEA (882 mg, 8.72 mmol, 1.21 mL, 2.20 eq) and Boc₂O (1.73 g, 7.93 mmol, 1.82 mL, 2.00 eq) . The mixture was stirred at 20℃ for 4 hrs. LC-MS showed the starting material was consumed completely and the desired mass was found. The mixture was diluted with EtOAc (15.0 mL) and water (10.0 mL) , and then separated. The organic layer was washed with brine (10.0 mL) , dried over Na₂SO₄, filtered and concentrated in vacuo. Compound 43-6 (1.10 g, 3.12 mmol, 78.7%yield) was obtained as yellow oil.

Step 5. ethyl 5- (4-fluorophenyl) -1-methyl-4-oxo-1, 4-dihydropyridazine-3-carboxylate

To a solution of compound 43-6 (1.00 g, 2.84 mmol, 1.00 eq) in toluene (10.0 mL) was added DMF-DMA (675 mg, 5.66 mmol, 752 μL, 2.00 eq) . The mixture was stirred at 60℃ for 10 hrs. LC-MS showed the starting material was consumed completely. The mixture was concentrated in vacuo. The residue was purified by column chromatography (SiO₂, PE: EtOAc = 100: 0 to 100: 30) (PE: EtOAc = 1: 1, R_f (P1) = 0.400) . Compound 43-7 (0.500 g, 1.81 mmol, 63.7%yield) was obtained as a yellow solid, which was confirmed by 2D NMR.

Step 6. 5- (4-fluorophenyl) -1-methyl-4-oxo-1, 4-dihydropyridazine-3-carboxylic acid

To a solution of compound 43-7 (100 mg, 361 μmol, 1.00 eq) in MeOH (1.00 mL) was added NaOH (28.9 mg, 723 μmol, 2.00 eq) in H₂O (1.00 mL) at 0 ℃. The mixture was stirred at 20 ℃ for 6 hrs. TLC (DCM: MeOH = 10: 1, R_f (R1) = 0.800) showed the starting material was consumed completely. The mixture was adjusted PH = 3 with HCl (1M) at 0 ℃, and extracted with DCM (50.0 mL *3) . The combined organic layers were dried over Na₂SO₄, and the solvent was evaporated under reduced pressure. The residue was purified by prep-HPLC (FA condition; column: Unisil 3-100 C18 Ultra 150 *50.0 mm *3.00 um; mobile phase: [water (FA) -ACN] ; B%: 12.0%-42.0%, 7 min) . Compound 43-8 (50.0 mg, 201 μmol, 55.6%yield, 100%purity) was obtainbed as a white solid, confirmed by H NMR and LC-MS.

H NMR: (400 MHz, DMSO-d₆) δ 9.05 (s, 1H) , 7.95 -7.91 (m, 2H) , 7.38 - 7.34 (m, 2H) , 4.14 (s, 3H) .

LC-MS: (M+H) ⁺: 249.1

Step 7. N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1H-indazol-4-yl) phenyl) -5- (4-fluorophenyl) -1-methyl-4-oxo-1, 4-dihydropyridazine-3-carboxamide

To a solution of compound 43-9 (50.0 mg, 122 μmol, 92.7%purity, 1.00 eq) and 43-8 (27.4 mg, 110 μmol, 100%purity, 0.900 eq) in pyridine (3.00 mL) was added EDCI (94.1 mg, 491 μmol, 4.00 eq) . The mixture was stirred at 20 ℃ for 2 hrs. LC-MS showed the desired mass was detected. The reaction mixture was quenched by addition water (10.0 mL) , and then extracted with DCM (20.0 mL) . The organic layer was washed with brine (10.0 mL *3) . Then the combined aqueous phases were extracted with DCM (10.0 mL *3) . Then the combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (FA condition; column: Phenomenex Synergi C18 150 *25.0 mm *10.0 um; mobile phase: [water (FA) -ACN] ; B%: 35.0%-65.0%, 8 min) . Compound 43 (20.0 mg, 32.1 μmol, 26.1%yield, 97.7%purity) was obtained as confirmed by H NMR and LC-MS.

H NMR: (400 MHz, DMSO-d₆) δ 12.41 (s, 1H) , 11.62 (s, 1H) , 8.88 (s, 1H) , 7.96 -7.92 (m, 2H) , 7.86 -7.83 (m, 2H) , 7.51 -7.49 (m, 2H) , 7.37 -7.32 (m, 2H) , 7.08 (s, 1H) , 6.74 (s, 1H) , 4.59 (d, J = 13.6 Hz, 1H) , 4.30 -4.27 (m, 2H) , 4.12 (s, 3H) , 4.08 (d, J = 14.8 Hz, 1H) , 3.16 -3.13 (m, 1H) , 2.94 -2.88 (m, 2H) , 2.63 -2.57 (m, 1H) , 1.94 -1.86 (m, 2H) , 1.63 -1.48 (m, 2H) , 1.04 -1.00 (m, 6H) .

LC-MS: (M+H) ⁺: 608.4

HPLC: purity: 97.7% (220 nm)

Example 16

N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1-methyl-1H-indazol-4-yl) phenyl) -5- (4-fluorophenyl) -4-hydroxynicotinamide (Compound 44)

To a solution of compound 44-1 (30.0 mg, 75.1 μmol, 98.0%purity, 1.00 eq) , compound 44-2 (17.5 mg, 75.1 μmol, 100%purity, 1.00 eq) and HATU (42.8 mg, 112 μmol, 1.50 eq) in DMF (3.00 mL) was added DIEA (29.1 mg, 225 μmol, 39.2 μL, 3.00 eq) . The mixture was stirred at 20 ℃ for 2 hrs. LC-MS showed the desired mass was detected. The reaction mixture was quenched by water (50.0 mL) , and then extracted with EtOAc (10.0 mL *5) . The combined organic layers were washed by water (10.0 mL *4) and brine (10.0 mL *4) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (DCM: MeOH = 10: 1, R_f (P1) = 0.400) . Compound 44 (20.0 mg, 32.6 μmol, 43.4%yield, 99.0%purity) was obtained as confirmed by H NMR and LC-MS.

H NMR: (400 MHz, DMSO-d₆) δ 13.09 (s, 1H) , 12.64 (s, 1H) , 8.63 (d, J =1.2 Hz, 1H) , 8.09 (d, J = 1.6 Hz, 1H) , 7.84 -7.82 (m, 2H) , 7.74 -7.70 (m, 2H) , 7.47 -7.45 (m, 2H) , 7.29 -7.25 (m, 2H) , 7.22 (s, 1H) , 6.74 (s, 1H) , 4.60 (d, J = 11.2 Hz, 1H) , 4.35 (s, 2H) , 4.08 (d, J = 10.4 Hz, 1H) , 3.77 (s, 3H) , 3.17 -3.10 (m, 1H) , 2.94 -2.88 (m, 2H) , 2.63 -2.60 (m, 1H) , 1.93 -1.86 (m, 2H) , 1.67 -1.55 (m, 2H) , 1.05 -1.00 (m, 6H) .

LC-MS: (M+H) ⁺: 607.4

HPLC: purity: 99.0% (220 nm)

Example 17

N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1-methyl-1H-pyrazolo [4, 3-c] pyridin-4-yl) phenyl) -1-isopropyl-2, 4-dioxo-3- (pyridin-2-yl) -1, 2, 3, 4-tetrahydropyrimidine-5-carboxamide (Compound 46)

Step 1. 1- (4- (3-amino-4-bromo-1-methyl-1H-pyrazolo [4, 3-c] pyridin-6-yl) piperidin-1-yl) -2-methylpropan-1-one

To a solution of compound 46-1 (35.0 mg, 95.5 μmol, 1.00 eq) and K₂CO₃ (26.4 mg, 191 μmol, 2.00 eq) in DMF (2.00 mL) was added CH₃I (13.5 mg, 95.5 μmol, 5.95 μL, 1.00 eq) . The mixture was stirred at 45 ℃ for 2 hrs. LC-MS showed desired mass detected. The reaction mixture was diluted with H₂O 10.0 mL and extracted with EtOAc 40.0 mL (20.0 mL *2) . The combined organic layers were washed with brine 60.0 mL (20.0 mL *3) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiO₂, DCM: MeOH = 10: 1) . (Plate 1: DCM: MeOH = 10: 1) . Compound 46-2 (10.0 mg, 26.3 μmol, 27.5%yield) was obtained as a white solid. The structure was confirmed by H NMR.

H NMR: (400 MHz, DMSO-d6) δ 7.27 (s, 1H) , 5.52 (s, 2H) , 4.58 -4.54 (m, 1H) , 4.07 -4.04 (m, 1H) , 3.74 (s, 3H) , 3.16 -3.10 (m, 2H) , 2.92 -2.88 (m, 2H) , 1.95 -1.86 (m, 2H) , 1.62 -1.49 (m, 2H) , 1.03 -0.98 (m, 6H) .

Step 2. N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1-methyl-1H-pyrazolo [4, 3-c] pyridin-4-yl) phenyl) -1-isopropyl-2, 4-dioxo-3- (pyridin-2-yl) -1, 2, 3, 4-tetrahydropyrimidine-5-carboxamide

Compound 46-2 (5.00 mg, 13.5 μmol, 1.00 eq) , compound 46-3 (18.7 mg, 39.4 μmol, 3.00 eq) and DIEA (5.10 mg, 39.4 μmol, 6.87 μL, 3.00 eq) , Pd (t-Bu₃P) ₂ (671 ug, 1.31 μmol, 0.100 eq) were taken up into a microwave tube in dioxane (1.50 mL) , H₂O (0.100 mL) . The sealed tube was heated at 130 ℃ for 60 mins under microwave. LC-MS showed desired mass detected. The reaction mixture was concentrated to give a residue. The residue was purified by prep-TLC (SiO₂, DCM: MeOH = 10: 1) . (Plate 1: DCM: MeOH = 10: 1) . The residue was purified by prep-HPLC (basic condition, column: Waters Xbridge 150 *25 mm *5 um; mobile phase: [water (ammonia hydroxide v/v) -ACN] ; B%: 30%-60%, 9 mins) . Compound 46 (1.71 mg, 2.39 μmol, 18.2%yield, 91.0%purity) was obtained as confirmed by H NMR, LC-MS and HPLC.

H NMR: (400 MHz, CDCl₃) δ 10.87 (s, 1H) , 8.76 -8.72 (m, 2H) , 8.01 -7.97 (m, 1H) , 7.82 -7.80 (m, 2H) , 7.72 -7.70 (m, 2H) , 7.53 -7.49 (m, 1H) , 7.41 -7.40 (m, 1H) , 6.83 (s, 1H) , 5.03 -4.96 (m, 1H) , 4.87 -4.84 (m, 1H) , 4.08 -4.03 (m, 3H) , 3.84 (s, 3H) , 3.20 -3.08 (m, 2H) , 2.93 -2.80 (m, 1H) , 2.72 -2.67 (m, 1H) , 2.21 -2.09 (m, 2H) , 1.80 -1.74 (m, 2H) , 1.51 (d, J = 6.8 Hz, 6H) , 1.16 (t, J = 6.8 Hz, 7.6H) .

LC-MS: M+H: 650.4

HPLC: 91.0%purity

Example 18

N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1H-indazol-4-yl) -3-fluorophenyl) -5- (4-fluorophenyl) -4-hydroxynicotinamide (Compound 48)

To a solution of compound 48-1 (50.0 mg, 107 umol, 85.0%purity, 1.00 eq) and compound 48-2 (25.0 mg, 107 μmol, 1.00 eq) in DMF (3.00 mL) was added HATU (122 mg, 322 μmol, 3.00 eq) and DIEA (41.7 mg, 322 umol, 56.2 uL, 3.00 eq) . The mixture was stirred at 20℃ for 2 hrs. LC-MS showed the desired mass was detected. The reaction mixture was quenched by water (50.0 mL) , and then extracted with EtOAc (10.0 mL *5) . The combined organic layers were washed by water (10.0 mL *4) and brine (10.0 mL *4) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The crude product was purified by prep-TLC (plate1, DCM: MeOH = 10: 1, R_f (P1) = 0.400) and further purified by prep-HPLC (HCl condition; column: 3_Phenomenex Luna C18.0 75.0 *30.0 mm *3.00 um; mobile phase: [water (HCl) -ACN] ; B%: 28.0%-48.0%, 8 min) . Then the mixture was concentrated under reduced pressure to remove MeCN and adjusted by sat. NaHCO₃ until pH was around 8. The aqueous part was extracted with DCM: MeOH = 10: 1 (20.0 mL *3) . The combined organic layers were dried over anhydrous Na₂SO₄, then filtered and concentrated under reduced pressure to give a residue. Compound 48 (9.31 mg, 14.8 umol, 13.8%yield, 97.4%purity) was obtained as confirmed by H NMR and LC-MS.

H NMR: (400 MHz, DMSO-d₆) : δ 13.67 (s, 1H) , 11.61 (s, 1H) , 8, 63 (d, J =1.2 Hz, 1H) , 8.07 (d, J = 0.8 Hz, 1H) , 7.96 -7.92 (m, 1H) , 7.73 -7.70 (m, 2H) , 7.44 -7.42 (m, 1H) , 7.38 -7.33 (m, 1H) , 7.26 -7.22 (m, 2H) , 7.10 (s, 1H) , 6.74 (s, 1H) , 4.58 (d, J = 11.2 Hz, 1H) , 4.20 (s, 2H) , 4.07 (d, J = 11.6 Hz, 1H) , 3.16 -3.09 (m, 1H) , 2.94 -2.85 (m, 2H) , 2.62 -2.59 (m, 1H) , 1.93 -1.85 (m, 2H) , 1.61 -1.45 (m, 2H) , 1.03 -0.99 (m, 6H) .

LC-MS: (M+H) ⁺: 611.3

HPLC: purity: 97.4% (215 nm)

Example 19

N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1H-indazol-4-yl) phenyl) -5- (4-fluorophenyl) -1-isopropyl-6-methyl-4-oxo-1, 4-dihydropyridine-3-carboxamide (Compound 49)

To a solution of compound 49-2 (50.0 mg, 122 μmol, 92.7%purity, 1.00 eq) and compound 49-1 (32.3 mg, 110 μmol, 98.9%purity, 0.900 eq) in DMF (2.00 mL) was added HATU (70.0 mg, 184 μmol, 1.50 eq) and DIEA (47.6 mg, 368 μmol, 64.1 μL, 3.00 eq) . The mixture was stirred at 20℃ for 2hrs. LC-MS showed the desired mass was detected. The reaction mixture was quenched by water (50.0 mL) , and then extracted with EtOAc (10.0 mL *5) . The combined organic layers were washed by water (10.0 mL *4) and brine (10.0 mL *4) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The crude product was purified by prep-TLC (DCM: MeOH = 10: 1, R_f (P1) = 0.400) and further purified by prep-HPLC (FA condition; column: Phenomenex luna C18.0 150 *25.0 mm *10.0 um;mobile phase: [water (FA) -ACN] ; B%: 41.0%-71.0%, 10 min) . Then the mixture was concentrated under reduced pressure to remove MeCN and adjusted by sat. NaHCO₃ until pH was around 8. The aqueous part was extracted with DCM (20.0 mL *3) . The combined organic layers were dried over anhydrous Na₂SO₄, then filtered and concentrated under reduced pressure to give a residue. Compound 49 (18.9 mg, 28.7 μmol, 23.4%yield, 99.2%purity) was obtained as confirmed by H NMR and LC-MS.

H NMR: (400 MHz, DMSO-d₆) δ 13.01 (s, 1H) , 11.60 (s, 1H) , 8.71 (s, 1H) , 7.81 -7.90 (m, 2H) , 7.48 -7.44 (m, 2H) , 7.29 -7.27 (m, 4H) , 7.06 (s, 1H) , 6.72 (s, 1H) , 4.81 -4.75 (m, 1H) , 4.59 (d, J = 4.8 Hz, 1H) , 4.29 (s, 2H) , 4.07 (d, J = 11.2 Hz, 1H) , 3.16 -3.09 (m, 1H) , 2.94 -2.85 (m, 2H) , 2.62 -2.59 (m, 1H) , 2.29 (s, 3H) , 1.93 -1.85 (m, 2H) , 1.62 -1.47 (m, 8H) , 1.04 -0.99 (m, 6H) .

LC-MS: (M+H) ⁺: 649.5

HPLC: purity: 99.2% (215 nm)

Example 20

N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1-methyl-1H-pyrazolo [3, 4-b] pyridin-4-yl) phenyl) -1-isopropyl-2, 4-dioxo-3- (pyridin-2-yl) -1, 2, 3, 4-tetrahydropyrimidine-5-carboxamide (Compound 50)

Step 1. 2- (4-iodo-1H-pyrazolo [3, 4-b] pyridin-3-yl) isoindoline-1, 3-dione

A mixture of compound 50-1 (9.00 g, 34.6 mmol, 1.00 eq) and compound 50-2 (7.69 g, 51.9 mmol, 1.50 eq) in HOAc (90.0 mL) was stirred at 120 ℃ for 12 hrs. LC-MS showed the desired mass was detected. The reaction mixture was filtered and the cake was washed with 100 mL MTBE, then concentrated under reduced pressure to give a residue. Compound 50-3 (11.3 g, 28.9 mmol, 83.6%yield) was obtained as an off-white solid, confirmed by H NMR.

H NMR: (400 MHz, DMSO-d₆) δ 14.47 (s, 1H) , 8.27 -8.25 (m, 1H) , 8.13 -8.10 (m, 2H) , 8.02 -7.99 (m, 2H) , 7.80 -7.79 (m, 1H) .

Step 2. 3- (1, 3-dioxoisoindolin-2-yl) -4-iodo-1H-pyrazolo [3, 4-b] pyridine 7-oxide

To a solution of compound 50-3 (4.00 g, 10.2 mmol, 1.00 eq) in HOAc (50.0 mL) was added m-CPBA (7.08 g, 34.8 mmol, 85.0%purity, 3.40 eq) at 0 ℃. The mixture was stirred at 70 ℃ for 10 hrs. LC-MS showed the desired mass was detected. The reaction mixture was concentrated to give a residue. The residue was adjusted pH to 7 by sat. aq. NaHCO₃. Then the mixture was extracted with DCM (30.0 mL *3) . The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The crude product was purified by reversed-phase HPLC (0.100%FA condition) . Compound 50-4 (1.50 g, 3.69 mmol, 36.0%yield) was obtained as a yellow solid, confirmed by H NMR.

H NMR: (400 MHz, DMSO-d₆) δ 13.39 (s, 1H) , 7.90 -7.89 (m, 3H) , 7.70 -7.69 (m, 2H) , 7.57 -7.53 (m, 1H) .

Step 3. 2- (6-chloro-4-iodo-1H-pyrazolo [3, 4-b] pyridin-3-yl) isoindoline-1, 3-dione

To a solution of compound 50-4 (1.50 g, 3.69 mmol, 1.00 eq) in MeCN (20.0 mL) was added POCl₃ (2.26 g, 14.8 mmol, 1.37 mL, 4.00 eq) . The mixture was stirred at 20 ℃ for 3 hrs. LC-MS showed the desired mass was detected. The mixture was filtered and the cake was concentrated under reduced pressure to give a residue. And the filtrate was concentrated under reduced pressure to remove. The residue was adjusted pH to 9 by sat. aq NaHCO₃ and extracted with DCM 150 mL (50.0 mL *3) . The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. Compound 50-5 (1.00 g, 2.36 mmol, 63.7%yield) was obtained as a yellow solid.

Step 4. 6-chloro-4-iodo-1H-pyrazolo [3, 4-b] pyridin-3-amine

To a solution of compound 50-5 (1.00 g, 2.36 mmol, 1.00 eq) in MeOH (30.0 mL) was added N₂H₄·H₂O (2.09 g, 40.8 mmol, 2.03 mL, 98.0%purity, 17.3 eq) . Then the mixture was stirred at 20 ℃ for 1 hr. LC-MS showed the desired mass was detected. The mixture was filtered and the cake was concentrated under reduced pressure to give a residue. And the filtrate was quenched by water (20.0 mL) , and then extracted with DCM (30.0 mL *3) . The organic layers were washed by water (20.0 mL *3) and brine (20.0 mL *3) . The organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. Compound 50-6 (250 mg, 0.849 mmol, 36.0%yield) was obtained as a yellow solid.

Step 5. 6-chloro-4-iodo-1-methyl-1H-pyrazolo [3, 4-b] pyridin-3-amine

To a solution of compound 50-6 (140 mg, 475 μmol, 1.00 eq) in DMF (3.00 mL) was added K₂CO₃ (131 mg, 950 μmol, 2.00 eq) and MeI (85.9 mg, 605 μmol, 37.7 uL, 1.27 eq) at 0 ℃. Then the mixture was stirred at 20 ℃ for 3 hrs under N₂ atmosphere. LC-MS showed the desired mass was detected. The reaction mixture was quenched by water (10.0 mL) , and then extracted with EtOAc (10.0 mL *3) . The combined organic layers were washed by water (10.0 mL *4) and brine (10.0 mL *4) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (DCM: MeOH = 10: 1, R_f (P1) = 0.600) . Compound 50-7 (80.0 mg, 214 μmol, 45.0%yield, 82.5%purity) was obtained as a yellow solid, confirmed by LC-MS.

LC-MS: (M+H) ⁺: 309.0

Step 6. 6-chloro-1-methyl-4- (4-nitrophenyl) -1H-pyrazolo [3, 4-b] pyridin-3-amine

Four batches was in parallel. To a solution of compound 7 (20.0 mg, 53.5 μmol, 82.5%purity, 1.00 eq) and compound 50-8 (7.15 mg, 42.8 μmol, 0.800 eq) in dioxane (1.50 mL) and H₂O (0.100 mL) was added DIEA (20.7 mg, 160 μmol, 27.9 μL, 3.00 eq) and Pd (t-Bu₃P) ₂ (2.74 mg, 5.35 μmol, 0.100 eq) . The mixture was stirred at 130 ℃ for 1 hr under N₂ atmosphere under microwave. LC-MS showed the desired mass was detected. The mixture was purified by prep-TLC (PE: THF = 1: 1, R_f (P1) = 0.600) . Compound 50-9 (44.0 mg, 144 μmol, 67.6%yield) was obtained as a yellow solid.

Step 7. 1- (4- (3-amino-1-methyl-4- (4-nitrophenyl) -1H-pyrazolo [3, 4-b] pyridin-6-yl) -3, 6-dihydropyridin-1 (2H) -yl) -2-methylpropan-1-one

Two reactions were carried out in parallel. To a solution of compound 50-9 (22.0 mg, 72.4 μmol, 1.00 eq) and compound 50-10 (87.0 mg, 289 umol, 92.9%purity, 4.00 eq) in dioxane (1.50 mL) and H₂O (0.100 mL) was added DIEA (28.0 mg, 217 μmol, 37.8 μL, 3.00 eq) and Pd (t-Bu₃P) ₂ (3.70 mg, 7.24 μmol, 0.100 eq) . The mixture was stirred at 120 ℃ for 1 hr under N₂ atmosphere under microwave. LC-MS showed the desired mass was detected. The mixture was purified by prep-TLC (DCM: MeOH = 10: 1, R_f (P1) = 0.600) . Crude 50-11 (70.0 mg) was obtained as a yellow solid.

Step 8. 1- (4- (3-amino-4- (4-aminophenyl) -1-methyl-1H-pyrazolo [3, 4-b] pyridin-6-yl) piperidin-1-yl) -2-methylpropan-1-one

To a solution of compound 50-11 (70.0 mg, 166 μmol, 1.00 eq) in THF (8.00 mL) was added Pd/C (700 mg, 10.0%purity) , degassed and purged with H₂ (15 Psi) for 3 times, then stirred at 20 ℃ for 2 hrs. LC-MS showed the desired mass was detected. The mixture was filtered and filtrate was concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (DCM: MeOH =10: 1, R_f (P1) = 0.400) . Compound 50-12 (16.0 mg, 40.7 μmol, 24.4%yield) was obtained as a yellow solid.

Step 9. N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1-methyl-1H-pyrazolo [3, 4-b] pyridin-4-yl) phenyl) -1-isopropyl-2, 4-dioxo-3- (pyridin-2-yl) -1, 2, 3, 4-tetrahydropyrimidine-5-carboxamide

To a solution of compound 50-12 (13.0 mg, 33.1 umol, 1.00 eq) and compound 50-13 (8.21 mg, 29.8 μmol, 0.900 eq) in pyridine (5.00 mL) was added EDCI (25.4 mg, 132 μmol, 4.00 eq) . Then the mixture was stirred at 20 ℃ for 1 hr. LC-MS showed the desired mass was detected. The reaction mixture was quenched by water (20.0 mL) , and then extracted with DCM (10.0 mL *3) . The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (DCM: MeOH = 10: 1, R_f (P1) = 0.400) and prep-HPLC (ammonia hydroxide condition; column: Waters Xbridge 150 *25.0 mm *5.00 um; mobile phase: [water (ammonia hydroxide v/v) -ACN] ; B%: 33.0%-63.0%, 9 min) . Compound 50 (4.00 mg, 5.92 μmol, 17.8%yield, 96.1%purity) was obtained as confirmed by H NMR and LC-MS.

H NMR: (400 MHz, CDCl₃) δ 10.89 (s, 1H) , 8.76 -8.75 (m, 1H) , 8.72 (s, 1H) , 8.02 -7.97 (m, 1H) , 7.81 -7.79 (m, 2H) , 7.56 -7.50 (m, 3H) , 7.41 -7.39 (m, 1H) , 6.75 (s, 1H) , 5.02 -4.96 (m, 1H) , 4.81 (d, J = 13.6 Hz, 1H) , 4.10 (d, J = 10.0 Hz, 1H) , 3.94 (s, 3H) , 3.88 (s, 2H) , 3.24 -3.18 (m, 1H) , 3.08 -3.02 (m, 1H) , 2.90 -2.84 (m, 1H) , 2.75 -2.69 (m, 1H) , 2.09 -2.02 (m, 2H) , 1.90 -1.84 (m, 2H) , 1.52 -1.50 (m, 6H) , 1.19 -1.15 (m, 6H) .

LC-MS: (M+H) ⁺: 650.2

HPLC: purity: 96.1% (215 nm)

Example 21

N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1H-indazol-4-yl) phenyl) -5- (4- fluorophenyl) -1-methyl-4-oxo-1, 4-dihydropyridine-3-carboxamide (Compound 51)

Step 1. 5- (4-fluorophenyl) -1-methyl-4-oxo-1, 4-dihydropyridine-3-carboxylic acid

To a solution of compound 51-1 (100 mg, 406 μmol, 1.00 eq) and compound 51-2 (113 mg, 812 μmol, 2.00 eq) in dioxane (1.00 mL) was added Na₂CO₃ (2.00 M, 609 uL, 3.00 eq) and Pd (PPh₃) ₄ (46.9 mg, 40.6 μmol, 0.100 eq) . The mixture was stirred at 90℃ for 12 hrs under N₂ atmosphere. LC-MS showed desired mass detected. The reaction mixture was diluted with H₂O 20.0 mL and adjusted pH to 3 by sat. aq citric acid. Then xtracted with DCM: IPA (3: 1, 40.0 mL *2) . The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (DCM: MeOH = 10: 1, R_f (P1) = 0.400) . Compound 51-3 (60.0 mg, 242 umol, 59.7%yield) was obtained as a yellow solid.

Step 2. N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1H-indazol-4-yl) phenyl) -5- (4-fluorophenyl) -1-methyl-4-oxo-1, 4-dihydropyridine-3-carboxamide

To a solution of compound 51-4 (50.0 mg, 122 μmol, 92.7%purity, 1.00 eq) and compound 51-3 (27.3 mg, 110 μmol, 0.900 eq) in DMF (2.00 mL) was added HATU (70.0 mg, 184 μmol, 1.50 eq) and DIEA (47.6 mg, 368 μmol, 64.1 μL, 3.00 eq) . Then the mixture was stirred at 20℃ for 2 hrs. LC-MS showed the desired mass was detected. The reaction mixture was quenched by water (50.0 mL) , and then extracted with EtOAc (10.0 mL *5) . The combined organic layers were washed by water (10.0 mL *4) and brine (10.0 mL *4) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The crude product was purified by prep-TLC (DCM: MeOH = 10: 1, R_f (P1) = 0.400) . Compound 51 (12.7 mg, 20.1 μmol, 15.8%yield, 95.9%purity) was obtained as confirmed by H NMR and LC-MS.

H NMR: (400 MHz, DMSO-d₆) δ 13.10 (s, 1H) , 11.61 (s, 1H) , 8.70 (d, J =2.0 Hz, 1H) , 8.16 (d, J = 2.0 Hz, 1H) , 7.84 -7.82 (m, 2H) , 7.76 -7.72 (m, 2H) , 7.48 -7.46 (m, 2H) , 7.31 -7.27 (m, 2H) , 7.07 (s, 1H) , 6.73 (s, 1H) , 4.59 (d, J = 11.6 Hz, 1H) , 4.30 (s, 2H) , 4.07 (d, J = 12.8 Hz, 1H) , 3.93 (s, 3H) , 3.16 -3.10 (m, 1H) , 2.94 -2.86 (m, 2H) , 2.63 -2.58 (m, 1H) , 1.93 -1.85 (m, 2H) , 1.62 -1.45 (m, 2H) , 1.04 -0.99 (m, 6H) .

LC-MS: (M+H) ⁺: 607.1

HPLC: purity: 95.9% (215 nm)

Example 22

N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1H-indazol-4-yl) phenyl) -5- (4-fluorophenyl) -1-isopropyl-4-oxo-1, 4-dihydropyridine-3-carboxamide (Compound 52)

Step 1. methyl 5-bromo-4-hydroxynicotinate

To a solution of compound 52-1 (0.500 g, 2.29 mmol, 1.00 eq) in MeOH (5.00 mL) was added H₂SO₄ (1.84 g, 18.7 mmol, 1.00 mL, 8.18 eq) . The mixture was stirred at 80℃ for 12 hrs. LC-MS showed desired mass detected. The reaction mixture was diluted with H₂O 30.0 mL and extracted with DCM 100 mL (50.0 mL *2) . The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. Compound 52-2 (0.400 g, 1.72 mmol, 75.1%yield) was obtained as a white solid.

Step 2. methyl 5-bromo-1-isopropyl-4-oxo-1, 4-dihydropyridine-3-carboxylate

To a solution of compound 52-2 (200 mg, 861 μmol, 1.00 eq) in DMF (2.00 mL) was added Cs₂CO₃ (337 mg, 1.03 mmol, 1.20 eq) . The mixture was stirred at 75℃ for 30 min. The reaction mixture was cooled to 25℃, compound 52-3 (219 mg, 1.29 mmol, 129 μL, 1.50 eq) was added to the mixture. Then the mixture was stirred at 55℃ for 12 hrs. LC-MS showed desired mass detected. The reaction mixture was filtered. The filtrate was directly used for further purification. The residue was purified by prep-HPLC (basic condition, column: Waters Xbridge 150 *25.0 mm *5.00 um; mobile phase: [water (ammonia hydroxide v/v) -ACN] ; B%: 7.00%-37.0%, 9 min) . Compound 52-4 (70.0 mg, 255 μmol, 29.6%yield) was obtained as a white solid. The structure was confirmed by H NMR.

H NMR: (400 MHz, DMSO-d₆) δ 8.47 (d, J = 2.0 Hz, 1H) , 8.40 (d, J = 2.0 Hz, 1H) , 4.51 -4.39 (m, 1H) , 3.73 (s, 3H) , 1.41 -1.39 (m, 6H) .

Step 3. 5- (4-fluorophenyl) -1-isopropyl-4-oxo-1, 4-dihydropyridine-3-carboxylic acid

To a solution of compound 52-4 (70.0 mg, 255 μmol, 1.00 eq) and compound 52-5 (71.4 mg, 510 μmol, 2.00 eq) in dioxane (1.00 mL) was added Pd (PPh₃) ₄ (29.5 mg, 25.5 μmol, 0.100 eq) and Na₂CO₃ (2.00 M, 383 μL, 3.00 eq) . The mixture was stirred at 90℃ for 12 hrs under N₂ atmosphere. LC-MS showed desired mass detected. The reaction mixture was concentrated to give a residue. The residue was purified by prep-TLC (DCM: MeOH = 10: 1, R_f (P1) = 0.300) . Compound 52-6 (40.0 mg, 145 μmol, 56.9%yield) was obtained as a yellow solid. The structure was confirmed by H NMR.

H NMR: (400 MHz, CDCl₃) δ 8.61 (d, J = 2.0 Hz, 1H) , 7.68 (d, J = 2.0 Hz, 1H) , 7.62 -7.58 (m, 2H) , 7.19 -7.15 (m, 2H) , 4.39 -4.33 (m, 1H) , 1.63 -1.62 (m, 6H) .

Step 4. N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1H-indazol-4-yl) phenyl) -5- (4-fluorophenyl) -1-isopropyl-4-oxo-1, 4-dihydropyridine-3-carboxamide

To a solution of compound 52-7 (46.0 mg, 113 μmol, 92.7%purity, 1.00 eq) and compound 52-6 (28.0 mg, 101 μmol, 0.900 eq) in DMF (3.00 mL) was added HATU (64.4 mg, 169 μmol, 1.50 eq) and DIEA (43.8 mg, 339 umol, 59.0 uL, 3.00 eq) . Then the mixture was stirred at 20℃ for 2 hrs. LC-MS showed the desired mass was detected. The reaction mixture was quenched by water (50.0 mL) , and then extracted with EtOAc (10.0 mL *5) . The combined organic layers were washed by water (10.0 mL *4) and brine (10.0 mL *4) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The crude product was purified by prep-TLC (DCM: MeOH = 10: 1, R_f (P1) = 0.400) and further purified by prep-HPLC (HCl condition; column: 3_Phenomenex Luna C18.0 75.0 *30.0 mm *3.00 um; mobile phase: [water (HCl) -ACN] ; B%: 38.0%-58.0%, 8 min) . Then the mixture was concentrated under reduced pressure to remove MeCN and adjusted by sat. NaHCO₃ until pH was around 8. The aqueous part was extracted with DCM (20.0 mL *3) . The combined organic layers were dried over anhydrous Na₂SO₄, then filtered and concentrated under reduced pressure to give a residue. Compound 52 (8.59 mg, 13.4 μmol, 11.7%yield, 98.7%purity) was obtained as confirmed by H NMR and LC-MS.

H NMR: (400 MHz, CDCl₃) δ 12.86 (s, 1H) , 8.73 (s, 1H) , 7.91 -7.89 (m, 2H) , 7.59 -7.55 (m, 3H) , 7.52 -7.49 (m, 2H) , 7.20 -7.16 (m, 2H) , 7.09 (s, 1H) , 6.82 (s, 1H) , 4.86 (d, J = 10.0 Hz, 1H) , 4.36 -4.33 (m, 1H) , 4.10 (d, J = 10.0 Hz, 1H) , 3.91 (s, 2H) , 3.20 -3.14 (m, 1H) , 2.91 -2.86 (m, 2H) , 2.68 -2.62 (m, 1H) , 2.04 -1.97 (m, 2H) , 1.76 -1.71 (m, 2H) , 1.63 -1.62 (m, 6H) , 1.17 -1.15 (m, 6H) .

LC-MS: (M+H) ⁺: 635.1

HPLC: purity: 98.7% (215 nm)

Example 23

N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1H-pyrazolo [3, 4-b] pyridin-4-yl) phenyl) -5- (4-fluorophenyl) -4-hydroxynicotinamide (Compound 54)

Step 1. 2-fluoro-4-iodonicotinonitrile

To a solution of compound 54-1 (25.0 g, 204 mmol, 1.00 eq) in THF (300 mL) was added TMPMgCl^. LiCl (1.00 M, 307 mL, 1.50 eq) at -60 ℃. The mixture was stirred at -60 ℃ for 1 hr. I₂ (57.1 g, 225 mmol, 45.3 mL, 1.10 eq) in THF (100 mL) was added to the reaction mixture at -60 ℃, and stirred at 25 ℃ for 12 hrs. TLC indicated compound 54-1 was consumed completely and two new spots formed. The reaction was clean according to TLC. (Petroleum ether: Ethyl acetate = 3: 1) . The reaction mixture was quenched by addition sat. aq NH₄Cl 250 mL at 0 ℃, and then diluted with H₂O 200 mL and extracted with EtOAc 400 mL (200 mL *2) . The combined organic layers were washed with brine 400 mL (200 mL *2) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether: Ethyl acetate = 100: 1 to 0: 1) . Compound 54-2 (10.0 g, 40.3 mmol, 19.6%yield) was obtained as a yellow solid. The structure was confirmed by H NMR.

H NMR: (400 MHz, CDCl₃) δ 8.09 (d, J = 5.2 Hz, 1H) , 7.80 (d, J = 5.6 Hz, 1H) .

Step 2. 2-hydrazineyl-4-iodonicotinonitrile

To a solution of compound 54-2 (9.20 g, 37.1 mmol, 1.00 eq) in MeOH (100 mL) was added N₂H₄ ^. H₂O (15.1 g, 296 mmol, 14.2 mL, 98.0%purity, 8.00 eq) . The mixture was stirred at 15 ℃ for 0.5 hr. LC-MS showed desired mass detected. The reaction mixture was filtered. The cake was washed with 100 mL EtOH, then concentrated under reduced pressure to give a residue. Compound 54-3 (9.00 g, 34.6 mmol, 93.3%yield) was obtained as a yellow solid.

Step 3. 4-iodo-1H-pyrazolo [3, 4-b] pyridin-3-amine

To a solution of compound 54-3 (9.00 g, 34.6 mmol, 1.00 eq) in DCM (50.0 mL) was added TFA (81.0 g, 710 mmol, 52.6 mL, 20.5 eq) in anisole (5.00 mL) . The mixture was stirred at 20 ℃ for 0.5 hr. LC-MS showed desired mass detected. The reaction mixture was concentrated to give a residue. The residue was adjusted pH to 9 by NH₃·H₂O, then filtered. The cake was washed by 20.0 mL MTBE, then concentrated to obtain product. Compound 54-4 (8.50 g, 32.6 mmol, 94.4%yield) was obtained as a yellow solid. The structure was confirmed by H NMR.

H NMR: (400 MHz, DMSO-d6) δ 12.34 (brs, 1H) , 7.92 (d, J = 4.4 Hz, 1H) , 7.43 (d, J = 4.8 Hz, 1H) , 5.16 (s, 2H)

Step 4. 2- (4-iodo-1H-pyrazolo [3, 4-b] pyridin-3-yl) isoindoline-1, 3-dione

A mixture of compound 54-4 (8.50 g, 32.6 mmol, 1.00 eq) and compound 54-5 (7.26 g, 49.0 mmol, 1.50 eq) in HOAc (80.0 mL) was stirred at 120 ℃ for 12 hrs. LC-MS showed desired mass detected. The reaction mixture was filtered and the cake was washed with 100 mL MTBE, then concentrated under reduced pressure to give a residue. Compound 54-6 (11.0 g, 28.2 mmol, 86.2%yield) was obtained as a yellow solid. The structure was confirmed by H NMR.

H NMR: (400 MHz, DMSO-d6) δ 14.47 (s, 1H) , 8.25 (d, J = 4.8 Hz, 1H) , 8.13 -8.11 (m, 2H) , 8.02 -7.99 (m, 2H) , 7.79 (d, J = 4.8 Hz, 1H) .

Step 5. 3- (1, 3-dioxoisoindolin-2-yl) -4-iodo-1H-pyrazolo [3, 4-b] pyridine 7-oxide

To a solution of compound 54-6 (8.00 g, 20.5 mmol, 1.00 eq) in HOAc (100 mL) was added m-CPBA (16.6 g, 82.0 mmol, 85.0%purity, 4.00 eq) at 0 ℃. The mixture was stirred at 60 ℃ for 12 hrs. LC-MS showed desired mass detected. The reaction mixture was filtered. The filtrate was concentrated to remove HOAc, then diluted with 100 mL H₂O, and adjusted pH to 9 by sat. aq NaHCO₃, then extracted with EtOAc 300 mL (100 mL *3) . The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The crude product was triturated with MTBE 50.0 mL at 25 ℃ for 20 mins. The filtrate was purified by column chromatography (SiO₂, Petroleum ether: EtOAc = 100: 1 to 0: 1) . (Plate 1: DCM: MeOH = 10: 1) . Compound 54-7 (4.00 g, 9.85 mmol, 48.0%yield) was obtained as a yellow solid.

Step 6. 2- (6-chloro-4-iodo-1H-pyrazolo [3, 4-b] pyridin-3-yl) isoindoline-1, 3-dione

To a solution of compound 54-8 (4.00 g, 9.85 mmol, 1.00 eq) in MeCN (40.0 mL) was added POCl₃ (15.1 g, 98.4 mmol, 9.15 mL, 10.0 eq) at 0 ℃. The mixture was stirred at 25 ℃ for 12 hrs. LC-MS showed desired mass detected. The reaction mixture was filtered and the cake was washed with 100 mL MeOH, then concentrated under reduced pressure to give a residue. Compound 54-9 (2.00 g, 4.71 mmol, 47.8%yield) was obtained as a yellow solid.

Step 7. 6-chloro-4-iodo-1H-pyrazolo [3, 4-b] pyridin-3-amine

To a solution of compound 54-8 (2.00 g, 4.71 mmol, 1.00 eq) in MeOH (4.00 mL) was added N₂H₄ ^. H₂O (7.22 g, 141 mmol, 7.01 mL, 98%purity, 30.0 eq) . The mixture was stirred at 25 ℃ for 2 hrs. LC-MS showed desired mass detected. The reaction mixture was added 50.0 mL H₂O and extracted with EtOAc 150 mL (50 mL *3) . The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. Compound 54-9 (1.30 g, 4.41 mmol, 93.7%yield) was obtained as a yellow solid. The structure was confirmed by H NMR.

H NMR: (400 MHz, DMSO-d6) δ 7.51 (s, 1H) , 5.27 (s, 2H) .

Step 8. 6-chloro-4- (4-nitrophenyl) -1H-pyrazolo [3, 4-b] pyridin-3-amine

To a solution of compound 54-9 (200 mg, 679 μmol, 1.00 eq) and compound 54-10 (119 mg, 713 μmol, 1.05 eq) in dioxane (8.00 mL) was added K₂CO₃ (281 mg, 2.04 mmol, 3.00 eq) in H₂O (2.00 mL) and Pd (PPh₃) ₄ (78.4 mg, 67.9 μmol, 0.100 eq) . The mixture was stirred at 90℃ for 3hrs under N₂ atmosphere. TLC (PE: THF = 2: 1, R_f (P1) = 0.400) showed the starting material was consumed completely and there were new spots. The reaction mixture was quenched by water (20.0 mL) , and then extracted with EtOAc (30.0 mL) . The organic layers were washed by water (10.0 mL *3) and brine (10.0 mL *3) , and the combined aqueous layers were extracted with EtOAc (30.0 mL *3) . The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (PE: THF = 1: 1, R_f (P1) = 0.500) . Compound 54-11 (195 mg, 394 μmol, 58.0%yield, 58.5%purity) was obtained as a yellow solid, confirmed by LC-MS.

LC-MS: (M+H) ⁺: 290.2

Step 9. 1- (4- (3-amino-4- (4-nitrophenyl) -1H-pyrazolo [3, 4-b] pyridin-6-yl) -3, 6-dihydropyridin-1 (2H) -yl) -2-methylpropan-1-one

To a solution of compound 54-11 (175 mg, 353 μmol, 58.5%purity, 1.00 eq) and compound 54-12 (493 mg, 1.77 mmol, 5.00 eq) in dioxane (12.0 mL) was added K₃PO₄ (225 mg, 1.06 mmol, 3.00 eq) in H₂O (3.00 mL) and Xphos-Pd-G₂ (55.6 mg, 70.7 μmol, 0.200 eq) . The mixture was stirred at 100℃ for 2hrs under N₂ atmosphere. LC-MS showed the desired mass was detected. The reaction mixture was quenched by water (20.0 mL) , and then extracted with EtOAc (30.0 mL) . The organic layers were washed by water (10.0 mL *3) and brine (10.0 mL *3) , and the combined aqueous layers were extracted with EtOAc (30.0 mL *3) . The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (DCM: MeOH =10: 1, R_f (P1) = 0.400) . Compound 54-13 (85.0 mg, 173 μmol, 48.9%yield, 82.8%purity) was obtained as a yellow solid, confirmed by LC-MS.

LC-MS: (M+H) ⁺: 407.2

Step 10. 1- (4- (3-amino-4- (4-aminophenyl) -1H-pyrazolo [3, 4-b] pyridin-6-yl) piperidin-1-yl) -2-methylpropan-1-one

To a solution of compound 54-13 (80.0 mg, 163 μmol, 82.8%purity, 1.00 eq) in THF (30.0 mL) was added Pd/C (800 mg, 10.0%purity) , degassed and purged with H₂ (15 Psi) for 3 times, then stirred at 20 ℃ for 2 hrs. LC-MS showed the desired mass was detected. The mixture was filtered and filter was concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (DCM: MeOH = 10: 1, R_f (P1) = 0.400) . Compound 54-14 (35.0 mg, 91.4 μmol, 56.0%yield, 98.8%purity) was obtained as a yellow solid, confirmed by LC-MS.

LC-MS: (M+H) ⁺: 379.4

Step 11. N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1H-pyrazolo [3, 4-b] pyridin-4-yl) phenyl) -5- (4-fluorophenyl) -4-hydroxynicotinamide

To a solution of compound 54-14 (30.0 mg, 78.3 μmol, 98.8%purity, 1.00 eq) and compound 54-15 (16.4 mg, 70.5 μmol, 0.900 eq) in DMF (5.00 mL) was added HATU (44.6 mg, 117 μmol, 1.50 eq) and DIEA (30.3 mg, 235 μmol, 40.9 μL, 3.00 eq) . Then the mixture was stirred at 20℃ for 1hr. LC-MS showed the desired mass was detected. The reaction mixture was quenched by sat. NaHCO₃ (15.0 mL) , and then extracted with EtOAc (10.0 mL *5) . The combined organic layers were washed by water (10.0 mL *4) and brine (10.0 mL *4) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The crude product was purified by prep-TLC (DCM: MeOH = 10: 1, R_f (P1) = 0.400) and further purified by prep-HPLC (NH₃·H₂O condition; column: Waters Xbridge 150 *25.0 mm *5.00 um; mobile phase: [water (ammonia hydroxide v/v) -ACN] ; B%: 20.0%-50.0%, 32min) . Then the mixture was concentrated under reduced pressure to remove MeCN and freeze-dried to give a residue. Compound 54 (7.06 mg, 11.6 μmol, 14.9%yield, 98.2%purity) was obtained as confirmed by H NMR, LC-MS and HPLC.

H NMR: (400 MHz, MeOD) δ 8.71 (d, J = 1.6 Hz, 1H) , 7.97 (d, J = 1.6 Hz, 1H) , 7.91 -7.89 (m, 2H) , 7.67 -7.60 (m, 4H) , 7.20 -7.16 (m, 2H) , 6.93 (s, 1H) , 4.71 (d, J = 14.4 Hz, 1H) , 4.21 (d, J = 14.0 Hz, 1H) , 3.27 -3.24 (m, 1H) , 3.16 -3.10 (m, 1H) , 3.05 -2.98 (m, 1H) , 2.82 -2.75 (m, 1H) , 2.08 -2.00 (m, 2H) , 1.92 -1.78 (m, 2H) , 1.16 -1.11 (m, 6H) .

LC-MS: (M+H) ⁺: 594.4

HPLC: purity: 98.2% (215 nm)

Example 24

N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1-methyl-1H-pyrazolo [3, 4-b] pyridin-4-yl) phenyl) -N- (4-fluorophenyl) cyclopropane-1, 1-dicarboxamide (Compound 56)

Step 1. 3- (1, 3-dioxoisoindolin-2-yl) -4-iodo-1H-pyrazolo [3, 4-b] pyridine 7-oxide

To a solution of compound 56-1 (5.86 g, 15.0 mmol, 1.00 eq) in DCM (60.0 mL) was added UHP (5.09 g, 54.0 mmol, 3.60 eq) at 0℃, then added TFAA (9.46 g, 45.0 mmol, 6.27 mL, 3.00 eq) dropwise at 0℃. The reaction mixture was stirred at 25℃ for 4 hrs under N₂ atmosphere. LC-MS showed the desired mass was detected. The mixture was added MeCN (300 mL) , stirred at 20℃ for 10 min. Then the mixture was filtered. The cake was washed by MeCN (50.0 mL *3) and concentrated under reduced pressure to give a residue. Compound 56-2 (5.45 g, 13.4 mmol, 89.3%yield) was obtained as an off-white solid, confirmed by H NMR.

H NMR: (400 MHz, DMSO-d₆) δ 15.42 (s, 1H) , 8.23 (d, J = 6.4 Hz, 1H) , 8.14 -8.11 (m, 2H) , 8.03 -8.00 (m, 2H) , 7.67 (d, J = 6.4 Hz, 1H) .

Step 2. 2- (6-chloro-4-iodo-1H-pyrazolo [3, 4-b] pyridin-3-yl) isoindoline-1, 3-dione

To a solution of compound 56-2 (5.45 g, 13.4 mmol, 1.00 eq) in MeCN (60.0 mL) was added POCl₃ (45.2 g, 295 mmol, 27.4 mL, 22.0 eq) at 0 ℃. The mixture was stirred at 20℃ for 3 hrs. LC-MS showed the desired mass was detected. The mixture was added MTBE (100 mL) and filtered. And the cake was washed by MTBE (20.0 mL *3) , concentrated under reduced pressure to give a residue. Compound 56-3 (5.23 g, 12.3 mmol, 91.7%yield) was obtained as a white solid, confirmed by H NMR.

H NMR: (400 MHz, DMSO-d₆) δ 14.65 (s, 1H) , 8.13 -8.11 (m, 2H) , 8.02 -8.00 (m, 2H) , 7.93 (s, 1H) .

Step 3. 6-chloro-4-iodo-1H-pyrazolo [3, 4-b] pyridin-3-amine

To a solution of compound 56-3 (5.65 g, 13.3 mmol, 1.00 eq) in MeOH (60.0 mL) was added N₂H₄·H₂O (11.5 g, 226 mmol, 11.2 mL, 17.0 eq) . Then the mixture was stirred at 20 ℃ for 1 hr. LC-MS showed the desired mass was detected. The mixture was filtered and the cake was concentrated under reduced pressure to give a residue. And the filter was quenched by water (60.0 mL) , and then extracted with DCM (60.0 mL *3) and DCM: MeOH = 10: 1 (60 mL *3) . The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. Compound 56-4 (2.50 g, 8.49 mmol, 63.8%yield) was obtained as a yellow solid.

Step 4. 6-chloro-4-iodo-1-methyl-1H-pyrazolo [3, 4-b] pyridin-3-amine

To a solution of compound 56-4 (1.00 g, 3.40 mmol, 1.00 eq) in DMF (20.0 mL) was added K₂CO₃ (938 mg, 6.79 mmol, 2.00 eq) and MeI (1.37 g, 9.64 mmol, 0.600 mL, 2.84 eq) at 0℃. Then the mixture was stirred at 20 ℃ for 10 hrs under N₂ atmosphere. LC-MS showed the desired mass was detected. The reaction mixture was quenched by water (60.0 mL) , and then extracted with EtOAc (50.0 mL *5) . The combined organic layers were washed by water (100 mL *4) and brine (100 mL *4) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue (DCM: MeOH = 10: 1, R_f (P1) = 0.800) was purified by column chromatography (SiO₂, DCM: MeOH = 100: 0 to 100: 1) . Compound 56-5 (450 mg, 1.46 mmol, 42.9%yield) was obtained as a yellow solid, confirmed by H NMR.

H NMR: (400 MHz, DMSO-d₆) δ 7.51 (s, 1H) , 5.37 (s, 2H) , 3.72 (s, 3H) .

Step 5. 6-chloro-1-methyl-4- (4-nitrophenyl) -1H-pyrazolo [3, 4-b] pyridin-3-amine

Four batches was in parallel. To a solution of compound 56-5 (100 mg, 324 μmol, 1.00 eq) and compound 56-6 (40.5 mg, 243 μmol, 0.750 eq) in dioxane (1.50 mL) and H₂O (0.100 mL) was added DIEA (125 mg, 972 μmol, 169 uL, 3.00 eq) and Pd (t-Bu₃P) ₂ (16.5 mg, 32.4 μmol, 0.100 eq) . The mixture was stirred at 125℃ for 30 min under N₂ atmosphere under microwave. LC-MS showed the desired mass was detected. The reaction mixture was quenched by water (20.0 mL) , and then extracted with EtOAc (30.0 mL) . The organic layers were washed by water (10.0 mL *3) and brine (10.0 mL *3) , and the combined aqueous layers were extracted with EtOAc (30.0 mL *3) . The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The crude product was purified by prep-TLC (Petroleum ether: THF = 1: 1, R_f (P1) = 0.500) . Compound 56-7 (124 mg, 408 μmol, 31.4%yield) was obtained as a yellow solid.

Step 6. 1- (4- (3-amino-1-methyl-4- (4-nitrophenyl) -1H-pyrazolo [3, 4-b] pyridin-6-yl) -3, 6-dihydropyridin-1 (2H) -yl) -2-methylpropan-1-one

Two batches was in parallel. To a solution of compound 56-7 (62.0 mg, 204 μmol, 1.00 eq) and compound 56-8 (214 mg, 714 μmol, 92.9%purity, 3.50 eq) in dioxane (1.50 mL) and H₂O (0.100 mL) was added DIEA (79.1 mg, 612 umol, 106 uL, 3.00 eq) and Pd (t-Bu₃P) ₂ (10.4 mg, 20.4 μmol, 0.100 eq) . The mixture was stirred at 120℃ for 1 hr under N₂ atmosphere under microwave. LC-MS showed the desired mass was detected. The reaction mixture was quenched by water (20.0 mL) , and then extracted with EtOAc (30.0 mL) . The organic layers were washed by water (10.0 mL *3) and brine (10.0 mL *3) , and the combined aqueous layers were extracted with EtOAc (30.0 mL *3) . The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The crude product was purified by prep-TLC (DCM: MeOH = 10: 1, R_f (P1) = 0.500) . Compound 56-9 (100 mg, 237 μmol, 29.1%yield) was obtained as a yellow solid.

Step 7. 1- (4- (3-amino-4- (4-aminophenyl) -1-methyl-1H-pyrazolo [3, 4-b] pyridin-6-yl) piperidin-1-yl) -2-methylpropan-1-one

To a solution of compound 56-9 (100 mg, 237 μmol, 1.00 eq) in THF (10.0 mL) was added Pd/C (10.0 mg, 10.0%purity) , degassed and purged with H₂ (15 Psi) for 3 times, then stirred at 20℃ for 2 hrs. LC-MS showed the desired mass was detected. The mixture was filtered and filter was concentrated under reduced pressure to give a residue. Compound 56-10 (90.0 mg, 229 μmol, 96.4%yield) was obtained as a brown solid.

Step 8. N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1-methyl-1H-pyrazolo [3, 4-b] pyridin-4-yl) phenyl) -N- (4-fluorophenyl) cyclopropane-1, 1-dicarboxamide

To a solution of compound 56-10 (50.0 mg, 127 μmol, 1.00 eq) in pyridine (1.00 mL) was added compound 56-11 (28.4 mg, 127 μmol, 1.00 eq) , EDCI (97.6 mg, 509 μmol, 4.00 eq) . Then the mixture was stirred at 20℃ for 1 hr. LC-MS showed the desired mass was detected. The reaction mixture was quenched by water (10.0 mL) , and then extracted with DCM (30.0 mL) . The organic layers were washed by water (10.0 mL *3) and brine (10.0 mL *3) , and the combined aqueous layers were extracted with DCM (10.0 mL *3) . The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The crude product was purified by prep-TLC (DCM: MeOH = 10: 1, R_f (P1) = 0.400) and further purified by prep-HPLC (ammonia hydroxide condition; column: Waters Xbridge 150 *25.0 mm *5.00 um; mobile phase: [water (ammonia hydroxide v/v) -ACN] ; B%: 40.0%-70.0%, 9 min) . Then the mixture was concentrated under reduced pressure to remove MeCN. And the mixture was freeze dried to give a product. Compound 56 (9.50 mg, 14.9 μmol, 11.7%yield, 94.1%purity) was obtained as confirmed by H NMR and LC-MS.

H NMR: (400 MHz, CDCl₃) δ 9.64 (s, 1H) , 8.62 (s, 1H) , 7.73 –7.71 (m, 2H) , 7.57 –7.55 (m, 2H) , 7.49 –7.46 (m, 2H) , 7.09 –7.04 (m, 2H) , 6.75 (s, 1H) , 4.81 (d, J = 15.2 Hz, 1H) , 4.11 (d, J = 13.2 Hz, 1H) , 3.94 (s, 3H) , 3.24 –3.18 (m, 1H) , 3.09 –3.03 (m, 1H) , 2.89 –2.84 (m, 1H) , 2.75 –2.69 (m, 1H) , 2.10 –2.02 (m, 2H) , 1.91 –1,89 (m, 2H) , 1.77 –1.72 (m, 4H) , 1.19 –1.15 (m, 6H) .

LC-MS: (M+H) ⁺: 598.3

HPLC: purity: 94.1% (215 nm)

Example 25

N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1-methyl-1H-indazol-4-yl) phenyl) -4-ethoxy-1- (4-fluorophenyl) -2-oxo-1, 2-dihydropyridine-3-carboxamide (Compound 57)

To a solution of compound 57-1 (30 mg, 76.63 μmol, 1 eq) and compound 57-2 (22 mg, 79.35 μmol, 1.04 eq) in Py (2 mL) was added EDCI (30.00 mg, 156.49 μmol, 2.04 eq) . The mixture was stirred at 25℃ for 2 hrs. An aliquot was taken from the reaction mixture and added to MeOH, LCMS showed compound 57-1 was consumed and desired MS was detected. Dilute the reaction mixture with H₂O (10 mL) and extracted it with EtOAc (15 mL *2) . Wash the combined organic layers with H₂O (40 mL *3) followed by NH₄Cl (sat., 50 mL) , dry it over Na₂SO₄, filter and concentrate it under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge 150*25mm*5um; mobile phase: [water (ammonia hydroxide v/v) -ACN] ; B%: 34%-64%, 9min) , then concentrated to remove MeCN and freeze-dried to get Compound 57 (25 mg, 37.11 μmol, 41.39%yield, 96.6%purity) as confirmed by H NMR.

H NMR: (400 MHz, DMSO-d6) δ 10.38 (s, 1H) , 7.88 -7.74 (m, 3H) , 7.55 -7.30 (m, 6H) , 7.22 (s, 1H) , 6.71 (s, 1H) , 6.50 (d, J = 7.6 Hz, 1H) , 4.60 (d, J = 12.4 Hz, 1H) , 4.34 (brs, 1H) , 4.30 -4.15 (m, 2H) , 4.08 (d, J = 12.4 Hz, 1H) , 3.76 (s, 3H) , 3.20 - 3.05 (m, 1H) , 2.95 -2.80 (m, 2H) , 2.65 -2.55 (m, 1H) , 2.05 -1.80 (m, 2H) , 1.80 - 1.45 (m, 2H) , 1.30 (t, J = 7.2 Hz, 3H) , 1.10 -0.85 (m, 6H) .

Example 26

N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1-methyl-1H-indazol-4-yl) phenyl) -2, 5-dioxo-1-phenyl-1, 2, 5, 6, 7, 8-hexahydroquinoline-3-carboxamide (Compound 58)

To a solution of compound 58-1 (30 mg, 76.63 μmol, 1 eq) in Py (2 mL) was added compound 58-2 (24.00 mg, 84.72 μmol, 1.11 eq) and EDCI (30 mg, 156.49 μmol, 2.04 eq) . The mixture was stirred at 25℃ for 2 hrs. An aliquot was taken from the reaction mixture and added to MeOH, LCMS showed compound 58-1 was consumed and desired MS was detected. Dilute the reaction mixture with H₂O (5 mL) and extracte it with EtOAc (5 mL *2) . Wash the combined organic layers with H₂O (15 mL *3) followed by NH₄Cl (sat., 30 mL) , dry it over Na₂SO₄, filter and concentrate it under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex luna C18 150*25mm*10um; mobile phase: [water (FA) -ACN] ; B%: 53%-73%, 9min) , then concentrated to remove MeCN, and adjust to pH = 8 with NaHCO₃ (sat. ) , then extracted with EtOAc (20 ml*2) , dried it over Na₂SO₄, filtered and concentrated it under reduced pressure to get desired Compound 58 (25 mg, 37.95 μmol, 49.53%yield, 99.7%purity) as confrimed by H NMR.

H NMR: (400 MHz, DMSO-d₆) δ 11.57 (s, 1H) , 8.96 (s, 1H) , 7.98 -7.80 (m, 2H) , 7.75 -7.55 (m, 3H) , 7.53 -7.38 (m, 4H) , 7.22 (s, 1H) , 6.72 (s, 1H) , 4.70 -4.53 (m, 2H) , 4.40 -4.25 (m, 2H) , 4.15 -4.00 (m, 1H) , 3.76 (s, 3H) , 3.23 -3.05 (m, 1H) , 2.98 -2.80 (m, 2H) , 2.63 -2.54 (m, 3H) , 2.10 -1.80 (m, 4H) , 1.75 -1.48 (m, 2H) , 1.10 -0.95 (m, 6H) .

Example 27

N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1-methyl-1H-indazol-4-yl) phenyl) -5- (4-fluorophenyl) -1-methyl-4-oxo-1, 4-dihydropyridine-3-carboxamide (Compound 59)

To a solution of compound 59-2 (30 mg, 76.63 μmol, 1 eq) in DMF (3 mL) was added HATU (43.70 mg, 114.94 umol, 1.5 eq) , DIEA (19.81 mg, 153.25 μmol, 26.69 μL, 2 eq) and compound 59-1 (19 mg, 76.85 μmol, 1 eq) and then the mixture was stirred at 25℃ for 2 hr. An aliquot was taken from the reaction mixture and added to MeOH, LCMS showed compound 59-2 was consumed and desired MS was detected. Dilute the reaction mixture with H₂O (10 mL) and extracte it with EtOAc (5 mL *2) . Wash the combined organic layers with H₂O (10 mL *3) followed by brine (20 mL) , dry it over Na₂SO₄, filter and concentrate it under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge 150*25mm*5um; mobile phase: [water (ammonia hydroxide v/v) -ACN] ; B%: 38%-68%, 9min) and concentrated to remove MeCN and freeze-dried to get Compound 59 (18 mg, 28.71 umol, 37.47%yield, 99%purity) as confirmed by H NMR.

H NMR: (400 MHz, DMSO-d₆) δ 13.10 (s, 1H) , 8.70 (d, J = 6.4 Hz, 1H) , 8.15 (d, J = 2.0 Hz, 1H) , 7.83 (d, J = 8.8 Hz, 2H) , 7.79 -7.68 (m, 2H) , 7.46 (d, J = 8.4 Hz, 2H) , 7.33-7.20 (m, 3H) , 6.73 (s, 1H) , 4.60 (d, 1H, J = 12.8 Hz) , 4.36 (brs, 2H) , 4.17 -4.00 (m, 1H) , 3.93 (s, 3H) , 3.77 (s, 3H) , 3.21 -3.05 (m, 1H) , 2.99 -2.84 (m, 2H) , 2.63 -2.57 (m, 1H) , 1.97 -1.81 (m, 2H) , 1.69 -1.60 (m, 1H) , 1.60 -1.52 (m, 1H) , 1.07-0.97 (m, 6H) .

Example 28

N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1-methyl-1H-indazol-4-yl) phenyl) -1- (4-fluorophenyl) -2-oxo-1, 2-dihydropyridine-3-carboxamide (Compound 60)

To a solution of compound 60-1 (30 mg, 76.63 μmol, 1 eq) in Py (2 mL) was added compound 60-2 (20 mg, 85.77 μmol, 1.12 eq) and EDCI (30 mg, 156.49 μmol, 2.04 eq) , then the mixture was stirred at 25℃ for 2 hrs. An aliquot was taken from the reaction mixture and added to MeOH, LCMS showed compound 60-1 was consumed and desired MS was detected. Dilute the reaction mixture with H₂O (5mL) and extracte it with EtOAc (10 mL *2) . Wash the combined organic layers with H₂O (20 mL *3) followed by NH₄Cl (sat., 50 mL) , dry it over Na₂SO₄, filter and concentrate it under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Phenomenex luna C18 150*25mm*10um; mobile phase: [water (FA) -ACN] ; B%: 48%-78%, 10min) , then concentrated to remove MeCN, and adjust to pH = 8 with NaHCO₃ (sat. ) , then extracted with EtOAc (20 ml*2) , dried it over Na₂SO₄, filtered and concentrated it under reduced pressure to get desired Compound 60 (20 mg, 32.87 μmol, 42.89%yield, 99.7%purity) as confirmed by H NMR.

H NMR: (400 MHz, DMSO-d6) δ 12.04 (s, 1H) , 8.88 -8.53 (m, 1H) , 8.20 -8.05 (m, 1H) , 7.93 -7.77 (m, 2H) , 7.73 -7.57 (m, 2H) , 7.55 -7.38 (m, 4H) , 7.23 (s, 1H) , 6.77 -6.68 (m, 2H) , 4.68 -4.57 (m, 1H) , 4.40 -4.26 (m, 2H) , 4.17 -4.05 (m, 1H) , 3.77 (s, 3H) , 3.22 -3.05 (m, 1H) , 2.97 -2.80 (m, 2H) , 2.65-2.57 (m, 1H) , 1.97 -1.81 (m, 2H) , 1.74 -1.48 (m, 2H) , 1.08 -0.86 (m, 6H) .

Example 29

N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1-methyl-1H-indazol-4-yl) phenyl) -2-oxo-2H- [1, 2'-bipyridine] -3-carboxamide (Compound 61)

Step 1. methyl 2-oxo-2H- [1, 2'-bipyridine] -3-carboxylate

To a solution of compound 61-1 (500 mg, 3.24 mmol, 1.00 eq) in THF (4.00 mL) and DMF (1.00 mL) was added compound 61-2 (305 mg, 3.24 mmol, 1.00 eq) . And the mixture was stirred at 20℃ for 2 hrs. Then EDCI (746 mg, 3.89 mmol, 1.20 eq) and DMAP (198 mg, 1.62 mmol, 0.500 eq) were added to the mixture, and the mixture was stirred at 20℃ for 14 hrs. LC-MS showed the desired mass was detected. The mixture was quenched with 4.00 M HCl (10.0 mL) at 20℃ and stir for 30 min. Then the mixture was adjusted by Na₂CO₃ until pH was around 9. The aqueous part was extracted with EtOAc (100 mL *8) . The combined organic layers were dried over anhydrous Na₂SO₄, then filtered and concentrated under reduced pressure to give a residue. The residue (DCM: MeOH = 10: 1, Rf (P1) = 0.400) was purified by column chromatography (SiO₂, DCM: MeOH = 100: 0 to 100: 1) . Compound 61-3 (500 mg, 2.17 mmol, 66.9%yield) was obtained as a yellow solid, confirmed by H NMR.

H NMR: (400 MHz, CDCl₃) δ 8.58 -8.57 (m, 1H) , 8.25 -8.23 (m, 1H) , 8.13 -8.11 (m, 1H) , 7.98 -7.96 (m, 1H) , 7.87 -7.83 (m, 1H) , 7.37 -7.27 (m, 1H) , 6.41 -6.37 (m, 1H) , 3.92 (s, 3H) .

Step 2. 2-oxo-2H- [1, 2'-bipyridine] -3-carboxylic acid

To a solution of compound 61-3 (500 mg, 2.17 mmol, 1.00 eq) in MeOH (3.00 mL) was added NaOH (2.00 M, 2.88 mL, 2.65 eq) at 0℃. The mixture was stirred at 20 ℃ for 10 min. LC-MS showed the desired mass was detected. The mixture was concentrated under reduced pressure to remove MeOH. Then the mixture was dissolved into water (50.0 mL) and adjusted by 2.00 M HCl until pH was around 2. Then the mixture was filtered to give the residue. The mixture was dissolved into MeOH (50 mL) and concentrated under reduced pressure to give the product. Compound 61-4 (320 mg, 1.48 mmol, 68.1%yield) was obtained as a yellow solid.

Step 3. N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1-methyl-1H-indazol-4-yl) phenyl) -2-oxo-2H- [1, 2'-bipyridine] -3-carboxamide

To a solution of compound 61-5 (25.0 mg, 63.8 μmol, 1.00 eq) and compound 61-4 (12.4 mg, 57.4 μmol, 0.900 eq) in DCM (2.00 mL) was added HATU (36.4 mg, 95.7 μmol, 1.50 eq) and DIEA (41.2 mg, 319 μmol, 55.6 μL, 5.00 eq) . Then the mixture was stirred at 20℃ for 1 hr. LC-MS showed the desired mass was detected. The reaction mixture was quenched by sat. aq. NaHCO₃ (20.0 mL) , and then extracted with DCM (30.0 mL *3) . The organic layers were washed by sat. aq. NaHCO₃ (40.0 mL *2) . The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The crude product was purified by prep-TLC (DCM: MeOH = 10: 1, Rf (P1) = 0.500) . Compound 61 (27.5 mg, 45.3 μmol, 70.9%yield, 97.0%purity) was obtained as confirmed by H NMR, LC-MS and HPLC.

H NMR: (400 MHz, CDCl₃) δ 11.94 (s, 1H) , 8.80 -8.77 (m, 1H) , 8.68 -8.67 (m, 1H) , 8.06 -8.04 (m, 1H) , 7.99 -7.95 (m, 1H) , 7.88 -7.86 (m, 3H) , 7.51 -7.46 (m, 3H) , 6.97 (s, 1H) , 6.77 (s, 1H) , 6.69 -6.65 (m, 1H) , 4.87 (d, J = 12.0 Hz, 1H) , 4.11 (d, J = 12.4 Hz, 1H) , 3.85 (s, 3H) , 3.21 -3.14 (m, 1H) , 2.93 -2.83 (m, 2H) , 2.69 -2.63 (m, 1H) , 2.05 -1.98 (m, 2H) , 1.78 -1.72 (m, 2H) , 1.19 -1.15 (m, 6H) .

LC-MS: (M+H) ⁺: 590.3

HPLC: purity: 97.0% (215 nm)

Example 30

N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1-methyl-1H-indazol-4-yl) phenyl) -5'-fluoro-2-oxo-2H- [1, 2'-bipyridine] -3-carboxamide (Compound 66)

Step 1. methyl 5'-fluoro-2-oxo-2H- [1, 2'-bipyridine] -3-carboxylate

To a solution of compound 66-1 (1.00 g, 6.49 mmol, 1.00 eq) in THF (10.0 mL) and DMF (2.50 mL) was added compound 66-2 (727 mg, 6.49 mmol, 1.00 eq) , the mixture was stirred at 25℃ for 2 hrs, then EDCI (1.87 g, 9.73 mmol, 1.50 eq) , DMAP (396 mg, 3.24 mmol, 0.500 eq) was added, the mixture was stirred at 25℃ for 10 hrs. LC-MS showed compound 66-1 was consumed and the desired mass detected. The mixture was diluted with 4M HCl (10.0 mL) and stirred at 25℃ for 0.5 h, the mixture was filtered and the filter cake was the desired product compound 66-3 (660 mg, 2.66 mmol, 40.9%yield) as yellow solid.

Step 2. 5'-fluoro-2-oxo-2H- [1, 2'-bipyridine] -3-carboxylic acid

To a solution of compound 66-3 (660 mg, 2.66 mmol, 1.00 eq) in THF (10.0 mL) , MeOH (4.00 mL) was added a solution of LiOH^. H₂O (334 mg, 7.98 mmol, 3.00 eq) in H₂O (2.00 mL) at 0℃, the mixture was stirred at 25℃ for 3 hrs. LC-MS showed compound 66-3 was consumed and the desired mass detected. The mixture was concentrated to remove solvent and diluted with water (20.0 mL) , extracted with EtOAc (20.0 mL *2) , the organic layer was discard and the aqueous phase was adjusted the pH = 2 by 1M HCl at 0℃, extracted with DCM (30.0 mL *3) , the combined organic layer was washed with brine (50.0 mL) , dried over Na₂SO₄, filtered and concentrated to remove solvent to get the desired product compound 66-4 (500 mg, 2.14 mmol, 80.3%yield) as yellow solid.

Step 3. N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1-methyl-1H-indazol-4-yl) phenyl) -5'-fluoro-2-oxo-2H- [1, 2'-bipyridine] -3-carboxamide

To a solution of compound 66-5 (25.0 mg, 63.8 μmol, 1.00 eq) , compound 66-5 (13.4 mg, 57.4 μmol, 0.900 eq) in DCM (2.00 mL) was added DIEA (41.2 mg, 319 μmol, 55.6 μL, 5.00 eq) , HATU (36.4 mg, 95.8 μmol, 1.50 eq) , the mixture was stirred at 25℃ for 1 h. LC-MS showed the desired mass detected. The reaction mixture was diluted with sat. aq. NaHCO₃ (3.00 mL) and water (3.00 mL) , extracted with DCM (5.00 mL *3) , the combined organic layer was washed with brine (10.0 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by Prep-TLC (SiO₂, DCM: MeOH = 10: 1, Plate 1, DCM: MeOH = 10: 1, R_f = 0.55) to get the desired product Compound 66 (22.73 mg, 35.16 μmol, 55.06%yield, 94%purity) as confirmed by H NMR, LC-MS, and HPLC.

H NMR: (400 MHz, CDCl₃) δ 11.86 (s, 1H) , 8.79 –8.76 (m, 1H) , 8.49 (d, J = 2.8 Hz, 1H) , 8.03 –8.00 (m, 1H) , 7.92 –7.89 (m, 1H) , 7.87 –7.85 (m, 2H) , 7.69 –7.65 (m, 1H) , 7.51 –7.49 (m, 2H) , 6.97 (s, 1H) , 6.76 (s, 1H) , 6.67 (t, J = 7.2 Hz, 1H) , 4.86 (d, J = 13.6 Hz, 1H) , 4.10 (d, J = 12.4 Hz, 1H) , 3.87 –3.79 (m, 5H) , 3.17 (t, J =12.8 Hz, 1H) , 2.93 –2.83 (m, 2H) , 2.65 (t, J = 12.0 Hz, 1H) , 2.04 –1.98 (m, 2H) , 1.76 –1.70 (m, 2H) , 1.19 –1.14 (m, 6H) .

LC-MS: (M+H) ⁺: 608.4

HPLC: purity: 94.0% (220 nm)

Example 31

N- (4- (3-amino-6- (1-isobutyrylpiperidin-4-yl) -1-methyl-1H-pyrazolo [3, 4-b] pyridin-4-yl) phenyl) -4-ethoxy-5'-fluoro-2-oxo-2H- [1, 2'-bipyridine] -3-carboxamide (Compound 67)

To a solution of compound 67-1 (13.0 mg, 33.1 μmol, 1.00 eq) and compound 67-2 (8.29 mg, 29.8 μmol, 0.900 eq) in pyridine (1.00 mL) was added EDCI (25.4 mg, 132 μmol, 4.00 eq) . The mixture was stirred at 25 ℃ for 1 hr. LC-MS showed the desired mass was detected. The reaction mixture was quenched by water (10.0 mL) , and then extracted with DCM (30.0 mL *3) . The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The crude product was purified by prep-TLC (DCM: MeOH = 10: 1, Rf (P1) = 0.400) . Compound 67 (7.44 mg, 11.1 μmol, 33.6%yield, 97.8%purity) was obtained.

H NMR: (400 MHz, CDCl₃) δ 11.20 (s, 1H) , 8.45 (d, J = 2.8 Hz, 1H) , 7.97 (d, J = 8.0 Hz, 1H) , 7.91 -7.86 (m, 3H) , 7.66 -7.61 (m, 1H) , 7.53 -7.51 (m, 2H) , 8.76 (s, 1H) , 6.43 (d, J = 8.0 Hz, 1H) , 4.82 (d, J = 13.2 Hz, 1H) , 4.42 -4.36 (m, 2H) , 4.10 (d, J = 13.2 Hz, 1H) , 3.93 (s, 3H) , 3.90 (s, 2H) , 3.24 -3.18 (m, 1H) , 3.08 -3.02 (m, 1H) , 2.91 -2.84 (m, 1H) , 2.76 -2.70 (m, 1H) , 2.09 -2.03 (m, 2H) , 1.90 -1.84 (m, 2H) , 1.61 -1.57 (m, 3H) , 1.20 -1.15 (m, 6H) .

LC-MS: (M+H) ⁺: 653.4

Example 32

N- (4- (3-amino-1-methyl-6- ( (7R, 8aS) -3-oxooctahydroindolizin-7-yl) -1H-indazol-4-yl) phenyl) -4-ethoxy-1- (4-fluorophenyl) -2-oxo-1, 2-dihydropyridine-3-carboxamide, N- (4- (3-amino-1-methyl-6- ( (7S, 8aR) -3-oxooctahydroindolizin-7-yl) -1H-indazol-4-yl) phenyl) -4-ethoxy-1- (4-fluorophenyl) -2-oxo-1, 2-dihydropyridine-3-carboxamide, N- (4- (3-amino-1-methyl-6- ( (7R, 8aR) -3-oxooctahydroindolizin-7-yl) -1H-indazol-4-yl) phenyl) -4-ethoxy-1- (4-fluorophenyl) -2-oxo-1, 2-dihydropyridine-3-carboxamide, N- (4- (3-amino-1-methyl-6- ( (7S, 8aS) -3-oxooctahydroindolizin-7-yl) -1H-indazol-4-yl) phenyl) -4-ethoxy-1- (4-fluorophenyl) -2-oxo-1, 2-dihydropyridine-3-carboxamide (Compounds 68a, 68b, 68c and 68d)

Step 1. 2-amino-4-bromo-6-fluorobenzonitrile

To a solution of compound 68-1 (50.0 g, 229 mmol, 1.00 eq) in i-PrOH (300 mL) was added NH₃·H₂O (172 g, 1.38 mol, 189 mL, 28.0%purity, 6.00 eq) . The mixture was stirred under 15 Psi at 80 ℃ for 20 hrs in autoclave. TLC (Petroleum ether: Ethyl acetate = 3: 1, Rf (P1) = 0.500) showed there were new spots. The mixture was concentrated under reduced pressure to remove the i-PrOH. Then the mixture was extracted with EtOAc (300 mL *3) . The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue (Petroleum ether: Ethyl acetate = 3: 1, Rf (P1) = 0.500) was purified by column chromatography (SiO₂, Petroleum ether: Ethyl acetate = 100: 30 to 100: 45) . Compound 68-2 (43.2 g, 200 mmol, 86.4%yield) was obtained as a white solid.

H NMR: (400 MHz, DMSO-d₆) δ 6.81 (s, 1H) , 6.78 -6.75 (m, 3H) .

Step 2. 2-amino-6-fluoro-4- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzonitrile

To a solution of compound 68-2 (5.00 g, 23.2 mmol, 1.00 eq) and BPD (11.8 g, 46.5 mmol, 2.00 eq) in dioxane (50.0 mL) was added Pd (dppf) Cl₂ (1.70 g, 2.33 mmol, 0.100 eq) , KOAc (6.85 g, 69.7 mmol, 3.00 eq) at 25 ℃ under N₂. And the mixture was stirred at 100 ℃ for 12 hrs. TLC (PE: EtOAc = 3: 1) showed compound 68-2 was consumed and new spot formed. The mixture was filtered and concentrated to remove solvent. The crude was purified by column (SiO₂, PE: EtOAc = 1: 0 to 10: 1, PE: EtOAc = 3: 1, Rf (P1) = 0.400) . Compound 68-3 (6.00 g, 22.8 mmol, 98.4%yield) was obtained as a white solid.

H NMR: (400 MHz, CDCl₃) δ 6.94 (s, 1H) , 6.86 -6.84 (m, 1H) , 1.34 (s, 12H) .

Step 3. (3-amino-4-cyano-5-fluorophenyl) boronic acid

To a solution of compound 68-3 (6.00 g, 22.8 mmol, 1.00 eq) in DCM (40.0 mL) was added TFA (7.43 g, 65.1 mmol, 4.84 mL, 2.85 eq) and compound 68-4 (6.85 g, 114 mmol, 5.00 eq) , the mixture was stirred at 25 ℃ for 1 hr. LC-MS showed the desired mass was detected. The mixture was filtered, and the cake was concentrated to give a crude product. The crude was purified by column (SiO₂, EtOAc: MeOH =1: 0 to 20: 1) . Compound 68-5 (3.79 g, 21.0 mmol, 92.0%yield) was obtained as a white solid.

H NMR: (400 MHz, DMSO-d₆) δ 7.07 (s, 1H) , 6.75 -6.70 (m, 1H) , 6.34 (m, 2H) .

Step 4. 1- (but-3-en-1-yl) pyrrolidine-2, 5-dione

To a solution of compound 68-14 (50.0 g, 504 mmol, 1.00 eq) in toluene (250 mL) was added compound 68-15 (102 g, 756 mmol, 76.8 mL, 1.50 eq) , K₂CO₃ (76.7 g, 555 mmol, 1.10 eq) and 18-crown-6 (1.33 g, 5.05 mmol, 0.0100 eq) . Then the mixture was stirred at 120 ℃ for 10 hrs under N₂. TLC (Petroleum ether: Ethyl acetate = 1: 1, Rf (P1) = 0.500) showed the starting material was consumed completely and there was new spot. The mixture was filtered. And the cake was dissolved into water (200 mL) , extracted with EtOAc (100 mL *3) . The combined organic layers were poured into filtrate. The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue (Petroleum ether: Ethyl acetate = 1: 1, Rf (P1) = 0.500) was purified by column chromatography (SiO₂, Petroleum ether: Ethyl acetate = 100: 0 to 100: 20) . Compound 68-16 (56.5 g, 368 mmol, 73.1%yield) was obtained as light yellow oil.

H NMR: (400 MHz, CDCl₃) δ 5.74 -5.65 (m, 1H) , 5.05 -4.99 (m, 2H) , 3.58 -3.54 (m, 2H) , 2.67 (s, 4H) , 2.34 -2.29 (m, 2H) .

Step 5. 1- (but-3-en-1-yl) -5-hydroxypyrrolidin-2-one

Compound 68-16 (10.0 g, 65.2 mmol, 1.00 eq) was dissolved in MeOH (100 mL) , and NaBH4 (3.70 g, 97.9 mmol, 1.50 eq) was added portionwise at 10 ℃. Then the mixture was stirred at 20 ℃ for 1 hr. TLC (DCM: MeOH = 10: 1, Rf (P1) = 0.300) showed the starting material was consumed completely and there was new spot. LC-MS showed the desired mass was detected. The reaction mixture was adjusted pH to 7 by sat. aq. NH₄Cl (200 mL) . Then the mixture was concentrated to remove MeOH (100 mL) . And the mixture was extracted with DCM (100 mL *3) . The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue (DCM: MeOH = 10: 1, Rf (P1) = 0.300) was purified by column chromatography (SiO₂, DCM: MeOH = 100: 0 to 100: 3) . Compound 68-17 (5.00 g, 32.2 mmol, 49.3%yield) was obtained as light-yellow oil.

H NMR: (400 MHz, CDCl₃) δ 5.81 -5.74 (m, 1H) , 5.22 -5.21 (m, 1H) , 5.11 -5.04 (m, 2H) , 3.71 (s, 1H) , 3.59 -3.52 (m, 1H) , 3.29 -3.24 (m, 1H) , 2.55 -2.51 (m, 1H) , 2.39 -2.25 (m, 4H) , 1.93 -1.85 (m, 1H) .

Step 6. 3-oxooctahydroindolizin-7-yl formate

A solution of compound 68-17 (5.00 g, 32.2 mmol, 1.00 eq) in HCOOH (50.0 mL) was stirred at 20 ℃ for 16 hrs. LC-MS (EW33258-707-P1A1) showed the desired mass was detected. TLC (DCM: MeOH = 10: 1, Rf (P1) = 0.600) showed the starting material was consumed completely and there were new spots. The mixture was concentrated under reduced pressure to give a residue. Compound 68-18 (8.48 g, crude) was obtained as yellow oil.

H NMR: (400 MHz, CDCl₃) δ 8.01 (s, 1H) , 5.02 -4.97 (m, 1H) , 4.21 -4.16 (m, 1H) , 3.64 -3.62 (m, 1H) , 2.77 -2.76 (m, 1H) , 2.50 -2.45 (m, 2H) , 2.28 -2.26 (m, 2H) , 2.12 -2.02 (m, 1H) , 1.73 -1.63 (m, 1H) , 1.55 -1.45 (m, 1H) , 1.38 -1.29 (m, 1H) .

Step 7. 7-hydroxyhexahydroindolizin-3 (2H) -one

To a solution of compound 68-18 (8.48 g, 46.2 mmol, 1.00 eq) in MeOH (40.0 mL) was added LiOH·H₂O (3.88 g, 92.5 mmol, 2.00 eq) in H₂O (12.0 mL) at 0 ℃. Then the mixture was stirred at 20 ℃ for 2 hrs. LC-MS showed the desired mass was detected. TLC (DCM: MeOH = 10: 1, Rf (P1) = 0.300) showed the starting material was consumed completely and there were new spots. The reaction mixture was quenched with 1.00 M HCl to pH = 7. Then the reaction solution was concentrated under reduced pressure at 50 ℃ to give a residue. Then the residue was added DCM: MeOH = 1: 1 (200 mL) and stirred at 20 ℃ for 1 hr. The mixture was filtered and filtrate was concentrated under reduced pressure. The residue (DCM: MeOH = 10: 1, Rf (P1) = 0.300) was purified by column chromatography (SiO₂, DCM: MeOH = 100: 0 to 100: 10) . Compound 68-19 (4.18 g, 26.9 mmol, 58.1%yield) was obtained as a light yellow solid.

H NMR: (400 MHz, CDCl₃) δ 4.18 -4.13 (m, 1H) , 3.80 -3.76 (m, 1H) , 3.52 -3.49 (m, 1H) , 2.70 -2.63 (m, 1H) , 2.41 -2.37 (m, 2H) , 2.28 -2.20 (m, 2H) , 1.99 -1.96 (m, 1H) , 1.70 -1.62 (m, 1H) , 1.41 -1.29 (m, 1H) , 1.22 -1.14 (m, 1H) .

Step 8. hexahydroindolizine-3, 7-dione

To a solution of compound 68-19 (3.64 g, 23.4 mmol, 1.00 eq) in DCM (40.0 mL) was added DMP (19.9 g, 46.9 mmol, 14.5 mL, 2.00 eq) at 0 ℃. Then the mixture was stirred at 20 ℃ for 3 hrs. LC-MS showed the desired mass was detected. The mixture was filtered and filtrate was concentrated under reduced pressure to give a residue. The residue (DCM: MeOH = 10: 1, Rf (P1) = 0.400) was purified by column chromatography (SiO₂, DCM: MeOH = 100: 0 to 100: 10) . Compound 68-20 (4.05 g, crude) was obtained as a white solid.

H NMR: (400 MHz, CDCl₃) δ 4.43 -4.38 (m, 1H) , 3.85 -3.79 (m, 1H) , 3.02 -2.95 (m, 1H) , 2.62 -2.58 (m, 1H) , 2.51 -2.24 (m, 6H) , 1.79 -1.70 (m, 1H) .

Step 9. (Z) -4-methyl-N'- (3-oxohexahydroindolizin-7 (1H) -ylidene) benzenesulfonohydrazide

To a solution of compound 68-20 (4.05 g, 26.4 mmol, 1.00 eq) , TosNHNH₂ (5.91 g, 31.7 mmol, 1.20 eq) in EtOH (4.00 mL) , the mixture was stirred at 80 ℃ for 3.5 hrs. LC-MS showed the desired mass was detected. The mixture was filtered and concentrated to remove solvent. The crude was purified by column (SiO₂, DCM: MeOH = 1: 0 to 20: 1, DCM: MeOH = 10: 1, Rf (P1) = 0.500) . Compound 68-6 (8.00 g, 24.8 mmol, 94.1%yield) was obtained as a white solid.

H NMR: (400 MHz, DMSO-d₆) δ 10.36 (s, 1H) , 7.73 -7.71 (m, 2H) , 7.40 -7.38 (m, 2H) , 4.00 -3.97 (m, 1H) , 3.48 -3.47 (m, 1H) , 2.79 -2.78 (m, 1H) , 2.63 - 2.59 (m, 1H) , 2.38 (s, 3H) , 2.25 -2.06 (m, 5H) , 1.89 -1.87 (m, 1H) , 1.56 -1.53 (m, 1H) .

Step 10. 2-amino-6-fluoro-4- (3-oxooctahydroindolizin-7-yl) benzonitrile

To a solution of compound 68-5 (2.10 g, 11.6 mmol, 1.00 eq) and compound 68-6 (4.50 g, 14.0 mmol, 1.20 eq) in dioxane (20.0 mL) was added Cs₂CO₃ (5.70 g, 17.5 mmol, 1.50 eq) . The mixture was stirred at 110 ℃ for 6 hrs. LC-MS showed desired mass detected. The reaction mixture was diluted with H₂O (30.0 mL) and extracted with EtOAc (50.0 mL *4) . The combined organic layers were washed with brine (100 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether: Ethyl acetate = 100: 10 to 0: 100) . Compound 68-7 (1.00 g, 3.66 mmol, 31.3%yield) was obtained as yellow gum.

Step 11. 2-fluoro-6-iodo-4- (3-oxooctahydroindolizin-7-yl) benzonitrile

To a solution of compound 68-7 (1.00 g, 3.66 mmol, 1.00 eq) and HCl (4.00 M, 15.0 mL, 16.4 eq) in MeCN (10.0 mL) was added NaNO₂ (378 mg, 5.49 mmol, 1.50 eq) in H₂O (2.00 mL) at 0 ℃. The mixture was stirred at 0 ℃ for 0.5 hr. KI (1.52 g, 9.15 mmol, 2.50 eq) in H₂O (2.00 mL) was added to the reaction mixture at 0 ℃ and stirred at 25 ℃ for 1 hr. LC-MS showed desired mass detected. The reaction mixture was diluted with H₂O (20.0 mL) and extracted with EtOAc (40.0 mL *2) . The combined organic layers were washed with sat. aq Na₂SO₃ (40.0 mL *2) , then dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiO₂, DCM: MeOH = 10: 1) . Compound 68-8 (0.500 g, 1.30 mmol, 35.5%yield) was obtained as a white solid.

Step 12. 7- (3-amino-4-iodo-1-methyl-1H-indazol-6-yl) hexahydroindolizin-3 (2H) -one

To a solution of compound 68-8 (500 mg, 1.30 mmol, 1.00 eq) in n-BuOH (10.0 mL) was added compound 68-9 (1.24 g, 10.7 mmol, 1.42 mL, 40.0 purity, 8.27 eq) . The mixture was stirred at 110 ℃ for 5 hrs. LC-MS showed desired mass detected. The reaction mixture was concentrated to give a residue. The residue was purified by prep-TLC (SiO₂, DCM: MeOH = 10: 1) . Compound 68-10 (300 mg, 731 μmol, 56.1%yield) was obtained as a yellow solid.

Step 13. 7- (3-amino-4- (4-aminophenyl) -1-methyl-1H-indazol-6-yl) hexahydroindolizin-3 (2H) -one

Compound 68-10 (300 mg, 731 μmol, 1.00 eq) , compound 68-11 (208 mg, 950 μmol, 1.30 eq) and Pd (t-Bu₃P) ₂ (37.3 mg, 73.1 μmol, 0.100 eq) , DIEA (472 mg, 3.66 mmol, 636 μL, 5.00 eq) were taken up into a microwave tube in H₂O (1.00 mL) and dioxane (5.00 mL) . The sealed tube was heated at 120 ℃ for 30 min under microwave. LC-MS showed desired mass was detected. The reaction mixture was concentrated to give a residue. The residue was purified by prep-TLC (SiO₂, DCM: MeOH = 10: 1) . The residue was purified by prep-HPLC (basic condition, column: Waters Xbridge 150 *25.0 mm *5.00 um; mobile phase: [water (ammonia hydroxide v/v) -ACN] ; gradient: 16.0%-46.0%B over 9 min) . Compound 68-12 (160 mg, 426 μmol, 58.2%yield) was obtained as a white solid.

Step 14. (7R, 8aS) -7- (3-amino-4- (4-aminophenyl) -1-methyl-1H-indazol-6-yl) hexahydroindolizin-3 (2H) -one, (7S, 8aR) -7- (3-amino-4- (4-aminophenyl) -1-methyl-1H-indazol-6-yl) hexahydroindolizin-3 (2H) -one, (7R, 8aR) -7- (3-amino-4- (4-aminophenyl) -1-methyl-1H-indazol-6-yl) hexahydroindolizin-3 (2H) -one and (7S, 8aS) -7- (3-amino-4- (4-aminophenyl) -1-methyl-1H-indazol-6-yl) hexahydroindolizin-3 (2H) -one

Compound 68-12 (160 mg) was separated by SFC (column: DAICEL CHIRALPAK AD (250 mm *30.0 mm, 10.0 um) ; mobile phase: [CO₂-EtOH (0.100%NH₃·H₂O) ] ; 60.0%B isocratic elution mode) to give Compounds 68-12a, 68-12b, 68-12c and 68-12d.

Compound 68-12a was obtained as peak 1 (Rt = 1.575 min, 16.0 mg, 42.6 μmol, 10.0%yield) .

Compound 68-12b was obtained as peak 2 (Rt = 1.978 min, 24.0 mg, 63.9 μmol, 15.0%yield) .

Compound 68-12c was obtained as peak 3 (Rt = 2.203 min, 50.0 mg, 133 μmol, 31.2%yield) .

Compound 68-12d was obtained as peak 4 (Rt = 2.642 min, 50.0 mg, 133 μmol, 31.2%yield) .

Step 18. N- (4- (3-amino-1-methyl-6- ( (7R, 8aS) -3-oxooctahydroindolizin-7-yl) -1H-indazol-4-yl) phenyl) -4-ethoxy-1- (4-fluorophenyl) -2-oxo-1, 2-dihydropyridine-3-carboxamide, N- (4- (3-amino-1-methyl-6- ( (7S, 8aR) -3-oxooctahydroindolizin-7-yl) -1H-indazol-4-yl) phenyl) -4-ethoxy-1- (4-fluorophenyl) -2-oxo-1, 2-dihydropyridine-3-carboxamide, N- (4- (3-amino-1-methyl-6- ( (7R, 8aR) -3-oxooctahydroindolizin-7-yl) -1H-indazol-4-yl) phenyl) -4-ethoxy-1- (4-fluorophenyl) -2-oxo-1, 2-dihydropyridine-3-carboxamide, N- (4- (3-amino-1-methyl-6- ( (7S, 8aS) -3-oxooctahydroindolizin-7-yl) -1H-indazol-4-yl) phenyl) -4-ethoxy-1- (4-fluorophenyl) -2-oxo-1, 2-dihydropyridine-3-carboxamide

To a solution of 68-12a (16.0 mg, 42.6 μmol, 1.00 eq) in pyridine (2.00 mL) was added compound 68-13 (8.27 mg, 29.8 μmol, 0.700 eq) , EDCI (32.6 mg, 170 μmol, 4.00 eq) . Then the mixture was stirred at 20 ℃ for 0.5 hr. LC-MS showed the desired mass was detected. The reaction mixture was quenched by water (5.00 mL) , and then extracted with DCM (5.00 mL *3) . The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The crude product was purified by prep-TLC (DCM: MeOH = 10: 1, Rf (P1) = 0.500) . Compound 68a (9.83 mg, 15.3 μmol, 36.0%yield, 99.2%purity) was obtained.

H NMR: (400 MHz, CDCl₃) δ 11.41 (s, 1H) , 7.83 -7.81 (m, 2H) , 7.51 (d, J =8.0 Hz, 1H) , 7.46 -7.44 (m, 2H) , 7.40 -7.36 (m, 2H) , 7.25 -7.23 (m, 2H) , 7.07 (s, 1H) , 6.85 (s, 1H) , 6.37 (d, J = 8.0 Hz, 1H) , 4.40 -4.35 (m, 2H) , 4.07 -4.03 (m, 1H) , 3.86 (s, 3H) , 3.82 (s, 2H) , 3.74 -3.69 (m, 1H) , 3.41 (s, 1H) , 3.10 -3.04 (m, 1H) , 2.49 -2.46 (m, 1H) , 2.41 -2.31 (m, 3H) , 2.25 -2.20 (m, 1H) , 2.04 -1.96 (m, 1H) , 1.82 - 1.75 (m, 1H) , 1.67 -1.62 (m, 1H) , 1.59 -1.58 (m, 3H) .

LC-MS: (M+H) ⁺: 635.4

Compounds 68b, 68c and 68d were prepared in a similar way as Compound 68a except that compounds 68-12b, 68-12c and 68-12d were used instead of compound 68-12a.

Compound 68b

H NMR: (400 MHz, CDCl₃) δ 11.41 (s, 1H) , 7.83 -7.81 (m, 2H) , 7.51 (d, J =7.6 Hz, 1H) , 7.46 -7.44 (m, 2H) , 7.40 -7.37 (m, 2H) , 7.25 -7.23 (m, 2H) , 7.07 (s, 1H) , 6.85 (s, 1H) , 6.37 (d, J = 8.0 Hz, 1H) , 4.40 -4.35 (m, 2H) , 4.07 -4.02 (m, 1H) , 3.86 (s, 3H) , 3.82 (s, 2H) , 3.74 -3.69 (m, 1H) , 3.41 (s, 1H) , 3.11 -3.04 (m, 1H) , 2.49 -2.46 (m, 1H) , 2.41 -2.33 (m, 3H) , 2.27 -2.21 (m, 1H) , 2.03 -1.96 (m, 1H) , 1.83 - 1.75 (m, 1H) , 1.67 -1.64 (m, 1H) , 1.59 -1.58 (m, 3H) .

LC-MS: (M+H) ⁺: 635.4

Compound 68c

H NMR: (400 MHz, CDCl₃) δ 11.41 (s, 1H) , 7.83 -7.81 (m, 2H) , 7.51 (d, J =8.0 Hz, 1H) , 7.44 -7.42 (m, 2H) , 7.40 -7.36 (m, 2H) , 7.25 -7.23 (m, 2H) , 6.94 (s, 1H) , 6.75 (s, 1H) , 6.37 (d, J = 7.6 Hz, 1H) , 4.40 -4.34 (m, 2H) , 4.32 -4.28 (m, 1H) , 3.85 (s, 3H) , 3.65 -3.62 (m, 1H) , 2.92 -2.80 (m, 1H) , 2.46 -2.42 (m, 2H) , 2.32 -2.24 (m, 1H) , 2.19 -2.16 (m, 1H) , 2.01 -1.98 (m, 1H) , 1.72 -1.66 (m, 4H) , 1.61 -1.58 (m, 3H) .

LC-MS: (M+H) ⁺: 635.4

Compound 68d

H NMR: (400 MHz, CDCl₃) δ 11.41 (s, 1H) , 7.83 -7.81 (m, 2H) , 7.51 (d, J =8.0 Hz, 1H) , 7.44 -7.42 (m, 2H) , 7.40 -7.36 (m, 2H) , 7.25 -7.23 (m, 2H) , 6.94 (s, 1H) , 6.75 (s, 1H) , 6.37 (d, J = 7.6 Hz, 1H) , 4.40 -4.35 (m, 2H) , 4.32 -4.27 (m, 1H) , 3.85 (s, 3H) , 3.67 -3.61 (m, 1H) , 2.92 -2.83 (m, 1H) , 2.46 -2.42 (m, 2H) , 2.32 -2.26 (m, 1H) , 2.19 -2.16 (m, 1H) , 2.01 -1.98 (m, 1H) , 1.72 -1.67 (m, 4H) , 1.61 -1.58 (m, 3H) .

LC-MS: (M+H) ⁺: 635.4

Example 33

N- (4- (3-amino-1-methyl-6- ( (7R, 8aR) -3-oxooctahydroindolizin-7-yl) -1H-indazol-4-yl) phenyl) -4-ethoxy-5'-fluoro-2-oxo-2H- [1, 2'-bipyridine] -3-carboxamide (Compound 69)

To a solution of compound 69-1 (20.0 mg, 53.2 μmol, 1.00 eq) in pyridine (2.00 mL) was added compound 69-2 (13.3 mg, 47.9 μmol, 0.900 eq) and EDCI (40.8 mg, 213 μmol, 4.00 eq) . Then the mixture was stirred at 20 ℃ for 1 hr. LC-MS showed the desired mass was detected. The reaction mixture was quenched by water (10.0 mL) , and then extracted with DCM (30.0 mL *3) . The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The crude product was purified by prep-TLC (DCM: MeOH = 10: 1, Rf (P1) = 0.400) and further purified by prep-HPLC (ammonia hydroxide condition; column: Waters Xbridge 150 *25.0 mm *5.00 um; mobile phase: [water (ammonia hydroxide v/v) -ACN] ; gradient: 22.0%-52.0%B over 9 min) . Then the mixture was concentrated under reduced pressure to remove MeCN. And the mixture was freezedried to give a product. Compound 69 (13.9 mg, 21.8 μmol, 41.0%yield, 98.9%purity) was obtained.

H NMR: (400 MHz, CDCl₃) δ 11.06 (s, 1H) , 8.44 (d, J = 2.8 Hz, 1H) , 7.96 (d, J = 8.0 Hz, 1H) , 7.92 -7.88 (m, 1H) , 7.84 -7.81 (m, 2H) , 7.66 -7.61 (m, 1H) , 7.46 -7.44 (m, 2H) , 6.95 (s, 1H) , 6.76 (s, 1H) , 6.42 (d, J = 8.0 Hz, 1H) , 4.41 -4.35 (m, 2H) , 4.32 -4.28 (m, 1H) , 3.85 (s, 3H) , 3.65 -3.63 (m, 1H) , 2.89 -2.83 (m, 1H) , 2.46 -2.42 (m, 2H) , 2.33 -2.26 (m, 1H) , 2.20 -2.16 (m, 1H) , 2.01 -1.98 (m, 1H) , 1.74 -1.69 (m, 4H) , 1.60 -1.56 (m, 3H) .

LC-MS: (M+H) ⁺: 636.5

Example 34

(R) -N- (4- (3-amino-1-methyl-6- (6-oxohexahydropyrrolo [1, 2-a] pyrazin-2 (1H) -yl) -1H-indazol-4-yl) phenyl) -4-ethoxy-1- (4-fluorophenyl) -2-oxo-1, 2-dihydropyridine-3-carboxamide and (S) -N- (4- (3-amino-1-methyl-6- (6-oxohexahydropyrrolo [1, 2-a] pyrazin-2 (1H) -yl) -1H-indazol-4-yl) phenyl) -4-ethoxy-1- (4-fluorophenyl) -2-oxo-1, 2-dihydropyridine-3-carboxamide (Compounds 70a and 70b)

Step 1. (R) -2- (3-amino-4- (4-aminophenyl) -1-methyl-1H-indazol-6-yl) hexahydropyrrolo [1, 2-a] pyrazin-6 (2H) -one and (S) -2- (3-amino-4- (4-aminophenyl) -1-methyl-1H-indazol-6-yl) hexahydropyrrolo [1, 2-a] pyrazin-6 (2H) -one

To a solution of compound 70-1 (200 mg, 630 μmol, 1.00 eq) , compound 70-2 (441 mg, 3.15 mmol, 5.00 eq) in dioxane (2.00 mL) was added Cs₂CO₃ (410 mg, 1.26 mmol, 2.00 eq) and PEPPSI-iPent (61.3 mg, 63.0 μmol, 0.100 eq) , the mixture was stirred at 80 ℃ for 12 h. LC-MS showed the desired mass was detected. The mixture was filtered and filtrate was concentrated to remove solvent. The crude was purified by prep-TLC (DCM: MeOH = 10: 1, Rf = 0.400) . The product (SFC: EW38424-143-P1A_D13) was separated by SFC (column: DAICEL CHIRALPAK AD (250 mm *30.0 mm, 10.0 um) ; mobile phase: [CO₂ -ACN/i-PrOH (0.100%NH₃·H₂O) ] ; B%: 60.0%, isocratic elution mode) . Compound 70-3a (20.0 mg, 53.1 μmol, 8.43%yield) (SFC, peak 1: Rt = 1.73 min) was obtained as a white solid. Compound 70-3b (20.0 mg, 53.1 μmol, 8.43%yield) (SFC, peak 2: Rt = 2.08 min) was obtained as a white solid.

Step 2. (R) -N- (4- (3-amino-1-methyl-6- (6-oxohexahydropyrrolo [1, 2-a] pyrazin-2 (1H) -yl) -1H-indazol-4-yl) phenyl) -4-ethoxy-1- (4-fluorophenyl) -2-oxo-1, 2-dihydropyridine-3-carboxamide and (S) -N- (4- (3-amino-1-methyl-6- (6-oxohexahydropyrrolo [1, 2-a] pyrazin-2 (1H) -yl) -1H-indazol-4-yl) phenyl) -4-ethoxy-1- (4-fluorophenyl) -2-oxo-1, 2-dihydropyridine-3-carboxamide

To a solution of compound 70-3a (15.0 mg, 39.8 μmol, 1.00 eq) , compound 70-4 (11.0 mg, 39.8 μmol, 1.00 eq) in Py (1.00 mL) was added EDCI (15.2 mg, 79.6 μmol, 2.00 eq) , the mixture was stirred at 25 ℃ for 1 h. LC-MS showed the desired mass was detected. The mixture was diluted with water (10.0 mL) and NaHCO₃ (10.0 mL) , extracted with DCM (10.0 mL *3) , the combined organic layer was washed with brine (20.0 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The crude was purified by prep-TLC (DCM: MeOH = 10: 1, Rf = 0.400) . The crude was purified by prep-HPLC (column: Waters Xbridge 150 *25.0 mm *5.00 um; mobile phase: [water (ammoniahydroxide v/v) -ACN] ; gradient: 23.0%-53.0%B over 9 min) . Compound 70a (10.6 mg, 16.7 μmol, 100%yield, 99.8%purity) was obtained.

H NMR: (400 MHz, CDCl₃) δ 11.3 (s, 1H) , 7.80 (d, J = 8.4 Hz, 2H) , 7.50 (d, J = 8.0 Hz, 1H) , 7.43 (d, J = 8.4 Hz, 2H) , 7.39 -7.36 (m, 2H) , 7.24 -7.22 (m, 2H) , 6.61 -6.60 (m, 1H) , 6.48 -6.47 (m, 1H) , 6.36 (d, J = 8.0 Hz, 1H) , 4.39 -4.34 (m, 2H) , 4.17 -4.13 (m, 1H) , 3.86 -3.81 (m, 2H) , 3.79 (s, 3H) , 3.72 -3.70 (m, 1H) , 3.10 -3.04 (m, 1H) , 2.82 -2.75 (m, 1H) , 2.54 (t, J = 11.2 Hz, 1H) , 2.48 -2.44 (m, 2H) , 2.31 - 2.22 (m, 1H) , 1.75 -1.69 (m, 1H) , 1.59 (t, J = 7.2 Hz, 3H) .

LC-MS: (M+H) ⁺: 636.3

Compound 70b was prepared in similar way as Compound 70a except that compound 70-3b were used instead of compound 70-3a.

H NMR: (400 MHz, CDCl₃) δ 11.3 (s, 1H) , 7.80 (d, J = 8.4 Hz, 2H) , 7.50 (d, J = 8.0 Hz, 1H) , 7.43 (d, J = 8.4 Hz, 2H) , 7.39 -7.36 (m, 2H) , 7.24 -7.22 (m, 2H) , 6.61 -6.60 (m, 1H) , 6.48 -6.47 (m, 1H) , 6.36 (d, J = 8.0 Hz, 1H) , 4.39 -4.34 (m, 2H) , 4.17 -4.13 (m, 1H) , 3.86 -3.81 (m, 2H) , 3.79 (s, 3H) , 3.72 -3.70 (m, 1H) , 3.10 -3.04 (m, 1H) , 2.82 -2.75 (m, 1H) , 2.54 (t, J = 11.2 Hz, 1H) , 2.48 -2.44 (m, 2H) , 2.29 - 2.24 (m, 1H) , 1.75 -1.71 (m, 1H) , 1.59 (t, J = 7.2 Hz, 3H) .

LC-MS: (M+H) ⁺: 636.3

(R) -N- (4- (3-amino-1-methyl-6- (6-oxohexahydropyrrolo [1, 2-a] pyrazin-2 (1H) -yl) -1H-indazol-4-yl) phenyl) -4-ethoxy-5'-fluoro-2-oxo-2H- [1, 2'-bipyridine] -3- carboxamide and (S) -N- (4- (3-amino-1-methyl-6- (6-oxohexahydropyrrolo [1, 2-a] pyrazin-2 (1H) -yl) -1H-indazol-4-yl) phenyl) -4-ethoxy-5'-fluoro-2-oxo-2H- [1, 2'-bipyridine] -3-carboxamide (Compounds 79a and 79b)

Compounds 79a and 79b were prepared in similar way as Compounds 70a and 70b except that compound 67-2 was used instead of compound 70-4.

Compounds 79a

H NMR: (400 MHz, CDCl₃) δ 11.1 (s, 1H) , 8.44 (d, J = 2.8 Hz, 1H) , 7.96 (d, J = 8.0 Hz, 1H) , 7.91 -7.88 (m, 1H) , 7.82 (d, J = 8.0 Hz, 2H) , 7.65 -7.61 (m, 1H) , 7.44 (d, J = 8.4 Hz, 2H) , 6.63 -6.61 (m, 1H) , 6.47 -6.41 (m, 2H) , 4.41 -4.35 (m, 2H) , 4.19 -4.15 (m, 1H) , 3.89 -3.86 (m, 1H) , 3.80 (s, 3H) , 3.76 -3.72 (m, 1H) , 3.12 -3.06 (m, 1H) , 2.85 -2.77 (m, 1H) , 2.57 (t, J = 11.6 Hz, 1H) , 2.49 -2.45 (m, 2H) , 2.30 -2.25 (m, 1H) , 1.76 -1.70 (m, 2H) , 1.58 (t, J = 7.2 Hz, 3H) .

LC-MS: (M+H) ⁺: 637.3

Compound 79b

H NMR: (400 MHz, CDCl₃) δ (400 MHz, CDCl3) δ 11.1 (s, 1H) , 8.44 (d, J =2.8 Hz, 1H) , 7.96 (d, J = 8.0 Hz, 1H) , 7.91 -7.88 (m, 1H) , 7.82 (d, J = 8.0 Hz, 2H) , 7.65 -7.60 (m, 1H) , 7.43 (d, J = 8.4 Hz, 2H) , 6.64 -6.63 (m, 1H) , 6.47 -6.41 (m, 2H) , 4.41 -4.35 (m, 2H) , 4.18 -4.14 (m, 1H) , 3.89 -3.86 (m, 1H) , 3.80 (s, 3H) , 3.76 -3.73 (m, 1H) , 3.11 -3.04 (m, 1H) , 2.86 -2.79 (m, 1H) , 2.58 (t, J = 11.6 Hz, 1H) , 2.49 - 2.45 (m, 2H) , 2.32 -2.23 (m, 1H) , 1.76 -1.66 (m, 2H) , 1.58 (t, J = 7.2 Hz, 3H) .

LC-MS: (M+H) ⁺: 637.3

Example 35

N- (4- (3-amino-1-methyl-6- (3-oxooctahydroindolizin-7-yl) -1H-pyrazolo [3, 4-b] pyridin-4-yl) phenyl) -4-ethoxy-1- (4-fluorophenyl) -2-oxo-1, 2-dihydropyridine-3-carboxamide (Compound 71)

Step 1. methyl (E) -2- (3-ethoxy-3-oxoprop-1-en-1-yl) isonicotinate

To a solution of methyl compound 71-1 (5.00 g, 23.1 mmol, 1.00 eq) and compound 71-2 (5.76 g, 25.4 mmol, 1.10 eq) K₂CO₃ (9.60 g, 69.4 mmol, 3.00 eq) in dioxane (40.0 mL) and H₂O (10.0 mL) was added Pd (PPh₃) ₄ (2.67 g, 2.31 mmol, 0.100 eq) . The mixture was stirred at 90 ℃ for 3 hrs. TLC indicated compound 71-1 was consumed completely and many new spots formed. The reaction was messy according to TLC (Petroleum ether: Ethyl acetate = 5: 1) . The reaction mixture was filtered. The filtrate was concentrated to give a residue. The residue was purified by column chromatography (SiO₂, Petroleum ether: Ethyl acetate = 100: 1 to 10: 1) . Compound 71-3 (4.00 g, 17.0 mmol, 73.4%yield) was obtained as a yellow solid.

H NMR: (400 MHz, CDCl₃) δ 8.80 (d, J = 4.8 Hz, 1H) , 7.98 (s, 1H) , 7.82 -7.72 (m, 2H) , 6.98 (d, J = 16.0 Hz, 1H) , 4.32 -4.27 (m, 2H) , 3.98 (s, 3H) , 1.37 -1.33 (m, 3H) .

Step 2. methyl 2- (3-ethoxy-3-oxopropyl) piperidine-4-carboxylate

To a solution of compound 71-3 (4.00 g, 17.0 mmol, 1.00 eq) in HOAc (30.0 mL) was added Pd (OH) ₂ (1.00 g, 1.42 mmol, 20.0%purity, 8.38e-2 eq) under N₂. The suspension was degassed under vacuum and purged with H₂ several times. The mixture was stirred under H₂ (50 Psi) at 40 ℃ for 6 hrs. LC-MS showed desired mass detected. The reaction mixture was filtered and the cake was washed with 100 mL MeOH, then concentrated under reduced pressure to give a residue. Compound 71-4 (3.50 g, 14.3 mmol, 84.6%yield) was obtained as colorless oil.

H NMR: (400 MHz, DMSO-d₆) δ 4.07 -4.02 (m, 2H) , 3.59 (s, 3H) , 3.08 -3.05 (m, 1H) , 2.62 -2.58 (m, 2H) , 2.39 -2.35 (m, 3H) , 1.81 -1.78 (m, 2H) , 1.67 - 1.63 (m, 2H) , 1.46 -1.40 (m, 1H) , 1.19 -1.14 (m, 4H) .

Step 3. methyl 3-oxooctahydroindolizine-7-carboxylate

To a solution of compound 71-4 (3.50 g, 14.3 mmol, 1.00 eq) in MeOH (50.0 mL) was added TEA (2.33 g, 23.0 mmol, 3.20 mL, 1.60 eq) . The mixture was stirred at 65 ℃ for 12 hrs. LC-MS showed desired mass detected. The reaction mixture was concentrated to give a residue. The residue (DCM: MeOH = 10: 1) was purified by column chromatography (SiO₂, DCM: MeOH = 100: 1 to 50: 1) . Compound 71-5 (2.80 g, 14.2 mmol, 98.6%yield) was obtained as colorless oil.

H NMR: (400 MHz, CDCl₃) δ 4.22 -4.17 (m, 1H) , 3.69 (s, 3H) , 3.50 -3.45 (m, 1H) , 2.68 -2.66 (m, 1H) , 2.50 -2.48 (m, 1H) , 2.42 -2.37 (m, 2H) , 2.25 -2.15 (m, 2H) , 2.04 -1.98 (m, 1H) , 1.50 -1.47 (m, 1H) , 1.35 -1.32 (m, 1H) , 1.30 -1.27 (m, 1H) .

Step 4. 3-oxooctahydroindolizine-7-carboxylic acid

To a solution of compound 71-5 (2.80 g, 14.2 mmol, 1.00 eq) in MeOH (20.0 mL) was added NaOMe (5.11 g, 28.3 mmol, 30.0%purity, 2.00 eq) . The mixture was stirred at 50 ℃ for 2 hrs. LC-MS showed there was desired mass. The reaction mixture was concentrated to give a residue. The residue was diluted with H₂O 10.0 mL and added 1.00 M HCl to adjusted pH to 2. The mixture was extracted with EtOAc 500 mL (50.0 mL*10) . The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue (DCM: MeOH = 10: 1) was purified by column chromatography (SiO₂, DCM: MeOH =100: 1 to 10: 1) . Compound 71-6 (1.60 g, 8.73 mmol, 61.5%yield) was obtained as a white solid.

Step 5. N-methoxy-N-methyl-3-oxooctahydroindolizine-7-carboxamide

To a solution of compound 71-6 (1.00 g, 5.46 mmol, 1.00 eq) and compound 71-7 (798 mg, 8.19 mmol, 1.50 eq) in DCM (15.0 mL) was added HATU (3.11 g, 8.19 mmol, 1.50 eq) and DIEA (2.12 g, 16.3 mmol, 2.85 mL, 3.00 eq) . The mixture was stirred at 25 ℃ for 2 hrs. TLC (DCM: MeOH = 10: 1, Rf (R1) = 0.400) showed the starting material was consumed completely. The reaction mixture was quenched by sat. aq. NaHCO₃ (20.0 mL) , and then extracted with DCM (30.0 mL *2) . The combined organic layers were washed by sat. aq. NaHCO₃ (20.0 mL) and water (20.0 mL) . The organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. Compound 71-8 (800 mg, 3.54 mmol, 64.7%yield) was obtained as yellow oil.

H NMR: (400 MHz, CDCl₃) δ 4.19 -4.15 (m, 1H) , 3.69 (s, 3H) , 3.55 -3.51 (m, 1H) , 3.19 (s, 3H) , 2.89 -2.82 (m, 2H) , 2.76 -2.69 (m, 1H) , 2.40 -2.34 (m, 2H) , 2.27 -2.22 (m, 1H) , 1.99 -1.95 (m, 1H) , 1.83 -1.80 (m, 1H) , 1.67 -1.45 (m, 2H) .

Step 6. 7-acetylhexahydroindolizin-3 (2H) -one

To a solution of compound 71-8 (800 mg, 3.54 mmol, 1.00 eq) in THF (10.0 mL) was added MeMgBr (3.00 M, 2.95 mL, 2.50 eq) at 0 ℃. The mixture was stirred at 25 ℃ for 2 hrs under N₂. TLC (Petroleum ether: Ethyl acetate = 3: 1, Rf (P1) = 0.800) showed there were new spots. TLC (DCM: MeOH = 10: 1, Rf (R1) =0.600) showed the starting material was consumed completely and there were new spots. The reaction mixture was quenched by sat. aq. NH₄Cl (50.0 mL) , and then extracted with EtOAc (50.0 mL *3) . The combined organic layers were washed by brine (50.0 mL) . The organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. Compound 71-9 (840 mg, crude) was obtained as yellow oil.

H NMR: (400 MHz, CDCl₃) δ 4.25 -4.20 (m, 1H) , 3.75 -3.73 (m, 1H) , 3.51 -3.46 (m, 1H) , 3.21 -3.18 (m, 1H) , 2.55 -2.50 (m, 1H) , 2.42 -2.39 (m, 1H) , 2.38 - 2.36 (m, 1H) , 2.31 -2.26 (m, 1H) , 2.19 (s, 3H) , 2.13 -2.11 (m, 1H) , 1.99 -1.96 (m, 1H) , 1.70 -1.63 (m, 2H) .

Step 7. 2-hydroxy-4- (4-nitrophenyl) -6- (3-oxooctahydroindolizin-7-yl) nicotinonitrile

To a solution of compound 71-9 (533 mg, 3.53 mmol, 1.00 eq) , compound 71-10 (533 mg, 3.53 mmol, 1.00 eq) , compound 71-11 (349 mg, 3.53 mmol, 311 μL, 1.00 eq) in EtOH (10.0 mL) was added NH₄OAc (1.63 g, 21.1 mmol, 6.00 eq) . The mixture was stirred at 75 ℃ for 6 hrs. LC-MS showed the desired mass was detected. The mixture was concentrated under reduced pressure to give a residue. The residue (DCM: MeOH = 10: 1, Rf (P1) = 0.400) was purified by column chromatography (SiO₂, DCM: MeOH = 100: 0 to 100: 10) . Compound 71-12 (220 mg, 581 μmol, 16.4%yield) was obtained as a brown solid.

Step 8. 3-cyano-4- (4-nitrophenyl) -6- (3-oxooctahydroindolizin-7-yl) pyridin-2-yl trifluoromethanesulfonate

To a solution of compound 71-12 (200 mg, 528 μmol, 1.00 eq) in DCM (5.00 mL) was added pyridine (41.8 mg, 528 μmol, 42.6 μL, 1.00 eq) and Tf2O (223 mg, 792 μmol, 130 μL, 1.50 eq) at 0 ℃. Then the mixture was stirred at 25 ℃ for 2 hrs. LC-MS showed the desired mass was detected. The reaction mixture was quenched by water (10.0 mL) , and then extracted with DCM (10.0 mL *2) . The combined organic layers were washed by sat. aq. NH₄Cl (10.0 mL *2) and water (10.0 mL) . The organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. Compound 71-13 (220 mg, 431 μmol, 81.5%yield) was obtained as a brown solid.

Step 9. 7- (3-amino-1-methyl-4- (4-nitrophenyl) -1H-pyrazolo [3, 4-b] pyridin-6-yl) hexahydroindolizin-3 (2H) -one

To a solution of compound 71-13 (220 mg, 431 μmol, 1.00 eq) in i-PrOH (2.00 mL) was added Et3N (87.2 mg, 862 μmol, 119 μL, 2.00 eq) and compound 71-14 (148 mg, 1.29 mmol, 170 μL, 3.00 eq) . Then the mixture was stirred at 90 ℃ for 4 hrs. LC-MS showed the desired mass was detected. The mixture was concentrated under reduced pressure to give a residue. And the solution was quenched with NaClO (200 mL) at 0 ℃. The crude product was purified by prep-TLC (DCM: MeOH = 10: 1, Rf (P1) = 0.500) . Compound 71-15 (120 mg, 295 μmol, 68.5%yield) was obtained as a brown solid.

H NMR: (400 MHz, CDCl₃) δ 8.42 -8.40 (m, 2H) , 7.79 -7.77 (m, 2H) , 6.79 (s, 1H) , 4.34 -4.30 (m, 1H) , 3.99 (s, 3H) , 3.70 -3.63 (m, 1H) , 3.14 -3.06 (m, 1H) , 2.90 -2.82 (m, 1H) , 2.47 -2.43 (m, 2H) , 2.35 -2.22 (m, 2H) , 2.06 -2.03 (m, 1H) , 1.87 -1.79 (m, 1H) , 1.77 -1.71 (m, 1H) , 1.67 -1.60 (m, 1H) .

Step 10. 7- (3-amino-4- (4-aminophenyl) -1-methyl-1H-pyrazolo [3, 4-b] pyridin-6-yl) hexahydroindolizin-3 (2H) -one

To a solution of compound 71-15 (120 mg, 295 μmol, 1.00 eq) in THF (3.00 mL) , DCM (3.00 mL) and MeOH (3.00 mL) was added Pd/C (12.0 mg, 11.2 μmol, 10%purity, 3.82e-2 eq) . The mixture was degassed and purged with H₂ (15 Psi) for 3 times, then stirred at 20 ℃ for 2 hrs. LC-MS (EW43072-24-P1A1) showed the desired mass was detected. The mixture was filtered and filtrate was concentrated under reduced pressure to give a residue. Compound 71-16 (85.0 mg, 225 μmol, 76.4%yield) was obtained as a yellow solid.

H NMR: (400 MHz, CDCl₃) δ 7.40 -7.38 (m, 2H) , 6.83 -6.81 (m, 2H) , 6.72 (s, 1H) , 4.33 -4.28 (m, 1H) , 3.97 (s, 3H) , 3.69 -3.64 (m, 1H) , 3.11 -3.07 (m, 1H) , 2.89 -2.82 (m, 1H) , 2.46 -2.42 (m, 2H) , 2.33 -2.22 (m, 2H) , 2.06 -2.03 (m, 1H) , 1.84 -1.56 (m, 3H) .

Step 11. N- (4- (3-amino-1-methyl-6- (3-oxooctahydroindolizin-7-yl) -1H-pyrazolo [3, 4-b] pyridin-4-yl) phenyl) -4-ethoxy-1- (4-fluorophenyl) -2-oxo-1, 2-dihydropyridine-3-carboxamide

To a solution of compound 71-16 (15.0 mg, 39.8 μmol, 1.00 eq) in pyridine (2.00 mL) was added compound 71-17 (9.94 mg, 35.8 μmol, 0.900 eq) and EDCI (30.5 mg, 159 μmol, 4.00 eq) . Then the mixture was stirred at 20 ℃ for 1 hr. LC-MS showed the desired mass was detected. The reaction mixture was quenched by water (10.0 mL) , and then extracted with DCM (30.0 mL *3) . The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The crude product was purified by prep-TLC (DCM: MeOH = 10: 1, Rf (P1) = 0.400) . Compound 71 (7.87 mg, 11.6 μmol, 29.1%yield, 93.7%purity) was obtained.

H NMR: (400 MHz, CDCl₃) δ 11.55 (s, 1H) , 7.87 -7.85 (m, 2H) , 7.53 -7.49 (m, 3H) , 7.40 -7.36 (m, 2H) , 7.25 -7.23 (m, 2H) , 6.74 (s, 1H) , 6.38 (d, J = 7.6 Hz, 1H) , 4.41 -4.35 (m, 2H) , 4.33 -4.28 (m, 1H) , 3.93 (s, 3H) , 3.90 (s, 2H) , 3.67 -3.63 (m, 1H) , 3.05 -2.99 (m, 1H) , 2.87 -2.81 (m, 1H) , 2.47 -2.42 (m, 2H) , 2.31 -2.20 (m, 2H) , 2.06 -2.02 (m, 1H) , 1.83 -1.67 (m, 3H) , 1.62 -1.58 (m, 3H) .

LC-MS: (M+H) ⁺: 636.3

N- (4- (3-amino-1-methyl-6- (3-oxooctahydroindolizin-7-yl) -1H-pyrazolo [3, 4-b] pyridin-4-yl) phenyl) -4-ethoxy-1- (4-fluorophenyl) -2-oxo-1, 2-dihydropyridine-3-carboxamide (Compound 80)

Compound 80 was prepared in similar way as compound 71 except that compound 67-2 was used instead of compound 71-17.

H NMR: (400 MHz, CDCl₃) δ 11.21 (s, 1H) , 8.45 -8.44 (m, 1H) , 7.96 (d, J = 7.2 Hz, 1H) , 7.90 -7.85 (m, 3H) , 7.65 -7.61 (m, 1H) , 7.52 (d, J = 8.4 Hz, 2H) , 6.74 (s, 1H) , 6.42 (d, J = 8.0 Hz, 1H) , 4.41 -4.36 (m, 2H) , 4.32 -4.28 (m, 1H) , 3.93 (s, 3H) , 3.91 (s, 2H) , 3.68 -3.61 (m, 1H) , 3.08 -2.98 (m, 1H) , 2.88 -2.81 (m, 1H) , 2.46 -2.41 (m, 2H) , 2.32 -2.20 (m, 2H) , 2.05 -2.02 (m, 1H) , 1.86 -1.78 (m, 1H) , 1.76 -1.65 (m, 2H) , 1.58 (t, J = 9.2 Hz, 3H) .

LC-MS: (M+H) ⁺: 637.3

Example 36

N- (4- (3-amino-6- (4-isobutyrylpiperazin-1-yl) -1-methyl-1H-indazol-4-yl) phenyl) -4-ethoxy-1- (4-fluorophenyl) -2-oxo-1, 2-dihydropyridine-3-carboxamide (Compound 72)

Step 1. 1- (4- (3-amino-4- (4-aminophenyl) -1-methyl-1H-indazol-6-yl) piperazin-1-yl) -2-methylpropan-1-one

To a solution of compound 72-1 (200 mg, 630 μmol, 1.00 eq) in dioxane (2.00 mL) was added compound 72-2 (147 mg, 945 μmol, 1.50 eq) , Cs₂CO₃ (616 mg, 1.89 mmol, 3.00 eq) , PEPPSI-iPent (61.3 mg, 63.0 μmol, 0.100 eq) under N₂. The mixture was stirred at 100℃ for 2 h under N₂ atmosphere. LC-MS showed the desired mass was detected. The mixture was filtered and concentrated to remove solvent. The crude was purified by prep-HPLC (column: Waters Xbridge 150 *25 mm *5 um; mobile phase: [water (ammonia hydroxide v/v) -ACN] ; gradient: 20%-50%B over 9 min) to get the desired product compound 72-3 (20.0 mg, 50.9 μmol, 8.08%yield, -purity) as white solid.

Step 2. N- (4- (3-amino-6- (4-isobutyrylpiperazin-1-yl) -1-methyl-1H-indazol-4-yl) phenyl) -4-ethoxy-1- (4-fluorophenyl) -2-oxo-1, 2-dihydropyridine-3-carboxamide

To a solution of compound 72-3 (10.0 mg, 25.4 μmol, 1.0 eq) , compound 72-4 (7.06 mg, 25.4 μmol, 1.00 eq) in Py (1.00 mL) was added EDCI (9.77 mg, 50.9 μmol, 2.00 eq) , the mixture was stirred at 25℃ for 1 h. LC-MS showed the desired mass was detected. The mixture was diluted with water (10.0 mL) and NaHCO₃ (10.0 mL) , extracted with DCM (10.0 mL *3) , the combined organic layer was washed with brine (20.0 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The crude was purified by prep-TLC (SiO₂, plate 1, DCM: MeOH = 10: 1, Rf = 0.4) . The crude was purified by HPLC (column: Waters Xbridge 150 *25 mm *5um; mobile phase: [water (ammonia hydroxide v/v) -ACN] ; gradient: 29%-59%B over 9 min) to get the desired product Compound 72 (8.25 mg, 12.56 μmol, 49.31%yield, 99.24%purity) .

H NMR: (400 MHz, CDCl₃) δ 11.38 (s, 1H) , 7.80 (d, J = 8.4 Hz, 2H) , 7.50 (d, J = 7.6 Hz, 1H) , 7.43 (d, J = 9.6 Hz, 2H) , 7.39 -7.36 (m, 2H) , 7.26 -7.22 (m, 2H) , 6.61 (d, J = 2.0 Hz, 1H) , 6.46 (d, J = 2.0 Hz, 1H) , 6.36 (d, J = 8.0 Hz, 1H) , 4.38 -4.33 (m, 2H) , 3.82 (brs, 2H) , 3.78 (s, 3H) , 3.74 -3.70 (m, 4H) , 3.28 -3.25 (m, 4H) , 2.88 -2.81 (m, 1H) , 1.58 (t, J = 6.8 Hz, 3H) , 1.17 (d, J = 6.8 Hz, 6H) .

LC-MS: (M+H) ⁺: 652.4

Example 37

N- (4- (3-amino-6- (4-isobutyrylpiperazin-1-yl) -1-methyl-1H-pyrazolo [3, 4-b] pyridin-4-yl) phenyl) -4-ethoxy-5'-fluoro-2-oxo-2H- [1, 2'-bipyridine] -3-carboxamide (Compound 73)

Step1. 1- (4- (3-amino-1-methyl-4- (4-nitrophenyl) -1H-pyrazolo [3, 4-b] pyridin-6-yl) piperazin-1-yl) -2-methylpropan-1-one

To a solution of compound 73-1 (130 mg, 428 μmol, 1.00 eq) in dioxane (4.00 mL) was added compound 73-2 (334 mg, 2.14 mmol, 5.00 eq) , Cs₂CO₃ (418 mg, 1.28 mmol, 3.00 eq) and PEPPSI-iPent (41.6 mg, 42.8 μmol, 0.100 eq) . Then the mixture was stirred at 100 ℃ for 10 hrs under N₂. LC-MS showed the desired mass was detected. The mixture was filtered and filtrate was concentrated under reduced pressure to give a residue. The residue (DCM: MeOH = 10: 1, Rf (P1) =0.500) was purified by column chromatography (SiO₂, DCM: MeOH = 100: 0 to 100: 2) . Compound 73-3 (300 mg, crude) was obtained as yellow oil.

Step 2. 1- (4- (3-amino-4- (4-aminophenyl) -1-methyl-1H-pyrazolo [3, 4-b] pyridin-6-yl) piperazin-1-yl) -2-methylpropan-1-one

To a solution of compound 73-3 (300 mg, 708 μmol, 1.00 eq) in THF (6.00 mL) and DCM (2.00 mL) was added Pd/C (30.0 mg, 10.0%purity) , degassed and purged with H₂ (15 Psi) for 3 times, then stirred at 20 ℃ for 1 hr. LC-MS showed the desired mass was detected. The mixture was filtered and filtrate was concentrated under reduced pressure to give a residue. The crude product was purified by prep-TLC (DCM: MeOH = 10: 1, Rf (P1) = 0.400) and further purified by prep-HPLC (ammonia hydroxide condition; column: Waters Xbridge 150 *25.0 mm *5.00 um; mobile phase: [water (ammonia hydroxide v/v) -ACN] ; gradient: 20.0%-50.0%B over 9 min) . Then the mixture was concentrated under reduced pressure to remove MeCN. The aqueous part was extracted with DCM: MeOH = 10: 1 (20.0 mL *3) . The combined organic layers were dried over anhydrous Na₂SO₄, then filtered and concentrated under reduced pressure to give a residue. Compound 73-4 (42.0 mg, 106 μmol, 15.0%yield) was obtained as a white solid.

Step 3. N- (4- (3-amino-6- (4-isobutyrylpiperazin-1-yl) -1-methyl-1H-pyrazolo [3, 4-b] pyridin-4-yl) phenyl) -4-ethoxy-5'-fluoro-2-oxo-2H- [1, 2'-bipyridine] -3-carboxamide (Compound 73)

To a solution of compound 73-4 (15.0 mg, 38.1 μmol, 1.00 eq) in pyridine (2.00 mL) was added compound 73-5 (10.6 mg, 38.1 μmol, 1.00 eq) , EDCI (29.2 mg, 152 μmol, 4.00 eq) . Then the mixture was stirred at 20 ℃ for 1 hr. LC-MS showed the desired mass was detected. The reaction mixture was quenched by water (10.0 mL) , and then extracted with DCM (30.0 mL *3) . The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The crude product was purified by prep-TLC (DCM: MeOH = 10: 1, Rf (P1) = 0.400) and further purified by prep-HPLC (ammonia hydroxide condition; column: Waters Xbridge 150 *25.0 mm *5.00 um; mobile phase: [water (ammonia hydroxide v/v) -ACN] ; gradient: 27.0%-57.0%B over 9 min) . Then the mixture was concentrated under reduced pressure to remove MeCN. And the mixture was freezedried to give a product. Compound 73 (4.54 mg, 6.54 μmol, 17.1%yield, 94.2%purity) was obtained.

H NMR: (400 MHz, CDCl₃) δ 11.20 (s, 1H) , 8.45 (d, J = 2.8 Hz, 1H) , 7.97 (d, J = 8.4 Hz, 1H) , 7.91 -7.84 (m, 3H) , 7.66 -7.61 (m, 1H) , 7.50 -7.48 (m, 2H) , 6.43 (d, J = 8.0 Hz, 1H) , 6.33 (s, 1H) , 4.41 -4.36 (m, 2H) , 4.04 (s, 2H) , 3.82 (s, 3H) , 3.80 -3.77 (m, 4H) , 3.70 -3.68 (m, 4H) , 2.87 -2.84 (m, 1H) , 1.60 -1.57 (m, 3H) , 1.19 - 1.17 (m, 6H) .

LC-MS: (M+H) ⁺: 654.4

N- (4- (3-amino-6- (4-isobutyrylpiperazin-1-yl) -1-methyl-1H-pyrazolo [3, 4-b] pyridin-4-yl) phenyl) -4-ethoxy-1- (4-fluorophenyl) -2-oxo-1, 2-dihydropyridine-3-carboxamide (Compound 82)

Compound 82 was prepared in similar way as Compound 73 except that compound 72-4 was used instead of compound 73-5.

H NMR: (400 MHz, CDCl₃) δ 11.54 (s, 1H) , 7.86 -7.84 (m, 2H) , 7.52 (d, J = 8.0 Hz, 1H) , 7.48 -7.46 (m, 2H) , 7.40 -7.36 (m, 2H) , 7.25 -7.23 (m, 2H) , 6.38 (d, J = 7.6 Hz, 1H) , 6.32 (s, 1H) , 4.40 -4.35 (m, 2H) , 4.11 (s, 2H) , 3.81 (s, 3H) , 3.79 -3.77 (m, 4H) , 3.70 -3.68 (m, 4H) , 2.89 -2.82 (m, 1H) , 1.62 -1.58 (m, 3H) , 1.19 -1.17 (m, 6H) .

LC-MS: (M+H) ⁺: 653.3

Example 38

N- (4- (6- (4-acetylpiperazin-1-yl) -3-amino-1-methyl-1H-pyrazolo [3, 4-b] pyridin-4-yl) phenyl) -4-ethoxy-1- (4-fluorophenyl) -2-oxo-1, 2-dihydropyridine-3-carboxamide (Compound 74)

Step 1. 1- (4- (3-amino-1-methyl-4- (4-nitrophenyl) -1H-pyrazolo [3, 4-b] pyridin-6-yl) piperazin-1-yl) ethan-1-one

To a solution of compound 74-1 (80.0 mg, 263 μmol, 1.00 eq) and compound 74-2 (168 mg, 1.32 mmol, 5.00 eq) in dioxane (1.00 mL) was added Cs₂CO₃ (257 mg, 790 μmol, 3.00 eq) and PEPPSI-iPent (25.6 mg, 26.3 μmol, 0.100 eq) . The mixture was stirred at 100 ℃ for 2 hrs. LC-MS showed desired mass detected. The reaction mixture was concentrated to give a residue. The residue was purified by prep-TLC (SiO₂, DCM: MeOH = 10: 1) . Compound 74-3 (30.0 mg, 75.8 μmol, 28.8%yield) was obtained as a yellow solid.

Step 2. 1- (4- (3-amino-4- (4-aminophenyl) -1-methyl-1H-pyrazolo [3, 4-b] pyridin-6-yl) piperazin-1-yl) ethan-1-one

To a solution of compound 74-3 (30.0 mg, 75.8 μmol, 1.00 eq) in THF (2.00 mL) was added Pd/C (30.0 mg, 10.0%purity) under N₂. The suspension was degassed under vacuum and purged with H₂ several times. The mixture was stirred under H₂ (15 Psi) at 25 ℃ for 1 hr. LC-MS showed desired mass detected. The reaction mixture was filtered and the cake was washed with 100 mL MeOH. Then the filtrate was concentrated under reduced pressure to give a residue. Compound 74-4 (20.0 mg, 54.7 μmol, 72.1%yield) was obtained as a yellow solid.

Step 3. N- (4- (6- (4-acetylpiperazin-1-yl) -3-amino-1-methyl-1H-pyrazolo [3, 4-b] pyridin-4-yl) phenyl) -4-ethoxy-1- (4-fluorophenyl) -2-oxo-1, 2-dihydropyridine-3-carboxamide (Compound 74)

To a solution of compound 74-5 (13.6 mg, 49.2 μmol, 0.900 eq) and compound 74-4 (20.0 mg, 54.7 μmol, 1.00 eq) in pyridine (2.00 mL) was added EDCI (31.4 mg, 164 μmol, 3.00 eq) . The mixture was stirred at 25 ℃ for 1 hr. LC-MS showed desired mass detected. The reaction mixture was diluted with H₂O 30.0 mL and extracted with EtOAc 80.0 mL (40.0 mL *2) . The combined organic layers were washed with brine 100 mL (50.0 mL *2) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiO₂, DCM: MeOH = 10: 1) . (DCM: MeOH = 10: 1, Rf (P1) = 0.400) . And the residue was further purified by prep-HPLC (ammonia hydroxide condition; column: Waters Xbridge 150 *25.0 mm *5.00 um; mobile phase: [water (ammonia hydroxide v/v) -ACN] ; gradient: 20.0%-50.0%B over 9 min) . Then the mixture was concentrated under reduced pressure to remove MeCN. And the mixture was freezedried to give a product. Compound 74 (4.19 mg, 6.27 μmol, 11.4%yield, 93.5%purity) was obtained.

H NMR: (400 MHz, CDCl₃) δ 11.63 (s, 1H) , 7.88 -7.86 (m, 2H) , 7.53 (d, J = 7.6 Hz, 1H) , 7.45 -7.25 (m, 6H) , 6.39 -6.36 (m, 2H) , 4.41 -4.37 (m, 2H) , 3.85 - 3.63 (m, 11H) , 2.17 (m, 3H) , 1.62 -1.58 (m, 3H) .

LC-MS: (M+H) ⁺: 625.4

Example 39

N- (4- (3-amino-1-methyl-6- (4-methyl-3-oxopiperazin-1-yl) -1H-pyrazolo [3, 4-b] pyridin-4-yl) phenyl) -4-ethoxy-1- (4-fluorophenyl) -2-oxo-1, 2-dihydropyridine-3-carboxamide (Compound 75)

Step 1. 4- (3-amino-1-methyl-4- (4-nitrophenyl) -1H-pyrazolo [3, 4-b] pyridin-6-yl) -1-methylpiperazin-2-one

To a solution of compound 75-1 (80.0 mg, 263 μmol, 1.00 eq) in dioxane (3.00 mL) was added compound 75-2 (240 mg, 2.11 mmol, 8.00 eq) , Cs₂CO₃ (257 mg, 790 μmol, 3.00 eq) and PEPPSI-iPent (25.6 mg, 26.3 μmol, 0.100 eq) . Then the mixture was stirred at 100 ℃ for 10 hrs under N₂. LC-MS showed the desired mass was detected. The mixture was filtered and filtrate was concentrated under reduced pressure to give a residue. The crude product was purified by prep-TLC (DCM: MeOH = 10: 1, Rf (P1) = 0.500) . Compound 75-3 (35.0 mg, 91.7 μmol, 34.8%yield) was obtained as a yellow solid.

Step 2. 4- (3-amino-4- (4-aminophenyl) -1-methyl-1H-pyrazolo [3, 4-b] pyridin-6-yl) -1-methylpiperazin-2-one

To a solution of compound 75-3 (35.0 mg, 91.7 μmol, 1.00 eq) in THF (2.00 mL) and DCM (2.00 mL) was added Pd/C (5.00 mg, 10.0%purity) . The mixture was degassed and purged with H₂ (15 Psi) for 3 times, then stirred at 20 ℃ for 2 hrs. LC-MS showed the desired mass was detected. The mixture was filtered and filtrate was concentrated under reduced pressure to give a residue. The crude product was purified by prep-TLC (DCM: MeOH = 10: 1, Rf (P1) = 0.400) . Compound 75-4 (10.0 mg, 28.4 μmol, 31.01%yield) was obtained as a white solid

Step 3. N- (4- (3-amino-1-methyl-6- (4-methyl-3-oxopiperazin-1-yl) -1H-pyrazolo [3, 4-b] pyridin-4-yl) phenyl) -4-ethoxy-1- (4-fluorophenyl) -2-oxo-1, 2-dihydropyridine-3-carboxamide

To a solution of compound 75-4 (10.0 mg, 28.4 μmol, 1.00 eq) in pyridine (2.00 mL) was added compound 75-5 (7.10 mg, 25.6 μmol, 0.900 eq) and EDCI (21.8 mg, 113 μmol, 4.00 eq) . Then the mixture was stirred at 25 ℃ for 1 hr. LC-MS showed the desired mass was detected. The reaction mixture was quenched by water (10.0 mL) , and then extracted with DCM (10.0 mL *3) . The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The crude product was purified by prep-HPLC (ammonia hydroxide condition; column: Waters Xbridge 150 *25.0 mm *5.00 um; mobile phase: [water (ammonia hydroxide v/v) -ACN] ; gradient: 19.0%-49.0%B over 9 min) . Then the mixture was concentrated under reduced pressure to remove MeCN. And the mixture was freezedried to give a product. Compound 75 (4.14 mg, 6.60 μmol, 23.2%yield, 97.4%purity) was obtained.

H NMR: (400 MHz, CDCl₃) δ 11.56 (s, 1H) , 7.87 -7.85 (m, 2H) , 7.52 (d, J = 8.0 Hz, 1H) , 7.47 -7.45 (m, 2H) , 7.40 -7.36 (m, 2H) , 7.25 -7.23 (m, 2H) , 6.38 (d, J = 8.0 Hz, 1H) , 6.28 (s, 1H) , 4.41 -4.35 (m, 2H) , 4.32 (s, 2H) , 4.03 -4.01 (m, 2H) , 3.82 (s, 3H) , 3.52 -3.50 (m, 2H) , 3.07 (s, 3H) , 1.62 -1.58 (m, 3H) .

LC-MS: (M+H) ⁺: 611.3

Example 40

N- (4- (3-amino-1-methyl-6- (3-oxopiperazin-1-yl) -1H-pyrazolo [3, 4-b] pyridin-4-yl) phenyl) -4-ethoxy-1- (4-fluorophenyl) -2-oxo-1, 2-dihydropyridine-3-carboxamide (Compound 76)

Step 1. 4- (3-amino-1-methyl-4- (4-nitrophenyl) -1H-pyrazolo [3, 4-b] pyridin-6-yl) piperazin-2-one

Two reactions were carried out in parallel. To a solution of compound 76-1 (60.0 mg, 197 μmol, 1.00 eq) in dioxane (1.00 mL) was added compound 76-2 (158 mg, 1.58 mmol, 8.00 eq) , Cs₂CO₃ (193 mg, 592 μmol, 3.00 eq) , PEPPSI-iPent (19.2 mg, 19.7 μmol, 0.100 eq) , the mixture was stirred at 100 ℃ for 5 hrs under N₂. LC-MS showed the desired mass was detected. The mixture was filtered and filtrate was concentrated to remove solvent. The crude was purified by prep-TLC (SiO₂, DCM: MeOH = 10: 1, Rf (P1) = 0.400) . Compound 76-3 (15.0 mg, 43.4 μmol, 11.0%yield) was obtained as a yellow solid.

Step 2. 4- (3-amino-4- (4-aminophenyl) -1-methyl-1H-pyrazolo [3, 4-b] pyridin-6-yl) piperazin-2-one

To a solution of compound 76-3 (15.0 mg, 30.6 μmol, 1.00 eq) in DCM (4.00 mL) and THF (2.00 mL) was added Pd/C (5.00 mg, 10.0%purity) , the mixture was degassed and purged with H₂ three times, the mixture was stirred at 25 ℃ for 1 hr. LC-MS showed the desired mass was detected. The mixture was filtered and filtrate was concentrated under reduced pressure to give a residue. Compound 76-4 (11.0 mg, 32.6 μmol, 80.0%yield) was obtained as a brown solid.

Step 3. N- (4- (3-amino-1-methyl-6- (3-oxopiperazin-1-yl) -1H-pyrazolo [3, 4-b] pyridin-4-yl) phenyl) -4-ethoxy-1- (4-fluorophenyl) -2-oxo-1, 2-dihydropyridine-3-carboxamide

To a solution of compound 76-4 (11.0 mg, 32.6 μmol, 1.00 eq) , compound 76-5 (9.04 mg, 32.6 μmol, 1.00 eq) in pyridine (1.00 mL) was added EDCI (12.5 mg, 65.2 μmol, 2.00 eq) , the mixture was stirred at 25 ℃ for 1 hr. LC-MS showed the desired mass was detected. The mixture was diluted with water (10.0 mL) and NaHCO3 (10.0 mL) , extracted with DCM (10.0 mL *3) , the combined organic layer was washed with brine (20.0 mL) , dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The crude was purified by prep-TLC (SiO₂, DCM: MeOH = 10: 1, Rf (P1) = 0.400) . Compound 76 (5.56 mg, 9.08 μmol, 27.8%yield, 97.3%purity) was obtained.

H NMR: (400 MHz, CDCl₃) δ 11.51 (s, 1H) , 7.86 -7.83 (m, 2H) , 7.53 -7.48 (m, 3H) , 7.40 -7.36 (m, 2H) , 7.26 -7.25 (m, 2H) , 6.37 (d, J = 7.6 Hz, 1H) , 6.26 (s, 1H) , 5.98 (s, 1H) , 4.40 -4.37 (m, 2H) , 4.35 (s, 2H) , 4.00 -3.97 (m, 2H) , 3.81 (s, 3H) , 3.80 -3.78 (m, 2H) , 3.56 (s, 2H) , 1.62 -1.58 (m, 3H) .

LC-MS: (M+H) ⁺: 597.2

Example 41

N- (4- (3-amino-6- (6-isobutyryl-2, 6-diazaspiro [3.3] heptan-2-yl) -1-methyl-1H-pyrazolo [3, 4-b] pyridin-4-yl) phenyl) -4-ethoxy-1- (4-fluorophenyl) -2-oxo-1, 2-dihydropyridine-3-carboxamide (Compound 77)

Step 1. tert-butyl 6-isobutyryl-2, 6-diazaspiro [3.3] heptane-2-carboxylate

To a solution of compound 77-6 (1.00 g, 5.04 mmol, 1.00 eq) in DCM (10.0 mL) was added TEA (1.53 g, 15.1 mmol, 2.11 mL, 3.00 eq) , compound 77-7 (644 mg, 6.05 mmol, 634 μL, 1.20 eq) at 0 ℃, the mixture was stirred at 25 ℃ for 2 hrs. LC-MS showed the desired mass was detected. The mixture was concentrated to remove solvent. The crude was purified by column (SiO₂, DCM: MeOH = 1: 0 to 10: 1, DCM: MeOH = 10: 1, Rf (P1) = 0.400) , to get the desired product compound 77-8 (975 mg, 3.63 mmol, 72.0%yield) as a white solid.

H NMR: (400 MHz, CDCl₃) δ 4.24 -4.06 (m, 8H) , 2.44 -2.37 (m, 1H) , 1.44 (s, 9H) , 1.10 -1.08 (m, 6H) .

Step 2. 6-isobutyryl-2, 6-diazaspiro [3.3] heptan-2-ium, 2, 2, 2-trifluoroacetate

To a solution of compound 77-8 (975 mg, 3.63 mmol, 1.00 eq) in DCM (5.00 mL) was added TFA (7.68 g, 67.3 mmol, 5.00 mL, 18.5 eq) at 0 ℃. Then the mixture was stirred at 25 ℃ for 1 hr. TLC (DCM: MeOH = 10: 1, Rf (R1) = 0.700) showed the starting material was consumed completely and there was new spot. LC-MS (EW43072-35-P1B1) showed there was desired mass. The mixture was concentrated under reduced pressure to give a residue. Compound 77-2 (2.27 g, crude, TFA) was obtained as light yellow oil.

H NMR: (400 MHz, CDCl₃) δ 4.57 -4.19 (m, 9H) , 2.49 -2.43 (m, 1H) , 1.13 -1.01 (m, 6H) .

Step 3. 1- (6- (3-amino-1-methyl-4- (4-nitrophenyl) -1H-pyrazolo [3, 4-b] pyridin-6-yl) -2, 6-diazaspiro [3.3] heptan-2-yl) -2-methylpropan-1-one

Two reactions were carried out in parallel. To a solution of compound 77-1 (65.0 mg, 214 μmol, 1.00 eq) in dioxane (6.00 mL) was added compound 77-2 (966 mg, 3.42 mmol, 16.0 eq, TFA) , Cs₂CO₃ (1.88 g, 5.78 mmol, 27.0 eq) and PEPPSI-iPent (20.8 mg, 21.4 μmol, 0.100 eq) . Then the mixture was stirred at 100 ℃ for 2 hrs under N₂. LC-MS showed the desired mass was detected. The mixture was filtered and filtrate was concentrated under reduced pressure to give a residue. The residue (DCM: MeOH = 10: 1, Rf (P1) = 0.500) was purified by column chromatography (SiO₂, DCM: MeOH = 100: 0 to 100: 10) . Compound 77-3 (110 mg, 252 μmol, 59.0%yield) was obtained as a yellow solid.

Step 4. 1- (6- (3-amino-4- (4-aminophenyl) -1-methyl-1H-pyrazolo [3, 4-b] pyridin-6-yl) -2, 6-diazaspiro [3.3] heptan-2-yl) -2-methylpropan-1-one

To a solution of compound 77-3 (110 mg, 252 μmol, 1.00 eq) in THF (6.00 mL) , DCM (3.00 mL) and MeOH (3.00 mL) was added Pd/C (11.0 mg, 10.0%purity) . The mixture was degassed and purged with H₂ (15 Psi) for 3 times, then stirred at 20 ℃ for 2 hrs. LC-MS showed the desired mass was detected. The mixture was filtered and filtrate was concentrated under reduced pressure to give a residue. Compound 77-4 (80.0 mg, 197 μmol, 78.1%yield) was obtained as a yellow solid.

H NMR: (400 MHz, CDCl₃) δ 7.36 -7.34 (m, 2H) , 6.80 -6.78 (m, 2H) , 5.89 (s, 1H) , 4.35 (s, 2H) , 4.26 -4.25 (m, 4H) , 4.19 (s, 2H) , 3.80 (s, 3H) , 2.49 -2.42 (m, 1H) , 1.13 -1.11 (m, 6H) .

Step 5. N- (4- (3-amino-6- (6-isobutyryl-2, 6-diazaspiro [3.3] heptan-2-yl) -1-methyl-1H-pyrazolo [3, 4-b] pyridin-4-yl) phenyl) -4-ethoxy-1- (4-fluorophenyl) -2-oxo-1, 2-dihydropyridine-3-carboxamide

To a solution of compound 77-4 (20.0 mg, 49.3 μmol, 1.00 eq) in pyridine (2.00 mL) was added compound 77-5 (12.9 mg, 46.8 μmol, 0.950 eq) , EDCI (37.8 mg, 197 μmol, 4.00 eq) . Then the mixture was stirred at 20 ℃ for 1 hr. LC-MS showed the desired mass was detected. The reaction mixture was quenched by water (10.0 mL) , and then extracted with DCM (10.0 mL *3) . The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The crude product was purified by prep-TLC (DCM: MeOH = 10: 1, Rf (P1) = 0.400) . Compound 77 (10.2 mg, 14.1 μmol, 28.7%yield, 91.9%purity) was obtained.

H NMR: (400 MHz, CDCl₃) δ 11.49 (s, 1H) , 7.84 -7.82 (m, 2H) , 7.51 (d, J = 7.6 Hz, 1H) , 7.48 -7.46 (m, 2H) , 7.39 -7.36 (m, 2H) , 7.25 -7.23 (m, 2H) , 6.37 (d, J = 8.0 Hz, 1H) , 5.91 (s, 1H) , 4.40 -4.35 (m, 4H) , 4.25 -4.24 (m, 4H) , 4.19 (s, 2H) , 3.80 (s, 3H) , 3.76 (s, 2H) , 2.49 -2.42 (m, 1H) , 1.61 -1.59 (m, 3H) , 1.13 -1.11 (m, 6H) .

LC-MS: (M+H) ⁺: 665.4

Example 42

(S) -N- (4- (3-amino-1-methyl-6- (6-oxohexahydropyrrolo [1, 2-a] pyrazin-2 (1H) -yl) -1H-pyrazolo [3, 4-b] pyridin-4-yl) phenyl) -4-ethoxy-1- (4-fluorophenyl) -2-oxo-1, 2-dihydropyridine-3-carboxamide and (R) -N- (4- (3-amino-1-methyl-6- (6-oxohexahydropyrrolo [1, 2-a] pyrazin-2 (1H) -yl) -1H-pyrazolo [3, 4-b] pyridin-4-yl) phenyl) -4-ethoxy-1- (4-fluorophenyl) -2-oxo-1, 2-dihydropyridine-3-carboxamide (Compounds 78a and 78b)

Step 1. 2- (3-amino-1-methyl-4- (4-nitrophenyl) -1H-pyrazolo [3, 4-b] pyridin-6-yl) hexahydropyrrolo [1, 2-a] pyrazin-6 (2H) -one

To a solution of compound 78-1 (158 mg, 520 μmol, 1.00 eq) in dioxane (6.00 mL) was added compound 78-2 (364 mg, 2.60 mmol, 5.00 eq) , Cs₂CO₃ (339 mg, 1.04 mmol, 2.00 eq) and PEPPSI-iPent (50.6 mg, 52.0 μmol, 0.100 eq. Then the mixture was stirred at 80 ℃ for 10 hrs under N₂. LC-MS showed the desired mass was detected. The mixture was filtered and filtrate was concentrated under reduced pressure to give a residue. The residue (DCM: MeOH = 10: 1, R_f (P1) = 0.500) was purified by column chromatography (SiO₂, DCM: MeOH = 100: 0 to 100: 10) . Compound 78-3 (226 mg, crude) was obtained as a yellow solid.

Step 2. 2- (3-amino-4- (4-aminophenyl) -1-methyl-1H-pyrazolo [3, 4-b] pyridin-6-yl) hexahydropyrrolo [1, 2-a] pyrazin-6 (2H) -one

To a solution of compound 78-3 (226 mg, 554 μmol, 1.00 eq) in THF (4.00 mL) and DCM (1.00 mL) was added Pd/C (23.0 mg, 21.6 μmol, 10.0%purity, 3.90e-2 eq) , degassed and purged with H₂ (15 Psi) for 3 times, then stirred at 20 ℃ for 1 hr. LC-MS showed the desired mass was detected. The mixture was filtered and filtrate was concentrated under reduced pressure to give a residue. The residue was purified by Prep-TLC (DCM: MeOH = 10: 1, R_f (P1) = 0.500) and further purified by prep-HPLC (NH₃·H₂O condition; column: Waters Xbridge 150 *25.0 mm *5.00 um; mobile phase: [water (NH₃·H₂O) -ACN] ; gradient: 15.0%-45.0%B over 9 min) . Then the mixture was extracted with DCM (20.0 mL *3) . The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. Compound 78-4 (50.0 mg, 132 μmol, 23.8%yield) was obtained as a white solid.

Step 3. Separation of (S) -2- (3-amino-4- (4-aminophenyl) -1-methyl-1H-pyrazolo [3, 4-b] pyridin-6-yl) hexahydropyrrolo [1, 2-a] pyrazin-6 (2H) -one and (R) -2- (3-amino-4- (4-aminophenyl) -1-methyl-1H-pyrazolo [3, 4-b] pyridin-6-yl) hexahydropyrrolo [1, 2-a] pyrazin-6 (2H) -one (Compounds 78-5a and 78-5b)

Compound 78-4 (45.0 mg, 119 μmol, 1.00 eq) was purified by SFC (SFC: RT of peak 1: 1.341 min, RT of peak 2: 1.808 min, column: DAICEL CHIRALPAK IG (250 mm *30.0 mm, 10.0 um) ; mobile phase: [CO₂-ACN/MeOH (0.100%IPAm) ] ; B%: 65.0%, isocratic elution mode) . Compound 78-5a (21.0 mg, 55.6 μmol, 46.6%yield) was obtained as a white solid (peak 1, SFC: RT: 1.302 min) . Compound 78-5b (22.0 mg, 58.2 μmol, 48.8%yield) was obtained as a white solid (peak 2, SFC: RT: 1.770 min) .

Step 4. (S) -N- (4- (3-amino-1-methyl-6- (6-oxohexahydropyrrolo [1, 2-a] pyrazin-2 (1H) -yl) -1H-pyrazolo [3, 4-b] pyridin-4-yl) phenyl) -4-ethoxy-1- (4-fluorophenyl) -2-oxo-1, 2-dihydropyridine-3-carboxamide and (R) -N- (4- (3-amino-1-methyl-6- (6-oxohexahydropyrrolo [1, 2-a] pyrazin-2 (1H) -yl) -1H-pyrazolo [3, 4-b] pyridin-4-yl) phenyl) -4-ethoxy-1- (4-fluorophenyl) -2-oxo-1, 2-dihydropyridine-3-carboxamide

To a solution of compound 78-5a (9.00 mg, 23.8 μmol, 1.00 eq) in pyridine (2.00 mL) was added compound 78-6 (5.95 mg, 21.4 μmol, 0.900 eq) and EDCI (18.2 mg, 95.3 μmol, 4.00 eq) . Then the mixture was stirred at 20 ℃ for 1 hr. LC-MS showed the desired mass was detected. The reaction mixture was quenched by water (10.0 mL) , and then extracted with DCM (30.0 mL *3) . The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The crude product was purified by prep-HPLC (ammonia hydroxide condition; column: Waters Xbridge 150 *25.0 mm *5.00 um; mobile phase: [water (ammonia hydroxide v/v) -ACN] ; gradient: 20.0%-50.0%B over 9 min) . Then the mixture was concentrated under reduced pressure to remove MeCN. And the mixture was freezedried to give a product. Compound 78a (5.01 mg, 7.08 μmol, 29.7%yield, 89.9%purity) was obtained.

H NMR: (400 MHz, CDCl₃) δ 11.51 (s, 1H) , 7.85 -7.83 (m, 2H) , 7.53 -7.47 (m, 3H) , 7.40 -7.36 (m, 2H) , 7.25 -7.23 (m, 2H) , 6.38 (d, J = 7.6 Hz, 1H) , 6.32 (s, 1H) , 4.73 -4.68 (m, 1H) , 4.53 -4.50 (m, 1H) , 4.40 -4.35 (m, 2H) , 4.14 -4.11 (m, 1H) , 3.81 (s, 3H) , 3.77 (s, 2H) , 3.73 -3.70 (m, 1H) , 3.02 -2.89 (m, 2H) , 2.69 -2.63 (m, 1H) , 2.49 -2.45 (m, 2H) , 2.31 -2.26 (m, 1H) , 1.75 -1.70 (m, 1H) , 1.60 (t, J = 6.8 Hz, 3H) .

LC-MS: (M+H) ⁺: 637.2

Compound 78b was prepared in similar way as Compound 78a except that compound 78-5b were used instead of compound 78-5a.

H NMR: (400 MHz, CDCl₃) δ 11.51 (s, 1H) , 7.85 -7.83 (m, 2H) , 7.53 -7.48 (m, 3H) , 7.40 -7.36 (m, 2H) , 7.25 -7.23 (m, 2H) , 6.38 (d, J = 7.6 Hz, 1H) , 6.32 (s, 1H) , 4.73 -4.69 (m, 1H) , 4.53 -4.50 (m, 1H) , 4.40 -4.35 (m, 2H) , 4.14 -4.11 (m, 1H) , 3.81 (s, 3H) , 3.77 (s, 2H) , 3.74 -3.70 (m, 1H) , 2.99 -2.93 (m, 2H) , 2.69 -2.63 (m, 1H) , 2.49 -2.45 (m, 2H) , 2.31 -2.26 (m, 1H) , 1.75 -1.69 (m, 1H) , 1.60 (t, J = 6.8 Hz, 3H) .

LC-MS: (M+H) ⁺: 637.3

(S) -N- (4- (3-amino-1-methyl-6- (6-oxohexahydropyrrolo [1, 2-a] pyrazin-2 (1H) -yl) -1H-pyrazolo [3, 4-b] pyridin-4-yl) phenyl) -4-ethoxy-5'-fluoro-2-oxo-2H- [1, 2'-bipyridine] -3-carboxamide and (R) -N- (4- (3-amino-1-methyl-6- (6-oxohexahydropyrrolo [1, 2-a] pyrazin-2 (1H) -yl) -1H-pyrazolo [3, 4-b] pyridin-4-yl) phenyl) -4-ethoxy-1- (4-fluorophenyl) -2-oxo-1, 2-dihydropyridine-3-carboxamide (Compounds 81a and 81b)

Compounds 81a and 81b were prepared in similar way as Compounds 78a and 78b except that compound 67-2 was used instead of compound 78-6.

Compounds 81a

H NMR: (400 MHz, CDCl₃) δ 11.17 (s, 1H) , 8.45 (d, J = 2.8 Hz, 1H) , 7.96 (d, J = 8.0 Hz, 1H) , 7.91 -7.87 (m, 1H) , 7.86 -7.83 (m, 2H) , 7.66 -7.61 (m, 1H) , 7.51 -7.49 (m, 2H) , 6.43 (d, J = 8.0 Hz, 1H) , 6.33 (s, 1H) , 4.73 -4.70 (m, 1H) , 4.53 -4.50 (m, 1H) , 4.41 -4.36 (m, 2H) , 4.14 -4.12 (m, 1H) , 3.82 (s, 3H) , 3.78 (s, 2H) , 3.74 - 3.69 (m, 1H) , 3.03 -2.90 (m, 2H) , 2.69 -2.63 (m, 1H) , 2.49 -2.45 (m, 2H) , 2.33 -2.24 (m, 1H) , 1.78 -1.68 (m, 1H) , 1.60 -1.58 (m, 3H) .

LC-MS: (M+H) ⁺: 638.2

Compounds 81b

H NMR: (400 MHz, CDCl₃) δ 11.18 (s, 1H) , 8.45 (d, J = 2.8 Hz, 1H) , 7.97 (d, J = 8.0 Hz, 1H) , 7.91 -7.88 (m, 1H) , 7.86 -7.84 (m, 2H) , 7.66 -7.61 (m, 1H) , 7.52 -7.49 (m, 2H) , 6.43 (d, J = 8.0 Hz, 1H) , 6.33 (s, 1H) , 4.73 -4.70 (m, 1H) , 4.53 -4.51 (m, 1H) , 4.41 -4.36 (m, 2H) , 4.14 -4.12 (m, 1H) , 3.82 (s, 3H) , 3.78 (s, 2H) , 3.73 - 3.72 (m, 1H) , 3.03 -2.90 (m, 2H) , 2.69 -2.64 (m, 1H) , 2.50 -2.45 (m, 2H) , 2.31 -2.26 (m, 1H) , 1.78 -1.67 (m, 1H) , 1.60 -1.58 (m, 3H) .

LC-MS: (M+H) ⁺: 638.2

Example 43

Biochemical Assays

Phospho-MER Inhibition Assay in M2 Macrophages

The ability of compounds to inhibit MER phosphorylation were evaluated in M2 macrophages.

Human peripheral blood mononuclear cells (PBMCs) were purchased from SailyBio Co., LTD. PBMCs were washed with PBS and monocytes were isolated using Human CD14 Positive Selection Kit II (STEMCELL, #17858) following the manufacturer's protocol as specified. M0 macrophages were generated by culturing the monocytes at 37 ℃, 5 %CO₂ for 5 days in RPMI-1640 + 10%FBS (Gibco, #10099141) +100 U/mL penicillin + 100 ug/mL streptomycin (Gibco, #15140122) , supplemented with 100 ng/mL macrophage colony stimulating factor (M–CSF, Peprotech, #300-25) . M0 macrophages were polarized to M2 macrophages by culturing for additional 2 days in fresh media supplemented with 100 ng/mL M–CSF, Human IL-10 (Peprotech, #200-10) and Human TGF-β1 (Peprotech, #100-21) .

M2 macrophages were detached with Accutase solution (SIGMA, #A6964) at 37℃ for 20 minutes. Cells were seeded in 96-well plates at 3E5 cells/well in RPMI-1640 + 10%FBS. Compounds were prepared at 30μM and diluted to a 4-fold serial dilutions. The macrophages were treated with Compounds for 1 hours at 37 ℃, 5 %CO₂. Five ug/mL anti-MER activation antibody was added to the macrophages for an additional 30 minutes to induce MER phosphorylation, then the cells were washed with cold PBS (Adama#C8020-500mL) . All PBS was carefully aspirated from the wells. Macrophages were lysed with 150 uL/well of 1× NP40 cell lysis buffer (Invitrogen, #FNN0021) containing Protease/Phophatase Inhibitor (Cell Signaling, #5872S) for 30 mins on ice. The lysates were centrifuged at 2000 xg for 3 minutes at 4 ℃ and the supernatant were applied to phospho-Mer analysis using Human Phospho-Mer DuoSet IC ELISA kit (R&D, #DYC2579E) following the instructions included with the kit.

In vitro kinase assay

All compounds were initially diluted in DMSO to 50 × their final concentrations. 50 μL of the compound dilution was transferred to each well in a 384-well Echo plate (Labcyte PP-0200) . All compounds were transferred to one well in a 384-well Echo plate and serially dilute the compound by 4-fold dilution of 100%DMSO in the next well and so forth for a total of 10 concentrations by precision. 50μl of 100%DMSO were added to two empty wells for no compound control and no enzyme control in the same 384-well Echo plate. Mark the plate as a source plate. 400nL of each well from the 384-well Echo plate were transferred to a 384-well assay plate (Corning 3573) in duplicates. To each well, 10 μL of enzyme solution containing either AXL, MER or TYRO3 (final concentrations 6, 1 or 1.5 nM, respectively) in 1 × Kinase buffer (50 mM HEPES pH 7.5, 10 mM MgCl2, 2 mM DTT and 0.01%Brij-35) and the mix incubated at room temperature for 10 min. To initiate each reaction, 10 μL of peptide solution containing FAM-labelled peptide (final concentrations 3000 nM FAM-P8 for AXL, MER and TYRO3) and ATP (final concentrations 81, 32 or 33 μM for AXL, MER or TYRO3, respectively) in 1 × Kinase buffer was added to each well. All reactions were incubated at 28℃ for 1 h and then terminated by the addition of 25 μL stop buffer (100 mM HEPES pH 7.5, 50 mM EDTA, 0.2%Coating Reagent #3 and 0.015%Brij-35) .

All samples were then subjected to analysis using Caliper EZ ReaderⅡ (Down stream voltages: -500V, Up stream voltages: -2250V, Base pressure -0.5 PSI, Screen pressure -1.2 PSI) to read conversion values. Conversion values were transformed into %inhibition of kinase activity using the formula: %Inhibition = [ (MA –X) / (MA –MI) ] × 100%where MA = conversion value of DMSO only controls, MI =conversion value of no enzyme controls and X = conversion value at any given compound dose. IC50 values were then calculated by plotting dose-response curves and then using the Xlfit application in Excel software.

The compounds provided herein were found to be inhibitors of one or more of AXL, MER, and TYRO3. In particular, the compounds provided herein are selective for AXL and MER over TYRO3. IC₅₀ data of exemplary compounds of the present disclosure are provided below in Table 2.

Table 2

The foregoing description is considered as illustrative only of the principles of the present disclosure. Further, since numerous modifications and changes will be readily apparent to those skilled in the art, it is not desired to limit the invention to the exact construction and process shown as described above. Accordingly, all suitable modifications and equivalents may be considered to fall within the scope of the invention as defined by the claims that follow.

Claims

A compound having Formula (II) :

or a pharmaceutical acceptable salt thereof,

wherein

Ring A is phenyl or 6-membered heteroaryl comprising 1 or 2 nitrogen atoms;

Ring B is phenyl or 6-membered heteroaryl comprising 1 or 2 nitrogen atoms, each of which is optionally substituted with one or two R²;

Ring C is cycloalkyl, heterocyclyl, aryl or heteroaryl, each of which is optionally substituted with one or more R^a;

W is CH, N, O, or S;

R¹ is cycloalkyl, heterocyclyl, aryl or heteroaryl, each of which is optionally substituted with one or more R^b;

R² is halogen, hydroxyl, cyano, amino or alkyl;

R³ is null, hydrogen, oxo, alkyl or haloalkyl;

R⁴ is NH₂ or haloalkyl;

each R^a is independently selected from the group consisting of hydrogen, oxo, halogen, hydroxyl, cyano, amino, alkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, and –C (=O) R^c; or

two adjacent R^a together with the atoms they attached form a cycloalkyl or heterocyclyl;

each R^b is independently selected from oxo, hydroxyl, alkyl, alkoxyl, haloalkyl, haloalkoxyl, cycloalkyl, heterocyclyl, aryl, heteroaryl or –C (O) NH-R^c, wherein the cycloalkyl, heterocyclyl, aryl, and heteroaryl are optionally substituted with one or more groups independently selected from halogen, hydroxyl, cyano, amino or alkyl; and

R^c is alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein the cycloalkyl, heterocyclyl, aryl, and heteroaryl are optionally substituted with one or more groups independently selected from halogen, hydroxyl, cyano, amino or alkyl.
The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein Ring A is phenyl optionally substituted with one or two R².
The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein Ring A is a 6-membered heteroaryl comprising 1 nitrogen atom.
The compound of claim 3, or a pharmaceutically acceptable salt thereof, whereinis selected from the group consisting of:
The compound of any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, wherein W is N.
The compound of any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, wherein W is O or S.
The compound of any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, wherein W is CH.
The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein Ring B is phenyl.
The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein Ring B is 6-membered heteroaryl comprising 1 or 2 nitrogen atoms.
The compound of claim 9, or a pharmaceutically acceptable salt thereof, wherein Ring B is pyridinyl.
The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R¹ is cycloalkyl optionally substituted with one or more R^b.
The compound of claim 11, or a pharmaceutically acceptable salt thereof, wherein R¹ is cyclopropyl optionally substituted with one or more R^b.
The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R¹ is heterocyclyl optionally substituted with one or more R^b.
The compound of any one of claims 11-13, or a pharmaceutically acceptable salt thereof, wherein each R^b is independently selected from oxo, hydroxyl, alkyl, alkoxyl, haloalkyl, haloalkoxyl, aryl, heteroaryl or –C (O) NH-R^c, wherein the aryl and heteroaryl are optionally substituted with one or more groups independently selected from halogen, hydroxyl, or cyano.
The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R¹ is selected from the group consisting of:
The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R² is halogen.
The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R² is fluoro.
The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein Ring C is heterocyclyl or aryl, each of which is optionally substituted with one or more R^a.
The compound of claim 18, or a pharmaceutically acceptable salt thereof, wherein Ring C is 6-membered heterocyclyl optionally substituted with one or more R^a.
The compound of claim 19, or a pharmaceutically acceptable salt thereof, wherein Ring C is piperidinyl optionally substituted with one or more R^a.
The compound of claim 20, or a pharmaceutically acceptable salt thereof, wherein Ring C is 4-piperidinyl optionally substituted with one or more R^a.
The compound of claim 19, or a pharmaceutically acceptable salt thereof, wherein Ring C is piperazinyl optionally substituted with one or more R^a.
The compound of claim 18, or a pharmaceutically acceptable salt thereof, wherein Ring C is 7-to 9-membered heterocyclyl optionally substituted with one or more R^a.
The compound of claim 23, or a pharmaceutically acceptable salt thereof, wherein Ring C is octahydroindolizinyl, octahydropyrrolo [1, 2-a] pyrazinyl, or 2, 6-diazaspiro [3.3] heptanyl, each optionally substituted with one or more R^a.
The compound of any one of preceding claims , or a pharmaceutically acceptable salt thereof, wherein each R^a is independently selected from the group consisting of hydrogen, oxo, halogen, hydroxyl, cyano, amino, alkyl, and –C (=O) R^c.
The compound of any one of preceding claims, or a pharmaceutically acceptable salt thereof, wherein each R^a is independently selected from the group consisting of hydrogen, halogen, hydroxyl, cyano, amino, alkyl, and –C (=O) R^c.
The compound of claim 26, or a pharmaceutically acceptable salt thereof, wherein R^c is alkyl, C_3-8 cycloalkyl, 3-8 membered heterocyclyl or 5-6 membered heteroaryl.
The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein Ring C is aryl optionally substituted with one or more R^a.
The compound of claim 28, or a pharmaceutically acceptable salt thereof, wherein Ring C is phenyl optionally substituted with one or more R^a.
The compound of claim 28 or 29, or a pharmaceutically acceptable salt thereof, wherein each R^a is independently selected from the group consisting of halogen, cyano, alkyl, C_3-8 cycloalkyl and 5-6 membered heteroaryl.
The compound of claim 28 or 29, or a pharmaceutically acceptable salt thereof, wherein two adjacent R^a together with the atoms they attached form C_5-8 cycloalkyl or 5-8 membered heterocyclyl.
The compound of any one of preceding claims, or a pharmaceutically acceptable salt thereof, wherein Ring C is selected from the group consisting of:
The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R³ is null.
The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R³ is hydrogen.
The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R³ is oxo.
The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R³ is alkyl.
The compound of claim 36, or a pharmaceutically acceptable salt thereof, wherein R³ is methyl or ethyl.
The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R³ is haloalkyl.
The compound of claim 38, or a pharmaceutically acceptable salt thereof, wherein R³ is difluoromethyl, trifluoroethyl or difluoroethyl.
The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R⁴ is NH₂.
The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R⁴ is haloalkyl.
The compound of claim 41, or a pharmaceutically acceptable salt thereof, wherein R⁴ is difluoromethyl.
The compound of claim 1 selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.
A pharmaceutical composition comprising the compound of any one of claims 1-43, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.
A method for inhibiting a TAM kinase, comprising contacting the TAM kinase with the compound of any one of claims 1-43, or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 44.
A method for inhibiting AXL and MER kinases, comprising contacting the AXL and MER kinases with the compound of any one of claims 1-43, or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 44.
A method for treating a disease or disorder mediated by TAM in a subject in need thereof, comprising administering an effective amount of the compound of any one of claims 1-43, or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 44 to the subject.
The method of claim 47, wherein the disease or disorder mediated by TAM is cancer.
The method of claim 48, wherein the cancer is selected from hepatocellular cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, gastric cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, prostate cancer, esophageal cancer, gall bladder cancer, pancreatic cancer, thyroid cancer, skin cancer, leukemia, multiple myeloma, chronic lymphocytic lymphoma, adult T cell leukemia, B-cell lymphoma, acute myelogenous leukemia, Hodgkin’s or non-Hodgkin’s lymphoma, Waldenstrom’s Macroglobulinemia, hairy cell lymphoma, Burkett’s lymphoma, glioblastoma, melanoma, and rhabdomyosarcoma.
The method of claim 49, wherein the cancer is lung cancer, prostate cancer, colon cancer, breast cancer, melanoma, renal cell carcinoma, multiple myeloma, gastric cancer, or rhabdomyosarcoma.