CN117321055A

CN117321055A - Modulators of STING (interferon gene stimulators)

Info

Publication number: CN117321055A
Application number: CN202280036065.4A
Authority: CN
Inventors: K·S·加吉瓦拉; 许璨宇; M·贾拉伊; R·L·帕特曼; E·Y·芮; 孙建敏; M·J·怀瑟斯
Original assignee: Pfizer Inc
Current assignee: Pfizer Inc
Priority date: 2021-03-18
Filing date: 2022-03-15
Publication date: 2023-12-29

Abstract

Provided herein are compounds of formula (I) and pharmaceutically acceptable salts thereof, methods of preparing such compounds, compositions containing such compounds, and the use of such compounds as modulators of STING (interferon gene stimulators).

Description

Modulators of STING (interferon gene stimulators)

Technical Field

The present invention relates to compounds of formula (a), (I), (II), (III-a), (IV), (V), (VI), (VII), (VIII) or (IX) and pharmaceutically acceptable salts thereof, pharmaceutical compositions comprising such compounds or salts thereof, and compounds for use as medicaments. The compounds, salts and compositions of the invention are useful for treating or ameliorating diseases or disorders such as inflammatory diseases and disorders, allergic diseases, autoimmune diseases, infectious diseases, abnormal cell growth (including cancer), and as vaccine adjuvants.

Background

The innate immune system is the first line of defense, which is triggered by Pattern Recognition Receptors (PRRs) upon detection of ligands from pathogens and associated molecular patterns of injury. An increasing number of these receptors have been identified, including sensors for double-stranded DNA and unique nucleic acids known as Cyclic Dinucleotides (CDNs). Activation of PRR results in upregulation of genes involved in inflammatory responses, including type 1 interferons (also known as IFN or INF), pro-inflammatory cytokines and chemokines that inhibit pathogen replication and promote adaptive immunity.

The adapter protein STING (also known as TMEM 173) has been identified as a central signaling molecule in an innate immune sensing pathway responsive to cytosolic nucleic acids. STING is critical for responding to cytosolic DNA from pathogens or host-derived cytosolic DNA. Activation of STING by CDNs in response to cytosolic DNA results in up-regulation of IRF3 and nfkb pathways, leading to induction of interferon beta (INF- β) and other cytokines. Barber, "Sting: injection, inflammation and cancer," Nat. Rev. Immun.,2015,15, page 760.

CDNs were originally identified as bacterial messengers responsible for controlling numerous responses in prokaryotic cells. Bacterial CDNs (such as c-di-GMP) are symmetrical molecules characterized by two 3',5' phosphodiester linkages. Direct activation of STING by bacterial CDNs has recently been demonstrated by X-ray crystallography (Burdette d.l. and Vance r.e., nature Immunology, 2013:1419-26). Bacterial CDNs are therefore of interest as potential vaccine adjuvants (Libanova r. Et al Microbial Biotechnology 2012:5, 168-176). Recently, it has been demonstrated that the response to cytosolic DNA involves the production of endogenous CDNs by an enzyme called cyclic guanine adenine synthase (cGAS), thereby producing a novel mammalian CDN signaling molecule identified as cyclic guanine adenine monophosphate (cGAMP), which binds to and activates STING. The interaction of cGAMP with STING has also been demonstrated by X-ray crystallography. Unlike bacterial CDNs, cGAMP is an asymmetric molecule characterized by a mixture of 2',5' and 3',5' phosphodiester linkages. Like bacterial CDNs, cGAMP activates STING, resulting in induction of type 1 interferon (type 1 INF). The effect of type 1 INF in response to invading pathogens is well documented. Recombinant interferon alpha (ifnα) is the first biologic therapeutic to be approved and has become an important therapy for viral infections and cancers. INF is also known to be a potent regulator of immune response, acting on cells of the immune system.

Administration of small molecule compounds that can stimulate an innate immune response (including activation of type 1 INF and other cytokines) can be an important strategy for the treatment and prevention of human diseases including viral infections and cancers. Such immunomodulating strategies have the potential to identify compounds that are useful in the treatment of diseases and disorders such as inflammatory diseases and disorders, allergic diseases, autoimmune diseases, infectious diseases, abnormal cell growth (including cancer), and as vaccine adjuvants.

STING remains an attractive target for modulation with small molecules in view of its role in modulating various biological processes. There remains a need to identify other compounds that bind STING. There remains a need to identify other compounds that activate STING. There remains a need to identify other compounds that have sufficient cell permeability. Furthermore, there remains a need for compounds that bind to STING and/or activate STING and that can be used as therapeutic agents.

The human bioavailability of a therapeutic agent, including, for example, the oral bioavailability of a therapeutic agent, is determined by factors such as the absorption, distribution, metabolism, and excretion properties of the therapeutic agent. There remains a need to identify compounds that bind to STING and/or activate STING and that are bioavailable. There remains a need to identify compounds that bind to STING and/or activate STING and that have oral bioavailability. Thus, there remains a need to identify compounds that bind to STING and/or activate STING and that have suitable properties (such as, but not limited to, solubility, permeability, absorption, pharmacokinetics, etc.).

Summary of The Invention

The present invention provides, in part, novel compounds and pharmaceutically acceptable salts thereof. Such compounds, upon incubation with human Dendritic Cells (DCs), may bind STING, activate STING and/or induce type 1 INF and/or other cytokines and/or co-stimulatory factors and thus be useful in treating or ameliorating diseases or disorders such as inflammatory diseases and disorders, allergic diseases, autoimmune diseases, infectious diseases, abnormal cell growth (including cancer), and as vaccine adjuvants.

Also provided are pharmaceutical compositions and medicaments comprising the compounds or salts of the invention alone or in combination with other therapeutic or palliative agents. The invention also provides, in part, methods of preparing the novel compounds, salts and compositions thereof, as well as methods of using the foregoing.

In one aspect, the present invention provides a compound of formula (I):

or a pharmaceutically acceptable salt thereof, wherein

Represents two conjugated double bonds in a 5 membered heteroaryl ring;

X ¹ selected from CH and N;

X ² selected from CH and N;

R ¹ selected from C ₁ -C ₄ Alkyl, cyclopropyl, cyclobutyl, C ₁ -C ₂ Alkylene- (cyclopropyl) and C ₁ -C ₂ Alkylene- (cyclobutyl) radicals, C ₁ -C ₄ Alkyl, cyclopropyl, cyclobutyl, C ₁ -C ₂ Alkylene- (cyclopropyl) or C ₁ -C ₂ Alkylene- (cyclobutyl) is optionally substituted by 1, 2 or 3 substituents each independently selected from halogen, hydroxy and-OC ₁ -C ₄ An alkyl group;

Z ¹ 、Z ² and Z ³ Selected such that:

Z ¹ is C, Z ² Is NR ² And Z is ³ Is CR (CR) ⁴ The method comprises the steps of carrying out a first treatment on the surface of the Or (b)

Z ¹ Is N, Z ² Is CR (CR) ³ And Z is ³ Is CR (CR) ⁴ The method comprises the steps of carrying out a first treatment on the surface of the Or (b)

Z ¹ Is C, Z ² Is CR (CR) ³ And Z is ³ Is NR ² ；

R ² Selected from C ₁ -C ₄ Alkyl, cyclopropyl, cyclobutyl, oxetanyl, C ₁ -C ₂ Alkylene- (cyclopropyl), C ₁ -C ₂ Alkylene- (cyclobutyl) and C ₁ -C ₂ Alkylene- (oxetanyl), said C ₁ -C ₄ Alkyl, cyclopropyl, cyclobutyl, oxetanyl, C ₁ -C ₂ Alkylene- (cyclopropyl), C ₁ -C ₂ Alkylene- (cyclobutyl) or C ₁ -C ₂ Alkylene- (oxetanyl) groups are optionally substituted with 1, 2, 3, 4, 5 or 6 substituents each independently selected from halogen, hydroxy, -CN and-OC ₁ -C ₄ An alkyl group;

R ³ selected from halogen, hydroxy, -CN, -OC ₁ -C ₄ Alkyl, C ₁ -C ₄ Alkyl, cyclopropyl, cyclobutyl, oxetanyl, C ₁ -C ₂ Alkylene- (cyclopropyl), C ₁ -C ₂ Alkylene- (cyclobutyl) and C ₁ -C ₂ Alkylene- (oxetanyl), said-OC ₁ -C ₄ Alkyl, C ₁ -C ₄ Alkyl, cyclopropyl, cyclobutyl, oxetanyl, C ₁ -C ₂ Alkylene- (cyclopropyl), C ₁ -C ₂ Alkylene- (cyclobutyl) or C ₁ -C ₂ Alkylene- (oxetanyl) groups are optionally substituted with 1, 2, 3, 4, 5 or 6 substituents each independently selected from halogen, hydroxy, -CN and-OC ₁ -C ₄ An alkyl group;

R ⁴ selected from H, halogen, hydroxy, C ₁ -C ₄ Alkyl and-OC ₁ -C ₄ Alkyl, said C ₁ -C ₄ Alkyl or-OC ₁ -C ₄ Alkyl is optionally substituted with 1, 2 or 3 substituents each independently selected from halogen and hydroxy;

R ⁵ selected from H, halogen, hydroxy, -CN, C ₁ -C ₄ Alkyl and-OC ₁ -C ₄ Alkyl, said C ₁ -C ₄ Alkyl or-OC ₁ -C ₄ Alkyl is optionally substituted with 1, 2 or 3 substituents each independently selected from halogen and hydroxy; and is also provided with

R ⁶ Selected from C ₁ -C ₄ Alkyl, cyclopropyl, cyclobutyl, oxetanyl, C ₁ -C ₂ Alkylene- (cyclopropyl), C ₁ -C ₂ Alkylene- (cyclobutyl) and C ₁ -C ₂ Alkylene- (oxetanyl), said C ₁ -C ₄ Alkyl, cyclopropyl, cyclobutyl, oxetanyl, C ₁ -C ₂ Alkylene- (cyclopropyl), C ₁ -C ₂ Alkylene- (cyclobutyl) or C ₁ -C ₂ Alkylene- (oxetanyl) groups are optionally substituted with 1, 2 or 3 substituents each independently selected from halogen, hydroxy, -CN and-OC ₁ -C ₄ An alkyl group.

In another aspect, the invention provides a pharmaceutical composition comprising a compound of any one of the formulae described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

In another aspect, the invention provides a compound of any one of the formulae described herein, or a pharmaceutically acceptable salt thereof, for use as a medicament.

In another aspect, the invention provides methods of treatment and uses comprising administering a compound of any of the formulae described herein, or a pharmaceutically acceptable salt thereof.

Also embodied in the invention is a method for treating abnormal cell growth in a mammal, the method comprising administering to the mammal a therapeutically effective amount of a compound of any one of the formulae described herein, or a pharmaceutically acceptable salt thereof.

Still other embodiments of the invention include embodiments wherein a method of upregulating STING activity in a mammal is provided, said method comprising the step of administering to said mammal an effective amount of a compound or salt as described herein; and/or a method of increasing the level of interferon-beta in a mammal, comprising the step of administering to said mammal an effective amount of a compound or salt as described herein. In one embodiment, the mammal is a human. In such embodiments, the mammal is a human in need of treatment.

Still other embodiments of the present invention include embodiments wherein a method of activating STING in a mammal is provided, the method comprising the step of administering to the mammal an effective amount of a compound or salt described herein. Also provided are methods of stimulating an innate immune response in a mammal comprising the step of administering to the mammal an effective amount of a compound or salt described herein. In one embodiment, the mammal is a human. In such embodiments, the mammal is a human in need of treatment.

Detailed Description

The following terms, as used in the specification and claims, have the meanings discussed below unless otherwise indicated. Variables defined in this section, such as R, X, n, etc., are only for reference in this section and are not intended to have the same meaning as may be used outside of this defined section. Furthermore, many of the groups defined herein may be optionally substituted. The list of typical substituents in this definition section is exemplary and is not intended to limit the substituents defined elsewhere in this specification and claims.

As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. For example, a "substituent includes one or more substituents.

"alkoxy" means-O-alkyl, wherein, unless otherwise defined, alkyl is preferably C ₁ -C ₈ 、C ₁ -C ₇ 、C ₁ -C ₆ 、C ₁ -C ₅ 、C ₁ -C ₄ 、C ₁ -C ₃ 、C ₁ -C ₂ Or C ₁ Alkyl groups, and may be represented, for example, as-OC ₁ -C ₄ An alkyl group.

"alkyl" refers to a saturated monovalent aliphatic hydrocarbon radical comprising straight and branched chain groups having 1 to 20 carbon atoms ("C" unless otherwise defined ₁ -C ₂₀ Alkyl "), preferably 1 to 12 carbon atoms (" C ") ₁ -C ₁₂ Alkyl "), more preferably 1 to 8 carbon atoms (" C ") ₁ -C ₈ Alkyl "), or 1 to 6 carbon atoms (" C ₁ -C ₆ Alkyl "), or 1 to 4 carbon atoms (" C ₁ -C ₄ Alkyl "). Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl (also known as 2-propyl), n-butyl, isobutyl, t-butyl, pentyl, neopentyl and the like. Alkyl groups may be substituted or unsubstituted. In particular, typical substituents include cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halogen, carbonyl, thiocarbonyl, O-carbamoyl, unless otherwise indicatedN-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl, C-amido, N-amido, C-carboxyl, O-carboxyl, nitro, oxo, thio (thioxo), amino and-NR ^x R ^y Wherein R is ^x And R is ^y Is, for example, hydrogen, alkyl, cycloalkyl, aryl, carbonyl, acetyl, sulfonyl, trifluoromethanesulfonyl, and (when combined) a 5-or 6-membered heteroalicyclic ring. "haloalkyl" refers to an alkyl group having one or more halo substituents. In certain embodiments, haloalkyl has 1, 2, 3, 4, 5, or 6 halogen substituents. In certain embodiments, haloalkyl has 1, 2, or 3 halogen substituents. In certain embodiments, the haloalkyl is fluoroalkyl.

"alkylene" refers to a divalent hydrocarbon radical having the indicated number of carbon atoms, which may link two other groups together. In certain embodiments, the alkylene is- (CH) ₂ ) _n -, where n is 1 to 8. In certain embodiments, n is 1-4. In certain embodiments, n is 1-2. In the cases specified therein, the alkylene groups may also be substituted by other groups. Typical substituents include the same groups described herein as suitable for alkyl groups. The open valences of the alkylene groups need not be located at opposite ends of the chain. Where alkylene groups are described as optionally substituted, substituents include those typically present on alkyl groups as described herein. For example, "C ₁ -C ₂ Alkylene "means-CH ₂ -、-CH ₂ CH ₂ -or-CH (CH) ₃ ) -, which alkylene group may be substituted or unsubstituted as defined herein.

"amino" means-NH ₂ A group.

"cyano" refers to a-C.ident.N group. Cyano can be represented as-CN.

As used interchangeably herein, the term "cycloalkyl" or "carbocyclic" refers to a non-aromatic, monocyclic, fused or bridged bicyclic or tricyclic carbocyclic group, in certain embodiments, containing from 3 to 10 carbon atoms. The cyclic hydrocarbon groups used herein may optionally contain one or two double bonds. The term "cycloalkyl" also includes spiro carbocyclic groups, including Including polycyclic ring systems linked by a single atom. The term "C ₃ -C ₁₀ Cyclic hydrocarbon radicals "," C ₃ -C ₇ Cyclic hydrocarbon radicals "," C ₃ -C ₆ Cyclic hydrocarbon radicals "," C ₃ -C ₅ Cyclic hydrocarbon radicals "," C ₃ -C ₄ Cycloalkyl "and" C ₅ -C ₇ Cycloalkyl "contains 3 to 10, 3 to 7, 3 to 6, 3 to 5, 3 to 4, and 5 to 7 carbon atoms, respectively. Cyclic hydrocarbon groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, octahydropentalenyl, octahydro-1H-indenyl, bicyclo [2.2.1]Heptyl, bicyclo [3.2.1]Octyl, bicyclo [5.2.0]Nonylalkyl, adamantyl, cyclohexadienyl, adamantyl, cycloheptyl, cycloheptatrienyl, and the like. The cyclic hydrocarbon group may be substituted or unsubstituted. Typical substituents include the same groups described herein as suitable for alkyl groups.

"halogen" or the prefix "halo" refers to fluorine, chlorine, bromine and iodine. In certain embodiments, halogen or halo refers to fluoro or chloro. In certain embodiments, halogen or halo refers to fluoro.

The terms "heterocyclyl", "heterocyclic" or "heteroalicyclic" may be used interchangeably herein to refer to a non-aromatic, monocyclic, saturated or partially unsaturated, fused or bridged bi-or tri-or spiro-cyclic group containing, in certain embodiments, a total of 3-10 ring atoms, 3-7 ring atoms or 4-6 ring atoms, of which 1, 1-2, 1-3 or 1-4 ring atoms are heteroatoms. The heteroatoms are independently selected from nitrogen, oxygen and sulfur, and wherein the sulfur atoms may optionally be oxidized by one or two oxygen atoms, with the remaining ring atoms being carbon, provided that such ring systems may not contain two adjacent oxygen atoms or two adjacent sulfur atoms. The heterocycle may also be substituted with oxo (=o) groups at any available carbon atom. The ring may also have one or more double bonds. The heterocycle may be fused to one or more other heterocycles or carbocycles, and the fused ring may be saturated, partially unsaturated or aromatic. Furthermore, such groups may be bonded to the remainder of the compounds of the embodiments disclosed herein through carbon atoms or heteroatoms (if possible). Examples of heterocyclic groups include, but are not limited to:

The heterocyclic group may be optionally substituted. Typical substituents include those described herein as suitable for use with alkyl, aryl or heteroaryl groups. In addition, the ring N atom may be optionally substituted with groups suitable for amines (e.g., alkyl, acyl, carbamoyl, sulfonyl substituents).

"hydroxy" refers to an-OH group.

"oxo" refers to a ═ O group.

"thio" refers to the ═ S group.

"aryl" or "aromatic" refers to an optionally substituted monocyclic, biaryl, or fused bicyclic or polycyclic ring system having well known aromatic characteristics, wherein at least one ring contains a fully conjugated pi-electron system. Typically aryl groups contain 6 to 20 carbon atoms ("C ₆ -C ₂₀ Aryl ") as ring members, preferably 6 to 14 carbon atoms (" C) ₆ -C ₁₄ Aryl ") or more preferably 6 to 12 carbon atoms (" C ₆ -C ₁₂ Aryl "). The fused aryl group may include an aryl ring (e.g., a benzene ring) fused to another aryl ring or fused to a saturated or partially unsaturated carbocyclic or heterocyclic ring. The point of attachment to the base molecule on such a fused aryl ring system may be a C atom of the aromatic portion of the ring system or a C or N atom of a non-aromatic portion. Examples of aryl groups include, but are not limited to, phenyl, biphenyl, naphthyl, anthryl, phenanthryl, indanyl, indenyl, and tetrahydronaphthyl. The aryl group may be unsubstituted or substituted, as further described herein.

Similarly, "heteroaryl" or "heteroaromatic" refers to a monocyclic, heteroaryl or fused bicyclic or polycyclic ring system of well known aromatic character containing the indicated number of ring atoms and including at least one heteroatom selected from N, O and S as a ring member in an aromatic ring. The inclusion of heteroatoms allows for aromaticity of the 5-membered ring and the 6-membered ring. Typically, heteroaryl groups contain 5 to 20 ring atoms ("5 to 20 membered heteroaryl"), preferably 5 to 14 ring atoms ("5 to 14 membered heteroaryl"), and more preferably 5 to 12 ring atoms ("5 to 12 membered heteroaryl"). The heteroaryl ring is attached to the base molecule through a ring atom of the heteroaromatic ring, thereby preserving aromaticity. Thus, a 6 membered heteroaryl ring may be attached to the base molecule through a ring C atom, while a 5 membered heteroaryl ring may be attached to the base molecule through a ring C or N atom. Examples of unsubstituted heteroaryl groups often include, but are not limited to, pyrrole, furan, thiophene, pyrazole, imidazole, isoxazole, oxazole, isothiazole, thiazole, triazole, oxadiazole, thiadiazole, tetrazole, pyridine, pyridazine, pyrimidine, pyrazine, benzofuran, benzothiophene, indole, benzimidazole, indazole, quinoline, isoquinoline, purine, triazine, naphthyridine, and carbazole. In certain embodiments, the 5-or 6-membered heteroaryl is selected from the group consisting of pyrrolyl, furanyl, thienyl, pyrazolyl, imidazolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, triazolyl, pyridinyl, pyrimidinyl, pyrazinyl, and pyridazinyl rings. The heteroaryl group may be unsubstituted or substituted, as further described herein.

Illustrative examples of monocyclic heteroaryl groups include, but are not limited to:

illustrative examples of fused ring heteroaryl groups include, but are not limited to:

aryl and heteroaryl moieties described herein as optionally substituted may be substituted with one or more substituents independently selected unless otherwise indicated. The total number of substituents may be equal to the total number of hydrogen atoms on the aryl, heteroaryl or heterocyclyl moiety, provided that such substitution is of chemical significance and retains aromaticity in the case of aryl and heteroaryl rings. An optionally substituted aryl, heteroaryl, or heterocyclyl group typically contains 1 to 5 optional substituents, in certain embodiments 1 to 4 optional substituents, in certain embodiments 1 to 3 optional substituents, and in certain other embodiments 1 to 2 optional substituents. Typical substituents include alkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halogen, carbonyl, thiocarbonyl, C-carboxy, O-carbamoyl, N-carbamoyl, C-amido, N-amido, nitro, oxo, thio and amino.

"optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

The terms "optionally substituted" and "substituted or unsubstituted" may be used interchangeably to indicate that a particular group described may have no non-hydrogen substituents (i.e., unsubstituted), or that the group may have one or more non-hydrogen substituents (i.e., substituted). If not otherwise specified, the total number of substituents which may be present is equal to the H atom present on the unsubstituted form of the group describedThe number of subunits, provided that such substitution produces chemical significance. In the case of optional substituents linked by a double bond (such as oxo (=o) substituents), the group occupies two available valencies, such that the total number of other substituents that can be included is reduced by two. Where the optional substituents are independently selected from the list of alternatives, the selected groups may be the same or different. For example, "a heterocyclic group optionally substituted with an alkyl group" means that an alkyl group may be, but is not necessarily, present, and the description includes cases where the heterocyclic group is substituted with an alkyl group and cases where the heterocyclic group is not substituted with an alkyl group. In certain embodiments, the specified group is substituted with 1-6 non-hydrogen substituents. In certain embodiments, the specified group is substituted with 1-4 non-hydrogen substituents. In certain embodiments, the specified group is substituted with 1-2 non-hydrogen substituents. In certain embodiments, the optional substituents are independently selected from D, halogen, -CN, -NH ₂ 、-OH、＝O、-NH(CH ₃ )、-N(CH ₃ ) ₂ -NH (cyclopropyl), -CH ₃ 、-CH ₂ CH ₃ 、-CF ₃ 、-OCH ₃ and-OCF ₃ 。

In one aspect, the present invention provides a compound of formula (a):

or a pharmaceutically acceptable salt thereof, wherein

Represents two conjugated double bonds in a 5 membered heteroaryl ring;

X ¹ selected from CH and N;

X ² selected from CH and N;

R ¹ selected from C ₁ -C ₆ Alkyl, cyclopropyl, cyclobutyl, C ₁ -C ₂ Alkylene- (cyclopropyl) and C ₁ -C ₂ Alkylene- (cyclobutyl) radicals, C ₁ -C ₆ Alkyl groupCyclopropyl, cyclobutyl, C ₁ -C ₂ Alkylene- (cyclopropyl) or C ₁ -C ₂ Alkylene- (cyclobutyl) is optionally substituted by 1, 2 or 3 substituents each independently selected from halogen, hydroxy and-OC ₁ -C ₆ An alkyl group;

Z ¹ 、Z ² and Z ³ Selected such that:

Z ¹ Is C, Z ² Is CR (CR) ³ And Z is ³ Is NR ² ；

Z ⁴ Is N or NR ⁷ ；

R ² Selected from C ₁ -C ₆ Alkyl, cyclopropyl, cyclobutyl, oxetanyl, C ₁ -C ₂ Alkylene- (cyclopropyl), C ₁ -C ₂ Alkylene- (cyclobutyl) and C ₁ -C ₂ Alkylene- (oxetanyl), said C ₁ -C ₆ Alkyl, cyclopropyl, cyclobutyl, oxetanyl, C ₁ -C ₂ Alkylene- (cyclopropyl), C ₁ -C ₂ Alkylene- (cyclobutyl) or C ₁ -C ₂ Alkylene- (oxetanyl) groups are optionally substituted with 1, 2, 3, 4, 5 or 6 substituents each independently selected from halogen, hydroxy, oxo, amino, -CN, -OC ₁ -C ₆ Alkyl and-OC ₁ -C ₆ A haloalkyl group;

R ³ selected from H, halogen, hydroxy, -CN, -OC ₁ -C ₆ Alkyl, C ₁ -C ₆ Alkyl, cyclopropyl, cyclobutyl, oxetanyl, C ₁ -C ₂ Alkylene- (cyclopropyl), C ₁ -C ₂ Alkylene- (cyclobutyl) and C ₁ -C ₂ Alkylene- (oxetanyl), said-OC ₁ -C ₆ Alkyl, C ₁ -C ₆ Alkyl, cyclopropyl, cyclobutyl, oxetanyl, C ₁ -C ₂ Alkylene- (cyclopropyl), C ₁ -C ₂ Alkylene- (cyclobutyl) or C ₁ -C ₂ Alkylene- (oxetanyl) groups are optionally substituted with 1, 2, 3, 4, 5 or 6 substituents each independently selected from halogen, hydroxy, -CN and-OC ₁ -C ₆ An alkyl group;

R ⁴ selected from H, halogen, hydroxy, C ₁ -C ₆ Alkyl and-OC ₁ -C ₆ Alkyl, said C ₁ -C ₆ Alkyl or-OC ₁ -C ₆ Alkyl is optionally substituted with 1, 2 or 3 substituents each independently selected from halogen and hydroxy;

R ⁵ selected from H, halogen, hydroxy, -CN, C ₁ -C ₆ Alkyl and-OC ₁ -C ₆ Alkyl, said C ₁ -C ₆ Alkyl or-OC ₁ -C ₆ Alkyl is optionally substituted with 1, 2 or 3 substituents each independently selected from halogen and hydroxy;

R ⁶ selected from C ₁ -C ₆ Alkyl, cyclopropyl, cyclobutyl, oxetanyl, C ₁ -C ₂ Alkylene- (cyclopropyl), C ₁ -C ₂ Alkylene- (cyclobutyl) and C ₁ -C ₂ Alkylene- (oxetanyl), said C ₁ -C ₆ Alkyl, cyclopropyl, cyclobutyl, oxetanyl, C ₁ -C ₂ Alkylene- (cyclopropyl), C ₁ -C ₂ Alkylene- (cyclobutyl) or C ₁ -C ₂ Alkylene- (oxetanyl) groups are optionally substituted with 1, 2 or 3 substituents each independently selected from halogen, hydroxy, phenyl, -CN and-OC ₁ -C ₆ An alkyl group; and is also provided with

R ⁷ Is H or C ₁ -C ₆ Alkyl, said C ₁ -C ₆ The alkyl group is optionally substituted with 1, 2 or 3 substituents each independently selected from halogen, hydroxy and-OC ₁ -C ₆ An alkyl group.

In certain embodiments, Z ⁴ Is N. In certain embodiments, Z ⁴ Is NR ⁷ . In certain embodiments, R ⁷ Is H. In certain embodiments, R ⁷ Is C ₁ -C ₄ An alkyl group. In certain embodiments, R ⁷ is-CH ₃ . In certain embodiments, R ⁷ is-CH ₂ CH ₃ . In certain embodiments, R ⁷ is-CH ₂ F. In certain embodiments, R ⁷ is-CH ₂ CF ₃ 。

In another aspect, the present invention provides a compound of formula (I):

or a pharmaceutically acceptable salt thereof, wherein

Represents two conjugated double bonds in a 5 membered heteroaryl ring;

X ¹ selected from CH and N;

X ² selected from CH and N;

Z ¹ 、Z ² and Z ³ Selected such that:

Z ¹ Is C, Z ² Is CR (CR) ³ And Z is ³ Is NR ² ；

In one embodiment, the present invention provides a compound of formula (II):

or a pharmaceutically acceptable salt thereof, wherein

Represents two conjugated double bonds in a 5 membered heteroaryl ring; and wherein R is ¹ 、Z ¹ 、Z ² 、Z ³ 、R ² 、R ³ 、R ⁴ 、R ⁵ And R is ⁶ As defined for formula (I).

In one embodiment, the present invention provides a compound of formula (III):

or a pharmaceutically acceptable salt thereof, wherein R ¹ 、R ² 、R ⁴ 、R ⁵ And R is ⁶ As defined for formula (I).

In one embodiment, the present invention provides a compound of formula (IV):

/>

or a pharmaceutically acceptable salt thereof, wherein R ¹ 、R ³ 、R ⁴ 、R ⁵ And R is ⁶ As defined for formula (I).

In one embodiment, the present invention provides a compound of formula (V):

or a pharmaceutically acceptable salt thereof, wherein R ¹ 、R ² 、R ³ 、R ⁵ And R is ⁶ As defined for formula (I).

In one embodiment, the present invention provides a compound of formula (VI):

or a pharmaceutically acceptable salt thereof, wherein

In one embodiment, the present invention provides a compound of formula (VII):

In one embodiment, the present invention provides a compound of formula (VIII):

or a pharmaceutically acceptable salt thereof, wherein

In one embodiment, the invention provides a compound of formula (IX):

In one embodiment of the compounds of the invention (including those of formulas (A), (I), (II), (III), (IV), (V), (VI), (VII), (VIII) and (IX)) or pharmaceutically acceptable salts thereof, R ¹ Is C ₁ -C ₄ Alkyl radicals, e.g. -CH ₃ or-CH ₂ CH ₃ . In one embodiment of the compounds of the invention (including those of formulas (A), (I), (II), (III), (IV), (V), (VI), (VII), (VIII) and (IX)) or pharmaceutically acceptable salts thereof, R ¹ Selected from-CH ₃ and-CH ₂ CH ₃ . In certain embodiments, R ¹ is-CH ₃ . In certain embodiments，R ¹ is-CH ₂ CH ₃ 。

In one embodiment of the compounds of the invention (including those of formulas (A), (I), (II), (III), (IV), (V), (VI), (VII), (VIII) and (IX)) or pharmaceutically acceptable salts thereof, R ² Selected from C ₁ -C ₄ Alkyl and C ₁ -C ₂ Alkylene- (cyclopropyl) s, C ₁ -C ₄ Alkyl or C ₁ -C ₂ Alkylene- (cyclopropyl) is optionally substituted with 1, 2, 3, 4, 5 or 6 substituents each independently selected from halogen, hydroxy, -CN and-OC ₁ -C ₄ An alkyl group. In one embodiment of the compounds of the invention (including those of formulas (A), (I), (II), (III), (IV), (V), (VI), (VII), (VIII) and (IX)) or pharmaceutically acceptable salts thereof, R ² Is C ₁ -C ₄ Alkyl radicals, e.g. -CH ₃ 、-CH ₂ CH ₃ Or- (CH) ₂ ) ₂ CH ₃ The C is ₁ -C ₄ Alkyl is optionally substituted with 1, 2, 3, 4, 5 or 6 substituents each independently selected from halogen, hydroxy, -CN and-OC ₁ -C ₄ Alkyl groups to form, for example, -CH ₂ CF ₃ 、-(CH ₂ ) ₂ CF ₃ 、-(CH ₂ ) ₃ OH、-(CH ₂ ) ₂ OCH ₃ Or- (CH) ₂ ) ₃ OCH ₃ . In one embodiment of the compounds of the invention (including those of formulas (A), (I), (II), (III), (IV), (V), (VI), (VII), (VIII) and (IX)) or pharmaceutically acceptable salts thereof, R ² Is C ₁ -C ₂ Alkylene- (cyclopropyl), e.g. -CH ₂ (cyclopropyl), said C ₁ -C ₂ Alkylene- (cyclopropyl) is optionally substituted with 1, 2, 3, 4, 5 or 6 substituents each independently selected from halogen, hydroxy, -CN and-OC ₁ -C ₄ An alkyl group. In one embodiment of the compounds of the invention (including those of formulas (A), (I), (II), (III), (IV), (V), (VI), (VII), (VIII) and (IX)) or pharmaceutically acceptable salts thereof, R ² Selected from-CH ₃ 、-CH ₂ CH ₃ 、-(CH ₂ ) ₂ CH ₃ 、-CH ₂ CF ₃ 、-(CH ₂ ) ₂ CF ₃ 、-(CH ₂ ) ₃ OH、-(CH ₂ ) ₂ OCH ₃ 、-(CH ₂ ) ₃ OCH ₃ and-CH ₂ (cyclopropyl). In certain embodiments, R ² is-CH ₃ . In certain embodiments, R ² is-CH ₂ CH ₃ . In certain embodiments, R ² Is- (CH) ₂ ) ₂ CH ₃ . In certain embodiments, R ² is-CH ₂ CF ₃ . In certain embodiments, R ² Is- (CH) ₂ ) ₂ CF ₃ . In certain embodiments, R ² Is- (CH) ₂ ) ₃ OH. In certain embodiments, R ² Is- (CH) ₂ ) ₂ OCH ₃ . In certain embodiments, R ² Is- (CH) ₂ ) ₃ OCH ₃ . In certain embodiments, R ² is-CH ₂ (cyclopropyl).

In one embodiment of the compounds of the invention (including those of formulas (A), (I), (II), (III), (IV), (V), (VI), (VII), (VIII) and (IX)) or pharmaceutically acceptable salts thereof, R ³ Is C ₁ -C ₄ Alkyl radicals, e.g. -CH ₂ CH ₃ The C is ₁ -C ₄ Alkyl is optionally substituted with 1, 2, 3, 4, 5 or 6 substituents each independently selected from halogen, hydroxy, -CN and-OC ₁ -C ₄ An alkyl group.

In one embodiment of the compounds of the invention (including those of formulas (A), (I), (II), (III), (IV), (V), (VI), (VII), (VIII) and (IX)) or pharmaceutically acceptable salts thereof, R ⁴ Selected from H and C ₁ -C ₄ Alkyl radicals, e.g. -CH ₃ The C is ₁ -C ₄ The alkyl group is optionally substituted with 1, 2 or 3 substituents each independently selected from halogen and hydroxy. Compounds of the invention (including those of formulas (A), (I), (II), (III), (IV), (V), (VI), (VII), (VIII) and (IX)) or pharmaceutically acceptable thereofIn one embodiment of the subject salt, R ⁴ Selected from H and-CH ₃ . In certain embodiments, R ⁴ Is H. In certain embodiments, R ⁴ is-CH ₃ 。

In one embodiment of the compounds of the invention (including those of formulas (A), (I), (II), (III), (IV), (V), (VI), (VII), (VIII) and (IX)) or pharmaceutically acceptable salts thereof, R ⁵ Selected from H, halogen, such as fluorine or chlorine, and hydroxy. In one embodiment of the compounds of the invention (including those of formulas (A), (I), (II), (III), (IV), (V), (VI), (VII), (VIII) and (IX)) or pharmaceutically acceptable salts thereof, R ⁵ Selected from H, chlorine and hydroxyl. In certain embodiments, R ⁵ Is H. In certain embodiments, R ⁵ Is chlorine. In certain embodiments, R ⁵ Is a hydroxyl group.

In one embodiment of the compounds of the invention (including those of formulas (A), (I), (II), (III), (IV), (V), (VI), (VII), (VIII) and (IX)) or pharmaceutically acceptable salts thereof, R ⁶ Selected from C ₁ -C ₄ Alkyl radicals, e.g. -CH ₃ 、-CH ₂ CH ₃ or-CH (CH) ₃ ) ₂ And cyclopropyl, said C ₁ -C ₄ Alkyl or cyclopropyl is optionally substituted with 1, 2 or 3 substituents each independently selected from halogen, e.g., fluorine, to form, e.g., -CH ₂ CHF ₂ Hydroxy, -CN and-OC ₁ -C ₄ An alkyl group. In one embodiment of the compounds of the invention (including those of formulas (A), (I), (II), (III), (IV), (V), (VI), (VII), (VIII) and (IX)) or pharmaceutically acceptable salts thereof, R ⁶ Selected from-CH ₃ 、-CH ₂ CH ₃ 、-CH ₂ CHF ₂ 、-CH(CH ₃ ) ₂ And cyclopropyl. In certain embodiments, R ⁶ is-CH ₃ . In certain embodiments, R ⁶ is-CH ₂ CH ₃ . In certain embodiments, R ⁶ is-CH ₂ CHF ₂ . In certain embodiments, R ⁶ is-CH (CH) ₃ ) ₂ . In certain embodimentsWherein R is ⁶ Is cyclopropyl.

In another aspect, the present invention provides a compound of formula (III-A):

/>

or a pharmaceutically acceptable salt thereof, wherein

R ¹ Is C ₁ -C ₄ Alkyl or C ₁ -C ₄ A fluoroalkyl group;

R ² is C ₁ -C ₄ Alkyl or (C) ₁ -C ₄ Alkylene) -OC ₁ -C ₄ Alkyl, said C ₁ -C ₄ Alkyl or (C) ₁ -C ₄ Alkylene) -OC ₁ -C ₄ Alkyl is optionally substituted with 1, 2 or 3 substituents each independently selected from halogen, oxo and hydroxy;

R ⁴ is C ₁ -C ₄ An alkyl group;

R ⁵ is H; and is also provided with

R ⁶ Is C ₁ -C ₄ An alkyl group.

In certain embodiments of the compounds of formula (III-A), R ¹ Is C ₁ -C ₄ An alkyl group. In certain embodiments, R ¹ Is C ₁ -C ₂ An alkyl group. In certain embodiments, R ¹ Is C ₁ -C ₄ A fluoroalkyl group. In certain embodiments, R ¹ Is C ₁ -C ₂ A fluoroalkyl group. In certain embodiments, R ¹ is-CH ₃ 、-CH ₂ CH ₃ 、-CH ₂ F、-CHF ₂ 、-CF ₃ 、-CHFCH ₃ 、-CF ₂ CH ₃ 、-CH ₂ CH ₂ F、-CH ₂ CHF ₂ or-CH ₂ CF ₃ . In certain embodiments, R ¹ is-CH ₃ 、-CH ₂ CH ₃ or-CH ₂ F. In certain embodiments, R ¹ is-CH ₃ . In certain embodiments, R ¹ is-CH ₂ CH ₃ . In certain embodiments, R ¹ is-CH ₂ F. In certain embodiments, R ² Is C ₁ -C ₄ An alkyl group. In certain embodiments, R ² Is C ₁ -C ₂ An alkyl group. In certain embodiments, R ² Is (C) ₁ -C ₄ Alkylene) -OC ₁ -C ₂ An alkyl group. In certain embodiments, R ² is-CH ₃ 、-CH ₂ CH ₃ 、-(CH ₂ ) ₂ OCH ₃ 、-(CH ₂ ) ₃ OCH ₃ 、-(CH ₂ ) ₂ OCH ₂ CH ₃ Or- (CH) ₂ ) ₃ OCH ₂ CH ₃ . In certain embodiments, R ² is-CH ₃ 、-CH ₂ CH ₃ 、-(CH ₂ ) ₂ OCH ₃ Or- (CH) ₂ ) ₃ OCH ₃ . In certain embodiments, R ² is-CH ₃ . In certain embodiments, R ² is-CH ₂ CH ₃ . In certain embodiments, R ² Is- (CH) ₂ ) ₂ OCH ₃ . In certain embodiments, R ² Is- (CH) ₂ ) ₃ OCH ₃ . In certain embodiments, R ⁴ Is C ₁ -C ₃ An alkyl group. In certain embodiments, R ⁴ Is C ₁ -C ₂ An alkyl group. In certain embodiments, R ⁴ is-CH ₃ 、-CH ₂ CH ₃ 、-CH ₂ CH ₂ CH ₃ or-CH (CH) ₃ ) ₂ . In certain embodiments, R ⁴ is-CH ₃ or-CH ₂ CH ₃ . In certain embodiments, R ⁴ is-CH ₃ . In certain embodiments, R ⁴ is-CH ₂ CH ₃ . In certain embodiments, R ⁶ Is C ₁ -C ₃ An alkyl group. In certain embodiments, R ⁶ Is C ₁ -C ₂ An alkyl group. In certain embodiments, R ⁶ is-CH ₃ 、-CH ₂ CH ₃ 、-CH ₂ CH ₂ CH ₃ or-CH%CH ₃ ) ₂ . In certain embodiments, R ⁶ is-CH ₃ or-CH ₂ CH ₃ . In certain embodiments, R ⁶ is-CH ₃ . In certain embodiments, R ⁶ is-CH ₂ CH ₃ 。

Other embodiments of the invention include compounds selected from the group consisting of:

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or a pharmaceutically acceptable salt of any of these.

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or a pharmaceutically acceptable salt of any of these.

General schemes for synthesizing the compounds of the present invention can be found in the examples section herein.

Unless otherwise indicated, all references herein to compounds of the invention include references to salts, solvates, hydrates and complexes thereof, as well as references to solvates, hydrates and complexes of salts thereof, including tautomers, polymorphs, stereoisomers and isotopically-labeled forms thereof.

The term "pharmaceutically acceptable salt" as used herein includes salts of acidic or basic groups that may be present in the various compounds disclosed herein, unless otherwise specified.

For example, the basic nature of the compounds of the present invention can form a wide variety of salts with various inorganic and organic acids. While such salts must be pharmaceutically acceptable for administration to animals, in practice it is often desirable to initially separate the compounds of the invention from the reaction mixture into pharmaceutically unacceptable salts, then simply convert the pharmaceutically unacceptable salts back to the free base compounds by treatment with an alkaline reagent, and then convert the latter free base to a pharmaceutically acceptable acid addition salt. The acid addition salts of the basic compounds of the present invention can be prepared by treating the basic compound with a substantial equivalent of the selected inorganic or organic acid in an aqueous solvent medium or in a suitable organic solvent such as methanol or ethanol. After evaporation of the solvent, the desired solid salt is obtained. The desired acid salt may also be precipitated from a solution of the free base in an organic solvent by adding an appropriate mineral or organic acid to the solution.

Acids useful in preparing pharmaceutically acceptable acid addition salts of such basic compounds are those which form non-toxic acid addition salts, i.e., salts containing a pharmacologically acceptable anion such as hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, acid citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, mesylate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate [ i.e., 1' -methylene-bis- (2-hydroxy-3-naphthoate) ].

Examples of salts include, but are not limited to, acetates, acrylates, benzenesulfonates, benzoates (such as chlorobenzoates, methylbenzates, dinitrobenzoates, hydroxybenzoates and methoxybenzoates), bicarbonates, bisulphates, bisulfites, bitartates, borates, bromides, butynes-1, 4-dioates, calcium edetate, camphorsulfonates, carbonates, chlorides, caprates, caprylates, clavulanates, citrates, caprates, dihydrochloride, dihydrogen phosphate, edetates, ethanedisulfonates, etoates, ethanesulfonates, ethylsuccinates, formates, fumaric acid salts, glucoheptonates, glucates, glutamic acid salts, ethanolates, glycolylpara-aminophenylarsonates, heptanates hexyne-1, 6-dioates, hexylresorcinol salt, hydramine (hydrobromide), hydrochloride, gamma-hydroxybutyrate, iodide, isobutyrate, isothiosulphate (isothiosulfonate), lactate, lactobionate, laurate, malate, maleate, malonate, mandelate, methanesulfonate, metaphosphate, methanesulfonate, methylsulfate, monohydrogen phosphate, mucinate, naphthalenesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, nitrate, oleate, oxalate, pamoate (pamoate), palmitate, pantothenate, phenylacetate, phenylbutyrate, phenylpropionate, phthalate, phosphate/diphosphate, polygalacturonate, propanesulfonate, propionate, propiolate, pyrophosphate, pyrosulfate, salicylate, stearate, basic acetate, suberate, succinate, sulfate, sulfonate, sulfite, tannate, tartrate, 8-chlorotheophylline salt, tosylate, triedoode, and valerate.

Illustrative examples of suitable salts include organic salts derived from amino acids such as glycine and arginine, ammonia, primary, secondary and tertiary amines, and cyclic amines such as piperidine, morpholine and piperazine, and inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum and lithium.

In addition to the acids described above, the compounds of the present invention comprising a basic moiety such as an amino group may form pharmaceutically acceptable salts with various amino acids.

Those compounds of the invention which are acidic in nature are capable of forming base salts with a variety of pharmacologically acceptable cations. Examples of such salts include alkali or alkaline earth metal salts, particularly sodium and potassium salts. These salts are prepared by conventional techniques. Chemical bases useful as reagents to prepare pharmaceutically acceptable base salts of the invention are those that form non-toxic base salts with the acidic compounds herein. These salts may be prepared by any suitable method, for example, treating the free acid with an inorganic or organic base such as an amine (primary, secondary or tertiary), alkali metal hydroxide or alkaline earth metal hydroxide, or the like. These salts can also be prepared as follows: the corresponding acidic compound is treated with an aqueous solution containing the desired pharmacologically acceptable cation, and the resulting solution is then evaporated to dryness, preferably under reduced pressure. Alternatively, they may also be prepared as follows: the lower alkanol solution of the acidic compound and the desired alkali metal alkoxide are mixed together and the resulting solution is evaporated to dryness in the same manner as before. In certain embodiments, stoichiometric amounts of reagents are employed in order to ensure reaction completion and maximum yield of the desired end product.

Chemical bases that can be used as reagents to prepare pharmaceutically acceptable base salts of the compounds of the invention that are acidic in nature are those that form non-toxic base salts with such compounds. Such non-toxic base salts include, but are not limited to, those derived from such pharmacologically acceptable cations such as alkali metal cations (e.g., potassium and sodium) and alkaline earth metal cations (e.g., calcium and magnesium), ammonium or water-soluble amine addition salts such as N-methylglucamine- (meglumine), and other base salts of lower alkanolammonium and pharmaceutically acceptable organic amines.

Semi-salts of acids and bases, such as hemisulfate and hemicalcium salts, may also be formed.

For a review of suitable salts, see, for example, starl and Wermuth, handbook of Pharmaceutical Salts: properties, selection, and Use (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds of the invention are known to those skilled in the art.

The salts of the present invention may be prepared according to methods known to those skilled in the art. Pharmaceutically acceptable salts of the compounds of the invention can be readily prepared by mixing together a solution of the compound and optionally the desired acid or base. The salt may be precipitated from the solution and may be collected by filtration or may be recovered by evaporation of the solvent. The degree of ionization in the salt may range from fully ionized to almost unionized.

Those skilled in the art will appreciate that the compounds of the present invention in the free base form having basic functional groups can be converted to acid addition salts by treatment with a stoichiometric excess of the appropriate acid. The acid addition salts of the compounds of the present invention may be reconverted to the corresponding free base by treatment with a stoichiometric excess of a suitable base, such as potassium carbonate or sodium hydroxide, typically in the presence of an aqueous solvent and at room temperature of about 0 ℃ to about 100 ℃. The free base form may be isolated by conventional means, for example extraction with an organic solvent. Furthermore, the acid addition salts of the compounds of the present invention may be exchanged by exploiting the differential solubility of the salts, the volatility or acidity of the acid, or by treatment with an appropriately loaded ion exchange resin. For example, acid reaction of a salt of a compound of the invention with a slight stoichiometric excess of an acid component having a pK lower than the starting salt may affect the exchange. Such conversion is typically carried out at a temperature between about 0 ℃ and the boiling point of the solvent used as the medium for the procedure. Exchange with base addition salts is possible, typically via the free base form of the intermediate.

The compounds of the present invention may exist in unsolvated and solvated forms. When the solvent or water is tightly bound, the complex will have a well-defined stoichiometry, independent of humidity. However, when the solvent or water is weakly bound, as in channel solvates and hygroscopic compounds, the water/solvent content will depend on the humidity and drying conditions. In such a case, non-stoichiometrics may be present. The term "solvate" is used herein to describe a molecular complex comprising a compound of the invention and one or more pharmaceutically acceptable solvent molecules (e.g., ethanol). When the solvent is water, the term "hydration The term "solvate" may optionally be used interchangeably with the term "solvate". Pharmaceutically acceptable solvates according to the invention include hydrates and solvates in which the solvent of crystallization may be replaced by isotopes, e.g. D ₂ O、d ₆ Acetone, d ₆ -DMSO。

Also included within the scope of the invention are complexes, such as clathrate compounds, drug-host inclusion complexes, wherein, in contrast to the solvates described above, the drug and host are present in stoichiometric or non-stoichiometric amounts. Also included are complexes of drugs containing two or more organic and/or inorganic components in amounts that may be stoichiometric or non-stoichiometric. The resulting complex may be ionized, partially ionized, or non-ionized. For an overview of such complexes, see J Pharm Sci,64 (8), 1269-1288 (month 8 of 1975), the disclosure of which is incorporated herein by reference in its entirety.

Polymorphs, prodrugs and isomers (including optical isomers, geometric isomers and tautomers) of the compounds of the present invention are also within the scope of the present invention.

The derivatives of the compounds of the invention may themselves have little or no pharmacological activity, but may be converted to the compounds of the invention when administered to a subject or patient, for example, by hydrolytic cleavage. Such derivatives are referred to as "prodrugs". Further information about the use of prodrugs can be found in: ' Pro-drugs as Novel Delivery Systems, volume 14, ACS Symposium Series (T Higuchi and W stilla) and ' Bioreversible Carriers in Drug Design ', pergamon Press,1987 (E B Roche, inc., american Pharmaceutical Association), the disclosures of which are incorporated herein by reference in their entirety.

For example, prodrugs according to the invention may be produced as follows: the appropriate functional groups present in the compounds of the present invention are replaced with certain moieties known to those skilled in the art as 'prodrug moieties', as described, for example, in "Design of Prodrugs" of H bundegaard (Elsevier, 1985), the disclosure of which is incorporated herein by reference in its entirety.

Some non-limiting examples of prodrugs include:

(i) In the case of compounds containing carboxylic acid functions- (COOH), esters thereof, e.g. with (C) ₁ -C ₈ ) Alkyl replaces hydrogen;

(ii) In the case of compounds containing alcohol functions (-OH), ethers thereof are, for example, those which are described as (C) ₁ -C ₆ ) Alkanoyloxymethyl instead of hydrogen; and

(iii) Containing primary or secondary amino functions (-NH) in the compound ₂ or-NHR, where r+.h), for its amide, for example, one or both hydrogens are replaced with a suitable metabolically labile group (such as amide, carbamate, urea, phosphonate, sulfonate, etc.).

Other examples of surrogate groups and examples of other prodrug types according to the preceding examples can be found in the foregoing references.

Finally, certain compounds of the invention may themselves act as prodrugs of other compounds of the invention.

The compounds of the present invention containing one or more asymmetric carbon and/or phosphorus atoms may exist as two or more stereoisomers. Carbon-carbon bonds of the compounds of the invention may be depicted herein using solid lines, solid wedges, or dotted wedges. The depiction of bonds with asymmetric carbon atoms with solid lines is meant to include all possible stereoisomers (e.g., specific enantiomers, racemic mixtures, etc.) at that carbon atom. Depicting bonds to asymmetric carbon atoms with solid or dotted wedges is meant to include only the stereoisomers shown. The compounds of the invention may contain more than one asymmetric atom. In the case of compounds according to the invention having at least one chiral center, they can accordingly exist as enantiomers. Where the compounds have two or more chiral centers, they may additionally exist as diastereomers.

The compounds of the invention having a chiral center may exist as stereoisomers such as racemates, enantiomers or diastereomers.

Included within the scope of the present invention are all stereoisomers, geometric isomers and tautomeric forms of the compounds of the invention, including compounds that exhibit more than one type of isomerism, and mixtures of one or more thereof. Stereoisomers of the compounds of the formulae herein may include cis and trans (or Z/E) isomers, optical isomers such as (R) and (S) enantiomers, diastereomers, geometric isomers, rotamers, atropisomers, conformational isomers and tautomers of the compounds of the invention, including compounds that exhibit more than one type of isomerism; and mixtures thereof (such as racemates and diastereomeric pairs). Also included are acid addition salts or base salts wherein the counterion is optically active, e.g., D-lactic acid or L-lysine, or racemic, e.g., DL-tartaric acid or DL-arginine.

When the racemate is crystallized, two different types of crystals are possible. The first is the racemic compound (true racemate) mentioned above, in which crystals are produced in a uniform form containing equimolar amounts of both enantiomers. The second type is a racemic mixture or mixture, where the two forms of crystals are produced in equimolar amounts, each comprising a single enantiomer.

The compounds of the present invention may exhibit tautomerism and structural isomerism. For example, the compounds may exist in several tautomeric forms including the enol and imine forms, the ketone and enamine forms and geometric isomers and mixtures thereof. All such tautomeric forms are included within the scope of the compounds of the invention. Tautomers exist as a mixture of tautomeric collections in solution. In solid form, one tautomer is usually dominant. Even though one tautomer may be described, the present invention includes all tautomers of the provided formulae compounds.

In addition, some compounds of the invention may form atropisomers (e.g., substituted biaryls). Atropisomers are conformational stereoisomers that occur when rotation around a single bond in a molecule is prevented or greatly slowed down due to steric interactions with other parts of the molecule and asymmetry of substituents at both ends of the single bond. Interconversion of atropisomers is slow enough to allow separation and isolation under predetermined conditions. By steric hindrance of free rotation of one or more bonds forming the chiral axis, the energy barrier to thermal racemization can be determined.

In the case of compounds of the invention containing alkenyl or alkenylene groups, there may be geometric cis/trans (or Z/E) isomers. The cis/trans isomer may be isolated by conventional techniques well known to those skilled in the art, such as chromatography and fractional crystallization.

Conventional techniques for preparing/separating each enantiomer include chiral synthesis from suitable optically pure precursors, or resolution of the racemate (or of a salt or derivative) using, for example, chiral High Pressure Liquid Chromatography (HPLC) or Supercritical Fluid Chromatography (SFC).

Alternatively, the racemate (or a racemic precursor) may be reacted with a suitable optically active compound, for example an alcohol, or with an acid or base such as tartaric acid or 1-phenylethylamine, in the case of compounds containing acidic or basic moieties. The resulting diastereomeric mixture may be separated by chromatography and/or fractional crystallization, and one or both diastereomers are converted to the corresponding pure enantiomers by means well known to those skilled in the art.

The stereoisomer mixture may be isolated by conventional techniques known to those skilled in the art; see, e.g., el Eliel, "Stereochemistry of Organic Compounds" (Wiley, new York, 1994), the disclosure of which is incorporated herein by reference in its entirety.

The invention also includes isotopically-labeled compounds of the invention, which are identical to those shown in one of the formulae provided, except that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature.

Isotopically-labeled compounds of the present invention can generally be prepared by conventional techniques known to those skilled in the art, or by processes analogous to those described herein, using an appropriate isotopically-labeled reagent in place of an unlabeled reagent otherwise employed.

Examples of isotopes suitable for inclusion in the compounds of the invention include isotopes of the following elements: hydrogen, such as ² H and ³ h, carbon, e.g. ¹¹ C、 ¹³ C and C ¹⁴ C, chlorine, e.g. ³⁶ Cl, fluorine, such as ¹⁸ F, iodine, e.g. ¹²³ I and ¹²⁵ i, nitrogen, such as ¹³ N and ¹⁵ n, oxygen, e.g. ¹⁵ O、 ¹⁷ O and ¹⁸ o, phosphorus, e.g. ³² P, and sulfur, such as ³⁵ S, S. Certain isotopically-labeled compounds of the present invention, for example, those into which a radioisotope is incorporated, are useful in drug and/or substrate tissue distribution studies. The radioactive isotope tritium is convenient in view of easy incorporation and detection means ³ H) And carbon-14% ¹⁴ C) Is particularly useful for this purpose. Using heavier isotopes such as deuterium ² H) Substitutions may provide certain therapeutic advantages resulting from greater metabolic stability, e.g., increased in vivo half-life or reduced dosage requirements, and thus may be preferred in certain circumstances. With positron-emitting isotopes (such as ¹¹ C、 ¹⁸ F、 ¹⁵ O and ¹³ n) substitutions can be used in Positron Emission Tomography (PET) studies to examine substrate receptor occupancy.

The compounds of the present invention intended for pharmaceutical use may be administered as crystalline or amorphous products or mixtures thereof. They can be obtained as follows: for example, in the form of solid plugs, powders or films by precipitation, crystallization, freeze-drying, spray-drying or evaporative drying. Microwave or radio frequency drying may be used for this purpose.

Pharmaceutical compositions and routes of administration

In one embodiment, the present invention relates to a pharmaceutical composition comprising a compound of the present invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

By "pharmaceutically acceptable excipient" is meant that an inert substance is added to the pharmaceutical composition to further facilitate administration of the compound. Non-limiting examples of excipients include calcium carbonate, calcium phosphate, various sugars and types of starches, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols. The choice of excipient will depend to a large extent on factors such as: the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.

"pharmaceutical composition" refers to a mixture of one or more of the compounds described herein or a physiologically/pharmaceutically acceptable salt, solvate, hydrate or prodrug thereof, with other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration of the compound to an organism.

As used herein, "physiologically/pharmaceutically acceptable carrier" refers to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.

Pharmaceutical compositions suitable for delivery of the compounds of the present invention and methods for their preparation will be apparent to those skilled in the art. Such compositions and methods of making them can be found, for example, in ' Remington's Pharmaceutical Sciences ', 19 th edition (Mack Publishing Company, 1995), the disclosure of which is incorporated herein by reference in its entirety.

The pharmaceutically acceptable carrier may comprise any conventional pharmaceutical carrier or excipient. The choice of carrier and/or excipient will depend to a large extent on factors such as: the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.

Suitable pharmaceutically acceptable carriers include inert diluents or fillers, water and various organic solvents (such as hydrates and solvates). If desired, the pharmaceutical composition may contain additional ingredients such as flavoring agents, binders, excipients, and the like. Thus, for oral administration, tablets containing various excipients such as citric acid may be used with various disintegrants such as starch, alginic acid and certain complex silicates, as well as with binders such as sucrose, gelatin and acacia. Non-limiting examples of excipients include calcium carbonate, calcium phosphate, various sugars and types of starches, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols. Additionally, lubricants such as magnesium stearate, sodium lauryl sulfate, and talc are often used for tabletting purposes. Similar types of solid compositions can also be used in soft and hard filled gelatin capsules. Thus, non-limiting examples of materials include lactose or milk sugar and high molecular weight polyethylene glycols. When aqueous suspensions or elixirs are desired for oral administration, the active compounds therein may be combined with the following agents: various sweeteners or flavoring agents, coloring agents or dyes, and, if desired, emulsifying or suspending agents, and diluents such as water, ethanol, propylene glycol, glycerin or combinations thereof.

In one aspect, the invention provides a pharmaceutical composition comprising a compound of the invention (including those of formulae (a), (I), (II), (III-a), (IV), (V), (VI), (VII), (VIII) and (IX)), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient. In certain embodiments, the pharmaceutical composition comprises two or more pharmaceutically acceptable carriers and/or excipients. Optionally, such compositions may comprise a compound or salt as described herein, which is a component of an antibody-drug conjugate; and/or may comprise a compound as described herein that is a component of a particle-based delivery system.

In one embodiment, the compounds of the present invention (including those of formula (a), (I), (II), (III-a), (IV), (V), (VI), (VII), (VIII) or (IX)) or pharmaceutically acceptable salts thereof may be administered orally. Oral administration may include swallowing, such that the compound enters the gastrointestinal tract; alternatively, buccal or sublingual administration may be employed such that the compound enters the blood stream directly from the oral cavity. Thus, the pharmaceutical composition may be in a form suitable for oral administration, such as a tablet, capsule, pill, powder, sustained release formulation, solution or suspension, for example.

Formulations suitable for oral administration include solid formulations such as tablets, capsules containing microparticles, liquids or powders, lozenges (including liquid-filled lozenges), chews, multiparticulates and nanoparticles, gels, solid solutions, liposomes, films (including mucoadhesive films), ovules, sprays and liquid formulations.

Liquid formulations include suspensions, solutions, syrups and elixirs. Such formulations may be used as fillers in soft or hard capsules and typically include a carrier, for example, water, ethanol, polyethylene glycol, propylene glycol, methylcellulose or a suitable oil, and one or more emulsifying and/or suspending agents. Liquid formulations may also be prepared by reconstitution of a solid (e.g., from a sachet).

The compounds of the invention may also be used in fast dissolving, fast disintegrating dosage forms, such as those described in Liang and Chen (2001) Expert Opinion in Therapeutic Patents,11(6) Those described in 981-986, the disclosure of which is incorporated herein by reference in its entirety.

For tablet dosage forms, the active agent may comprise from 1% to 80% by weight of the dosage form, more typically from 5% to 60% by weight of the dosage form. In addition to the active agent, tablets typically contain a disintegrant. Examples of disintegrants include sodium starch glycolate, sodium carboxymethylcellulose, calcium carboxymethylcellulose, croscarmellose sodium, crospovidone, polyvinylpyrrolidone, methylcellulose, microcrystalline cellulose, lower alkyl-substituted hydroxypropyl cellulose, starch, pregelatinized starch and sodium alginate. Typically, the disintegrant may comprise from 1 to 25% by weight of the dosage form, and in certain embodiments from 5 to 20% by weight.

Binders are typically used to impart cohesiveness to tablet formulations. Suitable binders include microcrystalline cellulose, gelatin, sugars, polyethylene glycols, natural and synthetic gums, polyvinylpyrrolidone, pregelatinized starch, hydroxypropyl cellulose and hydroxypropyl methylcellulose. The tablets may also contain diluents such as lactose (monohydrate, spray-dried monohydrate, anhydrous lactose, and the like), mannitol, xylitol, dextrose, sucrose, sorbitol, microcrystalline cellulose, starch, and dibasic calcium phosphate dihydrate.

Tablets may also optionally include surfactants such as sodium lauryl sulfate and polysorbate 80, as well as glidants such as silicon dioxide and talc. When present, the amount of surfactant is typically from 0.2 to 5% by weight of the tablet, and the amount of glidant is typically from 0.2 to 1% by weight of the tablet.

Tablets also typically contain lubricants such as magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, and mixtures of magnesium stearate with sodium lauryl sulfate. The lubricant is typically present in an amount of 0.25 to 10% by weight of the tablet and in certain embodiments 0.5 to 3% by weight.

Other conventional ingredients include antioxidants, colorants, flavors, preservatives, and taste masking agents.

Exemplary tablets may contain up to about 80% by weight of the active agent, about 10% to about 90% by weight of the binder, about 0% to about 85% by weight of the diluent, about 2% to about 10% by weight of the disintegrant, and about 0.25% to about 10% by weight of the lubricant.

The tablet blend may be compressed directly or by rollers to form tablets. Alternatively, the tablet blend or a portion of the blend may be wet, dry or melt granulated, melt solidified or extruded and then tableted. The final formulation may include one or more layers and may be coated or uncoated; or encapsulation.

"Pharmaceutical Dosage Forms" in H.Lieberman and L.Lachman: the formulation of Tablets is discussed in detail in Tablets, vol.1", marcel Dekker, N.Y., N.Y.,1980 (ISBN 0-8247-6918-X), the disclosure of which is incorporated herein by reference in its entirety.

Solid formulations for oral administration may be formulated for immediate release and/or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, targeted, and programmed release.

Suitable modified release formulations are described in U.S. Pat. No. 6,106,864. Details of other suitable release techniques such as high energy dispersion and penetration and coating of particles can be found in Verma et al, pharmaceutical Technology On-line,25 (2), 1-14 (2001). The use of chewing gum (chewing gum) for achieving controlled release is described in WO 00/35298. The disclosures of these references are incorporated herein by reference in their entirety.

The compounds of the invention may also be administered directly into the blood stream, muscle or internal organs. Suitable means for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, intravesical (e.g., bladder), subcutaneous, and intratumoral. Suitable devices for parenteral administration include needle (including microneedle) syringes, needleless syringes and infusion techniques. Suitable formulations for parenteral administration include, but are not limited to, sterile solutions, suspensions or emulsions.

In one embodiment, the compounds of the invention (including those of formula (a), (I), (II), (III-a), (IV), (V), (VI), (VII), (VIII) or (IX)) or pharmaceutically acceptable salts thereof may be administered intravenously.

In one embodiment, the compounds of the invention (including those of formula (a), (I), (II), (III-a), (IV), (V), (VI), (VII), (VIII) or (IX)) or pharmaceutically acceptable salts thereof may be intravesically administered.

Parenteral formulations are typically aqueous solutions which may contain excipients such as salts, carbohydrates and buffers (e.g. to a pH of 3-9), but for some applications they may be formulated more suitably as sterile non-aqueous solutions or as dry forms to be used in combination with a suitable vehicle such as sterile, pyrogen-free water.

The preparation of parenteral formulations under sterile conditions can be readily accomplished using standard pharmaceutical techniques well known to those skilled in the art, for example, by lyophilization.

By using appropriate formulation techniques, such as the incorporation of solubilizers, it is possible to increase the solubility of the compounds of the present invention for use in the preparation of parenteral solutions.

Formulations for parenteral administration may be formulated for immediate release and/or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, targeted, and programmed release. Thus, the compounds of the present invention may be formulated as solid, semi-solid or thixotropic liquids for administration as an implanted depot providing modified release of the active compound. Examples of such formulations include drug coated stents and PGLA microspheres.

Exemplary forms of parenteral administration include solutions or suspensions of the active compounds in sterile aqueous solutions (e.g., aqueous propylene glycol or dextrose). Such dosage forms may be suitably buffered if desired.

The compounds of the invention may also be applied topically to the skin or mucous membranes, i.e. by dermal or transdermal application. Typical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, dusting powders, dressings, foams, films, skin patches, wafers, implants, sponges, fibers, bandages and microemulsions. Liposomes can also be used. Typical carriers include alcohols, water, mineral oils, liquid petrolatum, white petrolatum, glycerin, polyethylene glycol and propylene glycol. Penetration enhancers may be incorporated; see, e.g., finnin and Morgan, J Pharm Sci,88 (10), 955-958 (10, 1999). Other topical application means include by electroporation, iontophoresis, phonophoresis, sonophoresis, and micropin or needleless (e.g. Powderject) ^TM ，Bioject ^TM Etc.) for delivery by injection. The disclosures of these references are incorporated herein by reference in their entirety.

Formulations for topical administration may be formulated for immediate release and/or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, targeted, and programmed release.

The compounds of the invention may also be administered intranasally or by inhalation, typically in dry powder form (alone, as a mixture, e.g. dry blends with lactose, or as mixed component particles, e.g. mixed with phospholipids such as phosphatidylcholine) from a dry powder inhaler, or as an aerosol spray from a pressurized container, pump, nebulizer, atomizer (preferably one that produces a fine mist with electrohydrodynamic properties) or a spray dispenser, with or without a suitable propellant, such as 1, 2-tetrafluoroethane or 1,2, 3-heptafluoropropane. For intranasal use, the powder may include a bioadhesive, such as chitosan or cyclodextrin.

Pressurized containers, pumps, sprays, atomizers or spray applicators may contain solutions or suspensions of the compounds of the present invention containing, for example, ethanol, aqueous ethanol or suitable alternative agents (for dispersion, solubilization or prolonged release of the active), a propellant as a solvent and optionally a surfactant such as sorbitan trioleate, oleic acid or oligolactic acid.

The compounds may be micronised to a size suitable for delivery by inhalation (typically less than 5 microns) prior to use in dry powder or suspension formulations. This may be achieved by any suitable comminution method, such as spiral jet milling, fluid bed jet milling, supercritical fluid treatment to form nanoparticles, high pressure homogenization or spray drying.

Capsules (e.g., made of gelatin or HPMC), blisters and cartridges for use in an inhaler or insufflator may be formulated containing a powder mix of a compound of the invention, a suitable powder base such as lactose or starch and a performance modifier such as l-leucine, mannitol or magnesium stearate. Lactose may be in anhydrous or monohydrate form, the latter being preferred. Other suitable excipients include dextran, glucose, maltose, sorbitol, xylitol, fructose, sucrose and trehalose.

Suitable solution formulations for atomizers for generating fine mist using electrohydrodynamic may contain from 1 μg to 20mg of a compound of the invention per actuation and the actuation volume may vary from 1 μl to 100 μl. Typical formulations include the compounds of the present invention, propylene glycol, sterile water, ethanol, and sodium chloride. Alternative solvents that may be used in place of propylene glycol include glycerol and polyethylene glycol.

Suitable flavoring agents such as menthol and left menthol or sweetening agents such as saccharin or sodium saccharin may be added to those formulations of the invention intended for inhalation/intranasal administration.

Formulations for inhalation/intranasal administration may be formulated for release and/or modified release using, for example, poly (DL-lactic-co-glycolic acid) (PGLA). Modified release formulations include delayed, sustained, pulsed, controlled, targeted, and programmed release.

In the case of dry powder inhalers and aerosols, the dosage unit is determined by means of a valve delivering a metered amount. The unit according to the invention is generally arranged to administer a metered dose or "jet (puff)" containing the desired amount of a compound of the invention. The total daily dose may be administered in a single dose or, more typically, in divided doses throughout the day.

The compounds of the invention may be administered rectally or vaginally, for example in the form of suppositories, pessaries or enemas. Cocoa butter is a conventional suppository base, but various alternatives may be used where appropriate.

Formulations for rectal/vaginal administration may be formulated for immediate release and/or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, targeted, and programmed release.

The compounds of the invention may also be applied directly to the eye or ear, typically in the form of droplets of a micronized suspension or solution in isotonic, pH adjusted sterile saline. Other formulations suitable for ocular and otic administration may include ointments, biodegradable (e.g., absorbable gel sponges, collagen) and non-biodegradable (e.g., silicone) implants, wafers, lenses, and particulate or vesicular systems, such as liposomes or liposomes. Polymers such as crosslinked polyacrylic acid, polyvinyl alcohol, hyaluronic acid, cellulosic polymers (e.g., hydroxypropyl methylcellulose, hydroxyethyl cellulose, or methylcellulose), or heteropolysaccharide polymers (e.g., gellan gum) may be incorporated with a preservative such as benzalkonium chloride. Such formulations may also be delivered by iontophoresis.

Formulations for ocular/otic administration may be formulated for immediate release and/or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, targeted or programmed release.

The compounds of the present invention may be combined with a soluble macromolecular entity such as cyclodextrin and suitable derivatives thereof or polyethylene glycol containing polymers in order to improve its solubility, dissolution rate, taste masking, bioavailability and/or stability for use in any of the aforementioned modes of administration.

For example, the drug-cyclodextrin complexes can be used in different dosage forms and routes of administration. Inclusion and non-inclusion complexes may be used. As an alternative to direct complexation with the drug, the cyclodextrin may be used as an auxiliary additive, i.e. as a carrier, diluent or solubiliser. The most commonly used for these purposes are α -, β -and γ -cyclodextrins, examples of which can be found in PCT publication Nos. WO 91/11172, WO 94/02518 and WO 98/55148, the disclosures of which are incorporated herein by reference in their entirety.

Nanoparticles also represent drug delivery systems suitable for most routes of administration. Various natural and synthetic polymers have been explored for many years to prepare nanoparticles, wherein poly (lactic acid) (PLA), poly (glycolic acid) (PGA) and their copolymers (PLGA) have been widely studied because of their biocompatibility and biodegradability. Nanoparticles and other nanocarriers function as potential carriers for several classes of drugs such as anticancer agents, antihypertensive agents, immunomodulators and hormones, and macromolecules such as nucleic acids, proteins, peptides and antibodies. See, for example, crit.rev. Ter. Drug Carrier syst.21:387-422,2004; nanomedicine: nanotechnology, biology and Medicine I:22-30,2005.

The compounds and compositions of the invention may be administered as an antibody-drug conjugate or component of other targeted delivery modalities.

The pharmaceutical composition may be in unit dosage form suitable for single administration of precise amounts.

Therapeutic methods and uses

The invention further provides methods of treatment and uses comprising a compound of the invention (including those of formula (a), (I), (II), (III-a), (IV), (V), (VI), (VII), (VIII) or (IX)) or a pharmaceutically acceptable salt thereof, alone or in combination with one or more therapeutic or palliative agents.

The term "treatment" as used herein refers to a method of alleviating or eliminating a cellular disorder and/or its attendant symptoms. In particular for cancer, these terms simply refer to an increase in the life expectancy of an individual affected by the cancer, or a decrease in one or more symptoms of the disease.

"in vitro" refers to an operation performed in an artificial environment (e.g., without limitation, in a test tube or culture medium).

"in vivo" refers to an operation performed in a living organism (e.g., without limitation, mice, rats, rabbits, and/or humans).

An "organism" refers to any living entity that is made up of at least one cell. Living organisms can be as simple as, for example, a single eukaryotic cell, or as complex as mammals (including humans).

The term "subject" as used herein refers to a human or animal subject. In certain preferred embodiments, the subject is a human.

The term "patient" as used herein refers to a "subject" in need of treatment. In certain preferred embodiments, the patient is a human.

The terms "abnormal cell growth" and "hyperproliferative disorder" are used interchangeably. Unless otherwise indicated, "abnormal cell growth" refers to cell growth that does not rely on normal regulatory mechanisms (e.g., loss of contact inhibition). Abnormal cell growth may be benign (noncancerous) or malignant (cancerous).

As used herein, "cancer" refers to any malignant and/or invasive growth or tumor caused by abnormal cell growth. "cancer" as used herein refers to solid tumors, hematological, bone marrow or lymphatic system cancers, named for the type of cells that originally formed them. Examples of solid tumors include, but are not limited to, sarcomas and carcinomas. Examples of hematological cancers include, but are not limited to, leukemia, lymphoma, and myeloma. The term "cancer" includes, but is not limited to, primary cancer originating from a particular location in the body, metastatic cancer that has spread from where it began to other locations in the body, recurrence of primary cancer after alleviation, and secondary primary cancer, which is a new primary cancer in a person having a history of previous cancers of a different type than the latter. In particular, the compounds of the invention are useful for the prevention and treatment of a variety of hyperproliferative disorders in humans, such as malignant or benign abnormal cell growth.

The interferon gene Stimulator (STING) protein functions as a cytosolic DNA sensor and an adaptor protein in type 1 interferon signaling. The terms "STING" and "interferon gene stimulator" refer to any form of STING protein, as well as variants, subtypes and species homologs that retain at least a portion of the activity of STING. Unless otherwise indicated, such as by specific reference to human STING, STING includes all mammalian species of native sequence STING, e.g., human, monkey and mouse.

The term "STING activator" or "STING agonist" as used herein refers to a compound that, upon binding, (1) stimulates or activates STING and induces downstream signaling characterized by activation of molecules associated with STING function; (2) Enhancing, increasing, promoting, inducing or prolonging the activity, function or presence of STING, or (3) enhancing, increasing, promoting or inducing the expression of STING. Such effects include, but are not limited to, direct phosphorylation of STING, IRF3 and/or NF- κb, and may also include STAT6.STING pathway activation results in, for example, increased production of type 1 interferons (mainly IFN- α and IFN- β) and expression of interferon-stimulated genes (Chen H et al, "Activation of STAT by STING is Critical for Antiviral Innate Immunity". Cell,2011, volume 14: 433-446; and Liu S-y et al, "Systematic identification of type I and type II interferon-induced antiviral factors". Proc.Natl. Acad. Sci.2012: volume 109, 4239-4244).

The term "STING-mediated" as used herein refers to conditions that are directly affected by STING or are affected by STING pathways, including, but not limited to, inflammatory diseases and conditions, allergic diseases, autoimmune diseases, infectious diseases, abnormal cell growth (including cancer), and use as vaccine adjuvants.

In one embodiment, the compounds of the invention (including those of formulas (a), (I), (II), (III-a), (IV), (V), (VI), (VII), (VIII) and (IX)) or pharmaceutically acceptable salts thereof bind STING.

In one embodiment, the compounds of the invention (including those of formulas (a), (I), (II), (III-a), (IV), (V), (VI), (VII), (VIII) and (IX)) or pharmaceutically acceptable salts thereof activate STING, including, for example, as determined by interferon- β -induced modulation, phosphorylation of IRF3, and the like.

In one aspect, the invention provides compounds of the invention (including those of formulae (a), (I), (II), (III-a), (IV), (V), (VI), (VII), (VIII) and (IX)) or a pharmaceutically acceptable salt thereof for use as a medicament.

In one aspect, the invention provides methods of treatment and uses comprising administering a compound of the invention (including those of formulas (a), (I), (II), (III-a), (IV), (V), (VI), (VII), (VIII) and (IX)), or a pharmaceutically acceptable salt thereof.

In one aspect, the invention is a method for treating inflammatory diseases and conditions, allergic diseases, autoimmune diseases, infectious diseases, and abnormal cell growth in a mammal, comprising administering to the mammal a therapeutically effective amount of a compound of the invention (including those of formulas (a), (I), (II), (III-a), (IV), (V), (VI), (VII), (VIII), and (IX)), or a pharmaceutically acceptable salt thereof. The method may optionally employ a compound or salt as described herein as a component of an antibody-drug conjugate or as a component of a particle-based delivery system. One embodiment of the invention is a method for treating inflammatory diseases and conditions in a mammal. One embodiment of the invention is a method for treating an allergic disease in a mammal. One embodiment of the invention is a method for treating an autoimmune disease in a mammal. One embodiment of the invention is a method for treating an infectious disease in a mammal. In one embodiment, the mammal is a human. In such embodiments, the mammal is a human in need of treatment.

In one aspect, the invention is a method for treating abnormal cell growth in a mammal, comprising administering to the mammal a therapeutically effective amount of a compound of the invention (including those of formulas (a), (I), (II), (III-a), (IV), (V), (VI), (VII), (VIII), and (IX)), or a pharmaceutically acceptable salt thereof.

In another aspect, the invention provides a method for treating abnormal cell growth in a mammal, the method comprising administering to the mammal an amount of a compound of the invention (including those of formulae (a), (I), (II), (III-a), (IV), (V), (VI), (VII), (VIII) and (IX)) or a pharmaceutically acceptable salt thereof, effective to treat abnormal cell growth.

In such embodiments, the abnormal cell growth may be cancer. If the abnormal cell growth is cancer, the cancer to be treated may be lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, gastric cancer, colon cancer, breast cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, hodgkin's disease, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, sarcoma of soft tissue, cancer of the urethra, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, cancer of the kidney or ureter, renal cell carcinoma, renal pelvis cancer, tumors of the Central Nervous System (CNS), primary CNS lymphoma, spinal axis tumors, brain stem glioma or pituitary adenoma. In one embodiment, the cancer is bladder cancer. In one embodiment, the bladder cancer is urothelial cancer. In one embodiment, the bladder cancer is non-muscle invasive bladder cancer (NMIBC). In one embodiment, the bladder cancer is Muscle Invasive Bladder Cancer (MIBC). In one embodiment, the bladder cancer is a non-metastatic urothelial cancer. In one embodiment, the bladder cancer is metastatic urothelial cancer. In one embodiment, the bladder cancer is a non-urothelial cancer. In one embodiment, the mammal is a human. In such embodiments, the mammal is a human in need of treatment.

In yet another embodiment, the invention provides a method of inhibiting cancer cell proliferation in a subject, the method comprising administering to the subject an amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, effective to inhibit cancer cell proliferation.

The methods of the invention described herein may optionally employ a compound or salt as described herein as a component of an antibody-drug conjugate or as a component of a particle-based delivery system.

Also embodied in the present invention are compounds of the invention (including those of formulas (a), (I), (II), (III-a), (IV), (V), (VI), (VII), (VIII) and (IX)) or pharmaceutically acceptable salts thereof for use in treating abnormal cell growth in a mammal. In such embodiments, the abnormal cell growth may be cancer. In such embodiments, the mammal is a human. In such embodiments, the mammal is a human in need of treatment.

Also embodied in the present invention is the use of a compound of the invention (including those of formulae (a), (I), (II), (III-a), (IV), (V), (VI), (VII), (VIII) and (IX)) or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament useful for the treatment of abnormal cell growth in a mammal. In such embodiments, the abnormal cell growth may be cancer. In such embodiments, the mammal is a human. In such embodiments, the mammal is a human in need of treatment.

Still other embodiments of the present invention include embodiments wherein a method of activating STING in a mammal is provided, the method comprising the step of administering to the mammal an effective amount of a compound or salt described herein. Also provided is a method of stimulating an innate immune response in a mammal comprising the step of administering to the mammal an effective amount of a compound or salt described herein. In one embodiment, the mammal is a human. In such embodiments, the mammal is a human in need of treatment.

Dosing regimen

The amount of active compound administered will depend on the severity of the subject, disorder or condition being treated, the rate of administration, the disposition of the compound and the discretion of the prescribing physician.

The dosage regimen may be adjusted to provide the best desired response. For example, a single bolus dose may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the urgency of the treatment modality. As used herein, "dosage unit form" refers to physically discrete units suitable as unitary dosages for mammalian subjects to be treated, each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect, in association with the required pharmaceutical carrier.

The skilled artisan will appreciate based on the disclosure provided herein that the dosages and dosing regimens are adjusted according to methods well known in the therapeutic arts. That is, the maximum tolerated dose can be readily established, an effective amount to provide a detectable therapeutic benefit to the patient can be determined, and the time requirements for administration of each agent to provide a detectable therapeutic benefit to the patient can be determined.

It should be noted that the dosage value may vary with the type and severity of the condition to be alleviated, and may include single or multiple doses. It will further be appreciated that for any particular subject, the particular dosage regimen should be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the compositions, and that the dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed invention. For example, the dosage may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such as toxic effects and/or laboratory values. Thus, the present invention encompasses an increase in the dose in a patient as determined by the skilled artisan. Determining the appropriate dosage and regimen for administration of a chemotherapeutic agent is well known in the relevant art and, once the teachings disclosed herein are provided, the skilled artisan will understand to be encompassed.

One possible dose is in the range of about 0.001 to about 100 mg/kg body weight, administered daily, every other day, every third day, every fourth day, every fifth day, every sixth day, weekly, every other week, every third week, monthly or on other dosing schedules. In some cases, dosage levels below the lower limit of the above range may be more than adequate, while in other cases still larger doses may be used without causing any detrimental side effects, where such larger doses are typically divided into several smaller doses for administration during the day.

In one embodiment, the compositions described herein, alone or in combination with pharmaceutically acceptable excipients, are administered to a subject in an amount sufficient to induce, alter, or stimulate an appropriate immune response. The immune response may include, but is not limited to, a specific immune response, a non-specific immune response, specific and non-specific responses, an innate response, a primary immune response, adaptive immunity, a secondary immune response, a memory immune response, immune cell activation, immune cell proliferation, immune cell differentiation, and cytokine expression.

Combination therapy

The term "combination therapy" as used herein refers to the sequential or simultaneous administration of a compound of the invention (including those of formulae (a), (I), (II), (III-a), (IV), (V), (VI), (VII), (VIII) and (IX)) or a pharmaceutically acceptable salt thereof, together with at least one additional therapeutic agent (e.g., an anticancer agent) or therapy.

In one embodiment, the additional therapeutic agent or therapy is administered to a mammal (e.g., a human) prior to administration of the compounds of the invention. In another embodiment, the additional therapeutic agent or therapy is administered to a mammal (e.g., a human) after administration of the compounds of the invention. In another embodiment, the additional therapeutic agent or therapy is administered to a mammal (e.g., a human) concurrently with administration of the compounds of the invention.

The present invention also relates to a pharmaceutical composition for the treatment of abnormal cell growth (including cancer) in a mammal (including a human) comprising an amount of a compound of the invention as defined above together with one or more (preferably one, two or three) additional therapeutic agents and a pharmaceutically acceptable carrier, wherein the amounts of the active agent and the additional therapeutic agents are therapeutically effective as a whole for the treatment of the abnormal cell growth.

The compounds of the invention and compositions thereof may be administered as a primary treatment, or for the treatment of cancers that do not respond to conventional therapies. In addition, the compounds of the invention and compositions thereof may be used in combination with other therapies (e.g., surgical excision, radiation, additional anticancer agents, etc.), thereby causing additive or enhanced therapeutic effects and/or reducing cytotoxicity of certain anticancer agents. The compounds of the invention and compositions thereof may be co-administered or co-formulated with another agent or formulated for continuous administration with another agent in any order. For combination therapy, the compounds are administered within any time frame suitable for the intended therapy. Thus, single agents may be administered substantially simultaneously (i.e., as a single formulation or within minutes or hours) or sequentially in any order. For example, a single agent treatment may be administered within about one year of each other, such as within about 10, 8, 6, 4, or 2 months, or within 4, 3, 2, or 1 weeks, or within about 5, 4, 3, 2, or 1 days.

In certain embodiments, the methods described herein further comprise administering to the subject an amount of an anti-cancer therapeutic or palliative agent, particularly a standard of care agent suitable for a particular cancer, which together are effective to treat or ameliorate the abnormal cell growth.

In one embodiment, the additional therapeutic agent is one or more palliative agents.

In one embodiment, the additional therapeutic agent is one or more anti-cancer therapeutic agents. In certain embodiments, the one or more anti-cancer therapeutic agents are selected from the group consisting of an anti-tumor agent, an anti-angiogenic agent, a signal transduction inhibitor, and an anti-proliferative agent, in amounts that together are effective to treat the abnormal cell growth.

In one aspect of the invention, the methods described herein further comprise the step of treating the subject with an additional form of therapy. In one aspect, the additional therapeutic modality is an additional anti-cancer therapy, including, but not limited to, chemotherapy, radiation, surgery, hormonal therapy, and/or additional immunotherapy.

In certain embodiments, the compounds of the invention and compositions thereof are administered in combination with one or more additional compositions, including vaccines, that are intended to stimulate an immune response against one or more predetermined antigens or adjuvants.

The compounds of the invention and compositions thereof may be used in combination with other therapeutic agents including, but not limited to, therapeutic antibodies, antibody Drug Conjugates (ADCs), immunomodulators, cytotoxic agents and cytostatic agents. Cytotoxic effects refer to the depletion, elimination, and/or killing of target cells (i.e., tumor cells). Cytotoxic agents refer to agents that have cytotoxic and/or cytostatic effects on cells. Cytostatic action refers to the inhibition of cell proliferation. Cytostatic agents refer to agents that have a cytostatic effect on cells, thereby inhibiting the growth and/or proliferation of a particular subset of cells (i.e., tumor cells). Immunomodulators refer to agents such as: which stimulates an immune response by producing cytokines and/or antibodies and/or modulating T cell function, thereby directly or indirectly inhibiting or reducing the growth of a subset of cells (i.e., tumor cells) by making another agent more potent. The compounds of the invention and one or more other therapeutic agents may be administered as part of the same or separate dosage forms by the same or different routes of administration and according to the same or different administration schedules according to standard pharmaceutical practice known to those of ordinary skill in the art.

The compounds of the invention and compositions thereof may also be used in combination with other therapeutic agents including, but not limited to: b7 co-stimulatory molecules, interleukin-2, interferon-7, gm-CSF, CTLA-4 antagonists, PD-1 pathway antagonists, anti-41 BB antibodies, OX-40/OX-40 ligands, CD40/CD40 ligands, sargratin, levamisole, vaccinia virus, BCG, liposomes, alum, freund's complete or incomplete adjuvant, detoxified endotoxins, mineral oils, surface active substances such as lipolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, adjuvants, lipids, inter-bilayer cross-linked multilamellar vesicles, biodegradable poly (D, L-lactic-co-glycolic acid) [ PLGA ] or polyanhydride-based nanoparticles or microparticles, and nanoporous particle supported lipid bilayers such as liposomes, inactivated bacteria (e.g., inactivated or attenuated monocyte hyperplasia) inducing innate immunity, via Toll-like receptors (nld) -retinoic acid receptor (r) -based receptor (rill-like receptor-inducing gene-type receptor (rli), TLR-mediated aggregation-related pathogen therapy ("TLR-mediated receptor, chemical receptor-like receptor-mediated combination"), and the like. Vectors for inducing T cell immune responses that preferentially stimulate cytolytic T cell responses over antibody responses are preferred, although vectors that stimulate both types of responses may also be used. Where the agent is a polypeptide, the polypeptide itself or a polynucleotide encoding the polypeptide may be administered. The vector may be a cell, such as an Antigen Presenting Cell (APC) or a dendritic cell. Antigen presenting cells include cell types such as macrophages, dendritic cells, and B cells. Other specialized antigen presenting cells include monocytes, border zone coulomb cells, microglial cells, langerhans cells, staggered dendritic cells, follicular dendritic cells, and T cells. Facultative antigen presenting cells may also be used. Examples of facultative antigen presenting cells include astrocytes, follicular cells, endothelial cells, and fibroblasts. The vector may be a bacterial cell that is transformed to express the polypeptide or to deliver a polynucleotide that is subsequently expressed in cells of the vaccinated individual. Adjuvants, such as aluminum hydroxide or aluminum phosphate, may be added to increase the ability of the vaccine to trigger, enhance or prolong an immune response. Additional agents are also possible adjuvants such as cytokines, chemokines and bacterial nucleic acid sequences, e.g., cpG, toll-like receptor (TLR) 9 agonists, as well as other agonists of TLR 2, TLR 4, TLR 5, TLR 7, TLR 8, TLR9, including lipoproteins, LPS, lipid A monophosphate, lipoteichoic acid, imiquimod, requimod and additionally retinoic acid inducible gene I (RIG-I) agonists such as poly I: C, alone or in combination with the described compositions. Other representative examples of adjuvants include synthetic adjuvants QS-21 comprising homogeneous saponins purified from Quillaja saponaria (Quillaja saponaria) bark and Corynebacterium parvum (Colynebacterium parvum) (McCune et al, cancer,1979; 43:1619). It will be appreciated that the adjuvant is optimised. In other words, the skilled artisan can conduct routine experimentation to determine the optimal adjuvant to be used.

In one embodiment, the other therapeutic agent is an interferon. The term "interferon" or "IFN" or "INF" (each of which is used interchangeably) refers to any member of the highly homologous species-species protein family that inhibits viral replication and cellular proliferation and modulates immune responses. For example, human interferons are classified into three categories: type I, which includes interferon- α, interferon- β, and interferon- ω; type II, which includes interferon-gamma; and type III, which includes interferon-lambda. The term "interferon" as used herein encompasses recombinant forms of interferon that have been developed and are commercially available. Subtypes of interferon, such as chemically modified or mutated interferon, are also encompassed by the term "interferon" as used herein. Chemically modified interferons may include pegylated interferons and glycosylated interferons. Examples of interferons also include, but are not limited to, interferon- α -2a, interferon- α -2b, interferon- α -n1, interferon- β -1a, interferon- β -1b, interferon- λ -1, interferon- λ -2, and interferon- λ -3. Examples of pegylated interferons include pegylated interferon-alpha-2 a and pegylated interferon-alpha-2 b.

In one embodiment, the additional therapeutic agent is a CTLA-4 pathway antagonist.

In one embodiment, the additional therapeutic agent is an anti-4-1 BB antibody. The term "4-1BB antibody" as used herein refers to an antibody as defined herein which is capable of binding to the human 4-1BB receptor (also referred to herein as "anti-4-1 BB antibody"). The terms "4-1BB" and "4-1BB receptor" are used interchangeably herein and refer to any form of 4-1BB receptor, as well as variants, subtypes and species homologs thereof that retain at least a portion of the activity of the 4-1BB receptor. Thus, the binding molecules as defined and disclosed herein may also bind 4-1BB from a species other than human. In other cases, the binding molecule may be entirely specific for human 4-1BB, and may not exhibit species or other types of cross-reactivity. Unless otherwise indicated, such as by specific reference to human 4-1BB, 4-1BB includes all mammalian species of native sequence 4-1BB, e.g., human, canine, feline, equine, and bovine. An exemplary human 4-1BB is a 255 amino acid protein (accession numbers NM-001561; NP-001552). 4-1BB comprises a signal sequence (amino acid residues 1-17) followed by an extracellular domain (169 amino acids), a transmembrane region (27 amino acids) and an intracellular domain (42 amino acids) (Cheuk ATC et al 2004Cancer Gene Therapy 11:215-226). The receptor is expressed on the cell surface in monomeric and dimeric form and may trimerize with the 4-1BB ligand to signal. As used herein, a "4-1BB agonist" refers to any compound or biological molecule as defined herein that, upon binding 4-1BB, (1) stimulates or activates 4-1BB, (2) enhances, increases, promotes, induces or prolongs the activity, function or presence of 4-1BB, or (3) enhances, increases, promotes or induces the expression of 4-1BB. 4-1BB agonists useful in any of the therapeutic methods, medicaments and uses of the invention include monoclonal antibodies (mAbs) or antigen-binding fragments thereof that specifically bind 4-1BB. Alternative names or synonyms for 4-1BB include CD137 and TNFRSF9. In any of the therapeutic methods, medicaments and uses of the invention wherein a human individual is treated, a 4-1BB agonist increases a 4-1 BB-mediated response. In certain embodiments of the therapeutic methods, medicaments and uses of the invention, 4-1BB agonists significantly enhance the cytotoxic T-cell response, resulting in antitumor activity in several models. Human 4-1BB comprises a signal sequence (amino acid residues 1-17) followed by an extracellular domain (169 amino acids), a transmembrane region (27 amino acids) and an intracellular domain (42 amino acids) (Cheuk ATC et al 2004Cancer Gene Therapy 11:215-226). Receptors are expressed on the cell surface in monomeric and dimeric forms and may trimerize with 4-1BB ligands to signal. Examples of mabs that bind to human 4-1BB and are useful in the methods of treatment, medicaments and uses of the invention are described in US 8,337,850 and US20130078240. In certain embodiments, the anti-4-1 BB antibody has a VH as set forth in SEQ ID NO:17 of WO2017/130076 and a VL as set forth in SEQ ID NO: 18.

In one embodiment, the other therapeutic agent is a PD-1 pathway antagonist. In one embodiment, the other therapeutic agent is an anti-PD-1 antibody. In one embodiment, the other therapeutic agent is an anti-PD-L1 antibody. Programmed death 1 (PD-1) receptors and PD-1 ligands 1 and 2 (PD-L1 and PD-L2, respectively) play an indispensable role in immunomodulation. PD-1 expressed on activated T cells is activated by PD-L1 (also known as B7-H1) and PD-L2 expressed on stromal cells, tumor cells, or both, thereby eliciting T cell death and local immunosuppression (Dong et al, nat Med 1999;5:1365-69; freeman et al, J Exp Med 2000; 192:1027-34), potentially providing an immune tolerant environment for tumor development and growth. In contrast, inhibition of this interaction may enhance local T cell responses and mediate antitumor activity in non-clinical animal models (Iwai Y et al Proc Natl Acad Sci USA 2002; 99:12293-97). Examples of anti-PD-1 antibodies that may be used in the methods of treatment, medicaments and uses of the invention include BCD-100, carlizumab, cimetidine Li Shankang, jenomab (CBT-501), MEDI0680, nivolumab, pembrolizumab, RN888 (see WO 2016/092419), signal-di Li Shan antibody, swadazumab (spartalizumab), STI-a1110, tirelimumab and TSR-042. In certain embodiments, the anti-PD-1 antibody has a VH as set forth in SEQ ID NO:4 of US10155037 and a VL as set forth in SEQ ID NO: 8. Examples of anti-PD-L1 antibodies that can be used in the methods of treatment, medicaments and uses of the invention include alemtuzumab, cervacizumab, BMS-936559 (MDX-1105) and LY3300054.

The disclosed combination therapies may result in synergistic therapeutic effects, i.e., effects greater than the sum of their respective effects or therapeutic results. For example, the synergistic therapeutic effect may be at least about twice the effect, or at least about five times, or at least about ten times, or at least about twenty times, or at least about fifty times, or at least about one hundred times the sum of the therapeutic effects caused by a single agent or a given combination of single agents. Synergistic therapeutic effects may also be observed as an increase in therapeutic effect of at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or more, compared to the sum of therapeutic effects caused by a single agent or a given combination of single agents. The synergistic effect is also an effect that allows for reduced administration of one or more therapeutic agents when used in combination.

Kit of parts:

since it may be desirable to administer a combination of active compounds, for example, for the purpose of treating a particular disease or disorder, it is within the scope of the present invention to include: two or more pharmaceutical compositions, at least one of which contains a compound according to the invention, may conveniently be combined in a kit suitable for co-administration of the compositions. Thus, the kit of the invention comprises two or more separate pharmaceutical compositions, at least one of which contains a compound of the invention, and means for separately retaining the compositions, such as containers, separate bottles or separate foil packs. An example of such a kit is the familiar blister pack used to package tablets, capsules, etc.

The kit of the invention is particularly suitable for administration of different dosage forms, such as oral and parenteral dosage forms, for administration of separate compositions at different dosage intervals, or for mutual titration of separate compositions. To facilitate compliance, the kit typically includes administration guidelines and may be provided with a memory aid.

Examples

General procedure

Synthetic experimental procedure:

the experiments are generally carried out under an inert atmosphere (nitrogen or argon), in particular in the case of reagents or intermediates which are sensitive to oxygen or moisture. Commercially available solvents and reagents are generally used without further purification and are purified in molecular sieves (typically Sure-Seal from Aldrich Chemical Company, milwaukee, wisconsin ^TM The product) is dried. Mass spectrometry data are reported by liquid chromatography-mass spectrometry (LC-MS), atmospheric Pressure Chemical Ionization (APCI), electrospray ionization (ESI), or liquid chromatography-time of flight (LC-TOF). Chemical shifts of Nuclear Magnetic Resonance (NMR) data are expressed in parts per million (ppm) with reference to residual peaks from the deuterated solvent used.

For syntheses with reference to procedures in other examples or methods, the reaction scheme (reaction time length and temperature) may vary. Generally, the reaction is followed by thin layer chromatography, LC-MS or HPLC and work-up if appropriate. Purification may vary from experiment to experiment: in general, the solvent and solvent ratio for the eluent/gradient are selected to provide the appropriate retention time. The reverse phase HPLC fractions were concentrated by lyophilization/freeze drying unless otherwise indicated. The intermediates and final compounds were stored in closed vials or flasks at (0 ℃) or room temperature under nitrogen. Compound names were generated using Chemdraw or ACD Labs software.

Abbreviations for solvents and/or reagents are based on american society of chemistry guidelines and are highlighted below:

ac = acetyl; acOH = acetic acid; ac (Ac) ₂ O=acetic anhydride; ad=adamantyl; bipy=2, 2 '-bipyridine=2, 2' -bipyridyl; bn=benzyl; bu=butyl; cataCXium a = di- (1-adamantyl) -n-butylphosphine; cataxium a-Pd-g3= [ (di (1-adamantyl) -butylphosphine) -2- (2 '-amino-1, 1' -biphenyl)]Palladium (II) mesylate; co=carbon monoxide; DIAD = diisopropyl azodicarboxylate; dbu=1, 8-diazabicyclo [5.4.0]Undec-7-ene;DCE = 1, 2-dichloroethane; DCM = dichloromethane; dipea=n, N-diisopropylethylamine; DMA = dimethylacetamide; dmb=2, 4-dimethoxybenzyl; DMF = N, N-dimethylformamide; dmf·dma=n, N-dimethylformamide dimethyl acetal; DMSO = dimethylsulfoxide; dppf = 1,1' -ferrocenediyl-bis (diphenylphosphine); dtbbpy = 4,4 '-di-tert-butyl-2, 2' -bipyridine; et=ethyl; etOAc = ethyl acetate; h=hr=hour; HFIP = 1, 3-hexafluoro-2-propanol; HPLC = high performance liquid chromatography; [ Ir (cod) OMe] ₂ =bis (1, 5-cyclooctadiene) bis- μ -methoxydi-iridium (I) = [ Ir (OMe) (1, 5-cod) ] ₂ = (1, 5-cyclooctadiene) (methoxy) iridium (I) dimer; KOAc = potassium acetate; lc=liquid chromatography; LCMS = liquid chromatography mass spectrometry; m-cpba=3-chloroperoxybenzoic acid=m-chloroperoxybenzoic acid=mcpba; me=methyl; meoh=methanol; mecn=acn=acetonitrile; msoh=methanesulfonic acid; n-bu=n-butyl; n-buli=n-butyllithium; NCS = N-chlorosuccinimide; pin=pinacol=2, 3-dimethyl-2, 3-butanediol=tetramethyl glycol; pd (OAc) ₂ =palladium (II) acetate; pd (dppf) Cl ₂ = [1,1' -bis (diphenylphosphino) ferrocene]-palladium (II) dichloride; phen=1, 10-phenanthroline; ph=phenyl; pmb=p-methoxybenzyl; phme=tol=toluene; pivoh=pivalic acid; rt=room temperature; TEA = triethylamine; TFA = trifluoroacetic acid; tf (Tf) ₂ O = trifluoromethanesulfonic anhydride; THF = tetrahydrofuran; TMS = trimethylsilyl; ts=tosyl=tosyl; t3p=2, 4, 6-tripropyl-1,3,5,2,4,6-trioxatriphosphorocyclohexane (phosphane) -2,4, 6-trioxide; xantphos = 4, 5-bis (diphenylphosphino) -9, 9-dimethylxanthene.

General scheme I:

as shown in general scheme I, compounds of type GS1a can be prepared in a suitable solvent (such as THF, phMe or similar solvents) using a suitable activator (such as diisopropyl azodicarboxylate) and a trialkyl/triarylphosphine (such as tri-n-butylphosphine or the like) at a temperature in the range of-20℃to room temperature Triphenylphosphine) under Mitsunobu alkylation conditions with an alcohol (HO-R) ² ) Alkylation to provide compounds such as GS1b. A compound such as GS1b can be hydrolyzed in a suitable solvent (such as THF, meOH, water or similar solvents) using a suitable base (MOH, where m=li, na, K or Cs) under basic conditions followed by chlorination with a suitable chlorinating agent (such as oxalyl chloride or thionyl chloride) in a suitable solvent (such as THF, phMe, DCM, DCE or DMF) to provide a compound such as GS1c. A compound such as GS1c may be alkylated with diazomethane or its equivalent (such as trimethylsilyl diazomethane) in a suitable solvent (such as diethyl ether, THF, meCN or similar solvents) followed by bromination with a suitable brominating agent (such as HBr, ferric (III) bromide or similar reagents) in a suitable solvent (such as AcOH, DCM, diethyl ether, meCN, etOAc or similar solvents) to provide a compound such as GS1d. Compounds such as GS1d can be condensed in pure formamide, usually in>A temperature of 140 ℃ to provide a compound such as GS1e. Compounds such as GS1e can be treated with an appropriate base (such as Cs) in an appropriate solvent (DMF, DMSO, THF or similar solvents) ₂ CO ₃ MH, wherein m=na, K, or similar base) is substituted with an alkyl group (R) bearing a suitable Leaving Group (LG) (such as Cl, br, OTs or similar leaving group) ⁶ LG) to provide compounds such as GS1f. In a suitable catalyst system (such as Pd (dppf) Cl ₂ Or Pd (OAc) ₂ Or similar catalysts), sometimes in the presence of copper promoters such as CuCl, cuBr, cuI, cu (Xantphos) Cl, cu (MeCN) ₄ PF ₆ 、Cu(Phen)PPh ₃ Br or similar catalysts), sometimes in the presence of additional phosphine ligands (such as PPh ₃ 、cataCXium A、Xantphos、PCy ₃ ·HBF ₄ Or similar phosphine ligands) with a suitable base (such as CsOPiv, csOAc, K) ₂ CO ₃ + PivOH, TMPMgCl.LiCl or TMPZnCl LiCl, DBU, n-BuLi+ZnCl ₂ Or similar bases/combinations) in a suitable solvent (such as PhMe, dioxane, meCN, TFE, t-amyl alcohol or similar solvents) at a temperature ranging from room temperature to 150C, the compound of type GS1f can be activated by C-H with the compound of type GS1gThe species are cross-coupled to provide a compound such as GS1h. Compounds such as GS1h may contain acid labile protecting groups which can be removed at this stage using conditions known in the art such as TFA/DCM or MsOH/HFIP (Protective Groups in Organic Synthesis, a.wiley-Interscience Publication,1981 or Protecting Groups,10Georg Thieme Verlag,1994) to provide compounds such as GS1i. The compounds at each step may be purified by standard techniques such as column chromatography, crystallization or reverse phase SFC or HPLC. Separation of positional or stereoisomers of any product in the synthesis sequence may be performed, if necessary, under standard methods known in the art, such as chiral SFC or HPLC, to provide single positional or stereoisomers. Variables such as PG, LG and R ¹ -R ⁶ As defined and/or depicted in the embodiments, schemes, examples, and claims herein.

Preparation of Head (HG) intermediates：

Preparation of 4-bromo-N- (2, 4-dimethoxybenzyl) -1-methyl-1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (Int-HG-1) according to scheme HG-1.

Protocol HG-1

Step 1 Synthesis of ethyl 1-methyl-5- { [ (4Z) -2-methyl-5-oxo-1, 3-oxazol-4-ylidene ] methyl } pyrazole-4-carboxylate (HG-1 a)

To a solution of 5-formyl-1-methyl-1H-pyrazole-4-carboxylic acid ethyl ester (10.7 g,58.6 mmol) and N-acetylglycine (10.3 g,88.0mmol,1.5 eq.) in acetic anhydride (15 mL, 4M) was added potassium acetate (9.09 g,88.0mmol,1.5 eq.) at room temperature, and to the slurry was added an additional 5mL Ac ₂ O to re-induce agitation. An air condenser (Findenser) was then placed on top of it and heated to 100 ℃. During heating, the white cloudy suspension became a clear yellow solution and after 10 minutes became a brown solution. After 1 hour, the reaction was cooled to room temperature. TLC analysis (2:1 heptane/EtOAc, KMnO4 dye) showed starting material (rf=0.61)Consumption was accompanied by formation of the product (rf=0.29). The reaction was then transferred to a 100mL beaker, the reaction flask was rinsed with DCM and saturated aqueous sodium bicarbonate was added dropwise with magnetic stirring until effervescence ceased. Thereafter, the contents of the beaker were transferred to a separatory funnel where the organic layer was separated. Subsequently, the aqueous layer was extracted with 4X 100mL of 3:1DCM/iPrOH and 2X 150mL of DCM. The combined organic layers were dried over MgSO ₄ Dried, filtered, and the solvent removed under reduced pressure. The resulting dark brown residue was dissolved in about 5mL DCM. MTBE (about 5 mL) was added dropwise thereto, and then the mixture was poured into a flask containing 200mL of heptane. After sonication, a yellow solid precipitated from the solution and was filtered off under reduced pressure. The mother liquor was then left to stand at 0℃for 2 hours, after which another batch of product precipitated out and was filtered again under reduced pressure. The two batches were combined to give the title compound 1-methyl-5- { [ (4Z) -2-methyl-5-oxo-1, 3-oxazol-4-ylidene as a pale yellow solid]Methyl } pyrazole-4-carboxylic acid ethyl ester (HG-1 a) (15.2 g, 98%). ¹ H NMR (400 MHz, chloroform-d) delta 7.93 (s, 1H), 7.53 (s, 1H), 4.29 (q, j=7.1 hz, 2H), 3.98 (s, 3H), 1.34 (t, j=7.1 hz, 3H).

Step 2 Synthesis of 1-methyl-4-oxo-4, 5-dihydro-1H-pyrazolo [4,3-c ] pyridine-6-carboxylic acid (HG-1 b)

To 1-methyl-5- { [ (4Z) -2-methyl-5-oxo-1, 3-oxazol-4-ylidene in methanol (57.8 mL, 1M)]To ethyl methyl } pyrazole-4-carboxylate (HG-1 a) (15.2 g,57.8 mmol) was added potassium carbonate (16.8 g,116mmol,2 eq.) followed by capping the vessel and heating to 70 ℃. After stirring for 16 hours, the previous dark brown turbid solution became light to tan. Based on LCMS, all starting material was consumed, so the cooled mixture was filtered under reduced pressure and the filter cake was washed with MeOH and MTBE. MTBE was added to the resulting filtrate to cause precipitation of additional solids, which were re-filtered using the same apparatus. The solid filter cake is then suspended in H ₂ O and concentrated HCl is added to acidify to pH 1. The tan solid precipitated, which was filtered off under reduced pressure, after which the filtrate was diluted with 1:1MeOH/MTBE and filtered again under reduced pressure. These two batches were combined to provide the title compound 1-methyl-4-oxo-4, 5-dihydro-1H-pyrazolo [4,3-c ] as a tan solid]Pyridine-6-carboxylic acid (HG-1)b) (10.46 g,94% yield). ¹ H NMR(400MHz,DMSO-d6)δ10.56(s,1H),8.11(d,J＝0.9Hz,1H),7.40(d,J＝0.9Hz,1H),4.03(s,3H)。

Step 3 Synthesis of methyl 1-methyl-4-oxo-4, 5-dihydro-1H-pyrazolo [4,3-c ] pyridine-6-carboxylate (HG-1 c)

To 1-methyl-4-oxo-4, 5-dihydro-1H-pyrazolo [4,3-c ] in methanol (40 mL, 1.4M)]To pyridine-6-carboxylic acid (HG-1 b) (10.46 g,54.17 mmol) was added dropwise concentrated sulfuric acid (90 mmol,5mL,2 eq.). This results in an exotherm upon addition of each drop. The resulting yellow slurry was heated to 70 ℃. After 17 hours the reaction was cooled to room temperature, at which point the starting material appeared to have been consumed and white microcrystalline solids began to precipitate out of solution. The reaction mixture was filtered under reduced pressure and the filter cake was washed with water. The first batch was collected and the filtrate was then diluted with 5mL ACN, 5mL MTBE and 10mL EtOH and then allowed to stand at 0 ℃. After 2 hours, the white crystallites precipitated from the solution were collected by vacuum filtration and combined with the previous batch to provide the title compound 1-methyl-4-oxo-4, 5-dihydro-1H-pyrazolo [4,3-c ] as a white solid ]Pyridine-6-carboxylic acid methyl ester (HG-1 c) (11.1 g, 99.0%). ¹ H NMR (400 MHz, methanol-d 4) δ8.20 (d, j=0.9 hz, 1H), 7.56 (d, j=0.9 hz, 1H), 4.12 (s, 3H), 4.04 (s, 3H).

Step 4 Synthesis of methyl 4-bromo-1-methyl-1H-pyrazolo [4,3-c ] pyridine-6-carboxylate (HG-1 d)

To 1-methyl-4-oxo-4, 5-dihydro-1H-pyrazolo [4,3-c ] in acetonitrile (53.9 mL, 1.0M)]Pyridine-6-carboxylate (HG-1 c) (11.1 g) pyridine (6.51 mL,80.8mmol,1.5 eq.) was added in one portion followed by Tf in about 1mL portions ₂ O anhydride (13.6 mL,80.8mmol,1.5 eq.). After 6mL of the addition, the solution turned from yellow to red (although still cloudy) and after the addition of the remaining triflic anhydride the reaction turned yellow again and began to clear. After 45 minutes LCMS showed consumption of starting material and significant formation of triflate. Lithium bromide (23.4 g, 264 mmol,5 eq.) and trifluoroacetic acid (5.23 ml,59.3mmol,1.1 eq.) were then added to the reaction mixture to give an orange suspension. After 1 hour from this point on, LCM analysis showed the disappearance of triflate and conversion to bromide. The reaction mixture was then slowly poured under magnetic stirring into a solution containing 200mL of saturated NaHCO ₃ Is provided. After gas evolution ceased, the two phases were transferred to a separatory funnel containing 800mL of EtOAc, shaken, and the aqueous layer was discarded. The organic layer was then washed once with sodium thiosulfate to decolorize and the two layers were separated. The organic layer was dried over MgSO ₄ Dried, filtered and the solvent removed under reduced pressure. The resulting brown oil was dissolved in 10mL DCM and 10mL MeCN and 10mL acetone were added thereto. The cloudy solution was left overnight at 0 ℃ after which the product had precipitated and was collected by vacuum filtration to afford the title compound 4-bromo-1-methyl-1H-pyrazolo [4,3-c ] as a tan solid]Pyridine-6-carboxylic acid methyl ester (HG-1 d) (11.77 g, 81%). ¹ H NMR (400 MHz, chloroform-d) δ8.23 (1H, d, j=1 Hz), 8.14 (1H, d, j=1.0 Hz), 4.16 (3H, s), 4.05 (3H, s).

Step 5 Synthesis of 4-bromo-1-methyl-1H-pyrazolo [4,3-c ] pyridine-6-carboxylic acid (HG-1 e)

4-bromo-1-methyl-1H-pyrazolo [4,3-c]Pyridine-6-carboxylic acid ester (HG-1 d) (1333 mg,4.935 mmol) was added to a solution containing 5mL tetrahydrofuran and 2mL H ₂ O flask. To this solution was added lithium hydroxide (177 mg,7.40mmol,1.5 eq.) at room temperature and stirred. After 2 hours LCMS analysis indicated consumption of starting material with concomitant product formation. The reaction mixture was acidified to pH 1 with concentrated HCl, at which point the reaction became cloudy. The resulting acidic suspension was left at 0℃for 1 hour, after which the product was observed to have precipitated. The solid was collected using vacuum filtration to provide the title compound 4-bromo-1-methyl-1H-pyrazolo [4,3-c ] as a white semi-crystalline solid ]Pyridine-6-carboxylic acid (HG-1 e) (1.15 g, 90%). ¹ H NMR(400MHz,DMSO-d6)δ13.43(1H,br s),8.49(1H,d,J＝0.8Hz),8.32(1H,d,J＝0.8Hz),4.18(3H,s)

Step 6 Synthesis of 4-bromo-N- (2, 4-dimethoxybenzyl) -1-methyl-1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (Int-HG-1)

To 4-bromo-1-methyl-1H-pyrazolo [4,3-c]To a suspension of pyridine-6-carboxylic acid (HG-1 e) (1.90 g,9.79 mmol) in DMF (2 mL) was added a first portion of triethylamine (4.13 mL,29.4 mmol) followed by dimethoxybenzylamine (1.64 g,9.79 mmol), wherein the latter resulted inThe solution was clarified. Adding T to the solution ₃ P (8.60 mL,50% in EtOAc, 14.7 mmol) after which the solution became yellow and became significantly hotter. After 30 minutes LCMS analysis of the cloudy yellow suspension showed consumption of starting material and formation of product. It was diluted with 5mL of EtOAc under magnetic stirring, then filtered under reduced pressure. The solid was washed with EtOAc and dried to afford the title compound 4-bromo-N- (2, 4-dimethoxybenzyl) -1-methyl-1H-pyrazolo [4,3-c ] as a white solid]Pyridine-6-carboxamide (Int-HG-1) (3.18 g, 81%). ¹ H NMR (400 MHz, chloroform-d) delta 8.53-8.38 (1H, m), 8.26 (1H, d, j=1 Hz), 8.09 (1H, d, j=1.0 Hz), 7.28 (1H, s), 6.50 (2H, dd, j=8.2, 2.4 Hz), 6.45 (2H, dd, j=8.2, 2.4 Hz), 4.63 (2H, d, j=6.1 Hz), 4.13 (3H, s), 3.90 (3H, s), 3.80 (3H, s).

Preparation of 4, 6-dichloro-1-ethyl-1H-pyrazolo [4,3-c ] pyridine (Int-HG-2) according to scheme HG-2.

Protocol HG-2

Step 1 Synthesis of 4, 6-dichloro-1-ethyl-1H-pyrazolo [4,3-c ] pyridine (Int-HG-2)

Will contain 4, 6-dichloro-1H-pyrazolo [4,3-c ]]A reaction flask of a solution of pyridine (HG-2 a) (1000 mg,5.35 mmol) in THF (16 mL) was cooled to 0deg.C in an ice-water bath and loaded with NaH (60 wt%, in mineral oil, 428mg,10.7 mmol) in portions. The reaction mixture was stirred for 10 minutes, at which point a brown solution was obtained. To the reaction mixture was added ethyl iodide (917 mg,5.88 mmol) followed by stirring at 0℃for another 30 minutes. At this stage, the ice bath was removed and the reaction was allowed to warm gradually to room temperature and stirred for an additional 16 hours. LCMS analysis indicated that the starting material was still present, so the reaction was heated to 50 ℃ and stirred for an additional 2 hours. An additional aliquot of ethyl iodide (418 mg,2.66 mmol) was added and the reaction stirred at room temperature for 17 hours. The reaction was quenched with MeOH (5 mL) and the solution was concentrated in vacuo to afford a yellow oil. The crude residue was purified by flash column chromatography (40 g SiO ₂ Isco,0-20% etoac/petroleum ether) to afford the title compound 4, 6-dichloro-1-ethyl acetate as a yellow solid1H-pyrazolo [4,3-c ]Pyridine (Int-HG-2) (482.3 mg, 42%). LCMS [ M+H]Observed value = 215.9; ¹ h NMR (400 MHz, chloroform-d) δ=8.12 (d, j=0.8 hz, 1H), 7.30 (d, j=0.9 hz, 1H), 4.39 (q, j=7.3 hz, 2H), 1.54 (t, j=7.3 hz, 4H).

The intermediates in the following table were prepared according to scheme HG-2 according to the method used in step 1 for the synthesis of 4, 6-dichloro-1-ethyl-1H-pyrazolo [4,3-c ] pyridine (Int-HG-2) using commercially available 4, 6-dichloro-1H-pyrrolo [3,2-c ] pyridine as starting material and non-critical variations or permutations of the example procedure as would be recognized by those skilled in the art.

Preparation of 4-chloro-N- [ (2, 4-dimethoxyphenyl) methyl ] -1-methyl-1H-pyrazolo [3,4-d ] pyrimidine-6-carboxamide (Int-HG-3) according to scheme HG-3.

Protocol HG-3

Step 1 Synthesis of ethyl 1-methyl-4-oxo-4, 5-dihydro-1H-pyrazolo [3,4-d ] pyrimidine-6-carboxylate (HG-3 b)

To a reaction flask containing 5-amino-1-methyl-1H-pyrazole-4-carboxamide (HG-3 a) (1.5 g,10.70 mmol) was added diethyl oxalate (25 mL). The reaction was heated at 185 ℃ overnight. The flask was removed from heating and gradually cooled to room temperature, which resulted in precipitation of a grey solid. The grey solid was filtered and washed with petroleum ether. The solid was collected to afford the title compound 1-methyl-4-oxo-4, 5-dihydro-1H-pyrazolo [3,4-d ] as a gray solid ]Pyrimidine-6-carboxylic acid ethyl ester (HG-3 b) (552 mg, 25%). GC/MS m/z 222.1[ M]。 ¹ H NMR(400MHz,DMSO-d6)δ＝12.68-12.41(m,1H),8.27-7.99(m,1H),4.52-4.27(m,2H),4.03-3.82(m,3H),1.44-1.18(m,3H)。

Step 2 Synthesis of 4-hydroxy-1-methyl-1H-pyrazolo [3,4-d ] pyrimidine-6-carboxylic acid (HG-3 c)

To contain1-methyl-4-oxo-4, 5-dihydro-1H-pyrazolo [3,4-d]A reaction flask of pyrimidine-6-carboxylic acid ethyl ester (HG-3 b) (300.0 mg,1.35 mmol) was charged with a solution of THF (12 mL) and lithium hydroxide (80.8 mg,3.38 mmol) in water (3 mL). The reaction was stirred overnight at 25 ℃ and then heated at 50 ℃ for 2 hours. The reaction was removed from heating and gradually cooled to room temperature. The solution was concentrated under reduced pressure. The aqueous solution thus obtained was acidified by dropwise addition of HCl (1N) until a ph=2-3 was reached. The solution was diluted with water and transferred to a separatory funnel. The aqueous phase was extracted with 2 parts DCM/IPA (3:1, 60mL each). The combined organic extracts were dried (Na ₂ SO ₄ ) Filtered, and concentrated under reduced pressure. The residue thus obtained was further dried under high vacuum overnight to afford the title compound 4-hydroxy-1-methyl-1H-pyrazolo [3,4-d as a yellow solid]Pyrimidine-6-carboxylic acid (HG-3 c) (240 mg, 91%). LC/MS m/z 195.1[ M+1 ]]。 ¹ H NMR(400MHz,DMSO-d6)δ＝12.37-12.16(m,1H),8.23-8.03(m,1H),4.09-3.83(m,3H)。

Step 3 Synthesis of 4-chloro-1-methyl-1H-pyrazolo [3,4-d ] pyrimidine-6-carbonyl chloride (HG-3 d)

To a reaction flask containing 4-hydroxy-1-methyl-1H-pyrazolo [3,4-d ] pyrimidine-6-carboxylic acid (HG-3 c) (300 mg,1.55 mmol) was added phosphorus oxychloride (4.74 g,30.9mmol,2.88 ml). The flask was equipped with a reflux condenser and the reaction was heated at 90 ℃ for 4 hours. The reaction mixture was concentrated under reduced pressure, and then azeotropically distilled 2 times with PhMe to afford the title compound 4-chloro-1-methyl-1H-pyrazolo [3,4-d ] pyrimidine-6-carbonyl chloride (HG-3 d) (357 mg, 98%) as a brown solid. LC/MS m/z 227[ M-1] (methyl ester).

Step 4 Synthesis of 4-chloro-N- (2, 4-dimethoxybenzyl) -1-methyl-1H-pyrazolo [3,4-d ] pyrimidine-6-carboxamide (Int-HG-3)

To a compound containing 4-chloro-1-methyl-1H-pyrazolo [3,4-d ]]To a reaction flask of pyrimidine-6-carbonyl chloride (HG-3 d) (356 mg,1.55 mmol) was added DCM (8 mL). The solution was cooled to 0deg.C, followed by the addition of triethylamine (938 mg,9.27mmol,1.29 ml). To the mixture was added 1- (2, 4-dimethoxyphenyl) methylamine (775 mg,4.64mmol,0.696 ml). The reaction was stirred at 0℃for 3 hours. The solution was concentrated in vacuo and the crude residue was purified by flash column chromatography (12 g SiO ₂ Isco,3% meoh/DCM) to afford the title compound 4-chloro-N- (2, 4-dimethoxybenzyl) -1-methyl-1H-pyrazolo [3,4-d ] as a pale yellow solid]Pyrimidine-6-carboxamide (Int-HG-3) (224 mg, 40%). LC/MS m/z 362[ M+1 ]]。 ¹ H NMR(400MHz,DMSO-d6)δ＝9.22-9.13(m,1H),8.59-8.54(m,1H),7.17-7.10(m,1H),6.61-6.56(m,1H),6.52-6.46(m,1H),4.48-4.41(m,2H),4.18-4.11(m,3H),3.87-3.82(m,3H),3.77-3.74(m,3H)。

Preparation of 4, 6-dichloro-1-methyl-1H-pyrazolo [4,3-c ] pyridine (Int-HG-4) according to scheme HG-4.

Scheme HG-4:

step 1 Synthesis of 2,4, 6-trichloropyridine-3-carbaldehyde (HG-4 b).

At N ₂ A solution of 2,4, 6-trichloropyridine (HG-4 a) (9.00 g,49.3 mmol) in anhydrous THF was cooled to-68℃at internal temperature under atmosphere, and n-BuLi (2.5M in hexane, 20.7mL,51.8 mmol) was added dropwise, maintaining the reaction temperature below-63℃at internal temperature. The mixture was stirred at-68 ℃ (internal temperature) for 30 minutes. Ethyl formate (4.75 g,64.1 mmol) was added dropwise, maintaining the reaction temperature below-63 ℃ (internal temperature). The mixture was stirred at-68 ℃ (internal temperature) for 1 hour. TLC analysis indicated consumption of starting material. Pouring the mixture into ice and saturated NH ₄ In a 1:1 mixture of aqueous Cl (100 mL). The mixture was stirred for 10 min and then extracted with EtOAc (2×200 mL). The combined organic layers were washed with brine (2×100 mL), dried over anhydrous Na ₂ SO ₄ Dried, filtered, and concentrated to dryness. The residue was purified by flash chromatography (80 g SiO ₂ 0-5% etoac/petroleum ether). The combined fractions were purified by flash chromatography (20 g SiO ₂ 0-5% etoac/petroleum ether). The product batches were combined to provide the title compound 2,4, 6-trichloropyridine-3-carbaldehyde (HG-4 b) (8.62 g,83% yield) as a white solid. ¹ H NMR(400MHz,CDCl ₃ )δ10.42(s,1H),7.46(s,1H)。

Step 2 Synthesis of 4, 6-dichloro-1H-pyrazolo [4,3-c ] pyridine (HG-4 c).

At N ₂ A solution of 2,4, 6-trichloropyridine-3-carbaldehyde (HG-4 b) (4.00 g,19.0 mmol) and DIPEA (7.62 g,58.9 mmol) in EtOH (100 mL) was cooled to-20deg.C under an atmosphere and hydrazine monohydrate (3.81 g,76.0 mmol) was added dropwise. The mixture was stirred at-20℃for 24 hours and then at 30℃for 16 hours. LCMS analysis indicated the formation of the desired product quality. The solution was concentrated to dryness. The resulting solid was slurried with 1:2 EtOAc/petroleum ether (300 mL) for 30 minutes. The solid was collected by filtration. The filter cake was purified by flash chromatography (40 gSiO ₂ 8-50% etoac/petroleum ether) to afford the title compound 4, 6-dichloro-1H-pyrazolo [4,3-c ] as a white solid ]Pyridine (HG-4 c) (1.6 g,45% yield). ¹ H NMR(400MHz,DMSO-d ₆ )δ14.06(br s,1H),8.41(s,1H),7.78(d,J＝1.0Hz,1H)。

Step 3 Synthesis of 4, 6-dichloro-1-methyl-1H-pyrazolo [4,3-c ] pyridine (Int-HG-4).

To 4, 6-dichloro-1H-pyrazolo [4,3-c ] at 0 DEG C]To a solution of pyridine (HG-4 c) (1.25 g,6.65 mmol) in anhydrous THF was added NaH (60% dispersion in mineral oil, 500mg,12.5 mmol). The mixture was stirred at 0℃for 10 minutes, and methyl iodide (1.89 g,13.3 mmol) was then added dropwise at the same temperature. The mixture was stirred at 0℃for 1 hour and then at 25℃for 16 hours. TLC analysis (2:1 EtOAc/petroleum ether) showed complete consumption of starting material. The reaction was carried out by adding saturated NH ₄ Aqueous Cl (20 mL) was quenched and then concentrated to remove THF. The aqueous mixture was extracted with EtOAc (3×20 mL). The combined organic layers were taken up over Na ₂ SO ₄ Dried, filtered, and concentrated to dryness. The residue was purified by flash chromatography (40 g SiO ₂ 5-30% etoac/petroleum ether) to afford the title compound 4, 6-dichloro-1-methyl-1H-pyrazolo [4,3-c ] as an off-white solid]Pyridine (Int-HG-4) (510 mg,38% yield). ¹ H NMR(400MHz,DMSO-d ₆ )δ8.42(d,J＝1.0Hz,1H),8.05(d,J＝0.9Hz,1H),4.12(s,3H)。

Preparation of 4- (4-bromo-1-methyl-1H-imidazol-2-yl) -N- [ (2, 4-dimethoxyphenyl) methyl ] -1-methyl-1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (Int-HG-5) according to scheme HG-5.

Protocol HG-5

Step 1 Synthesis of 4- (4-bromo-1-methyl-1H-imidazol-2-yl) -N- [ (2, 4-dimethoxyphenyl) methyl ] -1-methyl-1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (Int-HG-5)

4-bromo-N- [ (2, 4-dimethoxyphenyl) methyl]-1-methyl-1H-pyrazolo [4,3-c]Pyridine-6-carboxamide (Int-HG-1) (7.23 g,17.8 mmol), bromo-1-methyl-imidazole (HG-5 a) (2.34 g,14.5 mmol), pd (OAc) ₂ (320 mg,1.43 mmol), cuIXantphos (3.29 g,4.27 mmol), dppf (397 mg, 0.719 mmol) and Cs ₂ CO ₃ (14.0 g,42.9 mmol) thick light brown suspension in PhMe (130 mL) with N ₂ Purge for 5 cycles and heat to 125 ℃ with stirring for 17 hours. The reaction was removed from heating and gradually cooled to room temperature. The suspension was filtered over a pad of celite and the filter cake was washed with DCM (100 mL) and EtOAc (100 mL). The filtrate was concentrated in vacuo and the crude residue was purified by flash column chromatography (330 gSiO ₂ Isco,0-100% etoac/petroleum ether) to provide the desired product contaminated with trace impurities. The material was purified by preparative HPLC (YMC Triart C18250x50mmx7um column, 36-76% MeCN/H ₂ O (containing 0.05% NH) ₄ OH), 60 mL/min). The product containing fractions were concentrated in vacuo and triturated with MeOH for 1 hour. The suspension was filtered and the solid was collected. The isolated material was further dried under vacuum to provide the title compound 4- (4-bromo-1-methyl-1H-imidazol-2-yl) -N- [ (2, 4-dimethoxyphenyl) methyl as a white solid ]-1-methyl-1H-pyrazolo [4,3-c]Pyridine-6-carboxamide (Int-HG-5) (2,47 g, 31%). LCMS [ M+H]Observed value = 486.1; ¹ h NMR (400 MHz, chloroform-d) δ=8.88 (d, j=1.0 hz, 1H), 8.26 (d, j=1.0 hz, 2H), 7.30 (d, j=8.2 hz, 1H), 7.04 (s, 1H), 6.50 (d, j=2.2 hz, 1H), 6.47 (dd, j=2.4, 8.3hz, 1H), 5.31 (s, 1H), 4.65 (d, j=6.0 hz, 2H), 4.16 (s, 3H), 4.13 (s, 3H), 3.88 (s, 3H), 3.81 (s, 3H).

Preparation of 4, 6-dichloro-1-cyclopropyl-1H-pyrazolo [4,3-c ] pyridine (Int-HG-7) according to scheme HG-7.

Protocol HG-7

Step 1 Synthesis of 4, 6-dichloro-1-cyclopropyl-1H-pyrazolo [4,3-c ] pyridine (Int-HG-7)

Will contain 4, 6-dichloro-1H-pyrazolo [4,3-c ] in 1, 2-dichloroethane (2.5 mL)]Pyridine (HG-2 a) (150 mg,0.798 mmol), cyclopropylboronic acid (129 mg,1.50 mmol), na ₂ CO ₃ (159mg,1.50mmol)、Cu(OAc) ₂ A reaction vessel of (136 mg,0.749 mmol) and 2,2' -bipyridine (117 mg,0.749 mmol) was heated to 70℃and stirred for 3 hours. The mixture was then cooled to room temperature, and water (20 mL), CH ₂ Cl ₂ (20 mL) was diluted and filtered through a pad of celite. The phases were separated and the aqueous phase was treated with CH ₂ Cl ₂ (10 mL. Times.2) extraction. The combined organic extracts were subjected to anhydrous Na ₂ SO ₃ Dried, filtered, and concentrated under vacuum. The crude residue was purified by flash column chromatography (12 g SiO ₂ Combi-flash,5-30% etoac/petroleum ether) to afford the title compound 4, 6-dichloro-1-cyclopropyl-1H-pyrazolo [4,3-c ] as a yellow solid ]Pyridine (Int-HG-7) (132 mg, 72%). LCMS [ M+H]Observed value = 227.9; ¹ h NMR (chloroform-d) delta 8.06 (s, 1H), 7.46 (s, 1H), 3.57-3.63 (m, 1H), 1.21-1.26 (m, 4H).

Preparation of 4, 6-dichloro-1- (difluoromethyl) -1H-pyrazolo [4,3-c ] pyridine (Int-HG-8) according to scheme HG-8

Protocol HG-8

Step 1 Synthesis of 4, 6-dichloro-1- (difluoromethyl) -1H-pyrazolo [4,3-c ] pyridine (Int-HG-8)

To 4, 6-dichloro-1H-pyrazolo [4,3-c ] at room temperature (30 ℃ C.)]To a solution of pyridine (HG-2 a) (300 mg,1.60 mmol) and KF (275 mg,4.73 mmol) in MeCN (10 mL) was added diethyl (bromodifluoromethyl) phosphonate (HG-8 a) (511 mg,1.91 mmol) and stirred for 18 hours.The reaction was then concentrated in vacuo and then purified by flash column chromatography (40 g SiO ₂ Combi-flash,5-20% EtOAc/petroleum ether) to afford the title compound 4, 6-dichloro-1- (difluoromethyl) -1H-pyrazolo [4,3-c ] as a yellow solid]Pyridine (Int-HG-8) (120 mg, 32%). LCMS [ M+H]Observed value =237.9; ¹ h NMR (chloroform-d) delta 8.24 (s, 1H), 7.67 (s, 1H), 7.46 (t, J=59.0 Hz, 1H).

Preparation of 4, 6-dichloro-1- (fluoromethyl) -1H-pyrazolo [4,3-c ] pyridine (Int-HG-9) according to scheme HG-9

Protocol HG-9

Step 1 Synthesis of 4, 6-dichloro-1- (fluoromethyl) -1H-pyrazolo [4,3-c ] pyridine (Int-HG-9)

To 4, 6-dichloro-1H-pyrazolo [4,3-c ]]Pyridine (HG-2 a) (150 mg,0.798 mmol) and Cs ₂ CO ₃ To a yellow suspension of (520 mg,1.60 mmol) in anhydrous DMF (3 mL) was added fluoro (iodo) methane (162.3 mg,1.015 mmol). The resulting dark grey mixture was stirred at room temperature (27 ℃) for 1.5 hours. TLC (petroleum ether: etoac=2:1, uv and I ₂ ) Indicating that the reaction was complete. The resulting mixture was diluted with water (10 mL) and extracted with EtOAc (10 mL x 3). The combined organic extracts were washed with brine (10 ml x 3), dried over anhydrous Na ₂ SO ₄ Dried, filtered, and concentrated under vacuum. The crude residue was purified by flash column chromatography (EtOAc/petroleum ether=0% to 12%,12g silica gel column) to afford the title compound 4, 6-dichloro-1- (fluoromethyl) -1H-pyrazolo [4,3-c ] as a pale yellow solid]Pyridine (Int-HG-9) (131 mg, 74.6%). ¹ HNMR (400 MHz, chloroform-d) δ=8.24 (s, 1H), 7.48 (s, 1H), 6.30 (d, j=53.8 hz, 2H).

Preparation of 6-chloro-4- (methylamino) -5-nitropyridine-2-carboxylic acid methyl ester (Int-HG-11) according to scheme HG-11

Protocol HG-11

Step 1 Synthesis of 2, 6-dichloro-N-methyl-3-nitropyridin-4-amine (HG-11 b)

To a solution containing 2, 6-dichloro-3-nitropyridin-4-amine (HG-11 a) (2.00 g,9.61 mmol) and K in MeCN (30 mL) at room temperature ₂ CO ₃ To a reaction vessel of (2.66 g,19.2 mmol) was charged methyl iodide (0.921 mL,14.8 mmol). The mixture was heated to 90 ℃ for 5 hours, then additional methyl iodide (0.898 ml,14.4 mmol) was added and stirred at 90 ℃ for 5 hours. Additional methyl iodide (1.20 mL,19.2 mmol) was then added to the reaction and the reaction was stirred at 90℃for 8 hours. The reaction was cooled to room temperature, filtered, and concentrated in vacuo. The crude residue was purified by flash column chromatography (80 g SiO ₂ Combi-flash,5-20% etoac/petroleum ether) to afford the title compound 2, 6-dichloro-N-methyl-3-nitropyridin-4-amine (HG-11 b) (960 mg, 45%) as a yellow solid. LCMS [ M+H]Observed value = 221.8; ¹ h NMR (chloroform-d) delta: 6.80 (br s, 1H), 6.68 (s, 1H), 3.02 (d, J=5.0 Hz, 3H).

Step 2 Synthesis of 2-chloro-6-vinyl-N-methyl-3-nitropyridin-4-amine (HG-11 c)

2, 6-dichloro-N-methyl-3-nitropyridin-4-amine (HG-11 b) (1.06 g,4.77 mmol), 4, 5-tetramethyl-2-vinyl-1, 3, 2-dioxaborolan (882 mg,5.73 mmol), csF (2.18 g,14.3 mmol), pd (PPh) ₃ ) ₂ Cl ₂ (335 mg,0.477 mmol) in 1, 4-dioxane (10.6 mL) and H ₂ N for solution in O (5.3 mL) ₂ Deaeration was carried out 3 times, heated to 90℃and stirred for 16 hours. The reaction was cooled to room temperature, diluted with EtOAc (20 mL), and taken up in Na ₂ SO ₄ Dried, filtered through a pad of celite, and concentrated in vacuo. The crude residue was purified by flash column chromatography (80 g SiO ₂ Combi-flash,3-15% etoac/petroleum ether) to afford the title compound 2-chloro-6-vinyl-N-methyl-3-nitropyridin-4-amine (HG-11 c) (385 mg, 38%) as a yellow solid. LCMS [ M+H]Observed value = 213.9; ¹ HNMR (chloroform-d) δ:6.96-7.08 (m, 2H), 6.60 (s, 1H), 6.53 (dd, j=16.6, 1.8hz, 1H), 5.68 (dd, j=10.5, 1.7hz, 1H), 2.99 (d, j=5.1 hz, 3H).

Step 3 Synthesis of 6-chloro-4- (methylamino) -5-nitropyridine-2-carboxylic acid (HG-11 d)

To a reaction vessel containing 2-chloro-6-vinyl-N-methyl-3-nitropyridin-4-amine (HG-11 c) (340 mg,1.59 mmol) in acetone (9 mL) was added NaHCO at 27 ℃ ₃ (67 mg,0.80 mmol) and KMnO ₄ (755 mg,4.77mmol, added in portions over 30 minutes). The solution was stirred for 4 hours, then with MeOH (3 mL), H ₂ O (3 mL) was diluted and basified with NaOH (2N) to pH 10. The phases were separated and the aqueous phase was extracted with EtOAc (10 ml x 3). The resulting aqueous layer was then acidified to pH 1-2 with HCl (2N) and filtered to remove the precipitate. The filtrate was extracted with EtOAc (20 mL. Times.3) and the combined organic phases were taken up in Na ₂ CO ₃ Dried, filtered, and concentrated in vacuo to afford the crude title compound 6-chloro-4- (methylamino) -5-nitropyridine-2-carboxylic acid (HG-11 d) (208 mg, 56%) as a yellow solid, which was used without further purification.

Step 4 Synthesis of 6-chloro-4- (methylamino) -5-nitropyridine-2-carboxylic acid methyl ester (Int-HG-11)

To a reaction vessel containing 6-chloro-4- (methylamino) -5-nitropyridine-2-carboxylic acid (HG-11 d) (178 mg,0.769 mmol) in DMF (1.8 mL) was added K ₂ CO ₃ (212 mg,1.54 mmol) and methyl iodide (0.057 mL,0.922 mmol). The reaction was stirred at room temperature for 2 hours, then with H ₂ O (5 mL) dilution. The phases were separated, the aqueous phase was extracted with EtOAc (10 ml x 3), and the combined organic phases were washed with brine (15 ml x 3). The organic phase was taken up in Na ₂ CO ₃ Dried, filtered, and concentrated under vacuum. The crude residue was purified by flash column chromatography (20 g SiO ₂ Combi-flash,5-30% etoac/petroleum ether) to afford the title compound 6-chloro-4- (methylamino) -5-nitropyridine-2-carboxylic acid methyl ester (Int-HG-11) (108 mg, three combined batches 43%) as a yellow solid. LCMS [ M+H]Observed value = 245.9; ¹ h NMR (chloroform-d) δ:8.07 (br s, 1H), 6.81 (s, 1H), 3.98 (s, 3H), 3.08 (d, j=5.0 hz, 3H).

Preparation of Tail Group (TG) intermediates：

1-Ethyl-5- (1H-imidazol-4-yl) -3-methyl-1H-pyrazole (Int-TG-1) was prepared according to scheme TG-1.

Scheme TG-1

Step 1 Synthesis of 2-bromo-1- (1-ethyl-3-methyl-1H-pyrazol-5-yl) ethan-1-one (TG-1 b)

To a yellow suspension of 1-ethyl-3-methyl-1H-pyrazole-5-carboxylic acid (TG-1 a) (3 g,19.4 mmol) in anhydrous DCM (100 mL) was added DMF (0.1 mL) followed by slow addition (COCl) ₂ (3.0 mL,35 mmol). The reaction was stirred at room temperature for 0.5 hours. The solution was concentrated in vacuo and the crude residue was co-evaporated 2 times with DCM (50 mL each). The product was used in the next reaction without further purification. The product was dissolved in MeCN (100 mL), cooled in an ice-water bath and TMSCHN was added at 0 °c ₂ (4890 mg,42.8 mmol). The reaction was stirred at room temperature for 2 hours, then HBr (33% solution in AcOH, 8.3ml,50 mmol) was added to the solution at a rate to maintain the internal temperature below 30 ℃. The reaction was stirred at room temperature for 2 hours. The reaction mixture was diluted with EtOAc (100 mL) and water (100 mL) and transferred to a separatory funnel. The phases were separated and the aqueous layer was extracted with EtOAc (100 mL). The combined organic extracts were washed with 3 parts brine (50 mL each), dried (Na ₂ SO ₄ ) Filtered and concentrated under vacuum. The crude residue was purified by flash column chromatography (220 g SiO ₂ Combi-flash,85-100% etoac/petroleum ether) to afford the title compound 2-bromo-1- (1-ethyl-3-methyl-1H-pyrazol-5-yl) ethan-1-one (TG-1 b) (2.65 g, 59%) as a yellow oil. ¹ H NMR (400 MHz, chloroform-d) δ=6.68 (s, 1H), 4.52 (q, j=7.1 hz, 2H), 4.29 (s, 2H), 2.31 (s, 3H), 1.39 (t, j=7.1 hz, 3H).

Step 2 Synthesis of 1-ethyl-5- (1H-imidazol-4-yl) -3-methyl-1H-pyrazole (Int-TG-1)

A pale yellow mixture of 2-bromo-1- (1-ethyl-3-methyl-1H-pyrazol-5-yl) ethan-1-one (TG-1 b) (3.20 g13.8 mmol) in formamide (14.0 mL) was heated to 140℃and stirred for 16 hours. The reaction mixture was diluted with DCM (40 mL) and transferred to a separatory funnel. The phases were separated and the formamide phase was extracted with 3 parts DCM (30 mL each). The combined DCM extracts were concentrated in vacuo. The crude residue was purified by flash column chromatography (80 g SiO ₂ Combi-flash,0-100% EtOAc/petroleum ether, then 0-5% meoh/EtOAc) to afford the desired product containing residual formamide. The mixture was dissolved in EtOAc, diluted with 1NHCl aqueous solution (4 mL), and transferred to a separatory funnel. The phases were separated and the aqueous phase was extracted with 3 parts of EtOAc (30 mL each). Then, the pH of the aqueous phase was adjusted with 2N NaOH aqueous solution until ph= -10. The aqueous phase was extracted with 3 parts of EtOAc (50 mL each). The combined organic extracts from this stage were dried (Na ₂ SO ₄ ) Filtered and concentrated under vacuum. The crude residue was purified by flash column chromatography (80 g SiO ₂ Combi-flash,0-5% meoh/EtOAc) to afford the title compound 1-ethyl-5- (1H-imidazol-4-yl) -3-methyl-1H-pyrazole (Int-TG-1) (79mg, 32%) as a yellow solid, contaminated with about 2 equivalents of formamide. The material thus obtained was used without further purification. ¹ H NMR (400 MHz, chloroform-d) δ=7.71 (d, j=0.9 hz, 1H), 7.18 (d, j=1.0 hz, 1H), 6.16 (s, 1H), 4.39 (q, j=7.1 hz, 2H), 2.28 (s, 3H), 1.41 (t, j=7.2 hz, 3H).

The intermediates in the following table were prepared according to the procedure used in step 1-2 for the synthesis of 1-ethyl-5- (1H-imidazol-4-yl) -3-methyl-1H-pyrazole (Int-TG-1) using commercially available 1-ethyl-3- (trifluoromethyl) -1H-pyrazole-5-carboxylic acid as starting material and non-critical variations or permutations of the exemplified procedure as can be appreciated by the skilled artisan.

2-Ethyl-1 ', 4-dimethyl-1 ' H-1,4' -biimidazole (Int-TG-2) was prepared according to scheme TG-2.

Scheme TG-2

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Step 1 Synthesis of 2-ethyl-1 ', 4-dimethyl-1 ' H-1,4' -biimidazole (Int-TG-2)

To 4-iodo-1-methylimidazole (TG-2 a) (500 mg,2.40 mmol) and 2-ethyl-5-methyl-1H-imidazole(TG-2 b) (530 mg,4.81 mmol) in anhydrous DMF (10 mL) was added Cs ₂ CO ₃ (3.13 g 9.62 mmol), cuI (458 mg,2.40 mmol), L-proline (336 mg,2.88 mmol). The resulting mixture was treated with N ₂ Rinse for 2 minutes, seal, heat to 120 ℃ (heating block), and stir for 40 hours. The reaction mixture was diluted with water (20 mL) and transferred to a separatory funnel containing EtOAc. The phases were separated and the aqueous phase was extracted with 3 parts of EtOAc (20 mL each). The aqueous phase was saturated with brine and extracted with an additional 3 parts of EtOAc (20 mL each). The combined organic extracts were washed with 3 parts brine (15 mL each). The combined aqueous brine washes were back-extracted with 3 parts EtOAc (10 mL each). The organic extracts were combined again and dried (Na ₂ SO ₄ ) Filtered and concentrated under vacuum. The crude residue was purified by flash column chromatography (20 g SiO ₂ Combi-flash,0-10% meoh/DCM) to yield the desired product as a yellow oil, contaminated with residual DMF. The oil was diluted with DCM/MeOH (10:1, 20 mL) and transferred to a separatory funnel. The solution was washed with 3 parts brine (15 mL each). The organic phase was dried (Na ₂ SO ₄ ) Filtered and concentrated under vacuum. The residue was purified by flash column chromatography (20 g SiO ₂ Combi-flash,0-10% MeOH/DCM) to afford the title compound 2-ethyl-1 ', 4-dimethyl-1 ' H-1,4' -biimidazole (Int-TG-2) (140 mg, 30%) as a yellow oil. ¹ H NMR (400 MHz, chloroform-d) δ=7.36 (s, 1H), 6.83 (d, j=1.5 hz, 1H), 6.80 (s, 1H), 3.74 (s, 3H), 2.77 (q, j=7.5 hz, 2H), 2.24 (d, j=0.8 hz, 3H), 1.28 (t, j=7.5 hz, 3H).

1- [3- (benzyloxy) propyl ] -5- (1H-imidazol-4-yl) -3-methyl-1H-pyrazole (Int-TG-3) was prepared according to scheme TG-3.

Scheme TG-3

Step 1 Synthesis of 1- [3- (benzyloxy) propyl ] -3-methyl-1H-pyrazole-5-carboxylic acid ethyl ester (TG-3 b)

3-methyl-1H-pyrazole-5-carboxylic acid ethyl ester (TG-3 a) (11.5 g,74.6 mmol) and 3- (benzyloxy) propan-1-ol (13.0 g,78.3 mmol) in THF (300 mL)The solution was cooled in an ice-water bath and then P (n-Bu) was added dropwise ₃ (33.2 g,164 mmol) and DIAD (31.7 g,157 mmol) while maintaining the internal reaction temperature below 10 ℃. The ice bath was removed and the colorless reaction solution was stirred at room temperature for 16 hours. The reaction mixture was concentrated in vacuo and the crude residue thus obtained was purified by flash column chromatography (330 g SiO ₂ Combi-flash,0-15% EtOAc/petroleum ether) to afford the title compound 1- [3- (benzyloxy) propyl as a colorless oil ]-3-methyl-1H-pyrazole-5-carboxylic acid ethyl ester (TG-3 b) (21.4 g, 94%). LCMS [ M+H]Observed value = 302.8; ¹ H NMR(400MHz,DMSO-d6)δ＝7.43-7.25(m,5H),6.64(s,1H),4.50(t,J＝7.1Hz,2H),4.44(s,2H),4.26(q,J＝7.1Hz,2H),3.41(t,J＝6.1Hz,2H),2.18(s,3H),2.06-1.95(m,2H),1.27(t,J＝7.1Hz,3H)。

step 2 Synthesis of 1- [3- (benzyloxy) propyl ] -3-methyl-1H-pyrazole-5-carboxylic acid (TG-3 c)

To 1- [3- (benzyloxy) propyl group]To a solution of ethyl-3-methyl-1H-pyrazole-5-carboxylate (TG-3 b) (21.4 g,70.8 mmol) in MeOH (70 mL) was added an aqueous 1N solution of THF (350 mL) and LiOH (4.45 g,106 mmol) (106 mL). The reaction was stirred at room temperature for 24 hours. The reaction mixture was concentrated under vacuum to remove volatile solvents. The aqueous suspension thus obtained was transferred to a separatory funnel containing EtOAc. The phases were separated and the aqueous phase was extracted with EtOAc (50 mL). The pH of the aqueous phase was then adjusted to pH = -1 with 1N aqueous HCl and extracted with 2 parts EtOAc (150 mL each). The organic extracts were combined, dried (Na ₂ SO ₄ ) Filtered and concentrated in vacuo to afford the title compound 1- [3- (benzyloxy) propyl ] as a white solid]-3-methyl-1H-pyrazole-5-carboxylic acid (TG-3 c) (18.3 g, 94%). LCMS [ M+H]Observed value = 274.8; ¹ H NMR(400MHz,DMSO-d6)δ＝13.20(br s,1H),7.39-7.23(m,5H),6.59(s,1H),4.50(t,J＝7.2Hz,2H),4.44(s,2H),3.41(t,J＝6.3Hz,2H),2.17(s,3H),2.00(quin,J＝6.7Hz,2H)。

step 3 Synthesis of 1- {1- [3- (benzyloxy) propyl ] -3-methyl-1H-pyrazol-5-yl } -2-bromoethan-1-one (TG-3 d)

The reaction was carried out in two batches of 6.6g of starting material. To 1- [3- (benzyloxy) propyl group ]-3-methyl-1H-pyrazole-5-carboxylic acid (TG-3 c) (6.60 g, 2)4.1 mmol) to a colorless solution in DCM (2000 mL) was added DMF (0.3 mL) followed by dropwise addition (COCl) ₂ (3.66 mL,43.3 mmol). The reaction was stirred at room temperature for 1 hour and then concentrated in vacuo. The crude residue was co-evaporated with DCM (100 mL each) an additional 3 times. The product was used in the next step without further purification. The crude product was dissolved in MeCN (200 mL), the solution cooled in an ice-water bath, followed by dropwise addition of TMSCHN at 0 ℃ under inert atmosphere ₂ (2M solution in hexane, 26.5mL,52.9 mmol). The reaction was stirred at room temperature for 18 hours. At this stage, HBr (33% solution in AcOH, 10.3ml,62.6 mmol) was added at a rate to maintain the internal temperature. The reaction was stirred at room temperature for 1.5 hours. The two reaction batches were combined, quenched with water (100 mL) and transferred to a separatory funnel containing EtOAc (200 mL). The phases were separated and the aqueous phase was extracted with EtOAc (100 mL). The combined organic extracts were washed with 1 part brine (200 mL), dried (Na ₂ SO ₄ ) Filtered and concentrated under vacuum. The crude residue was purified by flash column chromatography (330 g SiO ₂ Biotage,0-19% etoac/petroleum ether) to afford the title compound 1- {1- [3- (benzyloxy) propyl as a pale yellow oil ]-3-methyl-1H-pyrazol-5-yl } -2-bromoethan-1-one (TG-3 d) (11.3 g, 66%). LCMS [ M+H]Observed value =351.8; ¹ h NMR (400 MHz, chloroform-d) δ=7.37-7.34 (m, 4H), 7.33-7.29 (m, 1H), 6.68 (s, 1H), 4.63 (t, j=7.2 hz, 2H), 4.51 (s, 2H), 4.27 (s, 2H), 3.53 (t, j=6.1 hz, 2H), 2.32 (s, 3H), 2.16-2.09 (m, 2H).

Step 4 Synthesis of 1- [3- (benzyloxy) propyl ] -5- (1H-imidazol-4-yl) -3-methyl-1H-pyrazole (Int-TG-3)

The reaction was carried out in 10 batches of 1.13g of starting material. 1- {1- [3- (benzyloxy) propyl ]]A solution of-3-methyl-1H-pyrazol-5-yl } -2-bromoethyl-1-one (TG-3 d) in formamide (2.0 mL) was heated to 140℃and stirred for 16 hours. All batches were allowed to cool to room temperature over 24 hours and then combined. The combined solutions were diluted with DCM and transferred to a separatory funnel. The phases were separated and the formamide phase was extracted with DCM (30 mL). The combined DCM extracts were washed with water (50 mL), dried (Na ₂ SO ₄ ) Filtered and concentrated under vacuum. The crude residue was purified by flash column chromatography[220g SiO ₂ ,Biotage,0-7％MeOH/(EtOAc/DCM 1:1)]Purification to afford the title compound 1- [3- (benzyloxy) propyl as a brown solid upon standing]-5- (1H-imidazol-4-yl) -3-methyl-1H-pyrazole (Int-TG-3) (5.4 g, 47%). LCMS [ M+H]Observed value = 297.0; ¹ H NMR(400MHz,DMSO-d6)δ＝12.31(br s,1H),7.75(s,1H),7.44(s,1H),7.38-7.25(m,5H),6.16(s,1H),4.46(br t,J＝6.9Hz,2H),4.41(s,2H),3.42(t,J＝6.2Hz,2H),2.14(s,3H),2.04-1.95(m,2H)。

According to the procedure used for the synthesis of 1- [3- (benzyloxy) propyl ] -5- (1H-imidazol-4-yl) -3-methyl-1H-pyrazole (Int-TG-3), the intermediates in the following tables were prepared using non-critical variations or permutations of the exemplary procedure as can be appreciated by those skilled in the art.

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The intermediates in the following table were prepared according to the procedure used in step 2-4 for the synthesis of 1- [3- (benzyloxy) propyl ] -5- (1H-imidazol-4-yl) -3-methyl-1H-pyrazole (Int-TG-3) using ethyl 3-methyl-1- (2, 2-trifluoroethyl) -1H-pyrazole-5-carboxylate (PCT Int. Appl.,2017198341,2017, 11, 23) as starting material and non-critical variations or permutations of the exemplified procedure as can be appreciated by the skilled artisan.

Preparation of 1-ethyl-4- [ (4-methoxyphenyl) methoxy ] -3-methyl-5- (1-methyl-1H-imidazol-4-yl) -1H-pyrazole (Int-TG-10) according to scheme TG-10.

Scheme TG-10

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Step 1 Synthesis of 1-ethyl-4- [ (4-methoxyphenyl) methoxy ] -3-methyl-1H-pyrazole (TG-10 a)

To a 100mL flask containing 1-ethyl-3-methyl-1H-pyrazole-4-carbaldehyde (1.0 g,7.24 mmol) was added DCM and m-chloroperoxybenzoic acid (mCPBA) (3.24 g,77% purity, 14.5 mmol). The solution was heated to 40 ℃ for 1 hour. The reaction was cooled to room temperature, diluted with DCM and taken up in saturated Na ₂ SO ₃ Aqueous solution, 2 parts of saturated Na ₂ CO ₃ The mixture of aqueous solution and brine was washed, dried (Na ₂ SO ₄ ) Filtered and concentrated in vacuo to afford 1-ethyl-3-methyl-1H-pyrazol-4-yl formate (1 g), which was used without further purification. To a 100mL flask containing 1-ethyl-3-methyl-1H-pyrazol-4-yl formate (1 g,6.49 mmol) was added MeOH and Et ₃ N (0.9 mL,6.48 mmol). The solution was stirred at room temperature for 30 minutes. The solution was concentrated in vacuo to afford 1-ethyl-3-methyl-1H-pyrazol-4-ol as a pink oil, which was used without further purification. To a solution of 1-ethyl-3-methyl-1H-pyrazol-4-ol (546 mg,4.33 mmol) and PMBCl (749 mg,4.78 mmol) in DMF (8.5 mL) was added K ₂ CO ₃ (660 mg,4.77 mmol). The reaction was stirred at 25℃for 16 hWhen (1). Will react with H ₂ O (25 mL) was quenched and transferred to a separatory funnel containing EtOAc. The phases were separated and the aqueous phase was extracted with 3 parts of EtOAc (20 mL each). The combined organic extracts were dried (Na ₂ SO ₄ ) Filtered and concentrated under vacuum. The crude residue was purified by flash column chromatography (20 gSiO ₂ Combi-flash,60-100% etoac/petroleum ether) to afford the title compound 1-ethyl-4- [ (4-methoxyphenyl) methoxy as a white solid]-3-methyl-1H-pyrazole (TG-10 a) (903 mg, 84%). LCMS [ M+H]Observed value = 247.0; ¹ h NMR (400 MHz, chloroform-d) δ=7.33 (d, j=8.8 hz, 2H), 6.96 (s, 1H), 6.91 (d, j=8.8 hz, 2H), 4.81 (s, 2H), 4.00 (q, j=7.3 hz, 2H), 3.83 (s, 3H), 2.19 (s, 3H), 1.41 (t, j=7.4 hz, 3H).

Step 2 Synthesis of 1-ethyl-4- [ (4-methoxyphenyl) methoxy ] -3-methyl-5- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1H-pyrazole (TG-10 b)

At N ₂ To 1-ethyl-4- [ (4-methoxyphenyl) methoxy group under atmosphere]To a colorless solution of (1, 5-cyclooctadiene) (methoxy) iridium (I) dimer (18.0 mg,0.027 mmol), 4 '-di-tert-butyl-2, 2' -bipyridine (203 mg,0.756 mmol) and 4, 5-tetramethyl- [1,3,2 were added (186 mg, 0.751 mmol) in anhydrous THF (3.7 mL) ]Dioxaborole (246 mg,1.92 mmol). The reaction mixture was heated to 60 ℃ and stirred under an inert atmosphere for 18 hours. The reaction was removed from heating and gradually cooled to room temperature. The solution was concentrated in vacuo and the crude residue was purified by flash column chromatography (20 g SiO ₂ Combi-flash,5-30% EtOAc/petroleum ether) to afford the title compound 1-ethyl-4- [ (4-methoxyphenyl) methoxy as a colorless oil]-3-methyl-5- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1H-pyrazole (TG-10 b) (108 mg, 38%). LCMS [ M+H]Observed value = 373.2.

Step 3 Synthesis of 1-ethyl-4- [ (4-methoxyphenyl) methoxy ] -3-methyl-5- (1-methyl-1H-imidazol-4-yl) -1H-pyrazole (Int-TG-10)

To 1-ethyl-4- [ (4-methoxyphenyl) methoxy group]-3-methyl-5- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -1H-pyrazole (TG-10 b) (108 mg, 0.2910 mmol) and 4-iodo-1-methyl-1H-imidazole (89.3 mg,0.429 mmol) in DMF (2.0 mL)/H ₂ K was added to the mixture in O (0.50 mL) ₃ PO ₄ (185 mg,0.874 mmol) and cataCXium A-Pd-G3 (10.6 mg,0.015 mmol). Will react with N ₂ The mixture was rinsed for 2 minutes, sealed, heated to 80℃and stirred under an inert atmosphere for 16 hours. The reaction was removed from heating and gradually cooled to room temperature. The solution was treated with H ₂ O (5 mL) was diluted and transferred to a separatory funnel containing EtOAc. The phases were separated and the aqueous phase was extracted with 3 parts of EtOAc (10 mL each). The combined organic extracts were washed with 3 parts brine (15 mL each), dried (Na ₂ SO ₄ ) Filtered and concentrated under vacuum. The crude residue was purified by flash column chromatography (20 g SiO ₂ Combi-flash,0-7.5% MeOH/DCM) to afford the title compound 1-ethyl-4- [ (4-methoxyphenyl) methoxy as a pale yellow gum]-3-methyl-5- (1-methyl-1H-imidazol-4-yl) -1H-pyrazole (Int-TG-10) (45.6 mg, 48%). LCMS [ M+H]Observed value =327.2; ¹ h NMR (400 MHz, chloroform-d) δ=7.58 (br s, 1H), 7.26 (d, j=8.8 hz, 2H), 7.17 (d, j=0.8 hz, 1H), 6.87 (d, j=8.5 hz, 2H), 4.75 (s, 2H), 4.51 (q, j=7.0 hz, 2H), 3.82 (s, 3H), 3.72 (s, 3H), 2.14 (s, 3H), 1.39 (t, j=7.2 hz, 3H).

Preparation of 4-chloro-1-ethyl-3-methyl-5- (1-methyl-1H-imidazol-4-yl) -1H-pyrazole (Int-TG-11) according to scheme TG-11.

Scheme TG-11

Step 1 Synthesis of 4-chloro-1-ethyl-3-methyl-5- (1-methyl-1H-imidazol-4-yl) -1H-pyrazole (Int-TG-11)

To a yellow suspension of 1-ethyl-3-methyl-5- (1-methyl-1H-imidazol-4-yl) -1H-pyrazole (a-1) (100 mg,0.526 mmol) in anhydrous DMF (3.5 mL) was added NCS (105 mg,0.788 mmol). The reaction was stirred at room temperature for 10 hours. Will react with H ₂ O (5 mL) was quenched and transferred to a separatory funnel containing EtOAc. The phases were separated and the aqueous phase was extracted with 3 parts of EtOAc (10 mL each). The combined organic extracts were concentrated in vacuo and the crude residue was purified by preparative thin layer chromatography (SiO ₂ ,10% MeOH/DCM) to afford the title compound 4-chloro-1-ethyl-3-methyl-5- (1-methyl-1H-imidazol-4-yl) -1H-pyrazole (Int-TG-11) as an orange oil (101 mg, 85%). LCMS [ M+H]Observed value =225.0; ¹ h NMR (400 MHz, methanol-d) ₄ )δ＝8.31(s,1H),7.69(d,J＝1.0Hz,1H),4.25(q,J＝7.3Hz,2H),3.90(s,3H),2.24(s,3H),1.32(t,J＝7.2Hz,3H)。

Preparation of 5- (1-ethyl-1H-imidazol-4-yl) -1- (3-methoxypropyl) -3-methyl-1H-pyrazole (Int-TG-15) according to scheme TG-15.

Scheme TG-15

Step 1 Synthesis of 1- [3- (benzyloxy) propyl ] -5- (1-ethyl-1H-imidazol-4-yl) -3-methyl-1H-pyrazole (TG-15 a)

At 0 ℃ at N ₂ Downward 1- [3- (benzyloxy) propyl group]To a dark yellow partial solution of 5- (1H-imidazol-4-yl) -3-methyl-1H-pyrazole (Int-TG-3) (900 mg,3.04 mmol) in THF (25 mL) was added NaH (60 wt% mineral oil) (284 mg,9.11 mmol). The reaction was stirred at 0 ℃ for 15 minutes during which time gas evolution was observed and a dark yellow suspension formed. At this stage, a solution of iodoethane (616 mg,3.95 mmol) in THF (2 mL) was added. The reaction was stirred at 0 ℃ for 30 minutes at which point the ice bath was removed. The reaction was warmed to 25 ℃ and stirred for 16 hours. The reaction was cooled in an ice-water bath (0 ℃ C.) and H was added dropwise ₂ O (20 mL) was quenched. The reaction mixture was transferred to a separatory funnel containing EtOAc and the phases separated. The aqueous phase was extracted with 3 parts EtOAc (20 mL). The combined organic extracts were washed with 1 part brine (20 mL), dried (Na ₂ SO ₄ ) Filtered and concentrated under vacuum. The crude residue was purified by flash column chromatography (40 g SiO ₂ 0-10% MeOH/DCM) to afford the title compound 1- [3- (benzyloxy) propyl as a yellow oil]-5- (1-ethyl-1H-imidazol-4-yl) -3-methyl-1H-pyrazole (TG-15 a) (858 mg, 87%). LCMS [ M+H]Observed value=325.1; ¹ HNMR (400 MHz, chloroform-d) δ=7.50 (s, 1H), 7.38-7.30 (m, 5H), 7.18 (s, 1H), 6.24 (s, 1H), 4.54-4.46 (m, 4H), 3.89-3.80 (m,2H),3.55(t,J＝6.0Hz,2H),2.30(s,3H),2.24-2.16(m,2H),1.39(t,J＝7.3Hz,3H)。

step 2 Synthesis of 3- [5- (1-ethyl-1H-imidazol-4-yl) -3-methyl-1H-pyrazol-1-yl ] propan-1-ol (TG-15 b)

At 0 ℃ at N ₂ Downward 1- [3- (benzyloxy) propyl group]To a yellow solution of 5- (1-ethyl-1H-imidazol-4-yl) -3-methyl-1H-pyrazole (TG-15 a) (859 mg,2.65 mmol) in DCM (25 mL) was added dropwise BCl ₃ (931 mg,7.94 mmol). The resulting yellow suspension was warmed to room temperature (22 ℃) and stirred for 16 hours. The reaction was cooled in an ice-water bath (0 ℃) and quenched with MeOH (5 mL). The solution was purified by adding NH ₃ MeOH (7M) was neutralized to pH 7. The solution was removed from the ice bath and gradually warmed to room temperature over 30 minutes with stirring. The suspension was filtered and the filtrate was concentrated in vacuo. The crude residue was purified by flash column chromatography (20 g SIO ₂ 0-10% meoh/DCM) to afford the title compound 3- [5- (1-ethyl-1H-imidazol-4-yl) -3-methyl-1H-pyrazol-1-yl as a yellow solid]Propan-1-ol (TG-15 b) (180 mg, 29%). LCMS [ M+H]Observed value=235.2.

Step 3 Synthesis of 5- (1-ethyl-1H-imidazol-4-yl) -1- (3-methoxypropyl) -3-methyl-1H-pyrazole (Int-TG-15)

At 0 ℃ at N ₂ Downward 3- [5- (1-ethyl-1H-imidazol-4-yl) -3-methyl-1H-pyrazol-1-yl]To a solution of propan-1-ol (TG-15 b) (180 mg,0.768 mmol) in THF (2 mL) was added NaH (60% by weight mineral oil) (76.8 mg,1.92 mmol). The reaction was stirred at 0 ℃ for 15 minutes during which time gas evolution was observed and a dark yellow suspension formed. At this stage, a solution of methyl iodide (164 mg,1.15 mmol) in THF (1 mL) was added. The reaction was stirred at 0 ℃ for 30 minutes at which point the ice bath was removed. The reaction was warmed to 25 ℃ and stirred for 1 hour. The reaction was cooled in an ice-water bath (0 ℃ C.) and H was added dropwise ₂ O (15 mL) was quenched. The reaction mixture was transferred to a separatory funnel containing EtOAc and the phases separated. The aqueous phase was extracted with 3 parts EtOAc (20 mL). The combined organic extracts were washed with 1 part brine (20 mL), dried (Na ₂ SO ₄ ) Filtered and concentrated under vacuum. The crude residue was purified by flash column chromatography (4 g SiO ₂ 0-10% MeOH/DCM) to purifyThe title compound 5- (1-ethyl-1H-imidazol-4-yl) -1- (3-methoxypropyl) -3-methyl-1H-pyrazole (Int-TG-15) (148 mg, 77%) was provided as a brown oil. LCMS [ M+H]Observed value =249.0; ¹ h NMR (400 MHz, chloroform-d) δ=7.65 (s, 1H), 7.23 (s, 1H), 6.27 (s, 1H), 4.47 (t, j=7.1 hz, 2H), 4.07 (q, j=7.3 hz, 2H), 3.41 (t, j=6.1 hz, 2H), 3.31 (s, 3H), 2.30 (s, 3H), 2.14 (quin, j=6.6 hz, 2H), 1.54 (t, j=7.4 hz, 3H).

Preparation of 4- (benzyloxy) -1- (3-methoxypropyl) -3-methyl-5- (1-methyl-1H-imidazol-4-yl) -1H-pyrazole (Int-TG-16) according to scheme TG-16.

Scheme TG-16

Step 1 Synthesis of tert-butyl 2-allylhydrazine-1-carboxylate (TG-16 a)

To a solution of allyl bromide (35.8 mL,413 mmol) and t-butyl hydrazinecarboxylate (65.5 g,496 mmol) in DMSO (150 mL) at room temperature (15 ℃ C.) was added NEt ₃ (72.0 mL,413 mmol). The mixture was heated to 50 ℃ and stirred for 15 hours. The reaction was then diluted with EtOAc (400 mL) and with NaHCO ₃ The aqueous solution is alkalized to pH 8-9. The phases were separated and the aqueous phase was extracted with EtOAc (400 mL). The organic extracts were washed with brine (100 mL x 2), water (100 mL) and concentrated in vacuo. The crude residue was purified by flash column chromatography (220 g x 2 and 80g SiO ₂ Column, combi-flash,0-30% etoac/petroleum ether) to afford the title compound tert-butyl 2-allylhydrazine-1-carboxylate (TG-16 a) (31 g, 44%) as a colourless oil, which solidified upon standing. ¹ H NMR(DMSO-d ₆ )δ:8.16(br s,1H),5.71-5.83(m,1H),5.14(dq,J＝17.3,1.7Hz,1H),5.01-5.08(m,1H),4.41-4.49(m,J＝4.8Hz,1H),3.27-3.32(m,2H),1.38(s,9H)。

Step 2 Synthesis of allyl hydrazine (TG-16 b)

To tert-butyl 2-allylhydrazine-1-carboxylate (TG-16 a) (28 g,163 mmol) at 15℃in CH ₂ Cl ₂ To the solution in (100 mL) was added HCl/dioxane (224 mL,894mmol, 4M) and stirred at 25-30deg.C for 20 hours. MeOH (100 m) was then added to the mixtureL) and stirred at 25 ℃ for 4 hours. The reaction was concentrated to 3/4 volume and additional MeOH (50 mL) was added followed by HCl/MeOH (200 mL,800mmol, 4M) and HCl/dioxane (100 mL,400mmol, 4M). The mixture was stirred at 25-30 ℃ for an additional 20 hours and then concentrated in vacuo to afford the title compound allyl hydrazine (TG-16 b) (24 g, 100%) as a white solid. ¹ H NMR(DMSO-d ₆ )δ:5.79-5.93(m,1H),5.25-5.39(m,2H),3.49-3.56(m,2H)。

Step 3 Synthesis of 1-allyl-2- (propan-2-ylidene) hydrazine (TG-16 c)

Allyl hydrazine (TG-16 b) (24.0 g,165 mmol) at 15℃in CH ₂ Cl ₂ To a solution in (331 mL) were added acetone (14.0 mL,190 mmol) and K ₂ CO ₃ (80.0 g,579 mmol). The reaction was stirred at 20℃for 20 hours, then the mixture was filtered, and taken up in CH ₂ Cl ₂ (300 mL. Times.2) and the filtrate was concentrated in vacuo to afford the title compound 1-allyl-2- (prop-2-ylidene) hydrazine (TG-16 c) (16.8 g, 90%) as a yellow oil. ¹ H NMR (chloroform-d) delta: 5.92-6.04 (m, 1H), 5.09-5.24 (m, 2H), 4.45 (br s, 1H), 3.75-3.82 (m, 2H), 1.94 (s, 3H), 1.76 (s, 3H).

Step 4 Synthesis of 1-allyl-3-methyl-1H-pyrazole-4-carbaldehyde (TG-16 d)

POCl was added dropwise to a reaction vessel containing DMF (100 mL) at 0deg.C ₃ (37.1 mL,406 mmol) and stirred for 1 hour. The mixture was cooled to-20 to-30 ℃ and a solution of 1-allyl-2- (prop-2-ylidene) hydrazine (TG-16 c) (17.9 g, 1599 mmol) in DMF (100 mL) was added dropwise. The reaction was stirred at-15 ℃ for 1.5 hours, warmed to room temperature, then heated to 80 ℃ for 5 hours. The reaction was then cooled to room temperature, slowly poured into ice water (200 mL) and basified with 30% aqueous NaOH (about 70g of solid NaOH) to pH 9-10. The phases are then separated and the aqueous phase is treated with CH ₂ Cl ₂ (500mL x 1,200mL x 2) extraction, washing with brine (300 ml x 3) and concentration under vacuum. The crude residue was purified by flash column chromatography (120 g SiO ₂ Combi-flash,4-45% etoac/petroleum ether) to afford the title compound 1-allyl-3-methyl-1H-pyrazole-4-carbaldehyde (TG-16 d) (19 g, 79%) as a yellow oil. ¹ H NMR(DMSO-d ₆ )δ:9.81(s,1H),8.35(s,1H),5.95-6.06(m,1H),5.13-5.26(m,2H),4.74(dt,J＝5.9,1.4Hz,2H),2.35(s,3H)。

Step 5 Synthesis of 1-allyl-3-methyl-1H-pyrazol-4-ol (TG-16 f)

To 1-allyl-3-methyl-1H-pyrazole-4-carbaldehyde (TG-16 d) (19.0 g,126 mmol) at 10deg.C in CHCl ₃ To the solution in (316 mL) was added 3-chloroperoxybenzoic acid (benzoperoxoic acid) (25.7 g,127 mmol) and stirred at 25-30℃for 40 hours. The reaction was then filtered and the filtrate concentrated in vacuo. The crude residue was purified by flash column chromatography (120 g SiO ₂ Combi-flash,0-20% etoac/petroleum ether) to afford the compound 1-allyl-3-methyl-1H-pyrazol-4-yl formate (TG-16 e) (21 g) as a yellow semi-solid, which was used without further purification. LCMS [ M+H]Observed value = 167.0. 1-allyl-3-methyl-1H-pyrazol-4-yl formate (TG-16 e) (21 g) in MeOH (150 mL) and H at 15 ℃ ₂ NaHCO was added to the solution in O (20 mL) ₃ (12.7 g,152 mmol) and stirred for 5 hours. The reaction mixture was filtered, washed with MeOH, and concentrated in vacuo. The crude residue was purified by flash column chromatography (80 g SiO ₂ Combi-flash,10-100% etoac/petroleum ether) to afford the title compound 1-allyl-3-methyl-1H-pyrazol-4-ol (TG-16 f) (11 g, 63% over two steps) as a yellow oil. LCMS [ M+H]Observed value =138.9; ¹ h NMR (chloroform-d) delta 7.02 (s, 1H), 5.90-6.01 (m, 1H), 5.14-5.26 (m, 2H), 4.51-4.59 (m, 2H), 2.19 (s, 3H).

Step 6 Synthesis of 1-allyl-4- (benzyloxy) -3-methyl-1H-pyrazole (TG-16 g)

To 1-allyl-3-methyl-1H-pyrazol-4-ol (TG-16 f) (11.1 g,80.2 mmol) and K at 15 ℃ ₂ CO ₃ To a solution of (16.6 g,120 mmol) in DMF (186 mL) was added benzyl bromide (10.5 mL,88.2 mmol). The mixture was heated to 50 ℃ and stirred for 20 hours. The reaction was then cooled to room temperature, slowly poured into ice water (400 mL) and diluted with EtOAc (300 mL). The phases were then separated and the aqueous phase was extracted with EtOAc (200 ml x 2), washed with water (200 ml x 2), brine (200 ml x 2) and concentrated in vacuo. The crude residue was purified by flash column chromatography (120 g SiO ₂ Combi-flash,0-40% EtOAc/petroleum ether) to extractThe title compound 1-allyl-4- (benzyloxy) -3-methyl-1H-pyrazole (TG-16 g) (14.6 g, 80%) was provided as a yellow oil. LCMS [ M+H]Observed value = 229.0; ¹ h NMR (chloroform-d) delta 7.30-7.43 (m, 5H), 6.97 (s, 1H), 5.91-6.03 (m, 1H), 5.14-5.26 (m, 2H), 4.89 (s, 2H), 4.54-4.59 (m, 2H), 2.21 (s, 3H).

Step 7 Synthesis of 4- (benzyloxy) -1- (3- ((tert-butyldimethylsilyl) oxy) propyl) -3-methyl-1H-pyrazole (TG-16 i)

At 0 ℃ at N ₂ To a solution of 1-allyl-4- (benzyloxy) -3-methyl-1H-pyrazole (TG-16 g) (4.40 g,19.3 mmol) in THF (110 mL) was added dropwise 9-borabicyclo [ 3.3.1) ]Nonane (77.1 mL,38.5mmol,0.5M in THF). The mixture was heated to 20-30 ℃ and stirred for 3 hours. The reaction was then cooled to 0deg.C and aqueous NaOH (4.75 mL,71.3mmol, 6M) was added dropwise followed by H ₂ O ₂ (7.28 mL,71.3 mmol). The mixture was stirred at 0-15℃for 30 min. Cooling the reaction to 0-5 ℃ with Na ₂ SO ₃ Aqueous solution (30 g,150mL H) ₂ O) quench and stir for 15 minutes. The phases were then separated and the aqueous phase was extracted with MTBE/EtOAc (100 mL. Times.2, 1:1 v/v), and the combined organic phases were washed with brine (100 mL) and concentrated in vacuo. The crude residue was purified by flash column chromatography (40 g SiO ₂ Combi-flash,10-100% etoac/petroleum ether) to afford the compound 3- (4- (benzyloxy) -3-methyl-1H-pyrazol-1-yl) propan-1-ol (TG-16H) (5.30 g) as a yellow oil. ¹ H NMR (chloroform-d) δ:7.31-7.44 (m, 5H), 6.96 (s, 1H), 4.89 (s, 2H), 4.07-4.11 (m, 2H), 3.60 (q, j=5.5 hz, 2H), 2.91 (t, j=5.8 hz, 1H), 2.19 (s, 3H), 1.97 (quin, j=6.0 hz, 2H). 3- (4- (benzyloxy) -3-methyl-1H-pyrazol-1-yl) propan-1-ol (TG-16H) (5.30 g) at 0deg.C in CH ₂ Cl ₂ To a solution of (100 mL) was added imidazole (2.20 g,32.3 mmol) and t-butyldimethylchlorosilane (3.57 g,32.7 mmol). The mixture was warmed to room temperature (15-25 ℃) and stirred for 15 hours. Then the reaction is carried out using H ₂ O (100 mL) and quenched with CH ₂ Cl ₂ (50 mL) dilution. The phases are then separated and the aqueous phase is treated with CH ₂ Cl ₂ (50 mL) extraction. The combined organic phases were washed with brine (50 mL) and concentrated in vacuo. The crude residue was purified by flash column chromatography (80 gSiO ₂ Combi-flash,0-15% etoac/petroleum ether) to afford the title compound 4- (benzyloxy) -1- (3- ((tert-butyldimethylsilyl) oxy) propyl) -3-methyl-1H-pyrazole (TG-16 i) (5.2 g, 75%) as a colorless oil.

Step 8 Synthesis of 3- (4- (benzyloxy) -3-methyl-1H-pyrazol-1-yl) propan-1-ol (TG-16 j)

To a solution of 4- (benzyloxy) -1- (3- ((tert-butyldimethylsilyl) oxy) propyl) -3-methyl-1H-pyrazole (TG-16 i) (1.49 g,4.13 mmol) in THF (15 mL) was added tetrabutylammonium fluoride (4.2 mL,4.2mmol,1.0M in THF) at room temperature. The mixture was then concentrated in vacuo and the crude residue was purified by flash column chromatography (20 g SiO ₂ ,Combi-flash,0-5％MeOH/(1:1EtOAc:CH ₂ Cl ₂ ) Purification to afford the title compound 3- (4- (benzyloxy) -3-methyl-1H-pyrazol-1-yl) propan-1-ol (TG-16 j) (974 mg, 96%) as a yellow oil, which solidified upon standing. ¹ H NMR (chloroform-d) delta 7.30-7.44 (m, 5H), 6.96 (s, 1H), 4.89 (s, 2H), 4.06-4.14 (m, 2H), 3.60 (t, j=5.7 hz, 2H), 2.20 (s, 3H), 1.97 (quin, j=6.0 hz, 2H).

Step 9 Synthesis of 4- (benzyloxy) -1- (3-methoxypropyl) -3-methyl-1H-pyrazole (TG-16 k)

To a solution of 3- (4- (benzyloxy) -3-methyl-1H-pyrazol-1-yl) propan-1-ol (TG-16 j) (971 mg,3.94 mmol) in THF (13 mL) was added NaH (190 mg,4.70 mmol) at 0deg.C. The mixture was warmed to 20 ℃ and stirred for 15 minutes. A solution of methyl iodide (650 mg,4.62 mmol) in THF (2 mL) was then added dropwise to the reaction and stirred at 20deg.C for 1 hour. Will react with H ₂ O (5 mL) was quenched and the phases separated. The aqueous phase was extracted with EtOAc (5 mL x 3), and the organic layer was washed with brine (20 mL) over Na ₂ SO ₄ Dried, filtered, and concentrated in vacuo to afford the title compound 4- (benzyloxy) -1- (3-methoxypropyl) -3-methyl-1H-pyrazole (TG-16 k) (1.03 mg, 101%) as a yellow oil. ¹ H NMR (chloroform-d) delta 7.30-7.45 (m, 5H), 6.95 (s, 1H), 4.90 (s, 2H), 4.04 (t, J=6.8 Hz, 2H), 3.26-3.31 (m, 5H), 2.22 (s, 3H), 1.98-2.08 (m, 2H).

Step 10 Synthesis of 4- (benzyloxy) -1- (3-methoxypropyl) -3-methyl-5- (1-methyl-1H-imidazol-4-yl) -1H-pyrazole (Int-TG-16)

To a solution of 4- (benzyloxy) -1- (3-methoxypropyl) -3-methyl-1H-pyrazole (TG-16 k) (373 mg,1.43 mmol) in anhydrous THF (7.2 mL) was added n-BuLi (1.5 mL,3.8mmol,2.5m in hexane) dropwise at-65 ℃ (internal temperature) to maintain the internal temperature below-60 ℃ and the mixture was stirred for 1.5 hours. Triisopropyl borate (3.3 mL,14 mmol) was then added to the reaction, the reaction was removed from the cold bath, warmed gradually to room temperature, and stirred for 16 hours. Saturated NH for reaction ₄ Aqueous Cl (3 mL) followed by H ₂ And O quenching. The phases were separated, the aqueous phase was extracted with EtOAc (8 mL. Times.3), and the organic layer was washed with brine (10 mL) over Na ₂ SO ₄ Dried, filtered, and concentrated in vacuo to provide compound (4- (benzyloxy) -1- (3-methoxypropyl) -3-methyl-1H-pyrazol-5-yl) boronic acid (TG-16 l) (529 mg) as a yellow gum, which was used without further purification. LCMS [ M+H]Observed value=305.1. Will be contained in DMF (8 mL) and H ₂ (4- (benzyloxy) -1- (3-methoxypropyl) -3-methyl-1H-pyrazol-5-yl) boronic acid (TG-16 l) (529 mg), 4-iodo-1-methyl-1H-imidazole (325 mg,1.56 mmol), K in O (2 mL) ₃ PO ₄ (885 mg,4.17 mmol), cataCXium A Pd G3 (56 mg,0.077 mmol) for the reaction vessel N ₂ Backfilling, heating to 80 ℃ and stirring for 22 hours. Then the reaction is carried out using H ₂ O (20 mL) was diluted, the phases separated, and the aqueous phase was extracted with EtOAc (20 mL. Times.4). The combined organic phases were washed with brine (20 ml x 2), dried over Na ₂ SO ₄ Dried, filtered, and concentrated in vacuo. The crude residue was purified by flash column chromatography (20 g SiO ₂ Combi-flash,0-21% EtOAc/petroleum ether, then 20% MeOH/EtOAc) and re-purified by preparative thin layer chromatography (EtOAc/MeOH 10:1) to afford the title compound 4- (benzyloxy) -1- (3-methoxypropyl) -3-methyl-5- (1-methyl-1H-imidazol-4-yl) -1H-pyrazole (Int-TG-16) (31 mg, over two steps 6.4%) as a yellow gum. LCMS [ M+H ]Observed value = 341.1. ¹ H NMR (chloroform-d) δ:7.47 (s, 1H), 7.30-7.39 (m, 5H), 7.15 (s, 1H), 4.82 (s, 2H), 4.53 (t, j=7.0 hz, 2H), 3.69 (s, 3H), 3.36 (t, j=6.4 hz, 2H), 3.27 (s, 3H), 2.15 (s, 3H), 2.03-2.11 (m, 2H).

Preparation of 4- [ (4-methoxyphenyl) methoxy ] -3-methyl-5- (1-methyl-1H-imidazol-4-yl) -1-propyl-1H-pyrazole (Int-TG-17) according to scheme TG-17.

Scheme TG-17

Step 1 Synthesis of 4- [ (4-methoxyphenyl) methoxy ] -3-methyl-1-propyl-1H-pyrazole (TG-17 a)

Containing 4- (benzyloxy) -3-methyl-1- (prop-2-en-1-yl) -1H-pyrazole (TG-16 g) (505 mg,2.21 mmol), wet Pd/C (10%, 230mg,0.22 mmol), NEt in MeOH (10 mL) and THF (10 mL) ₃ (1.0 mL,7.2 mmol) reaction vessel at H ₂ (15 psi, balloon) at 20℃for 2 hours. The reaction was then filtered through a celite pad and the filtrate was concentrated in vacuo to afford 3-methyl-1- (prop-2-en-1-yl) -1H-pyrazol-4-ol (378 mg) as a gray oil, which was used without further purification. LCMS [ M+H]Observed value = 140.8; ¹ h NMR (chloroform-d) δ:6.99 (s, 1H), 3.88 (t, j=7.1 hz, 2H), 2.18 (s, 3H), 1.79 (sxt, j=7.3 hz, 2H), 0.88 (t, j=7.4 hz, 3H). To a solution of 3-methyl-1- (prop-2-en-1-yl) -1H-pyrazol-4-ol (378 mg) and 1- (chloromethyl) -4-methoxybenzene (390 mg,2.49 mmol) in DMF (5 mL) was added K ₂ CO ₃ (349mg, 2.48 mmol) and stirred at 20℃for 17 hours. The reaction was then heated to 50 ℃ for 30 minutes, then with H ₂ O (20 mL) dilution. The phases were separated and the aqueous phase was extracted with EtOAc (20 ml x 4). The organic extracts were washed with brine (20 ml x 3), over Na ₂ SO ₃ Dried, filtered, and concentrated under vacuum. The crude residue was purified by flash column chromatography (12 g SiO ₂ Combi-flash,0-80% EtOAc/petroleum ether) and purified by flash column chromatography (12 g SiO ₂ Combi-flash,0-60% EtOAc/petroleum ether) to afford the title compound 4- [ (4-methoxyphenyl) methoxy as a yellow oil]-3-methyl-1-propyl-1H-pyrazole (TG-17 a) (419 mg, 73% over two steps). LCMS [ M+H]Observed value = 260.9; ¹ h NMR (chloroform-d) δ:7.29-7.35 (m, 2H), 6.88-6.96 (m, 3H), 4.81 (s, 2H), 3.89 (t, j=7.1 hz, 2H), 3.82 (s, 3H), 2.18 (s, 3H), 1.80 (sxt, j=7.3 hz, 2H), 0.89 (t, j=7.4 hz, 3H).

Step 2 Synthesis of {4- [ (4-methoxyphenyl) methoxy ] -3-methyl-1-propyl-1H-pyrazol-5-yl } boronic acid (TG-17 b)

At-65 ℃ (internal temperature) to 4- [ (4-methoxyphenyl) methoxy]To a solution of 3-methyl-1-propyl-1H-pyrazole (TG-17 a) (337 mg,1.29 mmol) in anhydrous THF (6.0 mL) was added dropwise n-BuLi (1.4 mL,3.5mmol,2.5M in hexane) to maintain the internal temperature below-60℃and the mixture was stirred for 1.5H. Triisopropyl borate (3.0 mL,13 mmol) was then added to the reaction, the reaction was removed from the cold bath, warmed gradually to room temperature, and stirred for 16 hours. Will react with H ₂ O (5 mL) was quenched, the phases separated, and the aqueous phase extracted with EtOAc (5 mL. Times.3). The combined organic phases were taken up in Na ₂ SO ₄ Dried, filtered, and concentrated in vacuo to afford {4- [ (4-methoxyphenyl) methoxy ] as an off-white oily solid]-3-methyl-1-propyl-1H-pyrazol-5-yl } boronic acid (TG-17 b) (564 mg) was used without further purification. LCMS [ M+H]Observed value = 305.0.

Step 3 Synthesis of 4- [ (4-methoxyphenyl) methoxy ] -3-methyl-5- (1-methyl-1H-imidazol-4-yl) -1-propyl-1H-pyrazole (Int-TG-17)

Will contain a mixture of 1, 4-dioxane (8.8 mL) and H ₂ {4- [ (4-methoxyphenyl) methoxy group in O (2.2 mL)]-3-methyl-1-propyl-1H-pyrazol-5-yl } boronic acid (TG-17 b) (564 mg), 4-iodo-1-methyl-1H-imidazole (299 mg,1.44 mmol), K ₃ PO ₄ (834 mg,3.93 mmol), cataCXium A Pd G3 (95 mg,0.13 mmol) for reaction vessel N ₂ Backfilling, heating to 80 ℃ and stirring for 22 hours. Then the reaction is carried out using H ₂ O (20 mL) was diluted, the phases separated, and the aqueous phase was extracted with EtOAc (20 mL. Times.4). The combined organic phases were washed with brine (20 ml x 2), dried over Na ₂ SO ₄ Dried, filtered, and concentrated in vacuo. The crude residue was purified by preparative thin layer chromatography (SiO ₂ EtOAc: meOH 20:1) to afford the impure compound 4- [ (4-methoxyphenyl) methoxy as a yellow gum ]-3-methyl-5- (1-methyl-1H-imidazol-4-yl) -1-propyl-1H-pyrazole (Int-TG-17) (268 mg), which was used without further purification. LCMS [ M+H]Observed value = 341.1; ¹ h NMR (chloroform-d) delta 7.48 (s, 1H), 7.23-7.26 (m, 2H),7.12-7.16(m,1H),6.85-6.89(m,2H),4.74(s,2H),4.39-4.43(m,2H),3.82(s,3H),3.70(s,3H),2.13(s,3H),1.77-1.85(m,2H),0.86(t,J＝7.4Hz,3H)。

preparation of 1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carboxylic acid ethyl ester according to scheme TG-18 (Int-TG-18)

Scheme TG-18

Step 1 Synthesis of 1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carboxylic acid ethyl ester (Int-TG-18)

To a solution of ethyl 1-ethyl-3-methyl-1H-pyrazole-5-carboxylate (13.0 g,71.3 mmol) in MeCN (150 mL) was added Selectfluor (75.8 g,214 mmol), heated to 90 ℃ and stirred for 14 hours. The reaction was then cooled to room temperature, filtered, and concentrated in vacuo. The crude residue was purified by flash column chromatography (0-5% etoac/petroleum ether) and re-purified by flash column chromatography (0-5% etoac/petroleum ether) to afford ethyl 1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carboxylate (Int-TG-18) (11.5 g, 80%) as a colorless oil, which was used without further purification. LCMS [ M+H]Observed value = 201.0. ¹ H NMR (chloroform-d) delta 4.35-4.50 (m, 4H), 2.24 (s, 3H), 1.37-1.43 (m, 6H).

The intermediates in the following table were prepared according to the procedure used in step 2-4 of scheme TG-3 for the synthesis of 1- [3- (benzyloxy) propyl ] -5- (1H-imidazol-4-yl) -3-methyl-1H-pyrazole (Int-TG-3), using non-critical variations or permutations of the exemplary procedure that can be appreciated by those skilled in the art.

Preparation of 1-ethyl-4-iodo-1H-imidazole according to scheme TG-20 (Int-TG-20)

Scheme TG-20

Step 1 Synthesis of 1-ethyl-4, 5-diiodo-1H-imidazole (TG-20 b)

To a solution of 4, 5-diiodo-1H-imidazole (TG-20 a) (1.00 g,3.13 mmol) in THF (8.0 mL) was added NaH (138 mg,3.44mmol,60% in mineral oil) in small portions at 0deg.C. The mixture was warmed to 20 ℃ and stirred for 1 hour. Ethyl bromide (1.56 ml,20.9 mmol) was then added to the reaction and stirred for 18 hours. The mixture was concentrated in vacuo, the residue was dissolved in EtOAc (10 mL), filtered, and concentrated in vacuo. The residue was then triturated in EtOAc in petroleum ether (1:1, 10 ml) at room temperature for 15 min, filtered, and concentrated in vacuo to afford the title compound 1-ethyl-4, 5-diiodo-1H-imidazole (TG-20 b) (460 mg, 63%) as a colorless solid. LCMS [ M+H]Observed value = 348.8; ¹ h NMR (chloroform-d) δ:7.65 (s, 1H), 4.03 (q, j=7.3 hz, 2H), 1.42 (t, j=7.3 hz, 3H).

Step 2 Synthesis of 1-ethyl-4-iodo-1H-imidazole (Int-TG-20)

To a solution of 1-ethyl-4, 5-diiodo-1H-imidazole (TG-20 b) (690 mg,1.98 mmol) in THF (7.0 mL) was added dropwise magnesium isopropylchloride (0.992 mL,1.98mmol,2.0M in THF) at 0deg.C. The mixture was stirred at 0 ℃ and stirred for 20 minutes. H is then added to the reaction ₂ O (0.5 mL), warmed to 20℃and stirred for 1 hour. The mixture was concentrated in vacuo, and the residue was dissolved in EtOAc (5 mL) and filtered. The filtrate was washed with brine (10 mL), over Na ₂ SO ₃ Dried and concentrated under vacuum. The crude residue was purified by flash column chromatography (12 g SiO ₂ ,Combi-flash,0-30％MeOH/CH ₂ Cl ₂ ) Purification provided the title compound 1-ethyl-4-iodo-1H-imidazole (Int-TG-20) (300 mg, 68%) as a colorless oil. LCMS [ M+H]Observed value = 222.9; ¹ h NMR (chloroform-d) δ:7.55 (s, 1H), 7.04 (d, j=1.3 hz, 1H), 4.02 (q, j=7.3 hz, 2H), 1.47 (t, j=7.4 hz, 3H).

The intermediates in the following table were prepared according to the procedure used in step 3 of scheme TG-10 for the synthesis of 1-ethyl-4- [ (4-methoxyphenyl) methoxy ] -3-methyl-5- (1-methyl-1H-imidazol-4-yl) -1H-pyrazole (Int-TG-10), using non-critical variations or permutations of the exemplary procedure as can be appreciated by those skilled in the art.

Preparation of 3-methyl-5- (1-methyl-1H-imidazol-4-yl) -1- [ (oxetan-3-yl) methyl ] -1H-pyrazole (Int-TG-23) according to scheme TG-23

Scheme TG-23

Step 1 Synthesis of 5-bromo-3-methyl-1- [ (oxetan-3-yl) methyl ] -1H-pyrazole (TG-23 c) and 3-bromo-5-methyl-1- [ (oxetan-3-yl) methyl ] -1H-pyrazole (TG-23 c')

To a solution of 5-bromo-3-methyl-1H-pyrazole (TG-23 a) (1500 mg,9.317 mmol) and (oxetan-3-yl) methanol (TG-23 b) (1.5 mL,19 mmol) in dioxane (37.5 mL) was added (cyanomethylene) tributylphosphine (4500 mg,18.6 mmol) at room temperature (19 ℃ C.). The brown solution was stirred at room temperature (19 ℃) for 16 hours. LCMS analysis indicated that starting material remained. At this stage, additional aliquots of (cyanomethylene) tributylphosphine (1000 mg,4.143 mmol) and (oxetan-3-yl) methanol (TG-23 b) (334. Mu.L, 4.15 mmol) were added and the reaction stirred at room temperature (20 ℃) for 19 hours. The reaction mixture was diluted with EtOAc (50 mL) and transferred to a separatory funnel. The solution was washed with 3 parts brine (20 mL), dried (Na ₂ SO ₄ ) Filtered and concentrated under vacuum. The crude residue was purified by reverse phase prep HPLC (YMC triple C18250 x 50mm x 7um column, 11-51% mecn/water (0.05% nh) ₄ OH v/v), 60 mL/min). The product containing fractions were collected and extracted with 2 parts of EtOAc (100 mL). The combined organic extracts were dried (Na ₂ SO ₄ ) Filtered, and concentrated in vacuo to provide the title compound 5-bromo-3-methyl-1- [ (oxetan-3-yl) methyl as a yellow oil]-1H-pyrazole (TG-23 c) and 3-bromo-5-methyl-1- [ (oxetan-3-yl) methyl ]-1H-pyrazole (TG-23 c') (1.71 g, about 3:2r.r., 79%). TG-23c (major product) ¹ H NMR (400 MHz, chlorine)Imitation-d) δ=6.06 (s, 1H), 4.82 (br d, j=4.9 hz, 2H), 4.57 (t, j=6.2 hz, 2H), 4.39 (d, j=7.3 hz, 2H), 3.60-3.46 (m, 1H), 2.23 (s, 3H). TG-23c' (minor product) ¹ H NMR (400 MHz, chloroform-d) δ=6.02 (s, 1H), 4.87-4.77 (m, 2H), 4.49 (t, j=6.1 hz, 2H), 4.29 (d, j=7.5 hz, 2H), 3.61-3.43 (m, 1H), 2.29 (s, 3H). The mixture of regioisomeric products (1588 mg) was further purified by preparative SFC to provide the desired major regioisomer 5-bromo-3-methyl-1- [ (oxetan-3-yl) methyl as a yellow oil]-1H-pyrazole (TG-23 c) (950 mg). ¹ H NMR (400 MHz, chloroform-d) δ=6.07 (s, 1H), 4.81 (d, j=6.4 hz, 1H), 4.79 (d, j=6.5 hz, 1H), 4.57 (t, j=6.2 hz, 2H), 4.39 (d, j=7.4 hz, 2H), 3.62-3.43 (m, 1H), 2.23 (s, 3H).

Step 2 Synthesis of 3-methyl-5- (1-methyl-1H-imidazol-4-yl) -1- [ (oxetan-3-yl) methyl ] -1H-pyrazole (Int-TG-23)

To 5-bromo-3-methyl-1- [ (oxetan-3-yl) methyl at room temperature (20 ℃)]To a solution of 1H-pyrazole (TG-23 c) (325 mg,1.41 mmol) in toluene (9 mL) was added 1-methyl-4- (tributylstannyl) -1H-imidazole (TG-23 d) (650 mg,1.4 mmol) and Pd (PPh) ₃ ) ₄ (325 mg, 0.281mmol). After addition, the reaction mixture was taken up in N ₂ Stirring was carried out at 100℃for 16 hours. The reaction mixture was filtered and the filtrate was concentrated in vacuo. The crude residue was purified by flash column chromatography (20 g SiO ₂ Isco,0-5% meoh/DCM) to afford the title compound 3-methyl-5- (1-methyl-1H-imidazol-4-yl) -1- [ (oxetan-3-yl) methyl as a yellow oil]-1H-pyrazole (Int-TG-23) (199mg, 61%). LCMS [ M+H]Observed value = 233.2; ¹ h NMR (400 MHz, chloroform-d) δ=7.50 (s, 1H), 7.05 (s, 1H), 6.09 (s, 1H), 4.83-4.69 (m, 4H), 4.55 (t, j=6.2 hz, 2H), 3.74 (s, 3H), 3.65-3.51 (m, 1H), 2.25 (s, 3H).

According to the method for the synthesis of 3-methyl-5- (1-methyl-1H-imidazol-4-yl) -1- [ (oxetan-3-yl) methyl ] -1H-pyrazole (Int-TG-23), the intermediates in the following table were prepared using non-critical variations or permutations of the exemplary procedure as can be appreciated by those skilled in the art.

Preparation of 1- (3-methoxypropyl) -3-methyl-5- (1-methyl-1H-imidazol-4-yl) -1H-pyrazole (Int-TG-25) according to scheme TG-25

Scheme TG-25

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Step 1 Synthesis of 1- (3-methoxypropyl) -3-methyl-1H-pyrazole (TG-25 c)

To 3-methyl-1H-pyrazole (TG-25 a) (5.00 g,60.9 mmol) and 1-bromo-3-methoxypropane (TG-25 b) (18.6 g,122 mmol) in CH ₂ Cl ₂ Cs was added to the solution in (40 mL) ₂ CO ₃ (29.3 g,89.9 mmol). The reaction was heated to reflux and stirred for 14 hours. The mixture was then filtered and the filter cake was purified with CH ₂ Cl ₂ (60 mL) and the filtrate was concentrated in vacuo. Residual 1-bromo-3-methoxypropane (TG-25 b) was removed by distillation (about 0.1mpa,33-36 ℃) and the remaining crude 1- (3-methoxypropyl) -3-methyl-1H-pyrazole (TG-25 c) and 1- (3-methoxypropyl) -5-methyl-1H-pyrazole (TG-25 c') (7.50 g) were separated into about 3:2 positional isomer mixtures, which were used in the next step without further purification. LCMS [ M+H]Observed value=155.1.

Step 2 Synthesis of Potassium trifluoro [1- (3-methoxypropyl) -3-methyl-1H-pyrazol-5-yl ] borate (TG-25 d)

To a solution of 1- (3-methoxypropyl) -3-methyl-1H-pyrazole (TG-25 c) and 1- (3-methoxypropyl) -5-methyl-1H-pyrazole (TG-25 c') (1.65 g) in THF (43 mL) at 0deg.C was added n-BuLi (7.4 mL,18mmol,2.5M in hexane) dropwise. The reaction was stirred for 10 minutes and then warmed to room temperature for 2 hours. The reaction was then cooled to 0deg.C and triisopropyl borate (9.9 mL,43 mmol) was added dropwise. After the addition was complete, the reaction was warmed to room temperature and stirred for 2 hours. The reaction was then cooled to 0℃followed by the addition of KHF ₂ (3.35 g,42.9 mmol) and H ₂ O (3 mL). The reaction was warmed to 60 ℃ (internal temperature=45℃) and stirred for 2 hours. Then adding additional KHF ₂ (2.51 g,32.1 mmol) and H ₂ O (3 mL), and the reaction was stirred at 80 ℃ (internal temperature=60℃) for 1 hour. The crude was then decanted and concentrated in vacuo to afford the compound trifluoro [1- (3-methoxypropyl) -3-methyl-1H-pyrazol-5-yl as a brown oil]Potassium borate (TG-25 d) (2.20 g) was used without further purification.

Step 3 Synthesis of 1- (3-methoxypropyl) -3-methyl-5- (1-methyl-1H-imidazol-4-yl) -1H-pyrazole (Int-TG-25)

Will contain trifluoro [1- (3-methoxypropyl) -3-methyl-1H-pyrazol-5-yl]Potassium borate (TG-25 d) (2.20 g), 4-iodo-1-methyl-1H-imidazole (1.35 g,6.49 mmol), K ₃ PO ₄ (4.09g,19.3mmol)、cataCXium A-Pd-G3(237mg,0.326mmol)、H ₂ N for reaction vessel of O (6.0 mL) and 1, 4-dioxane (30 mL) ₂ Backfilled and stirred at 80 ℃ (internal temperature) for 13 hours. The reaction was filtered, the phases separated, and the aqueous phase was extracted with EtOAc (6 ml x 3). Brine (20 mL) and water (20 mL) were added to the combined organic phases, the phases were separated, and the aqueous phase was extracted with EtOAc (15 mL x 3). The combined organic phases were taken up in Na ₂ SO ₄ Dried, filtered, and concentrated under vacuum. The crude residue was purified by flash column chromatography (40 g SiO ₂ Combi-flash,0-100% EtOAc/petroleum ether, then 0-20% meoh/EtOAc) to afford the title compound 1- (3-methoxypropyl) -3-methyl-5- (1-methyl-1H-imidazol-4-yl) -1H-pyrazole (Int-TG-25) (498 mg, 26% over 3 steps) as a brown oil. LCMS [ M+H]Observed value = 235.4; ¹ h NMR (methanol-d) ₄ )δ:7.73(s,1H),7.41(d,J＝1.2Hz,1H),6.25(s,1H),4.37(t,J＝7.1Hz,2H),3.81(s,3H),3.32-3.36(m,2H),3.26(s,3H),2.26(s,3H),1.97-2.05(m,2H)。

Preparation of [5- (1H-imidazol-4-yl) -3-methyl-1H-pyrazol-1-yl ] acetonitrile according to scheme TG-27 (Int-TG-27)

Scheme TG-27

Step 1 Synthesis of 1- (cyanomethyl) -3-methyl-1H-pyrazole-5-carboxylic acid methyl ester (TG-27 b)

3-To a solution of methyl-1H-pyrazole-5-carboxylic acid ethyl ester (TG-3 a) (3.00 g,19.5 mmol) and 2-bromoacetonitrile (2.80 g,2.34 mmol) in MeCN (30 mL) was added K ₂ CO ₃ (5.38 g,38.9 mmol) was heated to 85℃and stirred for 5 hours. The mixture was then filtered and the filtrate concentrated in vacuo. The crude residue was purified by flash column chromatography (40 g SiO ₂ Combi-flash,0-30% etoac/petroleum ether) to afford the title compound 1- (cyanomethyl) -3-methyl-1H-pyrazole-5-carboxylic acid methyl ester (TG-27 b) (1.75 g, 46%) as an off-white solid. ¹ H NMR (chloroform-d) δ:6.71 (s, 1H), 5.45 (s, 2H), 4.38 (q, j=7.3 hz, 2H), 2.30 (s, 3H), 1.39 (t, j=7.0 hz, 3H).

Step 2 Synthesis of 1- (cyanomethyl) -3-methyl-1H-pyrazole-5-carboxylic acid (TG-27 c)

To 1- (cyanomethyl) -3-methyl-1H-pyrazole-5-carboxylic acid methyl ester (TG-27 b) (1.85 g,9.57 mmol) in THF (37 mL) and H at 0deg.C ₂ To a solution of O (9.25 mL) was added lithium hydroxide monohydrate (442 mg,10.5 mmol) and stirred for 4 hours. The mixture was then acidified to pH 1 with aqueous HCl (1N), the phases were separated, and the aqueous phase was extracted with EtOAc (20 ml x 3). The combined organic extracts were subjected to Na ₂ SO ₃ Dried, filtered, and concentrated in vacuo to afford the title compound 1- (cyanomethyl) -3-methyl-1H-pyrazole-5-carboxylic acid (TG-27 c) (1.55 g, 98%) as a yellow solid. LCMS [ M+H]Observation value=166.0; ¹ H NMR(DMSO-d ₆ )δ:6.73(s,1H),5.60(s,2H),2.20(s,4H)。

step 3 Synthesis of 1- (cyanomethyl) -3-methyl-1H-pyrazole-5-carboxylic acid (TG-27 d)

To a solution of 1- (cyanomethyl) -3-methyl-1H-pyrazole-5-carboxylic acid (TG-27 c) (1.38 g,8.36 mmol) and methoxyamine hydrochloride (978 mg,10.0 mmol) in anhydrous DMF (23 mL) was added HATU (4.77 g,12.5 mmol) and stirred for 10 min. N-N-diisopropylethylamine (2.98 mL,16.7 mmol) was then added to the reaction, and the reaction was stirred for 16 hours. Will react with H ₂ O (20 mL) was diluted, the phases separated, and the aqueous phase was taken up in CH ₂ Cl ₂ (20 mL. Times.3) extraction. The combined organic phases were treated with saturated NH ₄ Cl aqueous solution (20 mL. Times.3), saturated Na ₂ CO ₃ Aqueous (20 mL. Times.3), brine (20 mL. Times.3), washed with Na ₂ SO ₄ Dried, filtered, and concentrated in vacuo. The crude residue was purified by flash column chromatography (40 g SiO ₂ Combi-flash,12.5-75% etoac/petroleum ether) was purified to afford 1- (cyanomethyl) -3-methyl-1H-pyrazole-5-carboxylic acid (TG-27 d) (1.91 g, 94% over three combined batches) as a yellow solid, which contained some impurities. This material was used without further purification. LCMS [ M+H]Observed value=209.1. ¹ H NMR (chloroform-d) delta: 6.70 (s, 1H), 5.47 (s, 2H), 3.71 (s, 3H), 2.31 (s, 3H).

Step 4 Synthesis of (5-formyl-3-methyl-1H-pyrazol-1-yl) acetonitrile (TG-27 e)

at-10deg.C at N ₂ To a solution of 1- (cyanomethyl) -3-methyl-1H-pyrazole-5-carboxylic acid (TG-27 d) (1.60 g,7.68 mmol) in anhydrous THF (76.8 mL) was added dropwise dibutylaluminum hydride (15.4 mL,15.4mmol,1 m) to maintain the internal temperature below-5 ℃. The reaction was stirred at-5℃for 2 hours, then saturated NH ₄ Aqueous Cl (50 mL) was quenched, treated with celite, and stirred at room temperature for 15 min. The mixture was filtered and the filter cake was washed with EtOAc (20 ml x 5). The phases were separated and the aqueous phase was extracted with EtOAc (20 ml x 3). The combined organic phases were taken up in anhydrous Na ₂ SO ₄ Dried, filtered, and concentrated under vacuum. The crude residue was purified by flash column chromatography (40 g SiO ₂ Combi-flash,5-30% etoac/petroleum ether) to afford the title compound (5-formyl-3-methyl-1H-pyrazol-1-yl) acetonitrile (TG-27 e) (460 mg, 41%) as a yellow solid. ¹ H NMR (chloroform-d) delta 9.81 (s, 1H), 6.78 (s, 1H), 5.43 (s, 2H), 2.34 (s, 3H).

Step 5 Synthesis of N- { (E) - [1- (cyanomethyl) -3-methyl-1H-pyrazol-5-yl ] methylene } -2-methylpropan-2-sulfinamide (TG-27 f)

To a solution of (5-formyl-3-methyl-1H-pyrazol-1-yl) acetonitrile (TG-27 e) (385 mg,2.58 mmol) in anhydrous THF (7.7 mL) were added 2-methylpropane-2-sulfinamide (375 mg,3.10 mmol) and titanium tetraethoxide (1.18 g,5.16 mmol). The reaction was stirred at room temperature for 2 hours and then concentrated in vacuo. The crude residue was purified by flash column chromatography (20 gSiO ₂ Combi-flash,10-50% etoac/petroleum ether) to afford the title compound N- { as an off-white solid(E) - [1- (cyanomethyl) -3-methyl-1H-pyrazol-5-yl]Methylene } -2-methylpropane-2-sulfinamide (TG-27 f) (607 mg, 93%). LCMS [ M+H]=253.8 observations; ¹ h NMR (chloroform-d) δ:8.52 (s, 1H), 6.60 (s, 1H), 5.62 (d, j=17.1 hz, 1H), 5.33 (d, j=17.3 hz, 1H), 2.33 (s, 3H), 1.31 (s, 9H).

Step 6 Synthesis of [5- (1H-imidazol-4-yl) -3-methyl-1H-pyrazol-1-yl ] acetonitrile (Int-TG-27)

N- { (E) - [1- (cyanomethyl) -3-methyl-1H-pyrazol-5-yl ] at-5 ℃]To a solution of methylene } -2-methylpropane-2-sulfinamide (TG-27 f) (540 mg,2.14 mmol) in MeOH (6.8 mL) was added 1- ((isocyanomethyl) sulfonyl) -4-methylbenzene (460 mg,2.35 mmol) and K ₂ CO ₃ (355 mg,2.57 mmol) and stirred for 30 minutes. Saturated NH for reaction ₄ Aqueous Cl (10 mL) was quenched, the phases separated, and the aqueous phase extracted with EtOAc (15 mL. Times.3). The combined organic phases were taken up in Na ₂ SO ₄ Dried, filtered, and concentrated under vacuum. The crude residue was purified by flash column chromatography (40 g SiO ₂ Combi-flash,0-7% MeOH/EtOAc) to afford the title compound [5- (1H-imidazol-4-yl) -3-methyl-1H-pyrazol-1-yl ] as an off-white solid]Acetonitrile (Int-TG-27) (78 mg, 19%). LCMS [ M+H]Observed value=188.0; ¹ HNMR (methanol-d) ₄ )δ:7.81(s,1H),7.48(s,1H),6.33(s,1H),5.55(s,2H),2.26(s,3H)。

Preparation of examples：

Preparation of 4- [4- (1-ethyl-3-methyl-1H-pyrazol-5-yl) -1-methyl-1H-imidazol-2-yl ] -1-methyl-1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (example AIA 01) according to scheme A.

Scheme a:

step 1 Synthesis of 1-ethyl-3-methyl-5- (1-methyl-1H-imidazol-4-yl) -1H-pyrazole (A-1)

To 1-ethyl-5- (1H-imidazol-4-yl) -3-methyl-1H-pyrazole (Int-TG-1) (79mg, 2.9 mmol) and K ₂ CO ₃ (1.21 g,8.74 mmol) in anhydrous MeCN (8.0 mL) in yellow MeI (45 mg,3.21 mmol) was added dropwise to the compound. The reaction was stirred at room temperature for 3 hours. The reaction was filtered and the filtrate was concentrated in vacuo. The crude residue was purified by flash column chromatography (40 g SiO ₂ Combi-flash,50% etoac/DCM) to give the desired product contaminated with formamide. The product was re-purified by preparative thin layer chromatography (10% meoh/DCM) to provide the title compound 1-ethyl-3-methyl-5- (1-methyl-1H-imidazol-4-yl) -1H-pyrazole (a-1) as a yellow oil (869 mg, 55%). LCMS [ M+H]Observed value =191.3; ¹ HNMR (400 MHz, chloroform-d) δ=7.49 (d, j=0.7 hz, 1H), 7.04 (d, j=1.3 hz, 1H), 6.13 (s, 1H), 4.45 (q, j=7.2 hz, 2H), 3.74 (s, 3H), 2.29 (s, 3H), 1.43 (t, j=7.2 hz, 3H).

Step 2 Synthesis of N- [ (2, 4-dimethoxyphenyl) methyl ] -4- [4- (1-ethyl-3-methyl-1H-pyrazol-5-yl) -1-methyl-1H-imidazol-2-yl ] -1-methyl-1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (A-2)

The reaction vessel was charged with 1-ethyl-3-methyl-5- (1-methyl-1H-imidazol-4-yl) -1H-pyrazole (A-1) (660 mg,3.47 mmol), 4-bromo-N- (2, 4-dimethoxybenzyl) -1-methyl-1H-pyrazolo [4,3-c ]]Pyridine-6-carboxamide (Int-HG-1) (2.10 g,5.17 mmol), pd (OAc) ₂ (236mg,1.09mmol)、CuI(200mg,1.05mmol)、PPh ₃ (273mg,1.04mmol)、Cs ₂ CO ₃ (3401.7 mg,10.440 mmol), pivOH (385 mg,3.77 mmol) and PhMe (26 mL). The solution was treated with N ₂ Rinsed for 2 minutes, sealed, and heated to 110 ℃ for 27 hours. LCMS analysis indicated incomplete conversion of starting material, so Pd (OAc) was added ₂ (124mg,0.551mmol)、CuI(101mg,0.529mmol)、PPh ₃ (139mg,0.529mmol)、Cs ₂ CO ₃ (1.14 g,3.51 mmol) and additional aliquots of PivOH (184 mg,1.80 mmol). The reaction mixture was reused with N ₂ Rinsed for 2 minutes, sealed, and heated to 110 ℃ for 19 hours. The reaction was filtered through a pad of celite and the filter cake was washed with DCM (20 mL) and then 3 parts DCM/MeOH (10:1, 10mL each). The combined filtrates were concentrated in vacuo. The crude residue was purified by flash column chromatography (40 g SiO ₂ Isco,0-100% EtOAc/petroleum ether followed by 10% meoh/EtOAc) to afford the title compound N- [ (2, 4-dimethoxyphenyl) methyl as a yellow gum]-4- [4- (1-ethyl-3-methyl-1H-pyrazol-5-yl) -1-methyl-1H-imidazol-2-yl]-1-methyl-1H-pyrazolo [4,3-c]Pyridine-6-carboxamide (A-2) (1.15 g, 65%) which contains some residue (A-1) starting material. The material was used in the next step without further purification. LCMS [ M+H]Observed value=515.1.

Step 3 Synthesis of 4- [4- (1-ethyl-3-methyl-1H-pyrazol-5-yl) -1-methyl-1H-imidazol-2-yl ] -1-methyl-1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (example AIA 01)

To a methyl group containing N- [ (2, 4-dimethoxyphenyl) methyl group]-4- [4- (1-ethyl-3-methyl-1H-pyrazol-5-yl) -1-methyl-1H-imidazol-2-yl]-1-methyl-1H-pyrazolo [4,3-c]To a reaction vessel of pyridine-6-carboxamide (A-2) (1.15 g,1.60 mmol) was added HFIP (10 mL) followed by dropwise addition of MsOH (1.50 g,15.6 mmol). The reaction was stirred at room temperature for 1 hour, which was accompanied by the gradual formation of a dark red solution. The reaction was concentrated in vacuo and the residue was dissolved in DCM (8 mL). The solution was treated with 7M NH ₃ MeOH to adjust the pH to about 8, which resulted in precipitation of a solid. The suspension was concentrated in vacuo and the crude residue was diluted with DCM (20 mL) and water (20 mL). Undissolved solids were filtered off at this stage. The filtrate was transferred to a separatory funnel and the phases were separated. The aqueous phase was extracted with 3 parts DCM (10 mL each). The combined organic extracts were dried (Na ₂ SO ₄ ) Filtered and concentrated under vacuum. The crude residue was purified by preparative thin layer chromatography (EtOAc/MeOH/NH ₄ OH, 20:1:0.1) to afford the desired product contaminated with some residue (A-1). The material thus obtained was purified by flash column chromatography (SiO ₂ Isco, DCM/MeOH, 10:1) to afford the desired product, which still contained residue (a-1). The beige solid thus obtained was diluted with DMSO and filtered. The filtrate was purified by preparative HPLC (Boston Prime C18150x30mmx5um column, 27-57% MeCN/H ₂ O (containing 0.05% NH) ₄ OH), 25 mL/min) to afford the title compound 4- [4- (1-ethyl-3-methyl-1H-pyrazol-5-yl) -1-methyl-1H-imidazol-2-yl) as a fluffy white solid]-1-methyl-1H-pyrazolo [4,3-c]Pyridine-6-carboxamide (example AIA 01) (114 mg, 20%). LCMS [ M+H]Observed value = 365.3; ¹ H NMR(400MHz,DMSO-d ₆ )δ＝8.73(d,J＝0.9Hz,1H),8.35(d,J＝0.6Hz,1H),7.94(br s,1H),7.90(br s,1H),7.86(s,1H),6.32(s,1H),4.55(q,J＝7.1Hz,2H),4.24(s,3H),4.19(s,3H),2.18(s,3H),1.40(t,J＝7.1Hz,3H)。

the examples in the following table are prepared according to the procedure used to synthesize 4- [4- (1-ethyl-3-methyl-1H-pyrazol-5-yl) -1-methyl-1H-imidazol-2-yl ] -1-methyl-1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (example AIA 01) using non-critical variations or permutations of the example procedure that can be recognized by those skilled in the art.

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Preparation of 4- [ 1-cyclopropyl-4- (1-ethyl-3-methyl-1H-pyrazol-5-yl) -1H-imidazol-2-yl ] -1-methyl-1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (example AIB 01) according to scheme B.

Scheme B:

step 1 Synthesis of 5- (1-cyclopropyl-1H-imidazol-4-yl) -1-ethyl-3-methyl-1H-pyrazole (B-1)

To a reaction vessel containing 1-ethyl-5- (1H-imidazol-4-yl) -3-methyl-1H-pyrazole (Int-TG-1) (106 mg,0.430 mmol) was added 2,2' -bipyridine (64.3 mg,0.412 mmol), cu (OAc) ₂ (73.3 mg,0.404 mmol), cyclopropylboronic acid (103.6 mg,1.21 mmol), na ₂ CO ₃ (134.3 mg,1.27 mmol) and DCE (1.2 mL). The reaction was heated to 70 ℃ and stirred for 3 hours. The reaction was removed from heating and allowed to cool to room temperature. The solution was diluted with water (10 mL) and transferred to a separatory funnel containing DCM (10 mL). The phases were separated and the aqueous phase was extracted with 3 parts of DCM (10 mL each). The combined organic extracts were treated with 2 parts of saturated NH ₄ Cl aqueous solution (10 mL each), 1 part saltWashed with water (15 mL), dried (Na ₂ SO ₄ ) Filtered and concentrated under vacuum. The crude residue was purified by preparative thin layer chromatography (SiO ₂ DCM/MeOH 10:1) to afford the title compound 5- (1-cyclopropyl-1H-imidazol-4-yl) -1-ethyl-3-methyl-1H-pyrazole (B-1) (45.3 mg) as a dark brown oil, containing minor impurities. The material was used in the next step without further purification. LCMS [ M+H]Observed value =216.8.

Step 2 Synthesis of 4- [ 1-cyclopropyl-4- (1-ethyl-3-methyl-1H-pyrazol-5-yl) -1H-imidazol-2-yl ] -N- [ (2, 4-dimethoxyphenyl) methyl ] -1-methyl-1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (B-2)

To a reaction vessel containing 5- (1-cyclopropyl-1H-imidazol-4-yl) -1-ethyl-3-methyl-1H-pyrazole (B-1) (111 mg,0.293 mmol) was charged 4-bromo-N- (2, 4-dimethoxybenzyl) -1-methyl-1H-pyrazolo [4,3-c]Pyridine-6-carboxamide (Int-HG-1) (175.4 mg,0.433 mmol), pd (OAc) ₂ (6.6mg,0.029mmol)、CuI(Xantphos)(67.1mg,0.087mmol)、dppf(8.8mg,0.016mmol)、Cs ₂ CO ₃ (284 mg,0.876 mmol) and PhMe (2.7 mL). N for container ₂ Purging for 5 cycles. The reaction mixture was heated to 110 ℃ and stirred for 15 hours. LCMS analysis at this stage showed that the starting material had been depleted. Pd (OAc) is added ₂ (7.8 mg,0.035 mmol), 4-bromo-N- (2, 4-dimethoxybenzyl) -1-methyl-1H-pyrazolo [4,3-c ] ]Additional aliquots of pyridine-6-carboxamide (Int-HG-1) (60.5 mg,0.149 mmol), dppf (10.2 mg,0.018 mmol) and the reaction was heated to 110℃for 8 hours. The reaction was removed from heating and gradually cooled to room temperature. The solution was diluted with DCM (10 mL) and filtered through a pad of celite. The filter cake was washed with 3 parts DCM (5 mL each) and the filtrate was concentrated in vacuo. The crude residue was purified by preparative thin layer chromatography (SiO ₂ 100% etoac) to afford the desired product contaminated with trace impurities. The material was repurified by preparative thin layer chromatography (EtOAc/MeOH 10:1) to afford the title compound 4- [ 1-cyclopropyl-4- (1-ethyl-3-methyl-1H-pyrazol-5-yl) -1H-imidazol-2-yl]-N- [ (2, 4-dimethoxyphenyl) methyl]-1-methyl-1H-pyrazolo [4,3-c]Pyridine-6-carboxamide (B-2) (109 mg) contains a trace amount of residue (B-1). Subjecting the material to no treatmentFurther purification was used in the next step. LCMS [ M+H]Observed value = 541.2.

Step 3 Synthesis of 4- [ 1-cyclopropyl-4- (1-ethyl-3-methyl-1H-pyrazol-5-yl) -1H-imidazol-2-yl ] -1-methyl-1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (AIB 01)

To 4- [ 1-cyclopropyl-4- (1-ethyl-3-methyl-1H-pyrazol-5-yl) -1H-imidazol-2-yl ]-N- [ (2, 4-dimethoxyphenyl) methyl]-1-methyl-1H-pyrazolo [4,3-c]To a yellow solution of pyridine-6-carboxamide (B-2) (109 mg,0.12 mmol) in HFIP (2.0 mL) was added MsOH (118 mg,1.23 mmol). The reaction was stirred at room temperature for 2 hours, which was accompanied by the gradual formation of a deep purple solution. The solution was concentrated in vacuo and co-evaporated 3 times with DCM (5 mL each). The crude residue was suspended in DMSO and MeOH, followed by filtration. The filtrate was purified by preparative HPLC (Boston Prime C18150x30mmx5um column, 28-58% MeCN/H ₂ O (containing 0.05% NH) ₄ OH), 25 mL/min) to afford the title compound 4- [ 1-cyclopropyl-4- (1-ethyl-3-methyl-1H-pyrazol-5-yl) -1H-imidazol-2-yl) as a white solid]-1-methyl-1H-pyrazolo [4,3-c]Pyridine-6-carboxamide (AIB 01) (10 mg, 22%). LCMS [ M+H]Observed value =391.2; ¹ H NMR(400MHz,DMSO-d ₆ )δ＝8.67(d,J＝0.6Hz,1H),8.38(s,1H),7.98(br s,1H),7.87(s,1H),7.84(br d,J＝1.8Hz,1H),6.35(s,1H),4.55(q,J＝7.1Hz,2H),4.48-4.40(m,1H),4.19(s,3H),2.17(s,3H),1.39(t,J＝7.1Hz,3H),1.08-1.00(m,2H),1.00-0.93(m,2H)。

1-ethyl-4- [4- (1-ethyl-3-methyl-1H-pyrazol-5-yl) -1-methyl-1H-imidazol-2-yl ] -1H-pyrazolo [4,3-C ] pyridine-6-carboxamide (example AIC 01) was prepared according to scheme C.

Scheme C:

step 1 Synthesis of 6-chloro-1-ethyl-4- [4- (1-ethyl-3-methyl-1H-pyrazol-5-yl) -1-methyl-1H-imidazol-2-yl ] -1H-pyrazolo [4,3-C ] pyridine (C-1)

To a reaction vessel containing 1-ethyl-3-methyl-5- (1-methyl-1H-imidazol-4-yl) -1H-pyrazole (A-1) (196 mg,1.03 mmol) was charged THF (5 mL). The solution was cooled to-78 ℃ in a dry ice/AcMe bath. n-BuLi (505. Mu.L, 1.26 mmol) was added dropwise to the solution under an inert atmosphere. After the addition, the reaction mixture was stirred at-78 ℃ for 1 hour. Then ZnCl is added at-78 DEG C ₂ (1.4 mL,380mg,2.8 mmol) and then the ice bath was removed to allow the reaction to gradually warm to room temperature. At this stage, 4, 6-dichloro-1-ethyl-1H-pyrazolo [4,3-c ] is added]Pyridine (Int-HG-2) (235 mg,1.09 mmol) and Pd (PPh) ₃ ) ₄ (255 mg,0.221 mmol) and the mixture was heated to 60℃and stirred under an inert atmosphere for 14 hours. The solution was concentrated in vacuo and the crude residue was purified by flash column chromatography (40 g SiO ₂ Isco,100% DCM to 1% meoh/DCM) to afford the title compound 6-chloro-1-ethyl-4- [4- (1-ethyl-3-methyl-1H-pyrazol-5-yl) -1-methyl-1H-imidazol-2-yl as a yellow solid]-1H-pyrazolo [4,3-c]Pyridine (C-1) (115 mg, 30%). LCMS [ M+H]Observed value=370.1; ¹ HNMR (400 MHz, chloroform-d) δ=8.86 (s, 1H), 7.29 (s, 1H), 7.24 (s, 1H), 6.22 (s, 1H), 4.67 (q, j=7.2 hz, 2H), 4.41 (q, j=7.3 hz, 2H), 4.28 (s, 3H), 2.32 (s, 3H), 1.55 (t, j=7.2 hz, 6H).

Step 2 Synthesis of 1-ethyl-4- [4- (1-ethyl-3-methyl-1H-pyrazol-5-yl) -1-methyl-1H-imidazol-2-yl ] -1H-pyrazolo [4,3-C ] pyridine-6-carbonitrile (C-2)

To a composition containing 6-chloro-1-ethyl-4- [4- (1-ethyl-3-methyl-1H-pyrazol-5-yl) -1-methyl-1H-imidazol-2-yl]-1H-pyrazolo [4,3-c]A reaction vessel of pyridine (C-1) (115 mg,0.310 mmol) was charged with DMA (5 mL), zn (CN) ₂ (50.0 mg,0.426 mmol), zn powder (14.4 mg,0.220 mmol) and (t-Bu) ₃ P) ₂ Pd (32.7 mg,0.064 mmol). The reaction solution was treated with N ₂ Rinse for 2 minutes, seal, heat to 120 ℃ and stir for 16 hours. The reaction mixture was removed from heating and gradually cooled to room temperature. The solution was filtered over a pad of celite and the filter cake was washed with 2 parts EtOAc (5 mL each) and 2 parts H ₂ O (3 mL each) was washed. The filtrate was transferred to a separatory funnel and the phases were separated. The aqueous phase was extracted with 3 parts EtOAc (5 mL). The combined organic extracts were washed with 3 parts brine (10 mL each), dried (Na ₂ SO ₄ ) Filtered, and concentrated in vacuo to provide the title compound 1-ethyl-4- [4- (1-ethyl-3-methyl-1H-pyrazol-5-yl)) -1-methyl-1H-imidazol-2-yl]-1H-pyrazolo [4,3-c]Pyridine-6-carbonitrile (C-2) (143 mg) was used in the next step without further purification. LCMS [ M+H]Observed value = 361.1.

Step 3 Synthesis of 1-ethyl-4- [4- (1-ethyl-3-methyl-1H-pyrazol-5-yl) -1-methyl-1H-imidazol-2-yl ] -1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (example AIC 01)

To a composition containing 1-ethyl-4- [4- (1-ethyl-3-methyl-1H-pyrazol-5-yl) -1-methyl-1H-imidazol-2-yl]-1H-pyrazolo [4,3-c]Into a reaction vessel of pyridine-6-carbonitrile (C-2) (143 mg,0.311 mmol), DMSO (2.7 mL), meOH (5.5 mL), H were added ₂ O ₂ (243. Mu.L, 3.11 mmol) and NaOH (2M in H) ₂ In O, 777. Mu.L, 1.55 mmol). The reaction was stirred at 25℃for 16 hours, then taken up in saturated Na ₂ SO ₃ The aqueous solution (2 mL) was quenched. The solution was concentrated in vacuo and the DMSO suspension was filtered. The filtrate was subjected to preparative HPLC (Waters Xbridge BEH C18100X25mmX5um column, 21-61% MeCN/H) ₂ O,25 mL/min) to provide the desired product with trace impurities. The material was further purified by trituration with MTBE (2 mL) and stirred at room temperature for 10 min. The suspension was filtered and the filter cake was washed with MTBE (1 mL). The solid was collected and dried under vacuum to provide the title compound 1-ethyl-4- [4- (1-ethyl-3-methyl-1H-pyrazol-5-yl) -1-methyl-1H-imidazol-2-yl as a pale yellow solid]-1H-pyrazolo [4,3-c]Pyridine-6-carboxamide (example AIC 01) (37 mg, 31%). LCMS [ M+H]Observed value = 379.4; ¹ H NMR(400MHz,DMSO-d ₆ )δ＝8.75(s,1H),8.37(s,1H),7.94(br s,1H),7.89(br s,1H),7.85(s,1H),6.30(s,1H),4.60(q,J＝7.3Hz,2H),4.55(q,J＝7.0Hz,2H),4.24(s,3H),2.18(s,3H),1.45(t,J＝7.1Hz,3H),1.40(t,J＝7.1Hz,3H)。

the examples in the following table are prepared according to the procedure used for the synthesis of 1-ethyl-4- [4- (1-ethyl-3-methyl-1H-pyrazol-5-yl) -1-methyl-1H-imidazol-2-yl ] -1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (example AIC 01) using non-critical variations or permutations of the example procedure as would be recognized by one skilled in the art.

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4- (2-ethyl-1 ', 4-dimethyl-1' H- [1,4 '-biimidazole ] -2' -yl) -1-methyl-1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (example AID 01) was prepared according to scheme D.

Scheme D:

step 1 Synthesis of N- [ (2, 4-dimethoxyphenyl) methyl ] -4- (2-ethyl-1 ', 4-dimethyl-1' H- [1,4 '-biimidazole ] -2' -yl) -1-methyl-1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (D-1)

To a reaction vessel containing 2-ethyl-1 ', 4-dimethyl-1 ' H-1,4' -biimidazole (Int-TG-2) (135 mg,0.710 mmol) was added anhydrous THF (5.0 mL) and the solution was cooled to-78℃in a dry ice/AcMe bath. n-BuLi (0.6 mL,1.50 mmol) was added dropwise to the solution at-78deg.C under an inert atmosphere. The resulting mixture was stirred at-78 ℃ for 2 hours. At this stage ZnCl is added dropwise at-78 DEG C ₂ (2M in Me-THF, 0.88mL,1.8 mmol) and the reaction was stirred for 10 minutes at which point the dry ice/AcMe bath was removed and the solution was allowed to gradually warm to room temperature over 30 minutes. The vessel was then charged with 4-bromo-N- [ (2, 4-dimethoxyphenyl) methyl]-1-methyl-1H-pyrazolo [4,3-c]Pyridine-6-carboxamide (Int-HG-1) (317 mg,0.782 mmol) and Pd (PPh) ₃ ) ₄ (82.0 mg,0.071 mmol). The brown suspension obtained is treated with N ₂ Rinsed for 2 minutes, sealed, and heated at 80 ℃ for 18 hours with stirring. The reaction vessel was removed from heating and gradually cooled to room temperature. The solution was diluted with DCM/MeOH (10:1) and filtered through a pad of celite. The filtrate was concentrated in vacuo and the crude residue was purified by flash column chromatography (40 g SiO ₂ Combi-flash,2.5-15% meoh/DCM) to provide the desired product contaminated with trace impurities. The material was purified by flash column chromatography (20 g SiO ₂ Combi-flash,50-100% EtOAc/stoneOily ether) to afford the title compound N- [ (2, 4-dimethoxyphenyl) methyl as a yellow solid]-4- (2-ethyl-1 ', 4-dimethyl-1 ' H- [1,4' -biimidazole)]-2' -yl) -1-methyl-1H-pyrazolo [4,3-c]Pyridine-6-carboxamide (D-1) (162 mg, 44%) contains minor impurities. The material was used in the next step without further purification. LCMS [ M+H]Observed value =515.4.

Step 2 Synthesis of 4- (2-ethyl-1 ', 4-dimethyl-1' H- [1,4 '-biimidazole ] -2' -yl) -1-methyl-1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (example AID 01)

To a methyl group containing N- [ (2, 4-dimethoxyphenyl) methyl group]-4- (2-ethyl-1 ', 4-dimethyl-1 ' H- [1,4' -biimidazole)]-2' -yl) -1-methyl-1H-pyrazolo [4,3-c]A reaction vessel of pyridine-6-carboxamide (D-1) (160 mg,0.311 mmol) was charged with HFIP (3 mL) and MsOH (299 mg,3.11 mmol). The resulting reddish brown solution was stirred at room temperature for 2 hours, which resulted in the gradual formation of a purple solution. The solution was diluted with DCM (20 mL) and prepared by adding NH ₃ (7M solution in MeOH) to reach ph= -8, followed by concentration under vacuum. The crude solid was triturated with DCM/MeOH (10:1, 5 mL) for 5 minutes with stirring, filtered, and the solid was washed with 3 parts DCM/MeOH (10:1, 2mL each). The filtrate was concentrated in vacuo and the crude residue was purified by preparative HPLC (YMC Triart C18250X50mmx7um column, 16-56% MeCN/H ₂ O (containing 0.05% NH) ₄ OH), 60 mL/min) to afford the title compound 4- (2-ethyl-1 ', 4-dimethyl-1 ' H- [1,4' -biimidazole) as a white solid]-2' -yl) -1-methyl-1H-pyrazolo [4,3-c]Pyridine-6-carboxamide (example AID 01) (68 mg, 61%). LCMS [ M+H]Observed value = 365.1; ¹ H NMR(400MHz,DMSO-d ₆ )δ＝8.71(s,1H),8.36(s,1H),7.94(br s,1H),7.88(br s,1H),7.68(s,1H),7.16(s,1H),4.24(s,3H),4.18(s,3H),2.84(q,J＝7.5Hz,2H),2.13(s,3H),1.24(t,J＝7.5Hz,3H)。

preparation of 4- {4- [1- (3-hydroxypropyl) -3-methyl-1H-pyrazol-5-yl ] -1-methyl-1H-imidazol-2-yl } -1-methyl-1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (example AIE 01) according to scheme E.

Scheme E:

step 1 Synthesis of 1- [3- (benzyloxy) propyl ] -3-methyl-5- (1-methyl-1H-imidazol-4-yl) -1H-pyrazole (E-1)

To a catalyst containing 1- [3- (benzyloxy) propyl group]A reaction vessel of 5- (1H-imidazol-4-yl) -3-methyl-1H-pyrazole (Int-TG-3) (4.40 g,14.9 mmol) was charged with THF (140 mL). The solution was cooled to 0 ℃ in an ice-water bath, then NaH (60 wt% mineral oil, 830 mg,20.8 mmol) was added in portions. The reaction was stirred at 0 ℃ for 15 minutes, which resulted in the formation of a dark yellow suspension. To the solution was added MeI (3.08 g,21.7 mmol) and the reaction was stirred at 0deg.C for 30 min at which point the ice bath was removed. The reaction was gradually warmed to room temperature over 1 hour. The reaction was quenched by careful addition of water (50 mL) and transferred to a separatory funnel containing EtOAc. The phases were separated and the aqueous phase was extracted with 3 parts of EtOAc (100 mL each). The combined organic extracts were washed with brine (100 mL), dried (Na ₂ SO ₄ ) Filtered and concentrated under vacuum. The crude residue (3.5 g) was combined with crude material from another batch (1.16 g) and purified by flash column chromatography (120 g SiO ₂ Biotage,0-10% meoh/EtOAc) to afford the desired product contaminated with trace impurities. The material was purified by flash column chromatography (120 g SiO ₂ Combi-flash,0-10% MeOH/EtOAc) was re-purified to provide the title compound 1- [3- (benzyloxy) propyl as a brown oil]-3-methyl-5- (1-methyl-1H-imidazol-4-yl) -1H-pyrazole (E-1) (2.82 g, 52%). LCMS [ M+H]Observed value = 311.0; ¹ H NMR(400MHz,DMSO-d ₆ )δ＝7.66(s,1H),7.42(s,1H),7.37-7.24(m,6H),6.12(s,1H),4.43(t,J＝7.2Hz,2H),4.40(s,2H),3.63(s,3H),3.41(t,J＝6.2Hz,2H),2.13(s,3H),2.03-1.95(m,2H)。

step 2 Synthesis of 4- (4- {1- [3- (benzyloxy) propyl ] -3-methyl-1H-pyrazol-5-yl } -1-methyl-1H-imidazol-2-yl) -N- [ (2, 4-dimethoxyphenyl) methyl ] -1-methyl-1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (E-2)

To a catalyst containing 1- [3- (benzyloxy) propyl group]To a reaction vessel of (E-1) (1.08 g,3.48 mmol) 3-methyl-5- (1-methyl-1H-imidazol-4-yl) -1H-pyrazole (E-1) was charged 4-bromo-N- [ (2, 4-dimethoxyphenyl) methyl]-1-methyl-1H-pyrazolo [4,3-c]Pyridine-6-carboxamide (Int-HG-1) (2.82 g,6.96 mmol), phMe (36 mL), cs ₂ CO ₃ (3.40g,10.4mmol)、Pd(OAc) ₂ (391mg,1.74mmol)、PPh ₃ (458 mg,1.74 mmol), cuI (331 mg,1.74 mmol) and PivOH (711 mg,6.96 mmol). The resulting mixture was treated with N ₂ Deaeration was carried out for 3 cycles, sealed and heated to 130 ℃ with stirring for 18 hours. The reaction was removed from heating and gradually cooled to room temperature. The solution was diluted with DCM/MeOH (10:1, 30 mL), filtered through celite, and the filtrate concentrated in vacuo. The crude residue was purified by flash column chromatography (120 gSiO ₂ Combi-flash,20-100% EtOAc/petroleum ether) to afford the title compound 4- (4- {1- [3- (benzyloxy) propyl) as a pale yellow solid]-3-methyl-1H-pyrazol-5-yl } -1-methyl-1H-imidazol-2-yl) -N- [ (2, 4-dimethoxyphenyl) methyl]-1-methyl-1H-pyrazolo [4,3-c]Pyridine-6-carboxamide (E-2) (1.10 g, 49%) which is contaminated with some minor impurities. This material was used in the next step without further purification. LCMS [ M+H]Observed value = 635.5.

Synthesis of step 3:N- [ (2, 4-dimethoxyphenyl) methyl ] -4- {4- [1- (3-hydroxypropyl) -3-methyl-1H-pyrazol-5-yl ] -1-methyl-1H-imidazol-2-yl } -1-methyl-1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (E-3)

To a catalyst containing 4- (4- {1- [3- (benzyloxy) propyl)]-3-methyl-1H-pyrazol-5-yl } -1-methyl-1H-imidazol-2-yl) -N- [ (2, 4-dimethoxyphenyl) methyl]-1-methyl-1H-pyrazolo [4,3-c]To a reaction vessel of pyridine-6-carboxamide (E-2) (1.10 g,1.73 mmol) was added DCM (26 mL) and the solution was cooled to 0deg.C in an ice-water bath. BCl was added dropwise to the solution at 0 °c ₃ (1M in DCM, 5.2mL,5.20 mmol). The ice bath was removed and the reaction was gradually warmed to room temperature with stirring for 21 hours. The solution was cooled to 0 ℃ in an ice water bath and carefully quenched with MeOH (12 mL). The pH of the solution was treated with NH ₃ (7M solution in MeOH) was adjusted to pH = -8 and stirred for 30 minutes, resulting in the formation of a pale yellow suspension. The suspension was filtered and the filtrate was concentrated in vacuo. The crude residue was purified by flash column chromatography (40 gSiO ₂ Combi-flash,0-10% MeOH/DCM) to afford the title compound N- [ (2, 4-dimethoxy) as an off-white solidPhenyl) methyl group]-4- {4- [1- (3-hydroxypropyl) -3-methyl-1H-pyrazol-5-yl]-1-methyl-1H-imidazol-2-yl } -1-methyl-1H-pyrazolo [4,3-c]Pyridine-6-carboxamide (E-3) (680 mg, 72%). LCMS [ M+H]Observed value =545.4.

Step 4 Synthesis of 4- {4- [1- (3-hydroxypropyl) -3-methyl-1H-pyrazol-5-yl ] -1-methyl-1H-imidazol-2-yl } -1-methyl-1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (example AIE 01)

To a methyl group containing N- [ (2, 4-dimethoxyphenyl) methyl group]-4- {4- [1- (3-hydroxypropyl) -3-methyl-1H-pyrazol-5-yl]-1-methyl-1H-imidazol-2-yl } -1-methyl-1H-pyrazolo [4,3-c]A reaction vessel of pyridine-6-carboxamide (E-3) (680 mg,1.25 mmol) was charged with HFIP (12.5 mL) and MsOH (1.20 g,12.5 mmol). The reaction was stirred at room temperature for 2.5 hours, at which point a dark purple solution formed. The solution was concentrated in vacuo and diluted with DCM/MeOH (10:1, 30 mL). The pH of the solution is then treated with NH ₃ (7M solution in MeOH) was adjusted to pH = -8, which resulted in precipitation of a solid. The suspension was filtered and the filter cake was washed with 4 parts DCM/MeOH (10:1, 5mL each). The filtrate was concentrated in vacuo and the crude residue was purified by flash column chromatography (40 g SiO ₂ Combi-flash,0-10% MeOH/DCM). Fractions containing the desired product were collected, concentrated, and further lyophilized to provide the title compound 4- {4- [1- (3-hydroxypropyl) -3-methyl-1H-pyrazol-5-yl as a pale yellow solid]-1-methyl-1H-imidazol-2-yl } -1-methyl-1H-pyrazolo [4,3-c]Pyridine-6-carboxamide (example AIE 01) (400 mg, 82%). LCMS [ M+H]Observed value = 395.3; ¹ H NMR(400MHz,DMSO-d ₆ )δ＝8.77(d,J＝1.0Hz,1H),8.35(d,J＝0.9Hz,1H),7.94(br s,1H),7.87(br s,1H),7.85(s,1H),6.32(s,1H),4.65-4.53(m,3H),4.23(s,3H),4.19(s,3H),3.45(q,J＝6.1Hz,2H),2.18(s,3H),1.97(quin,J＝6.7Hz,2H)。

the examples in the following table are prepared according to scheme E following the procedure used to synthesize 4- {4- [1- (3-hydroxypropyl) -3-methyl-1H-pyrazol-5-yl ] -1-methyl-1H-imidazol-2-yl } -1-methyl-1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (example AIE 01) using non-critical variations or permutations of the example procedure as would be recognized by one of skill in the art.

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1-methyl-4- [ 1-methyl-4- (3-methyl-1-propyl-1H-pyrazol-5-yl) -1H-imidazol-2-yl ] -1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (example AIF 01) was prepared according to scheme F.

Scheme F:

step 1 Synthesis of 3-methyl-5- (1-methyl-1H-imidazol-4-yl) -1-propyl-1H-pyrazole (F-1)

To a reaction vessel containing 5- (1H-imidazol-4-yl) -3-methyl-1-propyl-1H-pyrazole (Int-TG-4) (417 mg,1.33 mmol) was charged K ₂ CO ₃ (463mg, 3.34 mmol), meCN (10 mL). To the solution was added MeI (91.4. Mu.L, 1.47 mmol) dropwise and the resulting yellow suspension was stirred at 25℃for 16 hours. The solution was treated with H ₂ O (10 mL) was diluted and transferred to a separatory funnel containing EtOAc. The phases were separated and the aqueous phase was extracted with 2 parts of EtOAc (10 mL each). The combined organic extracts were concentrated in vacuo and the crude residue was purified by preparative TLC (SiO 2,10% meoh/DCM) to afford the title compound 3-methyl-5- (1-methyl-1H-imidazol-4-yl) -1-propyl-1H-pyrazole (F-1) (233 mg, 63%) as a yellow oil, contaminated with minor impurities. This material was used in the next step without further purification. LCMS [ M+H]=205.0 observations.

Step 2 Synthesis of N- [ (2, 4-dimethoxyphenyl) methyl ] -1-methyl-4- [ 1-methyl-4- (3-methyl-1-propyl-1H-pyrazol-5-yl) -1H-imidazol-2-yl ] -1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (F-2)

To a catalyst containing 4-bromo-N- [ (2, 4-dimethoxyphenyl) methyl group]-1-methyl-1H-pyrazolo [4,3-c]To a reaction vessel of pyridine-6-carboxamide (Int-HG-1) (553 mg,1.36 mmol) was added 3-methyl-5- (1-methyl-1H-mi-ne in PhMe (7 mL) Azol-4-yl) -1-propyl-1H-pyrazole (F-1) (233 mg,0.91 mmol), cs ₂ CO ₃ (874mg,2.68mmol)、PivOH(94.3mg,0.923mmol)、PPh ₃ (59.2 mg,0.226 mmol), cuI (34.3 mg,0.180 mmol) and Pd (OAc) ₂ (53.1 mg,0.237 mmol). The resulting mixture was treated with N ₂ Rinsed for 0.5 minutes, sealed, heated to 110 ℃, and stirred for 16 hours. The reaction was removed from heating and gradually cooled to room temperature. The suspension was filtered and the filter cake was washed with 2 parts of DCM (10 mL each). The filtrate was concentrated in vacuo and the crude residue was purified by flash column chromatography (SiO ₂ Isco,0-3% meoh/DCM) to afford the title compound N- [ (2, 4-dimethoxyphenyl) methyl as a yellow oil]-1-methyl-4- [ 1-methyl-4- (3-methyl-1-propyl-1H-pyrazol-5-yl) -1H-imidazol-2-yl]-1H-pyrazolo [4,3-c]Pyridine-6-carboxamide (F-2) (435 mg, 90%) contaminated with trace impurities. This material was used in the next step without further purification. LCMS [ M+H]Observed value = 529.3.

Step 3 Synthesis of 1-methyl-4- [ 1-methyl-4- (3-methyl-1-propyl-1H-pyrazol-5-yl) -1H-imidazol-2-yl ] -1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (example AIF 01)

To a methyl group containing N- [ (2, 4-dimethoxyphenyl) methyl group]-1-methyl-4- [ 1-methyl-4- (3-methyl-1-propyl-1H-pyrazol-5-yl) -1H-imidazol-2-yl]-1H-pyrazolo [4,3-c ]A reaction vessel of pyridine-6-carboxamide (F-2) (435 mg,0.51 mmol) was charged with HFIP (6 mL) and MsOH (490 mg,5.10 mmol). The reaction was stirred at 25 ℃ for 1 hour during which time the solution gradually turned purple. The solution was concentrated in vacuo and passed through a preparative HPLC (Phenomenex Gemini-NX 80X40mmx3um column, 22-62% MeCN/H ₂ O (containing 0.05% NH) ₄ OH), 25 mL/min). The product-containing fractions were lyophilized to provide the title compound 1-methyl-4- [ 1-methyl-4- (3-methyl-1-propyl-1H-pyrazol-5-yl) -1H-imidazol-2-yl as a white solid]-1H-pyrazolo [4,3-c]Pyridine-6-carboxamide (example AIF 01) (86 mg, 38%). LCMS [ M+H]Observed value = 379.0; ¹ H NMR(400MHz,DMSO-d ₆ )δ＝8.73(s,1H),8.35(s,1H),7.93(br s,1H),7.88(br s,1H),7.84(s,1H),6.31(s,1H),4.50(t,J＝7.3Hz,2H),4.24(s,3H),4.19(s,3H),2.18(s,3H),1.84(sxt,J＝7.3Hz,2H),0.89(t,J＝7.4Hz,3H)。

the examples in the following table were prepared according to the procedure used for the synthesis of 1-methyl-4- [ 1-methyl-4- (3-methyl-1-propyl-1H-pyrazol-5-yl) -1H-imidazol-2-yl ] -1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (example AIF 01) using non-critical variations or permutations of the example procedure as would be recognized by one skilled in the art.

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The examples in the following table were prepared according to the procedure used in step 2-3 for the synthesis of 1-methyl-4- [ 1-methyl-4- (3-methyl-1-propyl-1H-pyrazol-5-yl) -1H-imidazol-2-yl ] -1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (example AIF 01) using 1-ethyl-4- [ (4-methoxyphenyl) methoxy ] -3-methyl-5- (1-methyl-1H-imidazol-4-yl) -1H-pyrazole (Int-TG-10) as starting material and non-critical variations or permutations of the exemplified procedure as would be recognized by one skilled in the art.

The examples in the following table were prepared according to the procedure used in step 2-3 for the synthesis of 1-methyl-4- [ 1-methyl-4- (3-methyl-1-propyl-1H-pyrazol-5-yl) -1H-imidazol-2-yl ] -1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (example AIF 01) using 4-chloro-1-ethyl-3-methyl-5- (1-methyl-1H-imidazol-4-yl) -1H-pyrazole (Int-TG-11) as starting material and non-critical variations or permutations of the exemplified procedure as would be recognized by one skilled in the art.

The examples in the following tables are prepared according to the procedure used in step 2-3 for the synthesis of 1-methyl-4- [ 1-methyl-4- (3-methyl-1-propyl-1H-pyrazol-5-yl) -1H-imidazol-2-yl ] -1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (example AIF 01) using non-critical variations or permutations of the example procedure as would be recognized by a person skilled in the art.

Preparation of 4- [4- (1-ethyl-3-methyl-1H-pyrazol-5-yl) -1-methyl-1H-imidazol-2-yl ] -1-methyl-1H-pyrazolo [3,4-d ] pyrimidine-6-carboxamide (example AIG 01) according to scheme G.

Scheme G:

step 1 Synthesis of N- [ (2, 4-dimethoxyphenyl) methyl ] -4- [4- (1-ethyl-3-methyl-1H-pyrazol-5-yl) -1-methyl-1H-imidazol-2-yl ] -1-methyl-1H-pyrazolo [3,4-d ] pyrimidine-6-carboxamide (G-1)

To 4-chloro-N- [ (2, 4-dimethoxyphenyl) methyl]-1-methyl-1H-pyrazolo [3,4-d]To a solution of pyrimidine-6-carboxamide (Int-HG-3) (173 mg, 0.178 mmol) and 1-ethyl-3-methyl-5- (1-methyl-1H-imidazol-4-yl) -1H-pyrazole (a-1) (70 mg,0.37 mmol) in PhMe (3.8 mL) was added Cs ₂ CO ₃ (360mg,1.10mmol)、Pd(OAc) ₂ (25mg,0.110mmol)、PPh ₃ (29 mg,0.110 mmol), cuI (21 mg,0.110 mmol) and PivOH (78 mg,0.736 mmol). The reaction mixture was heated at 110℃for 2 hours. The reaction was removed from heating and cooled to room temperature. The solution was diluted with DCM (30 mL), filtered over celite, and the celite cake was washed with 10% MeOH/DCM (30 mL) was washed. The filtrate was concentrated under reduced pressure and the crude residue was purified by column chromatography (12 g SiO ₂ MeOH/DCM 1:10) to afford the title compound N- [ (2, 4-dimethoxyphenyl) methyl as a yellow oil]-4- [4- (1-ethyl-3-methyl-1H-pyrazol-5-yl) -1-methyl-1H-imidazol-2-yl]-1-methyl-1H-pyrazolo [3,4-d]Pyrimidine-6-carboxamide (G-1) (22 mg, 12%). LC/MS m/z 516[ M+1]]。

Step 2 Synthesis of 4- [4- (1-ethyl-3-methyl-1H-pyrazol-5-yl) -1-methyl-1H-imidazol-2-yl ] -1-methyl-1H-pyrazolo [3,4-d ] pyrimidine-6-carboxamide (AIG 01)

To a reaction flask containing N- (2, 4-dimethoxybenzyl) -4- (4- (1-ethyl-3-methyl-1H-pyrazol-5-yl) -1-methyl-1H-imidazol-2-yl) -1-methyl-1H-pyrazolo [3,4-d ] pyrimidine-6-carboxamide (G-1) (22 mg,0.041 mmol) was added TFA (1.0 mL). The reaction was heated at 35 ℃ overnight. The reaction mixture was concentrated under reduced pressure and then azeotropically distilled with PhMe. The crude residue was dissolved in DMSO (0.7 mL) and purified by reverse phase chromatography to provide the title compound 4- [4- (1-ethyl-3-methyl-1H-pyrazol-5-yl) -1-methyl-1H-imidazol-2-yl ] -1-methyl-1H-pyrazolo [3,4-d ] pyrimidine-6-carboxamide (AIG 01) (2.6 mg, 17%) as a white solid. LC/MS m/z 366[ M+1].

Examples AIH01-AIH19 were prepared according to scheme H.

Scheme H

Step 1 Suzuki Cross-coupling general procedure

To each reaction flask was added the appropriate commercially available heteroaryl borate (120. Mu.M, 1.2 eq) under inert atmosphere followed by 4- (4-bromo-1-methyl-1H-imidazol-2-yl) -N- [ (2, 4-dimethoxyphenyl) methyl]-1-methyl-1H-pyrazolo [4,3-c]Pyridine-6-carboxamide (Int-HG-5) (100. Mu. Mol of 0.125M in dioxane, 1.0 eq.) K ₃ PO ₄ (300. Mu. Mol of 1.5M aqueous solution, 3.0 eq.) and Pd (dppf) Cl ₂ (5. Mu. Mol,0.05 eq). The bottle cap was capped, heated to 100 ℃, and stirred for 16 hours. The reaction solution is reactedConcentrate by Speedvac and use bottle H ₂ O (1 mL each) was diluted. The aqueous solution was extracted with 3 parts of EtOAc (1 mL each). The combined organic extracts were collected and concentrated by Speedvac.

Step 2 general procedure for deprotection of amide

TFA/H was added to each reaction flask containing the unique intermediate from step 1 ₂ O (10:1) solution. The bottle cap was capped, heated to 80 ℃, and stirred for 16 hours. The reaction solution was concentrated by Speedvac and the crude residue was purified by preparative HPLC to provide example AIH01-AIH19.

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1-ethyl-4- {4- [1- (3-hydroxypropyl) -3-methyl-1H-pyrazol-5-yl ] -1-methyl-1H-imidazol-2-yl } -1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (example AIJ 01) was prepared according to scheme J.

Scheme J:

step 1 Synthesis of 1-ethyl-4- {4- [1- (3-hydroxypropyl) -3-methyl-1H-pyrazol-5-yl ] -1-methyl-1H-imidazol-2-yl } -1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (example AIJ 01)

At N ₂ Downward cooling with ice water bath 1-ethyl-4- {4- [1- (3-methoxypropyl) -3-methyl-1H-pyrazol-5-yl]-1-methyl-1H-imidazol-2-yl } -1H-pyrazolo [4,3-c]Pyridine-6-carboxamide (AIC 03) (120 mg,0.284 mmol) in DCM (6 mL) in yellow was added dropwise BCl ₃ (99.8 mg, 0.850 mmol). The resulting yellow suspension was gradually warmed to room temperature (20 ℃) for 48 hours under stirring. LCMS analysis indicated incomplete reaction, so the solution was cooled in an ice water bath and concentrated in N ₂ Additional aliquots of BCl3 (99.8 mg, 0.850 mmol) were added drop-wise. The ice bath was removed and the resulting yellow suspension was gradually warmed to room temperature (20 ℃) for 21 hours under stirring. LCMS analysis indicated incomplete reaction, so the solution was cooled in an ice water bath and concentrated in N ₂ Additional aliquots of BCl3 (166 mg,1.42 mmol) were added dropwise. The ice bath was removed and the resulting yellow suspension was gradually warmed to room temperature (20 ℃) for 21 hours under stirring. The reaction mixture was cooled to 0deg.C, quenched with MeOH (2 mL), quenched with NH ₃ MeOH (7M) was basified to pH 7-8, then warmed to room temperature and stirred for 30 minutes. The resulting solution was concentrated under vacuum. The crude residue was diluted with water (5 mL) and transferred to a separatory funnel. The solution was extracted with 2 parts DCM/MeOH (10:1, 5 mL). The combined organic extracts were washed with brine (5 mL), dried over anhydrous Na ₂ SO ₄ Dried, filtered, and concentrated in vacuo to afford the crude product as a yellow solid. The crude residue was purified by preparative TLC (silica gel, DCM: meoh=10:1, rf-0.3) to provide a pale yellow solid which was further lyophilized for 16 hours to provide the title compound 1-ethyl-4- {4- [1- (3-hydroxypropyl) -3-methyl-1H-pyrazol-5-yl as a pale yellow solid]-1-methyl-1H-imidazol-2-yl } -1H-pyrazolo [4,3-c]Pyridine-6-carboxamide (example AIJ 01) (15.78 mg, 14%). LCMS [ M+H]Observed value=409.2; ¹ H NMR(DMSO-d ₆ )δ:8.80(s,1H),8.38(s,1H),7.84-8.01(m,3H),6.32(s,1H),4.55-4.68(m,4H),4.23(s,3H),3.46(q,J＝5.3Hz,2H),2.19(s,3H),1.93-2.03(m,2H),1.45(t,J＝7.3Hz,3H)。

the examples in the following table were prepared according to the procedure used in step 1-3 of scheme C and followed by non-critical variations or permutations of the example procedure used in the synthesis of 1-ethyl-4- {4- [1- (3-hydroxypropyl) -3-methyl-1H-pyrazol-5-yl ] -1-methyl-1H-imidazol-2-yl } -1H-pyrazolo [4,3-C ] pyridine-6-carboxamide (example AIJ 01) using non-critical variations or permutations of the example procedure as would be recognized by one of skill in the art.

Preparation of 4- { 1-ethyl-4- [1- (3-methoxypropyl) -3-methyl-1H-pyrazol-5-yl ] -1H-imidazol-2-yl } -1-methyl-1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (example AIK 01) according to scheme K.

Scheme K:

step 1 Synthesis of N- [ (2, 4-dimethoxyphenyl) methyl ] -4- { 1-ethyl-4- [1- (3-methoxypropyl) -3-methyl-1H-pyrazol-5-yl ] -1H-imidazol-2-yl } -1-methyl-1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (K-1)

To 5- (1-ethyl-1H-imidazol-4-yl) -1- (3-methoxypropyl) -3-methyl-1H-pyrazole (Int-TG-15) (510 mg,2.05 mmol) and 4-bromo-N- [ (2, 4-dimethoxyphenyl) methyl]-1-methyl-1H-pyrazolo [4,3-c]To a solution of pyridine-6-carboxamide (Int-HG-1) (916 mg,2.26 mmol) in dry toluene (16 mL) was added Pd (OAc) ₂ (46 mg,0.205 mmol), dppf (228 mg,0.411 mmol), ((thiophene-2-carbonyl) oxy) copper (157 mg,0.823 mmol) and cesium pivalate (961 mg,4.11 mmol). The mixture was treated with N ₂ Rinse for 2 minutes, seal, heat to 100 ℃, and stir for 16 hours. The reaction was then filtered, concentrated in vacuo, and taken up in CH ₂ Cl ₂ Dilute, filter, and concentrate under vacuum. The crude residue was purified by flash column chromatography (20 g SiO ₂ ,Combi-flash,20-80％CH ₂ Cl ₂ EtOAc) to afford the title compound N- [ (2, 4-dimethoxyphenyl) methyl as brown gum ]-4- { 1-ethyl-4- [1- (3-methoxypropyl) -3-methyl-1H-pyrazol-5-yl]1H-imidazoles2-yl } -1-methyl-1H-pyrazolo [4,3-c]Pyridine-6-carboxamide (K-1) (1.067 g, 91%). LCMS [ M+H]Observed value = 573.2; ¹ h NMR (chloroform-d) delta: 8.92 (s, 1H), 8.24 (s, 1H), 8.09 (br t, j=5.7 hz, 1H), 7.29 (s, 1H), 7.23 (s, 1H), 6.42-6.48 (m, 2H), 6.22 (s, 1H), 4.54-4.68 (m, 6H), 4.13 (s, 3H), 3.82 (s, 3H), 3.78 (s, 3H), 3.40 (t, j=6.1 hz, 2H), 3.25 (s, 3H), 2.28 (s, 3H), 2.11-2.23 (m, 2H), 1.40 (t, j=7.2 hz, 3H).

Step 2 Synthesis of 4- { 1-ethyl-4- [1- (3-methoxypropyl) -3-methyl-1H-pyrazol-5-yl ] -1H-imidazol-2-yl } -1-methyl-1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (example AIK 01)

N- [ (2, 4-Dimethoxyphenyl) methyl at room temperature]-4- { 1-ethyl-4- [1- (3-methoxypropyl) -3-methyl-1H-pyrazol-5-yl]-1H-imidazol-2-yl } -1-methyl-1H-pyrazolo [4,3-c]To a solution of pyridine-6-carboxamide (K-1) (1.067 g,1.86 mmol) in HFIP (15 mL) was added methanesulfonic acid (895 mg,9.32 mmol) and stirred for 1 hour. Then the reaction is carried out with NH ₃ MeOH alkalization to ph=8, concentration under vacuum, use CH ₂ Cl ₂ Dilute, filter, and concentrate under vacuum. The crude residue was purified by flash column chromatography (40 g SiO ₂ ,Combi-flash,97-100％CH ₂ Cl ₂ MeOH) and purified by flash column chromatography (40 g SiO ₂ ,Combi-flash,97-100％CH ₂ Cl ₂ MeOH) was repurified. The material was then dried by lyophilization, triturated with MTBE (50 mL) for 3 hours, and the solid was collected by filtration. The material was again purified by flash column chromatography (40 g SiO ₂ Combi-flash,99-100% EtOAc/MeOH) purification to afford compound 4- { 1-ethyl-4- [1- (3-methoxypropyl) -3-methyl-1H-pyrazol-5-yl as a gray solid]-1H-imidazol-2-yl } -1-methyl-1H-pyrazolo [4,3-c]Pyridine-6-carboxamide (example AIK 01) (559 mg, 56%)). LCMS [ M+H]Observed value = 423.3. ¹ HNMR(DMSO-d ₆ )δ:8.80(d,J＝0.8Hz,1H),8.36(d,J＝0.9Hz,1H),7.89-7.93(m,2H),7.75(br s,1H),6.33(s,1H),4.75(q,J＝7.3Hz,2H),4.62(t,J＝7.3Hz,2H),4.19(s,3H),3.36(t,J＝6.1Hz,2H),3.19(s,3H),2.18(s,3H),2.01-2.09(m,2H),1.45(t,J＝7.2Hz,3H)。

Preparation of 4- (4- {1- [3- (difluoromethoxy) propyl ] -3-methyl-1H-pyrazol-5-yl } -1-methyl-1H-imidazol-2-yl) -1-methyl-1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (example AIL 01) according to scheme L.

Scheme L:

step 1 Synthesis of 4- (4- {1- [3- (difluoromethoxy) propyl ] -3-methyl-1H-pyrazol-5-yl } -1-methyl-1H-imidazol-2-yl) -N- [ (2, 4-dimethoxyphenyl) methyl ] -1-methyl-1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (L-1).

To N- [ (2, 4-dimethoxyphenyl) methyl]-4- {4- [1- (3-hydroxypropyl) -3-methyl-1H-pyrazol-5-yl]-1-methyl-1H-imidazol-2-yl } -1-methyl-1H-pyrazolo [4,3-c]Pyridine-6-carboxamide (E-3) (195 mg,0.358 mmol) in CH ₂ Cl ₂ (0.4 mL) of the solution in H ₂ (bromodifluoromethyl) trimethylsilane (73 mg,0.36 mmol) and KOAc (70 mg,0.72 mmol) in O (0.4 mL) and stirred at 18℃for 16 h. MeOH was then added to the reaction and concentrated in vacuo. Dissolving the residue in CH ₂ Cl ₂ (15 mL) and H ₂ O (25 mL) and the phases separated. The aqueous phase was collected and concentrated in vacuo to give 4- (4- {1- [3- (difluoromethoxy) propyl) as a brown solid]-3-methyl-1H-pyrazol-5-yl } -1-methyl-1H-imidazol-2-yl) -N- [ (2, 4-dimethoxyphenyl) methyl]-1-methyl-1H-pyrazolo [4,3-c]Pyridine-6-carboxamide (L-1) (170 mg) was used without further purification. LCMS [ M+H]Observed value = 595.4.

Step 2 Synthesis of 4- (4- {1- [3- (difluoromethoxy) propyl ] -3-methyl-1H-pyrazol-5-yl } -1-methyl-1H-imidazol-2-yl) -1-methyl-1H-pyrazolo [4,3-c ] pyridine-6-carboxamide formate salt (example AIL 01).

To 4- (4- {1- [3- (difluoromethoxy) propyl ] at room temperature (18 ℃ C.)]-3-methyl-1H-pyrazol-5-yl } -1-methyl-1H-imidazol-2-yl) -N- [ (2, 4-dimethoxyphenyl) methyl]-1-methyl-1H-pyrazolo [4,3-c]To a solution of pyridine-6-carboxamide (L-1) (250 mg) in HFIP (2.5 mL) was added methanesulfonic acid (402 mg,4.18 mmol) and stirred for 1.5 hours. The crude mixture was purified by preparative HPLC (Boston Prime C18150 x 30mm x 5um column, 2-42% MeCN/H ₂ O (containing formic acid (0.225%)), 25mL/min to provide 4- (4- {1- [3- (difluoromethoxy) propyl) as a white solid]-3-methyl-1H-pyrazol-5-yl } -1-methyl-1H-imidazol-2-yl) -1-methyl-1H-pyrazolo [4,3-c]Pyridine-6-carboxamide formate (AIL 01) (38 mg, 17% over two batches). LCMS [ M+H]Observed value=445.4. ¹ H NMR (methanol-d) ₄ )δ:8.80(s,1H),8.52(s,1H),8.42(br s,2H),8.22(t,J＝54.0Hz,1H),7.26(s,1H),5.48(t,J＝7.5Hz,2H),4.37(s,3H),4.23(s,3H),3.66(t,J＝5.8Hz,2H),2.74(s,3H),2.14-2.26(m,2H)； ¹⁹ F NMR (methanol-d) ₄ )δ:-97.73(s,1F)。

1-methyl-4- [ 1-methyl-4- (3-methyl-1- { [ (2R) -oxetan-2-yl ] methyl } -1H-pyrazol-5-yl) -1H-imidazol-2-yl ] -1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (example AIM 01) was prepared according to scheme M.

Scheme M:

step 1 Synthesis of (rac) -1-methyl-4- (1-methyl-4- { 3-methyl-1- [ (oxetan-2-yl) methyl ] -1H-pyrazol-5-yl } -1H-imidazol-2-yl) -1H-pyrazolo [4,3-c ] pyridine-6-carboxylic acid methyl ester (M-1)

The reaction vessel was charged with (rac) -3-methyl-5- (1-methyl-1H-imidazol-4-yl) -1- [ (oxetan-2-yl) methyl]-1H-pyrazole (Int-TG-24) (297.8 mg,1.28 mmol), 4-bromo-1-methyl-1H-pyrazolo [4, 3-c)]Pyridine-6-carboxylic acid methyl ester (HG-1 d) (719 mg,1.92 mmol), pd (OAc) ₂ (57.6mg,0.256mmol)、CuI(48.8mg,0.256mmol)、PPh ₃ (67.3mg,0.256mmol)、Cs ₂ CO ₃ (1250 mg,3.85 mmol), pivOH (157 mg,1.54 mmol), and PhMe (8 mL). The solution was treated with N ₂ Rinsed for 2 minutes, sealed, and heated to 110 ℃ for 20 hours. The reaction was removed from heating and gradually cooled to room temperature. The suspension was filtered and the filtrate was concentrated in vacuo. The crude residue was purified by preparative HPLC (Boston Prime C18150x30mmx5 μm column, 20-60% MeCN/H ₂ O(0.05％NH ₄ OH v/v), 25 mL/min) to afford the title compound as a white solidThe (rac) -1-methyl-4- (1-methyl-4- { 3-methyl-1- [ (oxetan-2-yl) methyl)]-1H-pyrazol-5-yl } -1H-imidazol-2-yl) -1H-pyrazolo [4,3-c]Pyridine-6-carboxylic acid methyl ester (M-1) (80 mg, 15%). LCMS [ M+H]Observed value=422.3; ¹ h NMR (400 MHz, methanol-d) ₄ )δ＝8.76(s,1H),8.27(s,1H),7.61(s,1H),6.36(s,1H),5.27(quin,J＝6.4Hz,1H),5.00(dd,J＝6.2,14.1Hz,1H),4.83-4.80(m,1H),4.68-4.62(m,1H),4.55-4.48(m,1H),4.30(s,3H),4.15(s,3H),4.01(s,3H),2.77-2.60(m,2H),2.27(s,3H)。

Step 2 Synthesis of methyl 1-methyl-4- [ 1-methyl-4- (3-methyl-1- { [ (2R) -oxetan-2-yl ] methyl } -1H-pyrazol-5-yl) -1H-imidazol-2-yl ] -1H-pyrazolo [4,3-c ] pyridine-6-carboxylate (M-2) and methyl 1-methyl-4- [ 1-methyl-4- (3-methyl-1- { [ (2S) -oxetan-2-yl ] methyl } -1H-pyrazol-5-yl) -1H-imidazol-2-yl ] -1H-pyrazolo [4,3-c ] pyridine-6-carboxylate (M-3)

(rac) -1-methyl-4- (1-methyl-4- { 3-methyl-1- [ (oxetan-2-yl) methyl)]-1H-pyrazol-5-yl } -1H-imidazol-2-yl) -1H-pyrazolo [4,3-c]Racemic mixtures of pyridine-6-carboxylic acid methyl ester (M-1) (90 mg,0.214 mmol) were purified by chiral preparative SFC (Daicel Chiralpak IC 250 mm. Times.30 mm,10 μm column, 50% EtOH (0.1% NH) ₄ OH)/CO ₂ 80 mL/min) to afford the title compound 1-methyl-4- [ 1-methyl-4- (3-methyl-1- { [ (2R) -oxetan-2-yl) as a white solid, respectively ]Methyl } -1H-pyrazol-5-yl) -1H-imidazol-2-yl]-1H-pyrazolo [4,3-c]Pyridine-6-carboxylic acid methyl ester (M-2) (40 mg, 44%) (LCMS [ M+H)]=422.3 observations) and 1-methyl-4- [ 1-methyl-4- (3-methyl-1- { [ (2S) -oxetan-2-yl ]]Methyl } -1H-pyrazol-5-yl) -1H-imidazol-2-yl]-1H-pyrazolo [4,3-c]Pyridine-6-carboxylic acid methyl ester (M-3) (50 mg, 56%) (LCMS [ M+H)]=422.3 observations). The absolute stereochemistry of the enantiomers (elution peak 1 (R) and elution peak 2 (S)) is arbitrarily specified.

Step 3 Synthesis of 1-methyl-4- [ 1-methyl-4- (3-methyl-1- { [ (2R) -oxetan-2-yl ] methyl } -1H-pyrazol-5-yl) -1H-imidazol-2-yl ] -1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (example AIM 01)

To a composition containing 1-methyl-4- [ 1-methyl-4- (3-methyl-1- { [ (2R) -oxetan-2-yl]Methyl } -1H-picolineOxazol-5-yl) -1H-imidazol-2-yl]-1H-pyrazolo [4,3-c]Methyl pyridine-6-carboxylate (M-2) (40.0 mg,0.0949 mmol) was added to the reaction vessel ₃ (7M solution in MeOH) (5 mL,30 mmol). The reaction was stirred at 20 ℃ for 18 hours and then concentrated under vacuum. The crude residue was purified by preparative HPLC (Phenomenex Gemini-NX 80 x 40mm x 3 μm column, 13-53% mecn/H ₂ O(0.05％NH ₄ OH v/v), 25 mL/min) to afford the title compound 1-methyl-4- [ 1-methyl-4- (3-methyl-1- { [ (2R) -oxetan-2-yl as a white solid ]Methyl } -1H-pyrazol-5-yl) -1H-imidazol-2-yl]-1H-pyrazolo [4,3-c]Pyridine-6-carboxamide (example AIM 01) (28 mg, 73%). LCMS [ M+H]Observed value = 407.4; ¹ h NMR (400 MHz, methanol-d) ₄ )δ＝8.80(d,J＝1.0Hz,1H),8.31(d,J＝1.0Hz,1H),7.68(s,1H),6.38(s,1H),5.28(quin,J＝6.3Hz,1H),5.00(dd,J＝6.3,14.3Hz,1H),4.85-4.79(m,1H),4.68-4.62(m,1H),4.50(td,J＝6.0,9.0Hz,1H),4.27(s,3H),4.19(s,3H),2.78-2.58(m,2H),2.28(s,3H)。

The examples in the following table are prepared according to the method used in step 3 of scheme M for the synthesis of 1-methyl-4- [ 1-methyl-4- (3-methyl-1- { [ (2R) -oxetan-2-yl ] methyl } -1H-pyrazol-5-yl) -1H-imidazol-2-yl ] -1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (example AIM 01), using non-critical variations or permutations of the example procedure as can be appreciated by the person skilled in the art.

Preparation of 1-cyclopropyl-4- {4- [1- (3-methoxypropyl) -3-methyl-1H-pyrazol-5-yl ] -1-methyl-1H-imidazol-2-yl } -1H-pyrazolo [4,3-c ] pyridine-6-carboxamide according to scheme N (example AIN 01)

Scheme N

Step 1 Synthesis of 6-chloro-1-cyclopropyl-4- {4- [1- (3-methoxypropyl) -3-methyl-1H-pyrazol-5-yl ] -1-methyl-1H-imidazol-2-yl } -1H-pyrazolo [4,3-c ] pyridine (N-1)

To 4, 6-dichloro-1-cyclopropyl-1H-pyrazolo [4,3-c]To a yellow solution of pyridine (Int-HG-7) (129.4 mg,0.567 mmol) and 1- (3-methoxypropyl) -3-methyl-5- (1-methyl-1H-imidazol-4-yl) -1H-pyrazole (Int-TG-25) (160 mg,0.681 mmol) in anhydrous toluene (3 mL) was added Pd (OAc) ₂ (25.5 mg,0.113 mmol), dppf (62.9 mg,0.113 mmol), thiophene-2-carboxylic acid cuprous (I) (CuTC) (43.3 mg,0.227 mmol) and CsOPiv (266 mg,1.13 mmol). The resulting mixture was treated with N ₂ Rinse for 2 minutes, seal, and heat to 100 ℃ (heat block) for 16 hours. The reaction was removed from the heating block and gradually cooled to room temperature. The solution was diluted with 10% meoh/DCM, filtered through a pad of celite, and the filtrate concentrated in vacuo. The crude residue was purified by flash column chromatography (20 g SiO ₂ 12-75% etoac/petroleum ether) to afford the title compound 6-chloro-1-cyclopropyl-4- {4- [1- (3-methoxypropyl) -3-methyl-1H-pyrazol-5-yl as a yellow gum]-1-methyl-1H-imidazol-2-yl } -1H-pyrazolo [4,3-c]Pyridine (N-1) (160 mg, 66%). ¹ H NMR (400 MHz, chloroform-d) δ=8.84 (s, 1H), 7.44 (d, j=0.8 hz, 1H), 7.28 (s, 1H), 6.24 (s, 1H), 4.69 (t, j=7.2 hz, 2H), 4.25 (s, 3H), 3.64-3.55 (m, 1H), 3.43 (t, j=6.1 hz, 2H), 3.29 (s, 3H), 2.31 (s, 3H), 2.24-2.15 (m, 2H), 1.25-1.21 (m, 4H).

Step 2 Synthesis of 1-cyclopropyl-4- {4- [1- (3-methoxypropyl) -3-methyl-1H-pyrazol-5-yl ] -1-methyl-1H-imidazol-2-yl } -1H-pyrazolo [4,3-c ] pyridine-6-carbonitrile (N-2)

To 6-chloro-1-cyclopropyl-4- {4- [1- (3-methoxypropyl) -3-methyl-1H-pyrazol-5-yl ]-1-methyl-1H-imidazol-2-yl } -1H-pyrazolo [4,3-c]Pyridine (N-1) (160 mg,0.376 mmol) and Zn powder (21.1 mg,0.323 mmol), (t-Bu) ₃ P) ₂ Pd (38.4 mg,0.0751 mmol) in DMA (2 mL) to Zn (CN) ₂ (90.0 mg,0.766 mmol). The resulting mixture was treated with N ₂ Rinse for 2 minutes, seal, heat to 120 ℃, and stir for 18 hours. The reaction was removed from the heating block and gradually cooled to room temperature. The solution was diluted with EtOAc (5 mL) and filtered through a celite pad. The filtrate was transferred to a separatory funnel containing EtOAc and washed with H ₂ O (5 mL) dilution. The phases were separated and the aqueous phase was extracted with 3 parts EtOAc (5 mL).The combined organic extracts were washed with 3 parts brine (10 mL), dried (Na ₂ SO ₄ ) Filtered and concentrated in vacuo to afford crude 1-cyclopropyl-4- {4- [1- (3-methoxypropyl) -3-methyl-1H-pyrazol-5-yl as a yellow solid]-1-methyl-1H-imidazol-2-yl } -1H-pyrazolo [4,3-c]Pyridine-6-carbonitrile (N-2) (150 mg) was used in the next step without further purification. LCMS [ M+H]=417.3 observations.

Step 3 Synthesis of 1-cyclopropyl-4- {4- [1- (3-methoxypropyl) -3-methyl-1H-pyrazol-5-yl ] -1-methyl-1H-imidazol-2-yl } -1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (example AIN 01)

To crude 1-cyclopropyl-4- {4- [1- (3-methoxypropyl) -3-methyl-1H-pyrazol-5-yl at 5 °c]-1-methyl-1H-imidazol-2-yl } -1H-pyrazolo [4,3-c]To a pale yellow suspension of pyridine-6-carbonitrile (N-2) (150 mg,0.360 mmol) in DMSO (1.2 mL)/MeOH (3.6 mL) was added dropwise NaOH (72 mg,1.80mmol,2M in H) ₂ O) to maintain the internal temperature below 10 ℃. After the addition is finished, add H ₂ O ₂ (408 mg,3.60mmol,30% solution). At this stage, the ice bath was removed and the reaction was gradually warmed to room temperature (27 ℃) with stirring for 3 hours. The reaction was back quenched into ice-cold saturated Na ₂ SO ₃ (10 mL) in a flask. The solution was transferred to a separatory funnel containing EtOAc and the phases separated. The aqueous phase was extracted with 3 parts EtOAc (10 mL). The combined organic extracts were washed with 3 parts brine (10 mL), dried (Na ₂ SO ₄ ) Filtered and concentrated under vacuum. The crude residue was purified by preparative HPLC (YMC-Triart Prep C18150 x 40mm x 7 μm column, 21-61MeCN/H ₂ O(0.05％NH ₄ OH v/v), 60 mL/min) to afford the title compound 1-cyclopropyl-4- {4- [1- (3-methoxypropyl) -3-methyl-1H-pyrazol-5-yl as a white solid]-1-methyl-1H-imidazol-2-yl } -1H-pyrazolo [4,3-c]Pyridine-6-carboxamide (example AIN 01) (85 mg, 52% over 2 steps). LCMS [ M+H ]Observed value = 435.2; 1H NMR (DMSO-d 6) delta: 8.75 (s, 1H), 8.31 (s, 1H), 7.89-7.99 (m, 2H), 7.84 (s, 1H), 6.30 (s, 1H), 4.61 (t, J=7.3 Hz, 2H), 4.22 (s, 3H), 3.95-4.00 (m, 1H), 3.34-3.37 (m, 2H), 3.19 (s, 3H), 2.18 (s, 3H), 2.00-2.08 (m, 2H), 1.16-1.24 (m, 4H).

The examples in the following table are prepared according to the procedure used in steps 1-3 of scheme N for the synthesis of 1-cyclopropyl-4- {4- [1- (3-methoxypropyl) -3-methyl-1H-pyrazol-5-yl ] -1-methyl-1H-imidazol-2-yl } -1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (example AIN 01) using non-critical variations or permutations of the exemplified procedure that can be recognized by one skilled in the art.

The examples in the following table were prepared according to the procedure used for the synthesis of 1-cyclopropyl-4- {4- [1- (3-methoxypropyl) -3-methyl-1H-pyrazol-5-yl ] -1-methyl-1H-imidazol-2-yl } -1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (example AIN 01) for steps 1-3 of scheme N and the procedure used in step 3 following scheme B, using non-critical variations or permutations of the example procedure that can be recognized by one skilled in the art.

Preparation of 4- {4- [1- (cyanomethyl) -3-methyl-1H-pyrazol-5-yl ] -1-methyl-1H-imidazol-2-yl } -1-methyl-1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (example AIP 01) and 4- {4- [1- (2-amino-2-oxoethyl) -3-methyl-1H-pyrazol-5-yl ] -1-methyl-1H-imidazol-2-yl } -1-methyl-1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (example AIP 02) according to scheme P

Scheme P

Step 1 Synthesis of [ 3-methyl-5- (1-methyl-1H-imidazol-4-yl) -1H-pyrazol-1-yl ] acetonitrile (P-1)

To [5- (1H-imidazol-4-yl) -3-methyl-1H-pyrazole ]1-yl group]To a solution of acetonitrile (Int-TG-27) (70 mg,0.37 mmol) in anhydrous DMF (2.0 mL) was added K ₂ CO ₃ (129 mg,0.935 mmol) followed by dropwise addition of methyl iodide (66 mg,0.47 mmol). The reaction was stirred at room temperature for 16 hours. The mixture was then diluted with brine (5 mL), the phases separated, and the aqueous phase extracted with EtOAc (5 mL x 3). The combined organic extracts were washed with brine (5 ml x 3), dried over anhydrous Na ₂ SO ₃ Dried, filtered, and concentrated in vacuo to afford the title compound [ 3-methyl-5- (1-methyl-1H-imidazol-4-yl) -1H-pyrazol-1-yl ] as a yellow solid]Acetonitrile (P-1) (68 mg, 91%). LCMS [ M+H]=202.1 observations; ¹ h NMR (chloroform-d) δ:7.51 (s, 1H), 7.14 (d, j=1.0 hz, 1H), 6.16 (s, 1H), 5.65 (s, 2H), 3.76 (s, 3H), 2.28 (s, 3H).

Step 2 Synthesis of 4- {4- [1- (cyanomethyl) -3-methyl-1H-pyrazol-5-yl ] -1-methyl-1H-imidazol-2-yl } -N- [ (2, 4-dimethoxyphenyl) methyl ] -1-methyl-1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (P-2)

To [ 3-methyl-5- (1-methyl-1H-imidazol-4-yl) -1H-pyrazol-1-yl]Acetonitrile (P-1) (65 mg,0.32 mmol) and 4-bromo-N- (2, 4-dimethoxybenzyl) -1-methyl-1H-pyrazolo [4,3-c ] ]To a solution of pyridine-6-carboxamide (Int-HG-1) (144 mg,0.355 mmol) in dry toluene (5.0 mL) was added Pd (OAc) ₂ (27 mg,0.12 mmol), dppf (36 mg,0.065 mmol), copper (((thiophene-2-carbonyl) oxy) (25 mg,0.129 mmol) and cesium pivalate (151 mg, 0.640 mmol). The mixture was treated with N ₂ Rinse for 2 minutes, seal, heat to 100 ℃, and stir for 40 hours. The reaction was then filtered through a celite pad and concentrated in vacuo. The crude residue was purified by flash column chromatography (20 g SiO ₂ Combi-flash,20-100% EtOAc/petroleum ether) to afford the title compound 4- {4- [1- (cyanomethyl) -3-methyl-1H-pyrazol-5-yl as a yellow solid]-1-methyl-1H-imidazol-2-yl } -N- [ (2, 4-dimethoxyphenyl) methyl]-1-methyl-1H-pyrazolo [4,3-c]Pyridine-6-carboxamide (P-2) (30 mg, 18%). LCMS [ M+H]Observed value = 526.4; ¹ h NMR (chloroform-d) delta: 8.85 (s, 1H), 8.26-8.34 (m, 2H), 7.28-7.35 (m, 2H), 6.43-6.53 (m, 2H), 6.26 (s, 1H), 5.72 (s, 2H), 4.62-4.71 (m, 2H), 4.17-4.22 (m, 6H), 3.89 (s, 3H), 3.81 (s, 3H), 2.32 (s, 3H).

Step 3 Synthesis of 4- {4- [1- (cyanomethyl) -3-methyl-1H-pyrazol-5-yl ] -1-methyl-1H-imidazol-2-yl } -1-methyl-1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (example AIP 01) and 4- {4- [1- (2-amino-2-oxoethyl) -3-methyl-1H-pyrazol-5-yl ] -1-methyl-1H-imidazol-2-yl } -1-methyl-1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (example AIP 02)

To 4- {4- [1- (cyanomethyl) -3-methyl-1H-pyrazol-5-yl]-1-methyl-1H-imidazol-2-yl } -N- [ (2, 4-dimethoxyphenyl) methyl]-1-methyl-1H-pyrazolo [4,3-c]To a solution of pyridine-6-carboxamide (P-2) (32 mg,0.061 mmol) in HFIP (1.0 mL) was added methanesulfonic acid (58 mg,0.61 mmol) and stirred for 2 hours. The reaction was then concentrated in vacuo, using CH ₂ Cl ₂ MeOH (10:1 v: v,5 mL) with NH ₃ MeOH (7M) alkalization to pH7-8, filtration and cake filtration with CH ₂ Cl ₂ MeOH (10:1 v: v,1mL x 3) and the filtrate was concentrated in vacuo. The crude mixture was purified by preparative thin layer chromatography (CH ₂ Cl ₂ MeOH,10:1 v:v) to afford the title compound 4- {4- [1- (cyanomethyl) -3-methyl-1H-pyrazol-5-yl as a white solid]-1-methyl-1H-imidazol-2-yl } -1-methyl-1H-pyrazolo [4,3-c]Pyridine-6-carboxamide (example AIP 01) (9 mg, 39%). LCMS [ M+H]Observed value = 376.2; ¹ H NMR(DMSO-d ₆ ) Delta 8.84 (s, 1H), 8.38 (s, 1H), 7.85-8.02 (m, 3H), 6.46 (s, 1H), 5.81 (s, 2H), 4.17-4.29 (m, 6H), 2.22 (s, 3H). In addition, the title compound 4- {4- [1- (2-amino-2-oxoethyl) -3-methyl-1H-pyrazol-5-yl]-1-methyl-1H-imidazol-2-yl } -1-methyl-1H-pyrazolo [4,3-c]Pyridine-6-carboxamide (example AIP 02) (4 mg, 17%) isolated as a white solid. LCMS [ M+H ]Observed value = 394.4; ¹ H NMR(DMSO-d ₆ )δ:8.76(d,J＝1.0Hz,1H),8.35(d,J＝1.0Hz,1H),7.95(br s,1H),7.83-7.90(m,2H),7.27(s,1H),7.18(br s,1H),6.37(s,1H),5.24(s,2H),4.22(s,3H),4.19(s,3H),2.19(s,3H)。

the examples in the following table are prepared according to the procedure used for the synthesis of step 1-3 of scheme P of 4- {4- [1- (cyanomethyl) -3-methyl-1H-pyrazol-5-yl ] -1-methyl-1H-imidazol-2-yl } -1-methyl-1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (example AIP 01) using non-critical variations or permutations of the example procedure as can be appreciated by those skilled in the art.

Preparation of 4- {4- [1- (2-cyanoethyl) -3-methyl-1H-pyrazol-5-yl ] -1-methyl-1H-imidazol-2-yl } -1-methyl-1H-pyrazolo [4,3-c ] pyridine-6-carboxamide according to scheme Q (example AIQ 01)

Scheme Q

Step 1 Synthesis of N- [ (2, 4-dimethoxyphenyl) methyl ] -1-methyl-4- { 1-methyl-4- [ 3-methyl-1- (3-oxopropyl) -1H-pyrazol-5-yl ] -1H-imidazol-2-yl } -1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (Q-1)

To N- [ (2, 4-dimethoxyphenyl) methyl]-4- {4- [1- (3-hydroxypropyl) -3-methyl-1H-pyrazol-5-yl]-1-methyl-1H-imidazol-2-yl } -1-methyl-1H-pyrazolo [4,3-c]To an orange solution of pyridine-6-carboxamide (E-3) (600 mg,0.83 mmol) in DCM (20 mL) was added dess-martin oxidant (526 mg,1.24 mmol). The resulting mixture was stirred at 20℃for 16 hours. The resulting yellow suspension was diluted with DCM (20 mL), filtered over a pad of celite, and concentrated in vacuo. The crude residue was purified by flash column chromatography (40 g SiO ₂ 0-10% MeOH/DCM) to afford the title compound N- [ (2, 4-dimethoxyphenyl) methyl as a yellow solid]-1-methyl-4- { 1-methyl-4- [ 3-methyl-1- (3-oxopropyl) -1H-pyrazol-5-yl]-1H-imidazol-2-yl } -1H-pyrazolo [4,3-c]Pyridine-6-carboxamide (Q-1) (500 mg, 83%). LCMS [ M+H]Observed value = 543.1; ¹ h NMR (400 MHz, chloroform-d) δ=9.86 (t, j=1.2 hz, 1H), 8.80 (d, j=1.0 hz, 1H), 8.31 (br t, j=6.4 hz, 1H), 8.28 (d, j=0.9 hz, 1H), 7.31 (d, j=8.3 hz, 1H), 7.27 (br s, 1H), 6.51 (d, j=2.3 hz, 1H), 6.48 (dd, j=2.4, 8.3hz, 1H), 6.21 (s, 1H), 4.98 (t, j=1H)7.0Hz,2H),4.67(d,J＝6.0Hz,2H),4.20(s,3H),4.17(s,3H),3.89(s,3H),3.82(s,3H),3.14(dt,J＝1.3,7.0Hz,2H),2.30(s,3H)。

Step 2 Synthesis of 3- {5- [2- (6- { [ (2, 4-dimethoxyphenyl) methyl ] carbamoyl } -1-methyl-1H-pyrazolo [4,3-c ] pyridin-4-yl) -1-methyl-1H-imidazol-4-yl ] -3-methyl-1H-pyrazol-1-yl } propanoic acid (Q-2)

To N- [ (2, 4-dimethoxyphenyl) methyl]-1-methyl-4- { 1-methyl-4- [ 3-methyl-1- (3-oxopropyl) -1H-pyrazol-5-yl]-1H-imidazol-2-yl } -1H-pyrazolo [4,3-c]To a colorless mixture of pyridine-6-carboxamide (Q-1) (420 mg,0.774 mmol) in THF (10 mL) was added t-BuOH (5 mL) and 2-methyl-2-butene (1630 mg,23.2 mmol). The resulting solution was cooled in an ice water bath (0 ℃ C.), followed by slow addition of NaClO ₂ (700 mg,7.74 mmol) and NaH ₂ PO ₄ (929 mg,7.74 mmol) in H ₂ O (5 mL). After the addition was complete, the ice bath was removed and the reaction was stirred at room temperature (20 ℃ C.) for 16 hours. Na to be reacted ₂ S ₂ O ₃ Quench with aqueous solution (3 mL) by addition of saturated NaHSO ₄ The aqueous solution was adjusted to a pH of about 3-4 and the solution was transferred to a separatory funnel containing EtOAc. The phases were separated and the aqueous phase was extracted with 3 parts EtOAc (15 mL). The combined organic extracts were washed with brine, dried (Na ₂ SO ₄ ) Filtered and concentrated under vacuum. The crude residue was purified by flash column chromatography (SiO ₂ Isco,0-10% MeOH/DCM) to afford the title compound 3- {5- [2- (6- { [ (2, 4-dimethoxyphenyl) methyl ] as a white solid]Carbamoyl } -1-methyl-1H-pyrazolo [4,3-c ]]Pyridin-4-yl) -1-methyl-1H-imidazol-4-yl]-3-methyl-1H-pyrazol-1-yl } propionic acid (Q-2) (355 mg, 82%). LCMS [ M+H]Observed value = 559.1.

Step 3 Synthesis of 4- {4- [1- (3-amino-3-oxopropyl) -3-methyl-1H-pyrazol-5-yl ] -1-methyl-1H-imidazol-2-yl } -N- [ (2, 4-dimethoxyphenyl) methyl ] -1-methyl-1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (Q-3)

3- {5- [2- (6- { [ (2, 4-dimethoxyphenyl) methyl } -]Carbamoyl } -1-methyl-1H-pyrazolo [4,3-c ] ]Pyridin-4-yl) -1-methyl-1H-imidazol-4-yl]-3-methyl-1H-pyrazol-1-yl } propionic acid (Q-2) (355 mg,0.636 mmol)A solution in DMF (10 mL) was cooled to 0deg.C in an ice-water bath. DIPEA (246 mg,1.91 mmol), HATU (290 mg,0.763 mmol) were added to the solution and the reaction was stirred at 0deg.C for 15 min. At this stage, NH is added ₄ Cl (170 mg,3.18 mmol) and the reaction was stirred at 20℃for 16 h. The reaction was quenched into a flask containing ice water (5 mL) and the solution was transferred to a separatory funnel containing EtOAc. The phases were separated and the aqueous phase was extracted with 3 parts of EtOAc (10 mL). The combined organic extracts were treated with 1 part NH ₄ Aqueous Cl, 1 part brine, and dried (Na ₂ SO ₄ ) Filtered and concentrated under vacuum. The crude residue was purified by preparative TLC (SiO ₂ 10% MeOH/DCM) to afford the title compound 4- {4- [1- (3-amino-3-oxopropyl) -3-methyl-1H-pyrazol-5-yl as a yellow solid]-1-methyl-1H-imidazol-2-yl } -N- [ (2, 4-dimethoxyphenyl) methyl]-1-methyl-1H-pyrazolo [4,3-c]Pyridine-6-carboxamide (Q-3) (280 mg, 79%). LCMS [ M+H]Observed value=558.1.

Step 4 Synthesis of 4- {4- [1- (2-cyanoethyl) -3-methyl-1H-pyrazol-5-yl ] -1-methyl-1H-imidazol-2-yl } -N- [ (2, 4-dimethoxyphenyl) methyl ] -1-methyl-1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (Q-4)

To 4- {4- [1- (3-amino-3-oxopropyl) -3-methyl-1H-pyrazol-5-yl]-1-methyl-1H-imidazol-2-yl } -N- [ (2, 4-dimethoxyphenyl) methyl]-1-methyl-1H-pyrazolo [4,3-c]To a stirred suspension of pyridine-6-carboxamide (Q-3) (280 mg,0.502 mmol) in DCM (20 mL) was added methyl N- (triethylammonium sulfonyl) carbamate (Burgess reagent) (319 mg,1.51 mmol) and the reaction was carried out in N ₂ Stirred at 25℃for 16 hours. Will react with H ₂ O (10 mL) was diluted and transferred to a separatory funnel containing DCM. The phases were separated and the aqueous phase was extracted with 3 parts DCM (10 mL). The combined organic extracts were dried (Na ₂ SO ₄ ) Filtered and concentrated under vacuum. The crude residue was purified by preparative TLC (SiO ₂ 10% MeOH/DCM) to afford the title compound 4- {4- [1- (2-cyanoethyl) -3-methyl-1H-pyrazol-5-yl as a yellow solid]-1-methyl-1H-imidazol-2-yl } -N- [ (2, 4-dimethoxyphenyl) methyl]-1-methyl-1H-pyrazolo [4,3-c]Pyridine-6-carboxamide (Q-4) (160 mg, 59%). LCMS [ M+H]Observed value=540.1.

Step 5 Synthesis of 4- {4- [1- (2-cyanoethyl) -3-methyl-1H-pyrazol-5-yl ] -1-methyl-1H-imidazol-2-yl } -1-methyl-1H-pyrazolo [4,3-c ] pyridine-6-carboxamide (example AIQ 01)

To 4- {4- [1- (2-cyanoethyl) -3-methyl-1H-pyrazol-5-yl ]-1-methyl-1H-imidazol-2-yl } -N- [ (2, 4-dimethoxyphenyl) methyl]-1-methyl-1H-pyrazolo [4,3-c]To a pale yellow solution of pyridine-6-carboxamide (Q-4) (160 mg, 0.294 mmol) in HFIP (5 mL) was added MeSO ₃ H (214 mg,2.22 mmol). The reaction color turned purple and was stirred at room temperature (20 ℃) for 1 hour. The solution was concentrated in vacuo and the crude residue was purified by prep HPLC (Boston Prime C18150 x 30mm x 5 μm column, 15-45MeCN/H ₂ O(0.05％NH ₄ OH v/v), 30 mL/min). The product containing fractions were collected and lyophilized to provide the title compound 4- {4- [1- (2-cyanoethyl) -3-methyl-1H-pyrazol-5-yl as a white solid]-1-methyl-1H-imidazol-2-yl } -1-methyl-1H-pyrazolo [4,3-c]Pyridine-6-carboxamide (example AIQ 01) (18 mg, 31%). LCMS [ M+H]Observed value=390.3; ¹ H NMR(DMSO-d ₆ )δ:8.74(s,1H),8.37(s,1H),7.85-8.00(m,3H),6.37(s,1H),4.86(t,J＝6.6Hz,2H),4.16-4.28(m,J＝15.4Hz,6H),3.12(t,J＝6.5Hz,2H),2.20(s,3H)。

biological examples

Biochemical assay method

Scintillation Proximity Assay (SPA) competitive binding

A radioligand binding assay was developed to determine whether a compound interacted with a tritium-labeled form of the natural STING ligand ³ H-cyclic guanine (2 ', 5') monophosphate adenine (3 ', 5') monophosphate [ ] ³ H-cGAMP) contend. STING constructs (WT and H232R) consist of residues 155-341 with N-and C-terminal truncations; the N-terminal transmembrane domain (1-154) and the C-terminal tail (342-379) were removed. Enzymatic and containing high affinity biotinylated peptide AviTag with E.coli biotin ligase (BirA) ^TM High-specificity N-terminal biotinylation is realized. Immobilization of 100nM STING protein in 150mM NaCl, 25mM Hepes (pH 7.5), 0.1mM EDTA, 1mM DTT, 0.005% (v/v) Tween-20, 1% (v/v) DMSO in 20. Mu.g streptavidin polyvinylformamideBenzene (SA-PVT) beads. 100nM is added ³ H-cGAMP and compound and equilibrated at room temperature (20 min). Serial test compounds were diluted 3-fold from 100 μm starting concentration and relative to complete blocking ³ Positive control compounds and negative control DMSO to which H-cGAMP binds were normalized. Using the Cheng-Prusoff equation (Cheng&Prusoff, biochemical Pharmacology,22 (1973), pages 3099-3108) from IC ₅₀ Determination of competitive binding K _I . Used in the Cheng-Prusoff equation ³ K of H-cGAMP _D Values were empirically determined to be 1nM (for WT STING) and 750nM (for R232H STING). SPA competitive binding data are provided in Table 1.

Table 1:

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phosphorylation of IRF 3: THP-1 cell ELISA

STING activation results in recruitment of TBK1 and phosphorylation of IRF3 transcription factors, followed by induction of type I interferon. THP-1 cells (InvivoGen) were cultured in RPMI medium supplemented with 2mM L-glutamine, 10% fetal bovine serum, and 0.5% pen-Strep. Will 10 ⁴ Individual cells were seeded in 96-well plates at 37℃with 5% CO ₂ Incubate overnight. Serial dilutions of compounds in medium (final 0.5% dmso) were added to the cells and incubated for an additional 3 hours. After incubation, the plates were centrifuged at 2000rpm for 5min. Cells were then lysed in 100 μl RIPA buffer and vortexed for 30min at room temperature. Mu.l of lysate was then transferred to a clear polystyrene High Bind plate which had been previously coated with mouse anti-human IRF-3 capture antibody (BD Pharmigen) and incubated for 16 hours at 4 ℃. The plates were then washed and incubated with rabbit anti-phosphorylated IRF3 detection antibody (Cell Signaling Technologies) for 1.5 hours at room temperature. Finally, HRP-linked secondary antibody (Cell Signaling Technologies) was added for 30min and then Glo Su was usedbstrate reagent (R)&D Systems) generates a luminescent signal. Signals were measured using a Perkin-Elmer Envision microplate reader. Data were normalized to "% effect" using positive control STING agonist and negative control DMSO, which are known to maximize phosphorylated IRF3 signal. IRF3 phosphorylation data are provided in table 2.

Table 2:

interferon-beta induction THP-1ISG reporter cell line

THP-1Lucia ^TM ISG cells (invitrogen) express a secreted luciferase "Lucia" reporter gene under the control of an IRF inducible composite promoter consisting of 5 interferon response elements. Culturing THP-1Lucia in RPMI medium supplemented with 2mM L-glutamine, 10% fetal bovine serum, and 0.5% pen-Strep ^TM ISG cells. Hygromycin B and Zeocin were present to maintain stable transfection. Will 10 ⁴ Individual cells were seeded in 96-well plates and incubated at 37℃with 5% CO ₂ Incubate overnight. 50. Mu.L of serial dilutions of compound in medium were added and incubated for an additional 24 hours. After incubation, the plates were centrifuged at 2000rpm for 10min. Mu.l of cell culture supernatant from each well was transferred to a white opaque 96-well plate. Preparation of a bag of QUANTI-Luc in 25mL of endotoxin-free water ^TM (InvivoGen) powder, and 100. Mu.L of the prepared temperature QUANTI-Luc solution was added to each well containing the supernatant. Luminescence signals were measured using a Perkin-Elmer Envision microplate reader. Data were normalized to "% effect" using positive control STING agonist and negative control DMSO, which are known to maximize luciferase signal. Interferon- β induction data are provided in table 3.

Table 3:

these examples are provided for illustrative purposes only and do not limit the scope of the claims provided herein. It will be apparent to those of ordinary skill in the art that certain changes and modifications may be made thereto without departing from the spirit or scope of the claims.

All publications and patent applications cited in the specification are herein incorporated by reference in their entirety.

Claims

1. A compound of formula (I):

or a pharmaceutically acceptable salt thereof, wherein

Represents two conjugated double bonds in a 5 membered heteroaryl ring;

X ¹ selected from CH and N;

X ² selected from CH and N;

Z ¹ 、Z ² and Z ³ Selected such that:

Z ¹ Is C, Z ² Is CR (CR) ³ And Z is ³ Is NR ² ；

2. The compound of formula (II) according to claim 1:

or a pharmaceutically acceptable salt thereof.

3. The compound of formula (III) according to claim 1:

or a pharmaceutically acceptable salt thereof.

4. According to claim 1 to3 or a pharmaceutically acceptable salt thereof, wherein R ¹ Is C ₁ -C ₄ Alkyl, said C ₁ -C ₄ The alkyl group is optionally substituted with 1, 2 or 3 substituents each independently selected from halogen, hydroxy and-OC ₁ -C ₄ An alkyl group.

5. The compound according to claim 4, or a pharmaceutically acceptable salt thereof, wherein R ¹ Selected from-CH ₃ and-CH ₂ CH ₃ 。

6. The compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, wherein R ² Selected from C ₁ -C ₄ Alkyl and C ₁ -C ₂ Alkylene- (cyclopropyl) s, C ₁ -C ₄ Alkyl or C ₁ -C ₂ Alkylene- (cyclopropyl) is optionally substituted with 1, 2, 3, 4, 5 or 6 substituents each independently selected from halogen, hydroxy, -CN and-OC ₁ -C ₄ An alkyl group.

7. The compound according to claim 6, or a pharmaceutically acceptable salt thereof, wherein R ² Selected from-CH ₃ 、-CH ₂ CH ₃ 、-(CH ₂ ) ₂ CH ₃ 、-CH ₂ CF ₃ 、-(CH ₂ ) ₂ CF ₃ 、-(CH ₂ ) ₃ OH、-(CH ₂ ) ₂ OCH ₃ 、-(CH ₂ ) ₃ OCH ₃ and-CH ₂ (cyclopropyl).

8. The compound according to claim 1 or claim 2, or a pharmaceutically acceptable salt thereof, wherein R ³ Is C ₁ -C ₄ Alkyl, said C ₁ -C ₄ Alkyl is optionally substituted with 1, 2, 3, 4, 5 or 6 substituents each independently selected from halogen, hydroxy, -CN and-OC ₁ -C ₄ An alkyl group.

9. The compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, wherein R ⁴ Selected from H and C ₁ -C ₄ Alkyl, said C ₁ -C ₄ The alkyl group is optionally substituted with 1, 2 or 3 substituents each independently selected from halogen and hydroxy.

10. The compound according to claim 9, or a pharmaceutically acceptable salt thereof, wherein R ⁴ Selected from H and-CH ₃ 。

11. The compound according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, wherein R ⁵ Selected from H, halogen and hydroxy.

12. The compound according to claim 11, or a pharmaceutically acceptable salt thereof, wherein R ⁵ Selected from H, chlorine and hydroxyl.

13. The compound according to any one of claims 1 to 12, or a pharmaceutically acceptable salt thereof, wherein R ⁶ Selected from C ₁ -C ₄ Alkyl and cyclopropyl, said C ₁ -C ₄ Alkyl or cyclopropyl is optionally substituted with 1, 2 or 3 substituents each independently selected from halogen, hydroxy, -CN and-OC ₁ -C ₄ An alkyl group.

14. The compound according to claim 13, or a pharmaceutically acceptable salt thereof, wherein R ⁶ Selected from-CH ₃ 、-CH ₂ CH ₃ 、-CH ₂ CHF ₂ 、-CH(CH ₃ ) ₂ And cyclopropyl.

15. A compound selected from the group consisting of:

or a pharmaceutically acceptable salt of any of these.

16. A compound selected from the group consisting of:

or a pharmaceutically acceptable salt of any of these.

17. A pharmaceutical composition comprising a compound according to any one of claims 1-16, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

18. A method for treating abnormal cell growth in a mammal, the method comprising administering to the mammal a therapeutically effective amount of a compound according to any one of claims 1-16, or a pharmaceutically acceptable salt thereof.

19. The method of claim 18, wherein the abnormal cell growth is cancer.

20. The method of claim 19, wherein the cancer is lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, gastric cancer, colon cancer, breast cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, hodgkin's disease, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, sarcoma of soft tissue, cancer of the urethra, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, renal pelvis carcinoma, tumors of the Central Nervous System (CNS), primary CNS lymphoma, spinal axis tumors, brain stem glioma, or adenoma.

21. The method of any one of claims 18 to 20, wherein the mammal is a human.

22. The method of any one of claims 18 to 21, comprising administering an additional therapeutic agent.

23. The method of claim 22, wherein the additional therapeutic agent is selected from the group consisting of an interferon, a CTLA-4 pathway antagonist, an anti-4-1 BB antibody, an anti-PD-1 antibody, and an anti-PD-L1 antibody.