阅读说明：本技术 作为jak抑制剂的5至7元杂环酰胺 (5-to 7-membered heterocyclic amides as JAK inhibitors ) 是由 丹尼尔·D·朗 C·史密斯 C·汤普森 于 2019-09-03 设计创作，主要内容包括：本发明提供用作JAK激酶抑制剂的式(I)化合物：其中变量在本说明书中定义；或其医药学上可接受的盐。本发明还提供包含这些化合物的医药组合物和使用这些化合物治疗呼吸道疾病的方法。(The present invention provides compounds of formula (I) useful as JAK kinase inhibitors:)

1. A compound of the formula (I),

wherein:

a is a 6-to 7-membered monocyclic heterocycle containing two nitrogen atoms, wherein A is linked to the carbonyl group in (I) through a nitrogen atom, A is via 1 to 3R²Substituted by radicals and optionally two R²The radicals together with A form-(CH₂) -a bridging ring; or

A is pyrrolidinyl, wherein the pyrrolidinyl is connected to the carbonyl in (I) through a nitrogen atom and wherein the pyrrolidinyl is NR³R⁴Substitution;

R¹is C_1-3An alkyl group;

each R²Independently is C optionally substituted with-OH_1-3An alkyl group;

R³is C_1-3An alkyl group;

and R is⁴Is C_1-3An alkyl group;

or a pharmaceutically acceptable salt thereof.

2. A compound according to claim 1, or a pharmaceutically acceptable salt thereof, having formula (Γ):

3. a compound according to any one of claims 1 or 2, or a pharmaceutically acceptable salt thereof, wherein R¹Selected from the group consisting of: methyl, propyl and isopropyl.

4. A compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein a is selected from the group consisting of: piperazinyl, 2, 5-diazabicyclo [2.2.1]Heptyl and 1, 4-diazepanyl, each of which is interrupted by 1 or 2R²Is substituted by radicals in which each R²Independently is C optionally substituted with-OH_1-3Alkyl, or A is NR³R⁴Substituted pyrrolidinyl, wherein R³Is C_1-3Alkyl and R⁴Is C_1-3An alkyl group.

5. The compound of any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, wherein A is selected from the group consisting of:

wherein R is²Is C optionally substituted by-OH_1-3Alkyl radical, R³Is C_1-3Alkyl and R⁴Is C_1-3An alkyl group.

6. A compound of the formula,

or a pharmaceutically acceptable salt thereof.

7. A compound of the formula,

or a pharmaceutically acceptable salt thereof.

8. A compound of the formula,

or a pharmaceutically acceptable salt thereof.

9. A compound of the formula,

or a pharmaceutically acceptable salt thereof.

10. A compound of the formula,

or a pharmaceutically acceptable salt thereof.

11. A compound of the formula,

or a pharmaceutically acceptable salt thereof.

12. A pharmaceutical composition comprising a compound according to any one of claims 1 to 11 and a pharmaceutically acceptable carrier.

13. A compound according to any one of claims 1 to 11 for use in the treatment of a respiratory disease in a mammal.

14. The compound of claim 13, wherein the respiratory disease is selected from the group consisting of: asthma, chronic obstructive pulmonary disease, cystic fibrosis, pneumonia, idiopathic pulmonary fibrosis, acute lung injury, acute respiratory distress syndrome, bronchitis, emphysema, obstructive bronchiolitis, sarcoidosis, eosinophilic disease, helminthic infection, pulmonary hypertension, pulmonary lymphangioleiomycosis, bronchiectasis, infiltrative lung disease, drug-induced pneumonia, fungal-induced pneumonia, allergic bronchopulmonary aspergillosis, allergic pneumonia, eosinophilic granuloma with polyangiitis, idiopathic acute eosinophilic pneumonia, idiopathic chronic eosinophilic pneumonia, eosinophilic leukocytosis syndrome, Lvfler syndrome, obstructive bronchiolitis organizing pneumonia, lung graft-versus-host disease, and immune checkpoint inhibitor-induced pneumonia.

15. The compound of claim 13, wherein the respiratory disease is asthma or chronic obstructive pulmonary disease.

16. A compound according to any one of claims 1 to 11 for use in the treatment of lung transplant rejection in a mammal.

17. The compound of claim 16, wherein the lung transplant rejection is selected from the group consisting of: primary graft dysfunction, organized pneumonia, acute rejection, lymphocytic bronchiolitis, and chronic lung allograft dysfunction.

18. The compound of claim 16, wherein the lung transplant rejection is acute lung transplant rejection.

19. The compound of claim 16, wherein the lung transplant rejection is chronic lung allograft dysfunction.

20. The compound of claim 16, wherein the lung transplant rejection is selected from the group consisting of: obstructive bronchiolitis, restrictive chronic lung allograft dysfunction and neutrophil allograft dysfunction.

21. Use of a compound according to any one of claims 1 to 11 for the manufacture of a medicament for treating a respiratory disease in a mammal.

22. The use of claim 21, wherein the respiratory disease is selected from the group consisting of: asthma, chronic obstructive pulmonary disease, cystic fibrosis, pneumonia, idiopathic pulmonary fibrosis, acute lung injury, acute respiratory distress syndrome, bronchitis, emphysema, obstructive bronchiolitis, sarcoidosis, eosinophilic disease, helminthic infection, pulmonary hypertension, pulmonary lymphangioleiomycosis, bronchiectasis, infiltrative lung disease, drug-induced pneumonia, fungal-induced pneumonia, allergic bronchopulmonary aspergillosis, hypersensitivity pneumonitis, eosinophilic granuloma with polyangiitis, idiopathic acute eosinophilic pneumonia, idiopathic chronic eosinophilic pneumonia, eosinophilic leukocytosis syndrome, Luverner's syndrome, obstructive bronchiolitis organizing pneumonia, lung graft-versus-host disease, and immune checkpoint inhibitor-induced pneumonia.

23. The use of claim 21, wherein the respiratory disease is asthma or chronic obstructive pulmonary disease.

24. Use of a compound according to any one of claims 1 to 11 in the manufacture of a medicament for treating lung transplant rejection in a mammal.

25. The use of claim 24, wherein the lung transplant rejection is selected from the group consisting of: primary graft dysfunction, organized pneumonia, acute rejection, lymphocytic bronchiolitis, and chronic lung allograft dysfunction.

26. The use of claim 24, wherein the lung transplant rejection is acute lung transplant rejection.

27. The use of claim 24, wherein the lung transplant rejection is chronic lung allograft dysfunction.

28. The use of claim 24, wherein the lung transplant rejection is selected from the group consisting of: obstructive bronchiolitis, restrictive chronic lung allograft dysfunction and neutrophil allograft dysfunction.

29. A method of treating a respiratory disease in a mammal, the method comprising administering to the mammal a pharmaceutical composition comprising a compound according to any one of claims 1 to 11 and a pharmaceutically acceptable carrier.

30. The method of claim 29, wherein the respiratory disease is selected from the group consisting of: asthma, chronic obstructive pulmonary disease, cystic fibrosis, pneumonia, idiopathic pulmonary fibrosis, acute lung injury, acute respiratory distress syndrome, bronchitis, emphysema, obstructive bronchiolitis, sarcoidosis, eosinophilic disease, helminthic infection, pulmonary hypertension, pulmonary lymphangioleiomycosis, bronchiectasis, infiltrative lung disease, drug-induced pneumonia, fungal-induced pneumonia, allergic bronchopulmonary aspergillosis, allergic pneumonia, eosinophilic granuloma with polyangiitis, idiopathic acute eosinophilic pneumonia, idiopathic chronic eosinophilic pneumonia, eosinophilic leukocytosis syndrome, Lvfler syndrome, obstructive bronchiolitis organizing pneumonia, lung graft-versus-host disease, and immune checkpoint inhibitor-induced pneumonia.

31. The method of claim 29, wherein the respiratory disease is asthma or chronic obstructive pulmonary disease.

32. A method of treating lung transplant rejection in a mammal, said reaction comprising administering to said mammal a pharmaceutical composition comprising a compound according to any one of claims 1 to 11 and a pharmaceutically acceptable carrier.

33. The method of claim 32, wherein the lung transplant rejection is selected from the group consisting of: primary graft dysfunction, organized pneumonia, acute rejection, lymphocytic bronchiolitis, and chronic lung allograft dysfunction.

34. The method of claim 32, wherein the lung transplant rejection is acute lung transplant rejection.

35. The method of claim 32, wherein the lung transplant rejection is chronic lung allograft dysfunction.

36. The method of claim 32, wherein the lung transplant rejection is selected from the group consisting of: obstructive bronchiolitis, restrictive chronic lung allograft dysfunction and neutrophil allograft dysfunction.

Technical Field

The present invention relates to compounds useful as JAK kinase inhibitors. The invention also relates to pharmaceutical compositions comprising these compounds and methods of using these compounds in the treatment of respiratory diseases.

Background

Asthma is a chronic disease of the trachea, which has no preventive or curative means. The disease is characterized by airway inflammation, fibrosis, hyperreactivity, and remodeling, all contributing to airflow limitation. It is estimated that 3 million people worldwide suffer from asthma, and it is estimated that the number of people suffering from asthma will increase by more than 1 million by 2025. In the united states, about 6% to 8% of the population suffers from asthma, making it one of the most common chronic diseases in the united states. While most patients can achieve control of asthma symptoms with inhaled corticosteroids, which can be combined with leukotriene modifiers and/or long-acting beta agonists, the disease in a subset of patients with severe asthma is not controlled by conventional therapies. Severe persistent asthma is defined as a disease in which higher doses of inhaled corticosteroids remain uncontrolled. While severe asthma patients are estimated to account for approximately 5% of all asthma patients, they are at high risk for morbidity and mortality and are responsible for a disproportionate share of health care resource utilization in asthma patients. There remains a need for novel therapeutic agents to treat these patients.

Interleukins are intercellular signaling molecules that include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. Interleukins are critical for normal cell growth and regulation of immunity and also drive immune-mediated diseases and contribute to malignant cell growth. Elevated levels of various interleukins are involved in the pathology of asthma inflammation. For example, antibody-based therapeutics targeting Interleukin (IL) -5 and 13 have been shown to provide clinical benefit to a subset of patients with severe asthma. Among the interleukins involved in asthmatic inflammation, many act through signaling pathways depending on the Janus (Janus) family of Signal Transducers and Activators of Transcription (STAT) that conduct signals through the Signal Transducer and Activator of Transcription family of Transcription factors. Interleukins involved in asthma inflammation that signal through the JAK-STAT pathway include: IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-11, IL-13, IL-23, IL-31, IL-27, Thymic Stromal Lymphopoietin (TSLP), interferon- γ (IFN γ), and granulocyte-macrophage colony stimulating factor (GM-CSF).

The JAK family contains four members: JAK1, JAK2, JAK3 and tyrosine kinase 2(TYK 2). Binding of the interleukin to the JAK-dependent interleukin receptor induces receptor dimerization that phosphorylates tyrosine residues on JAK kinases, thereby affecting JAK activation. Phosphorylated JAKs in turn bind and phosphorylate various STAT proteins that dimerize, internalize, and directly regulate gene transcription in the cell nucleus, among other effects, leading to downstream effects associated with inflammatory diseases. JAKs are typically associated in pairs with interleukin receptors as homodimers or heterodimers. Specific interleukins are associated with specific JAK pairs. Each of the four members of the JAK family is involved in signaling of at least one of the interleukins associated with asthma inflammation. Thus, relative to all members of the JAK family, chemical inhibitors with pan-activity can modulate a wide range of pro-inflammatory pathways contributing to severe asthma.

However, the broader anti-inflammatory action of these inhibitors may inhibit normal immune cell function, potentially leading to increased risk of infection. Evidence of an increased risk of infection was observed with the JAK inhibitor tofacitinib, which was administered orally for the treatment of rheumatoid arthritis. In asthma, inflammation is localized to the respiratory tract. In addition to asthma, airway inflammation is characteristic of other respiratory diseases. Chronic Obstructive Pulmonary Disease (COPD), Cystic Fibrosis (CF), pneumonia, interstitial lung disease (including idiopathic pulmonary fibrosis), acute lung injury, acute respiratory distress syndrome, bronchitis, emphysema and sarcoidosis are also respiratory diseases in which the pathophysiology is believed to be associated with JAK signaling interleukins. Local administration of JAK inhibitors to the lung by inhalation offers the possibility of being therapeutically effective by delivering effective anti-interleukin agents directly to the site of action, limiting systemic exposure and thus the possibility of adverse systemic immunosuppression. There remains a need for effective JAK inhibitors suitable for topical administration to the lung for the treatment of respiratory diseases.

JAK signaling interleukins also play a major role in T cell activation, a subset of immune cells that are important to many immune processes. Pathological T cell activation is crucial for the etiology of various respiratory diseases. Autoreactive T cells play a role in obstructive bronchiolitis with organizing pneumonia (also known as COS). Similar to COS, the etiology of lung transplant rejection is associated with abnormal T cell activation of recipient T cells that transplant donor lungs. The Lung transplant rejection may occur early in the form of Primary Graft Dysfunction (PGD), Organizing Pneumonia (OP), Acute Rejection (AR), or Lymphocytic Bronchiolitis (LB), or it may occur several years after Lung transplantation in the form of Chronic Lung Allograft Dysfunction (CLAD). CLAD has previously been known as obstructive Bronchiolitis (BO), but is now thought to have a number of syndromes of different pathological manifestations, including BO, restrictive CLAD (rCLAD or RAS) and basophilic allograft dysfunction. Chronic Lung Allograft Dysfunction (CLAD) is a major challenge in the long-term management of lung transplant recipients because it causes the transplanted lung to gradually become dysfunctional (cauthier et al, current transplant representatives, 2016,3(3), 185-. CLAD responds poorly to treatment and, therefore, there remains a need for effective compounds capable of preventing or treating this condition. Several JAK-dependent interleukins such as IFN γ and IL-5 are up-regulated in CLAD and lung transplant rejection (Berastegui et al, & 2017,31, e12898 in clinical transplants). In addition, high lung content of CXCR3 chemokines (e.g., CXCL9 and CXCL10) downstream of JAK-dependent IFN signaling is associated with worsening outcome in lung transplant patients (cinono et al, "complex of the public library (PLOS One), 2017,12(7), e 0180281). Systemic JAK inhibition was shown to be effective in kidney transplant rejection (weinett (Vicenti), et al, Journal of American Transplantation, 2012,12, 2446-56). Therefore, JAK inhibitors are likely to be effective in treating or preventing lung transplant rejection and CLAD. Similar T cell activation events as described as the basis for lung transplant rejection are also considered to be a major driver of lung graft-versus-host disease (GVHD) that may occur following hematopoietic stem cell transplantation. Similar to CLAD, lung GVHD is a chronic progressive condition with very poor outcomes and no currently approved treatment. Retrospective multicenter studies of patients with steroid refractory acute or chronic GVHD, 95 receiving the systemic JAK inhibitor, luzotinib, as a remedial treatment, demonstrated that most patients, including those with pulmonary GVHD, were completely or partially responsive to luzotinib (Zeiser et al, Leukemia (leukamia), 2015,29,10, 2062-68). Since systemic JAK inhibition is associated with severe adverse events and a small therapeutic index, there remains a need for inhalation of pulmonary-directed, non-systemic JAK inhibitors to prevent and/or treat lung transplant rejection or lung GVHD.

Disclosure of Invention

In one aspect, the invention provides novel compounds having activity as JAK kinase inhibitors.

Accordingly, the present invention provides a compound of formula (I):

wherein:

a is a 6-to 7-membered monocyclic heterocycle containing two nitrogen atoms, wherein A is linked to the carbonyl group in (I) through a nitrogen atom, A is via 1 to 3R²Substituted by radicals and optionally two R²The radicals together with A form- (CH)₂) -a bridging ring; or

A is pyrrolidinyl, wherein the pyrrolidinyl is connected to the carbonyl in (I) through a nitrogen atom and wherein the pyrrolidinyl is NR³R⁴Substitution;

R¹is C_1-3An alkyl group;

each R²Independently is C optionally substituted with-OH_1-3An alkyl group;

R³is C_1-3An alkyl group;

and R is⁴Is C_1-3An alkyl group;

or a pharmaceutically acceptable salt thereof.

The invention also provides a pharmaceutical composition comprising a compound of the invention and a pharmaceutically acceptable carrier.

The invention also provides a method of treating a respiratory disease, particularly asthma or CLAD, in a mammal, the method comprising administering to the mammal a therapeutically effective amount of a compound or a pharmaceutical composition of the invention.

The present invention also provides a compound of the invention as described herein for use in medical therapy; and the use of a compound of the invention for the manufacture of a formulation or medicament for the treatment of a respiratory disease in a mammal.

Detailed Description

In one embodiment, the present invention provides novel compounds having activity as JAK kinase inhibitors. Accordingly, the present invention provides a compound of formula (I):

wherein:

a is a 6-to 7-membered monocyclic heterocycle containing two nitrogen atoms, wherein A is linked to the carbonyl group in (I) through a nitrogen atom, A is via 1 to 3R²Substituted by radicals and optionally two R²The radicals together with A form- (CH)₂) -a bridging ring; or

A is pyrrolidinyl, wherein the pyrrolidinyl is connected to the carbonyl in (I) through a nitrogen atom and wherein the pyrroleAlkyl through NR³R⁴Substitution;

R¹is C_1-3An alkyl group;

each R²Independently is C optionally substituted with-OH_1-3An alkyl group;

R³is C_1-3An alkyl group;

and R is⁴Is C_1-3An alkyl group;

or a pharmaceutically acceptable salt thereof.

In some embodiments, compound (I) has formula (I'):

in some embodiments, R¹Selected from the group consisting of: methyl, propyl and isopropyl.

In some embodiments, a is selected from the group consisting of: piperazinyl, 2, 5-diazabicyclo [2.2.1]Heptyl and 1, 4-diazepanyl, each of which is interrupted by 1 or 2R²Is substituted by radicals in which each R²Independently is C optionally substituted with-OH_1-3Alkyl, or A is NR³R⁴Substituted pyrrolidinyl, wherein R³Is C_1-3Alkyl and R⁴Is C_1-3An alkyl group.

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

wherein R is²Is C optionally substituted by-OH_1-3Alkyl radical, R³Is C_1-3Alkyl and R⁴Is C_1-3An alkyl group.

In another embodiment, the invention provides a compound of the formula:

or a pharmaceutically acceptable salt thereof.

In another embodiment, the invention provides a compound of the formula:

or a pharmaceutically acceptable salt thereof.

In another embodiment, the invention provides a compound of the formula:

or a pharmaceutically acceptable salt thereof.

In another embodiment, the invention provides a compound of the formula:

or a pharmaceutically acceptable salt thereof.

In another embodiment, the invention provides a compound of the formula:

or a pharmaceutically acceptable salt thereof.

In another embodiment, the invention provides a compound of the formula:

or a pharmaceutically acceptable salt thereof.

In another embodiment, the present disclosure provides a compound of formula (II):

wherein:

R¹is C_1-3An alkyl group;

A¹selected from the group consisting of:

wherein R is²Is C optionally substituted by-OH_1-3An alkyl group;

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of formula (II) has formula (II'):

in some embodiments, R¹Selected from the group consisting of methyl and propyl.

In some embodiments, A¹Selected from the group consisting of:

in some embodiments, A¹Selected from the group consisting of:

and R is¹Selected from the group consisting of methyl and propyl.

In another embodiment, the present disclosure provides a compound of formula (III):

wherein:

R¹is C_1-3An alkyl group;

A²selected from the group consisting of:

wherein each R²Independently is C optionally substituted with-OH_1-3Alkyl radical, R³Is C_1-3Alkyl and R⁴Is C_1-3An alkyl group;

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of formula (III) has formula (III'):

in some embodiments, R¹Selected from the group consisting of: methyl, isopropyl and propyl.

In some embodiments, A²Selected from the group consisting of:

in some embodiments, R¹Selected from the group consisting of: methyl, isopropyl and propyl, and A²Selected from the group consisting of:

in another embodiment, the present disclosure provides a compound of formula (IV):

wherein:

R¹is C_1-3An alkyl group;

A³selected from the group consisting of:

wherein each R²Independently is C optionally substituted with-OH_1-3Alkyl radical, R³Is C_1-3Alkyl and R⁴Is C_1-3An alkyl group;

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of formula (IV) has formula (IV'):

in some embodiments, R¹Selected from the group consisting of: methyl, isopropyl and propyl.

In some embodiments, A³Selected from the group consisting of:

in some embodiments, R¹Selected from the group consisting of: methyl, isopropyl and propyl, and A³Selected from the group consisting of:

furthermore, the imidazo portion of the tetrahydroimidazopiperidine portion exists in tautomeric forms, as illustrated below for fragments of the compounds of the present disclosure.

According to the IUPAC convention, these diagrams yield different numbering of the atoms of the imidazole moiety: (1H-indazol-3-yl) -4,5,6, 7-tetrahydro-1H-imidazo [4,5-c]Pyridine (Structure A) vs. (1H-indazol-3-yl) -4,5,6, 7-tetrahydro-3H-imidazo [4,5-c]Pyridine (structure B). It is to be understood that while structures are shown or named in specific forms, the invention also includes tautomers thereof.

The compounds of the present disclosure may contain one or more chiral centers, and thus, these compounds (and intermediates thereof) may exist as racemic mixtures, pure stereoisomers (that is, enantiomers or diastereomers), stereoisomerically-enriched mixtures, and the like. Unless otherwise indicated, chiral compounds shown or named herein that do not have a defined stereochemistry at a chiral center are intended to include any and all possible stereoisomeric variations at an undefined stereocenter. Unless otherwise indicated, description or naming of a particular stereoisomer means that the indicated stereocenter has the indicated stereochemistry, while it is understood that minor amounts of other stereoisomers may also be present, provided that the utility of the depicted or named compound is not diminished by the presence of the other stereoisomer.

The compounds of the present disclosure may also contain several bases (e.g., amino groups), and thus, these compounds may exist in the form of the free base or in various salt forms, such as the monoprotized salt form, the diprotized salt form, the triprotized salt form, or mixtures thereof. Unless otherwise indicated, all such forms are included within the scope of the present invention.

The present invention also includes isotopically-labelled compounds of formulae (I), (I '), (II'), (III '), (IV) and (IV'), that is, compounds of formulae (I), (I '), (II'), (III '), (IV) and (IV') in which one or more atoms are replaced or enriched by an atom having the same atomic number but an atomic mass different from the predominant atomic mass in nature. May be incorporated into the compounds of formulae (I), (I '), (II'), (III '), (IV) and (IV')Examples of isotopes in matter include (but are not limited to)²H、³H、¹¹C、¹³C、¹⁴C、¹³N、¹⁵N、¹⁵O、¹⁷O and¹⁸and O. Of particular interest are tritium-or carbon 14-enriched compounds of formula (I), (I '), (II'), (III '), (IV) and (IV'), which are useful, for example, in tissue distribution studies. Of further interest are compounds of formula (I), (I '), (II'), (III '), (IV) and (IV') enriched in deuterium, especially at the metabolic site, which are expected to have higher metabolic stability. Furthermore, it is of great interest to enrich for example¹¹C、¹⁵O and¹³compounds of formulae (I), (I '), (II'), (III '), (IV) and (IV') of Positron emitting isotopes of N useful, for example, in Positron Emission Tomography (PET) studies.

Definition of

Unless otherwise indicated, when describing the present invention (including various aspects and embodiments thereof), the following terms have the following meanings.

The term "alkyl" means a monovalent saturated hydrocarbon group that can be straight or branched chain or a combination thereof. Unless otherwise defined, these alkyl groups typically contain 1 to 10 carbon atoms. Representative alkyl groups include, for example, methyl (Me), ethyl (Et), n-propyl (n-Pr) or (nPr), isopropyl (i-Pr) or (iPr), n-butyl (n-Bu) or (nBu), sec-butyl, isobutyl, tert-butyl (t-Bu) or (tBu), n-pentyl, n-hexyl, 2-dimethylpropyl, 2-methylbutyl, 3-methylbutyl, 2-ethylbutyl, 2-dimethylpentyl, 2-propylpentyl, and the like.

When a particular number of carbon atoms is intended for a particular term, the number of carbon atoms is presented before the term. For example, the term "C_1-3Alkyl "means an alkyl group having 1 to 3 carbon atoms in any chemically acceptable configuration, including straight or branched chain configurations.

The term "cycloalkyl" means a monovalent saturated carbocyclic group that may be monocyclic or polycyclic. Unless otherwise defined, these cycloalkyl groups typically contain 3 to 10 carbon atoms. Representative cycloalkyl groups include, for example, cyclopropyl (cPr), cyclobutyl (cBu), cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, adamantyl, and the like.

The term "cyclopropyl (cpropyl)" means cyclopropyl (cyclopropyl).

The term "heterocyclyl" (heterocyclic) "," heterocyclic (heterocyclic) "or" heterocyclic (heterocyclic ring) "means a monovalent saturated or partially unsaturated cyclic nonaromatic group having a total of 3 to 10 ring atoms, wherein the ring contains 2 to 9 carbon ring atoms and 1 to 4 ring heteroatoms selected from nitrogen, oxygen and sulfur. Heterocyclyl groups may be monocyclic or polycyclic (that is, fused or bridged). Representative heterocyclyl groups include, by way of example, pyrrolidinyl, piperidinyl, piperazinyl, imidazolidinyl, morpholinyl, thiomorpholinyl, indolin-3-yl, 2-imidazolinyl, tetrahydropyranyl, 1,2,3, 4-tetrahydroisoquinolin-2-yl, quinuclidinyl, 7-azaboropanyl, nortropyl (nortropanyl), and the like, wherein the point of attachment is at any available carbon or nitrogen ring atom. Where the point of attachment of the heterocyclyl group is made apparent above and below, these groups may alternatively be referred to as non-valent species, that is to say pyrrolidine, piperidine, piperazine, imidazole, tetrahydropyran, and the like.

The term "halo" means fluoro, chloro, bromo, or iodo.

The term "therapeutically effective amount" means an amount sufficient to effect treatment when administered to a patient in need thereof.

The term "treating" means preventing, ameliorating or inhibiting a medical condition, disease or disorder (e.g., a respiratory disease) in a patient (particularly a human being) being treated; or alleviating a symptom of a medical condition, disease or disorder.

The term "pharmaceutically acceptable salt" means that an acceptable salt (e.g., a salt that is acceptably mammalian safe for a given dosage regimen) is administered to a patient or mammal (e.g., a human). Representative pharmaceutically acceptable salts include the following salts: acetic acid, ascorbic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, ethanedisulfonic acid, fumaric acid, gentisic acid, gluconic acid, glucuronic acid, glutamic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, lactobionic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalenesulfonic acid, naphthalene-1, 5-disulfonic acid, naphthalene-2, 6-disulfonic acid, nicotinic acid, nitric acid, orotic acid, pamoic acid, pantothenic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid, p-toluenesulfonic acid, and hydroxynaphthoic acid, and the like.

The term "salt thereof" means a compound formed when the hydrogen of an acid is replaced with a cation (e.g., a metal cation, an organic cation, or the like). For example, the cation may be a protonated form of a compound of formula (I), (I '), (II'), (III '), (IV) or (IV'), that is to say a form in which one or more amino groups are protonated by an acid. Typically, the salt is a pharmaceutically acceptable salt, but this is not required for salts of intermediate compounds that are not intended for administration to a patient.

General synthetic procedure

The compounds of the present invention and intermediates thereof can be prepared according to the following general methods and procedures using commercially available or conventionally prepared starting materials and reagents. Substituents and variables used in the following schemes (e.g., A, R) unless otherwise indicated¹、R²、R³Etc.) have the same meaning as defined elsewhere herein. Furthermore, unless otherwise indicated, compounds having acidic or basic atoms or functional groups may be used as or may be produced as salts (in some cases, the use of a salt in a particular reaction will require the use of conventional procedures to convert the salt to a non-salt form, e.g., the free base, prior to conducting the reaction).

While particular embodiments of the invention may be shown or described in the following procedures, those skilled in the art will recognize that other embodiments or aspects of the invention may also be prepared using these procedures or by using other methods, reagents, and starting materials known to those skilled in the art. In particular, it will be appreciated that the compounds of the invention may be prepared by a variety of process routes in which the reactants are combined in different orders to provide different intermediate alkene routes to prepare the final product.

The general procedure for preparing the final compounds of the invention is illustrated in the following scheme.

Reacting compound K-18 with a ketone or aldehyde in the presence of a reducing agent to give a compound of formula K-19. Compound K-19 is then reacted with amine a under typical amide bond forming conditions to give compound (I). Typically, the carboxylic acid is contacted with between about 1 and about 4 equivalents of amine a in the presence of an excess of base. Amide bond formation reactions may utilize coupling agents such as N, N' -tetramethyl-O- (7-azabenzotriazol-1-yl) uronium Hexafluorophosphate (HATU) or other amide coupling agents known in the art. Hydrazine may be added to decompose undesirable by-products. The reaction is typically carried out at room temperature for between about 5 minutes and about 24 hours or until the reaction is substantially complete.

Pharmaceutical composition

The compounds of the present invention and pharmaceutically acceptable salts thereof are typically used in the form of pharmaceutical compositions or formulations. These pharmaceutical compositions may advantageously be administered to a patient by inhalation. In addition, the pharmaceutical compositions may be administered by any acceptable route of administration, including, but not limited to, oral, rectal, nasal, topical (including transdermal) and parenteral modes of administration.

Thus, in one of its composition aspects, the present invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient and a compound of formula (I), (I '), (II'), (III '), (IV) or (IV'), wherein "a compound of formula (I), (I '), (II'), (III '), (IV) or (IV'), as defined above, means a compound of formula (I), (I '), (II'), (III '), (IV) or (IV'), or a pharmaceutically acceptable salt thereof. Optionally, these pharmaceutical compositions may contain other therapeutic agents and/or compounding agents as necessary. When discussing compositions and uses thereof, the "compounds of the invention" may also be referred to herein as "active agents". As used herein, the term "compounds of the invention" is intended to include all compounds encompassed by formula (I) as well as species embodied in the form of formula (II) and pharmaceutically acceptable salts thereof.

The pharmaceutical compositions of the present invention generally contain a therapeutically effective amount of a compound of the present invention. However, one skilled in the art will recognize that a pharmaceutical composition may contain more than a therapeutically effective amount (that is, the host composition) or less than a therapeutically effective amount (that is, individual unit doses designed for multiple administrations to obtain a therapeutically effective amount).

Typically, these pharmaceutical compositions will contain from about 0.01 to about 95 weight percent of the active agent; including for example, from about 0.05 to about 30 wt%; and about 0.1% to about 10% by weight of an active agent.

Any conventional carrier or excipient may be used in the pharmaceutical composition of the present invention. The choice of a particular carrier or excipient, or combination of carriers or excipients, will depend on the mode of administration used to treat a particular patient or type of medical condition or disease state. In this regard, it is well within the purview of the skilled medical artisan to prepare suitable pharmaceutical compositions for particular modes of administration. In addition, the carrier or excipient used in the pharmaceutical composition of the present invention may be commercially available. By way of further illustration, conventional compounding techniques are described in Remington: The Science and Practice of Pharmacy, 20 th edition, Lippincott Williams & White, Baltimore, Maryland, Pa. (2000); and h.c. ansel (h.c. ansel) et al, Pharmaceutical Dosage Forms and Drug Delivery Systems (Pharmaceutical Delivery Forms and Drug Delivery Systems), 7 th edition, in Lippincott Williams & White, Baltimore, Maryland (1999), by baldrin.

Representative examples of materials that can serve as pharmaceutically acceptable carriers include, but are not limited to, the following: sugars such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, such as microcrystalline cellulose and its derivatives, e.g., sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; no pyrogen water; isotonic saline; ringer's solution (Ringer's solution); ethanol; a phosphate buffer solution; and other non-toxic compatible materials employed in pharmaceutical compositions.

Pharmaceutical compositions are generally prepared by thoroughly and intimately mixing or blending the active agent with a pharmaceutically acceptable carrier and one or more optional ingredients. The resulting uniformly blended mixture may then be shaped into or loaded into tablets, capsules, pills, and the like using conventional procedures and equipment.

In one aspect, the pharmaceutical composition is suitable for administration by inhalation. Pharmaceutical compositions for administration by inhalation are generally in the form of aerosols or powders. These compositions are typically administered using an inhaler delivery device, such as a Dry Powder Inhaler (DPI), metered-dose inhaler (MDI), nebulizer inhaler or similar delivery device.

In a particular embodiment, the pharmaceutical composition is administered by inhalation using a dry powder inhaler. These dry powder inhalers typically administer the pharmaceutical composition as a free-flowing powder that disperses in the airflow of the patient during inhalation. To obtain a free-flowing powder composition, the therapeutic agent is typically formulated with a suitable excipient such as lactose, starch, mannitol, dextrose, polylactic acid (PLA), polylactide-co-glycolide (PLGA), or a combination thereof. Typically, the therapeutic agent is micronized and combined with a suitable carrier to form a composition suitable for inhalation.

A representative pharmaceutical composition for use in a dry powder inhaler comprises lactose in micronized form and a compound of the invention. These dry powder compositions can be made, for example, by combining dry milled lactose with the therapeutic agent, and then dry blending the components. The composition is then typically loaded into a dry powder dispenser or an inhalation sleeve or capsule for use with a dry powder delivery device.

Dry powder inhaler delivery devices suitable for administering therapeutic agents by inhalation are described in the art and examples of such devices are commercially available. By way of example, representative dry powder inhaler delivery devices or products include: aeolizer (novartis); airmax (ivax); clickhaler (innovata biomed); diskhaler (glaxosmithkline); Diskus/Accuhaler (GlaxoSmithKline); ellipta (glaxosmithkline); easyhaler (orion pharma); eclipse (aventis); flowcaps (hovione); handihaler (boehringer ingelheim); pulvinal (chiesi); rotahaler (glaxosmithkline); SkyeHaler/Certihaler (SkyePharma); twist (Schering-Plough); turbuhaler (astrazeneca); ultrahaler (aventis); and the like.

In another particular embodiment, the pharmaceutical composition is administered by inhalation using a metered dose inhaler. These metered dose inhalers typically use a compressed propellant gas to expel a measured amount of the therapeutic agent. Thus, pharmaceutical compositions for administration using metered dose inhalers typically comprise a solution or suspension of the therapeutic agent in a liquefied propellant. Any suitable liquefied propellant may be employed, including Hydrofluoroalkanes (HFAs), such as 1,1,1, 2-tetrafluoroethane (HFA 134a) and 1,1,1,2,3,3, 3-heptafluoro-n-propane (HFA 227); and chlorofluorocarbons, e.g. CCl₃F. In a particular embodiment, the propellant is a hydrofluoroalkane. In some embodiments, the hydrofluoroalkane formulation contains a co-solvent, such as ethanol or pentane; and/or surfactants such as sorbitan trioleate, oleic acid, lecithin and glycerol.

Representative pharmaceutical compositions for use in a metered dose inhaler comprise from about 0.01% to about 5% by weight of a compound of the present invention; about 0% to about 20% by weight of ethanol; and about 0 wt% to about 5 wt% of a surfactant; with the remainder being HFA propellant. These compositions are typically prepared by adding a cooled or pressurized hydrofluoroalkane to a suitable container containing the therapeutic agent, ethanol (if present), and surfactant (if present). To prepare the suspension, the therapeutic agent is micron sized and then combined with a propellant. The composition is then loaded into an aerosol canister, which typically forms part of a metered dose inhaler device.

Metered dose inhaler devices suitable for administering therapeutic agents by inhalation are described in the art and examples of such devices are commercially available. By way of example, representative metered dose inhaler devices or products include: AeroBid inhaler systems (Forest Pharmaceuticals); atrovent inhalation aerosol (Boehringer Ingelheim); flovent (GlaxoSmithKline); maxair inhaler (3M); a Proventil inhaler (Schering); serevent inhalation aerosol (GlaxoSmithKline); and the like.

In another particular aspect, the pharmaceutical composition is administered by inhalation using a nebulizer inhaler. These nebulizer devices typically produce a high velocity air stream that causes the pharmaceutical composition to be nebulized as a mist into the respiratory tract of the patient. Thus, when formulated for use in a nebulizer inhaler, the therapeutic agent can be dissolved in a suitable carrier to form a solution. Alternatively, the therapeutic agent may be micro-sized or nano-milled and combined with a suitable carrier to form a suspension.

Representative pharmaceutical compositions for use in nebulizer inhalers include solutions or suspensions comprising from about 0.05 μ g/mL to about 20mg/mL of a compound of the invention and an excipient compatible with the nebulized formulation. In one embodiment, the solution has a pH of about 3 to about 8.

Nebulizer devices suitable for administering therapeutic agents by inhalation are described in the art and examples of such devices are commercially available. For example, representative nebulizer devices or products include: respimat Softmist inhaler (Boehringer Ingelheim); the AERx pulmonary delivery system (Aradigm Corp.); PARI LC Plus reusable nebulizer (PARI GmbH); and the like.

In a further aspect, the pharmaceutical composition of the invention may alternatively be prepared in a dosage form intended for oral administration. Suitable pharmaceutical compositions for oral administration may be in the form of capsules, lozenges, pills, buccal tablets, cachets, dragees, powders, granules; or in the form of a solution or suspension in an aqueous or non-aqueous liquid; or in the form of an oil-in-water or water-in-oil liquid emulsion; or in the form of elixirs or syrups; etc.; each containing a predetermined amount of the compound of the present invention as an active ingredient.

When in solid dosage form intended for oral administration, the pharmaceutical compositions of the present invention will generally comprise an active agent and one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate. Optionally or alternatively, the solid dosage forms may further comprise: fillers or extenders, binders, humectants, solution retarding agents, absorption accelerators, humectants, adsorbents, lubricants, colorants, and buffers. Release agents, humectants, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants may also be present in the pharmaceutical composition according to the invention.

Alternative formulations may also include controlled release formulations, liquid dosage forms for oral administration, transdermal patches, and parenteral formulations. Conventional excipients and methods for preparing these alternative formulations are described, for example, in the aforementioned Remington reference.

The following non-limiting examples illustrate representative pharmaceutical compositions of the present invention.

Dry powder composition

Micron-sized compound of formula (I) (1g) was blended with milled lactose (25 g). This blended mixture is then loaded into individual blisters of a peelable blister package in an amount sufficient to provide between about 0.1mg to about 4mg of the compound of formula I per dose. The contents of the blisters were administered using a dry powder inhaler.

Dry powder composition

Micron-sized compound of formula (I) (1g) was blended with milled lactose (20g) to form a host composition with a weight ratio of compound to milled lactose of 1: 20. The blend composition is packaged in a dry powder inhalation device capable of delivering between about 0.1mg to about 4mg of a compound of formula I per dose.

Metered dose inhaler composition

Micron-sized compound of formula (I) (10g) was dispersed in a solution prepared by dissolving lecithin (0.2g) in demineralized water (200 mL). The resulting suspension is spray dried and then micron sized to form a micron sized composition comprising particles having an average diameter of less than about 1.5 μm. The micron-sized composition is then loaded into a metered dose inhaler cartridge containing pressurized 1,1,1, 2-tetrafluoroethane in an amount sufficient to provide from about 0.1mg to about 4mg of the compound of formula I per dose when administered by the metered dose inhaler.

Nebulizer composition

The compound of formula (I) (25mg) was dissolved in a solution containing 1.5-2.5 equivalents of hydrochloric acid, followed by addition of sodium hydroxide to adjust the pH to 3.5 to 5.5 and 3 wt% glycerol. The solution was stirred well until all components were dissolved. The solution is administered using a nebulizer device that provides from about 0.1mg to about 4mg of the compound of formula (I) per dose.

Utility of

The JAK inhibitors of the invention have been designed to treat inflammatory and fibrotic diseases of the respiratory tract. In particular, the compounds are designed to enable the delivery of effective anti-interleukin agents directly to the site of respiratory disease action in the lung, while limiting systemic exposure.

The compounds of the invention are shown to be potent inhibitors of the JAK family of enzymes: JAK1, JAK2, JAK3 and TYK 2. In addition, the compounds have been shown to be effective in inhibiting proinflammatory and profibrotic interleukins. It is recognized that the broader anti-inflammatory action of JAK inhibitors may inhibit normal immune cell function, potentially leading to increased risk of infection. The present compounds are therefore optimized to limit absorption from the lung into the plasma, thus minimizing the risk of immunosuppression.

As described in the experimental section below, the absorption and distribution of typical compounds has been depicted in preclinical analysis. Compounds 1 to 6 were tested in mice and showed high concentrations in lung tissue and low absorption into plasma 5 hours after administration. The compounds of the invention are shown to inhibit the action of the proinflammatory interleukin IL-13 in mouse lung tissue. In particular, the compounds were shown to inhibit IL-13 induced phosphorylation of STAT6 in lung tissue, which provides evidence for the involvement of local lung JAK targets in vivo. This effect was observed when the proinflammatory interleukin IL-13 was administered 4 hours after the test compound, providing additional evidence of significant retention in the lung.

The tested compounds were demonstrated to exhibit two potent inhibitory activities in terms of cellular levels and significant retention in lung tissue. The present inventors extensively investigated the assay and, although it was possible to identify compounds that are effective at the cellular level or that exhibit significant retention in the lung, it was more difficult to find compounds that exhibit both of the desired characteristics.

The anti-inflammatory activity of JAK inhibitors has been firmly demonstrated in preclinical models of asthma (malavia et al, [ journal of international immunopharmacology ], [ 2010,10,829, -836 ], [ Matsunaga ] (Matsunaga) et al, communication of biochemical and biophysical studies (biochem. and biophysis. res. commun.) ], 2011,404, 261-267; kudack (Kudlacz) et al, journal of european pharmacology (eur.j. rmacol) ], 2008,582, 154-161). Interleukins involved in asthma inflammation that signal through the JAK-STAT pathway include: IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-11, IL-13, IL-23, IL-31, IL-27, Thymic Stromal Lymphopoietin (TSLP), interferon- γ (IFN γ), and granulocyte-macrophage colony stimulating factor (GM-CSF). Thus, the compounds of the present invention are expected to be useful in the treatment of inflammatory respiratory disorders, in particular asthma. Inflammation and fibrosis of the lung are characterized by other respiratory diseases besides asthma, such as Chronic Obstructive Pulmonary Disease (COPD), Cystic Fibrosis (CF), pneumonia, interstitial lung diseases (including idiopathic pulmonary fibrosis), acute lung injury, acute respiratory distress syndrome, bronchitis, emphysema, obstructive bronchiolitis, and sarcoidosis. Thus, the compounds of the present invention are also expected to be useful in the treatment of chronic obstructive pulmonary disease, cystic fibrosis, pneumonia, interstitial lung diseases (including idiopathic pulmonary fibrosis), acute lung injury, acute respiratory distress syndrome, bronchitis, emphysema, obstructive bronchiolitis, and sarcoidosis.

Compounds 1 to 6 were shown to have similar JAK activity when compared to the corresponding fluoro analogs (compounds C-1, C-2, C-3, C-4, C-5 and C-6). However, it has the advantage of causing a significant reduction in sulphated metabolism, as demonstrated in assay 5. This is important because sulfation metabolism occurs in the lung, which may cause a rapid reduction in exposure of the active parent compound.

The compounds of the present disclosure are shown to inhibit the cytokines associated with inflammation. Thus, the compounds of the present disclosure may be useful in the treatment of certain specific respiratory diseases, as described in detail below.

Eosinophilic airway inflammation is a characteristic feature of diseases collectively known as eosinophilic pulmonary disease (Cottin et al, "clinical thoracic medicine (clin. chest. med.), 2016,37(3), 535-56). Eosinophilic global disease is associated with IL-4, IL-13 and IL-5 signaling. Eosinophilic pneumoconiosis includes infection, particularly helminth infection, drug-induced pneumonia (e.g., induced by therapeutic drugs such as antibiotics, phenytoin (phenytoin) or l-tryptophan), fungal-induced pneumonia (e.g., allergic bronchopulmonary aspergillosis), allergic pneumonia, and eosinophilic granuloma with polyangiitis (previously known as Churg-Strauss syndrome). Eosinophilic pneumopathy of unknown etiology includes idiopathic acute eosinophilic pneumonia, idiopathic chronic eosinophilic pneumonia, eosinophilic leukocytosis syndrome and Lufferler syndrome: (syndrome)。

Polymorphisms in the IL-6 gene are associated with elevated IL-6 levels and increased risk of developing Pulmonary Arterial Hypertension (PAH) (Fang et al, J.Am.Soc.Hypertens., J.Am., 2017,11(3), 171-. Confirming the role of IL-6 in PAH, inhibition of IL-6 receptor chain gp130 ameliorated disease in rat models of PAH (Huang) et al, journal of cardiology, canada, 2016,32(11),1356.e1-1356.e 10).

Interleukins such as IFN γ, IL-12 and IL-6 have been implicated in a range of non-allergic lung diseases (e.g. sarcoidosis) and pulmonary lymphangioleiomyelitis (El-Hashimite et al, J.Respir.Cell.mol.biol.), (am.J.Respir.Cell.biol.), (2005, 33, 227-. The compounds of the invention are also shown to inhibit IFN γ signaling.

Bronchiectasis and infiltrative lung disease are diseases associated with chronic neutrophilic inflammation.

Pathological T cell activation is crucial for the etiology of various respiratory diseases. Autoreactive T cells play a role in obstructive bronchiolitis with organizing pneumonia (also known as COS). Similar to COS, the etiology of lung transplant rejection is associated with abnormal T cell activation of recipient T cells that transplant donor lungs. Lung transplant rejection can occur early in the form of Primary Graft Dysfunction (PGD), Organized Pneumonia (OP), Acute Rejection (AR) or Lymphococcal Bronchiolitis (LB), or it can occur years after lung transplantation in the form of Chronic Lung Allograft Dysfunction (CLAD). CLAD was previously known as obstructive Bronchiolitis (BO), but is now considered to have syndromes of different pathological manifestations, including BO, restrictive CLAD (rCLAD or RAS) and centrotropic allograft dysfunction. Chronic Lung Allograft Dysfunction (CLAD) is a major challenge in the long-term management of lung Transplant recipients because it causes the transplanted lung to gradually become dysfunctional (gothie et al, current Transplant representative, 2016,3(3), 185-. CLAD responds poorly to treatment and, therefore, there remains a need for effective compounds capable of preventing or treating this condition. Several JAK-dependent interleukins such as IFN γ and IL-5 are up-regulated in CLAD and lung transplant rejection (Berastegui et al, & 2017,31, e12898 in clinical transplants). In addition, high lung content of CXCR3 chemokines (e.g., CXCL9 and CXCL10) downstream of JAK-dependent IFN signaling is associated with worsening outcome in lung transplant patients (cinono et al, "complex of the public library (PLOS One), 2017,12(7), e 0180281). Systemic JAK inhibition was shown to be effective in kidney transplant rejection (weinett (Vicenti), et al, Journal of American Transplantation, 2012,12, 2446-56). Therefore, JAK inhibitors are likely to be effective in treating or preventing lung transplant rejection and CLAD. Similar T cell activation events as described as the basis for lung transplant rejection are also considered to be a major driver of lung Graft Versus Host Disease (GVHD) that may occur following hematopoietic stem cell transplantation. Similar to CLAD, lung GVHD is a chronic progressive condition with extremely poor outcomes and currently there is no approved treatment. Retrospective multicenter studies of patients with steroid refractory acute or chronic GVHD, 95 receiving the systemic JAK inhibitor, luzotinib, as a remedial treatment, demonstrated that the majority of patients, including those with lung GVHD, responded to luzotinib either completely or partially (Zeiser et al, Leukemia (leukamia), 2015,29,10, 2062-68). Since systemic JAK inhibition is associated with severe adverse events and a small therapeutic index, there remains a need for inhalation of pulmonary-directed, non-systemic JAK inhibitors to prevent and/or treat lung transplant rejection or lung GVHD. The compounds of the present disclosure have desirable characteristics to meet this need. In recent years, pneumonia induced by immune checkpoint inhibitors (another T cell mediated lung disease) has emerged with increased use of immune checkpoint inhibitors. In cancer patients treated with these T cell stimulators, fatal pneumonia may develop.

Thus, in one aspect, the invention provides a method of treating a respiratory disease in a mammal (e.g., a human), the method comprising administering to the mammal a therapeutically effective amount of a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of the invention, or a pharmaceutically acceptable salt thereof.

In one aspect, the respiratory disease is asthma, chronic obstructive pulmonary disease, cystic fibrosis, pneumonia, Chronic Obstructive Pulmonary Disease (COPD), Cystic Fibrosis (CF), pneumonia, interstitial lung diseases (including idiopathic pulmonary fibrosis), acute lung injury, acute respiratory distress syndrome, bronchitis, emphysema, obstructive bronchiolitis, or sarcoidosis. In another aspect, the respiratory disease is asthma or chronic obstructive pulmonary disease.

In one aspect, the respiratory disease is a pulmonary infection, an eosinophilic disease, a helminthic infection, pulmonary hypertension, sarcoidosis, pulmonary lymphangioleiomyomatosis, bronchiectasis, an infiltrative lung disease, a drug-induced pneumonia, a fungus-induced pneumonia, an allergic bronchopulmonary aspergillosis, an allergic pneumonia, eosinophilic granuloma associated with polyangiitis, idiopathic acute eosinophilic pneumonia, idiopathic chronic eosinophilic pneumonia, eosinophilic leukocytosis syndrome, Lufferler syndrome, obstructive bronchiolitis with organized pneumonia, acute and chronic lung transplant rejection (including PGD, OP, LB, AR and CLAD, BO, restrictive CLAD and neutrophilic allograft dysfunction), lung graft-versus-host disease, obstructive bronchiolitis with organized pneumonia, pulmonary hypertension, bronchiectasis or pneumonia induced by immune checkpoint inhibitors.

The present invention further provides a method of treating asthma in a mammal, the method comprising administering to the mammal a therapeutically effective amount of a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of the present disclosure, or a pharmaceutically acceptable salt thereof.

When used to treat asthma, the compounds of the present disclosure will generally be administered in a single daily dose or in multiple daily doses, although other forms of administration may be used. The amount of active agent administered per dose or the total amount administered per day will generally be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered and its relative activity, the age, weight and response of the individual patient, the severity of the patient's symptoms, and the like.

The present invention further provides a method of treating a respiratory disease (including, but not limited to, the diseases described herein) in a mammal, the method comprising administering to the mammal a therapeutically effective amount of a compound of the present invention, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of the present invention, or a pharmaceutically acceptable salt thereof.

When used to treat respiratory diseases, including (but not limited to) those described herein, the compounds of the present disclosure will typically be administered in a single daily dose or in multiple daily doses, although other administration forms may be used. The amount of active agent administered per dose or the total amount administered per day will generally be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered and its relative activity, the age, weight and response of the individual patient, the severity of the patient's symptoms, and the like.

As JAK inhibitors, the compounds of the present disclosure are also applicable to a variety of other diseases. The compounds of the present disclosure may be useful in a variety of gastrointestinal inflammatory disorders, including, but not limited to, inflammatory bowel disease, ulcerative colitis (rectosigmoiditis, pancolitis, ulcerative proctitis, and left-sided colitis), Crohn's disease, collagenous colitis, lymphocytic colitis, Behcet's disease, celiac disease, immune checkpoint inhibitor-induced colitis, ileitis, eosinophilic esophagitis, graft-versus-host disease-associated colitis, and infectious colitis. Ulcerative colitis (Remund et al, & J.Clin.immunology & gt, 1996,16,144 & lt 150 & gt), Crohn's disease (Woywood & lt 150 & gt), Eur, gastroenteropathy and liver disease (Eur.J.gastroenterology Hepatolology & gt), 1999,11,267 & lt 276 & gt), collagenous colitis (Kumawait) et al, & lt molecular immunology & gt, Immunology, 2013,55,355 & lt 364 & gt, Lymphatic colitis (Kumawait) et al, 2013), eosinophilic esophagitis (Wendland-gobi grid (Weinbrand-gobi-g) et al, & lt & gt, Immunol.Res., Res.56, 56 & lt 260 & gt, Wendland & lt 76 & gt, Crohn's disease of Crohn's disease (Mach's disease & lt 76 & gt, Blood transplant & lt 3276, Mach & lt 76 & gt, Mach's disease of infectious colitis (Mach & lt 76, Mach & gt, Mach & lt & gt, Mach & gt, Haemono. 25 & gt, Haemono. Shih & gt, Haemono. 25, Haemono. Shih & gt, Hazar &, 2004,19, 308-; (Yanye (Yano), et al, J.TRANSLATION. Med.), (2014, 12,191), PD-1 or PD-L1 inhibitor-induced colitis) and ileitis (Yamamoto, et al, digestion and liver disease (dig. liver Dis.), (2008, 40,253-259) are characterized by elevated levels of certain proinflammatory cytokines. Because many proinflammatory interleukins signal through JAK activation, the compounds described in this application may be able to reduce inflammation and provide symptomatic relief. In particular, the compounds of the present disclosure may be useful for inducing and maintaining remission of ulcerative colitis, and for treating gastrointestinal adverse effects in crohn's disease, immune checkpoint inhibitor-induced colitis, and graft versus host disease. Thus, in one aspect, the present disclosure provides a method of treating a gastrointestinal inflammatory disease in a mammal (e.g., a human), the method comprising administering to the mammal a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of the present disclosure, or a pharmaceutically acceptable salt thereof.

Atopic dermatitis and other inflammatory skin diseases are associated with elevated proinflammatory interleukins that depend on the JAK-STAT pathway. Thus, the compounds of the present disclosure, or pharmaceutically acceptable salts thereof, may be beneficial in a number of skin inflammatory or pruritic conditions, including, but not limited to, atopic dermatitis, alopecia areata, vitiligo, psoriasis, dermatomyositis, cutaneous T-Cell lymphoma (netchipouluk et al, Cell Cycle (Cell Cycle), 2014; 13,3331-, Angiocentric lymphoma, blastic NK cell lymphoma), prurigo nodularis, lichen planus, primary local skin amyloidosis, bullous pemphigoid, skin manifestations of graft versus host disease, pemphigoid, discoid lupus, granuloma annulare, chronic lichen simplex, vulval/scrotal/perianal pruritus, lichen sclerosus, post-herpetic neuralgia, lichen planus, and pilocystitis. In particular, atopic dermatitis (Bao et al, JAK-STAT,2013,2, e24137), alopecia areata (Xing) et al, & Nature medicine (Nat. Med.) 2014,20,1043-1049), leukoderma (Craiglow) et al, & JAMA skin disease (JAMA Dermatol.) 2015,151,1110-1112), prurigo nodularis (Sonkoly) et al, & Immunol & clinical immunology & Immunol & J.Immunol. Res. & It.2006, 117,411-417), lichen planus (Welz-Kubil. Immunol & Italy. & J.Immunol. Res. & Italy. & gt 854747, J.Immunol. Immunol & Italy. & gt & Immunol & gt & Italy & gt & Immunol & gt, 12,55-61) and the cutaneous manifestations of graft-versus-host disease (Okiyama et al, J.invest.Dermatol.) -2014, 134, 992-. Thus, the compounds of the present disclosure, or pharmaceutically acceptable salts thereof, may be capable of reducing the associated dermatitis or pruritus driven by these interleukins. In particular, the compounds of the present disclosure, or pharmaceutically acceptable salts thereof, are expected to be useful in the treatment of atopic dermatitis and other inflammatory skin diseases. Accordingly, in one aspect, the present disclosure provides a method of treating an inflammatory skin disease in a mammal (e.g., a human), the method comprising applying to the skin of the mammal a pharmaceutical composition comprising a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, and a pharmaceutical carrier. In one aspect, the inflammatory skin disease is atopic dermatitis.

Many ocular diseases have been shown to be associated with elevated proinflammatory interleukins that depend on the JAK-STAT pathway. Accordingly, the compounds of the present disclosure, or pharmaceutically acceptable salts thereof, may be useful in the treatment of a number of ocular diseases including, but not limited to, uveitis, diabetic retinopathy, diabetic macular edema, dry eye, age-related macular degeneration, and atopic keratoconjunctivitis. In particular, uveitis (cavernous wells (Horai) and Casti leather (Caspi), "J.Interferon Cytokine Res.)," 2011,31,733-, diabetic macular edema (Sohn et al, American Journal of ophthalmology (2011, 152, 686) 694), xerophthalmia (Stevenson et al, ophthalmoll literature (arch), 2012,130,90-100), and age-related macular degeneration (Knickelbein), et al, international ophthalmic clinic (int. ophthalmol. clin), 2015,55(3),63-78) are characterized by elevations of certain proinflammatory interleukins that transmit signals through the JAK-STAT pathway. Thus, a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, may be capable of reducing the associated ocular inflammation and reversing disease progression or providing symptomatic relief. Accordingly, in one aspect, the present disclosure provides a method of treating an ocular disease in a mammal, the method comprising administering to the eye of the mammal a pharmaceutical composition comprising a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, and a pharmaceutical carrier. In one aspect, the ocular disease is uveitis, diabetic retinopathy, diabetic macular edema, dry eye, age-related macular degeneration, or atopic keratoconjunctivitis. In one aspect, the method comprises administering a compound of the disclosure, or a pharmaceutically acceptable salt thereof, by intravitreal injection. The compounds of the present disclosure, or pharmaceutically acceptable salts thereof, may also be used in combination with one or more compounds suitable for ocular diseases.

The compounds of the present disclosure, or pharmaceutically acceptable salts thereof, may also be useful in the treatment of other diseases, such as other inflammatory diseases, autoimmune diseases, or cancer. A compound of the present disclosure, or a pharmaceutically acceptable salt thereof, may be used to treat one or more of the following: arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, transplant rejection, dry eye, psoriatic arthritis, diabetes, insulin-dependent diabetes mellitus, motor neuron disease, myelodysplastic syndrome, pain, sarcopenia, cachexia, septic shock, systemic lupus erythematosus, leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia, acute myelogenous leukemia, ankylosing spondylitis, myelofibrosis, B-cell lymphoma, hepatocellular carcinoma, Hodgkin's disease (Hodgkins disease), breast cancer, multiple myeloma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, clear cell carcinoma of the ovary, ovarian tumor, pancreatic tumor, polycythemia vera, Sjoegrens syndrome (Sjoegrens syndrome), soft tissue sarcoma, and bone marrow tumor, Sarcoma, splenomegaly, T cell lymphoma, and thalassemia major.

Combination therapy

A compound of the present disclosure or a pharmaceutically acceptable salt thereof may be used in combination with one or more agents that act by the same mechanism or different mechanisms to treat a disease. The different agents may be administered sequentially or simultaneously in separate compositions or in the same composition. Suitable classes of agents for combination therapy include (but are not limited to): beta 2 adrenoceptor agonists, muscarinic receptor antagonists, glucocorticoid agonists, G protein-coupled receptor-44 antagonists, leukotriene D4 antagonists, muscarinic M3 receptor antagonists, histamine H1 receptor antagonists, immunoglobulin E antagonists, PDE 4 inhibitors, IL-4 antagonists, muscarinic M1 receptor antagonists, histamine receptor antagonists, IL-13 antagonists, IL-5 antagonists, 5-lipoxygenase inhibitors, beta adrenoceptor agonists, CCR3 chemokine antagonists, CFTR stimulators, immunoglobulin modulators, interleukin 33 ligand inhibitors, PDE 3 inhibitors, phosphoinositide-3 kinase delta inhibitors, thromboxane A2 antagonists, elastase inhibitors, Kit tyrosine kinase inhibitors, leukotriene E4 antagonists, leukotriene antagonists, PGD2 antagonists, TNF α ligand inhibitors, TNF binders, complement cascade inhibitors, inhibitors of the eosin-taxin ligand, glutathione reductase inhibitors, histamine H4 receptor antagonists, IL-6 antagonists, IL2 gene stimulators, immunoglobulin gamma Fc receptor IIB modulators, interferon gamma ligands, interleukin 13 ligand inhibitors, interleukin 17 ligand inhibitors, L-selectin antagonists, leukocyte elastase inhibitors, leukotriene C4 antagonists, leukotriene C4 synthase inhibitors, membrane cuprammine oxidase inhibitors, metalloproteinase-12 inhibitors, metalloproteinase-9 inhibitors, mite allergen modulators, muscarinic receptor modulators, nicotinic acid acetylcholine receptor agonists, nuclear factor kappa B inhibitors, p-selectin antagonists, PDE 5 inhibitors, PDGF receptor antagonists, Phosphoinositide-3 kinase gamma inhibitors, TLR-7 agonists, TNF antagonists, Abl tyrosine kinase inhibitors, acetylcholine receptor antagonists, acid mammalian chitinase inhibitors, ACTH receptor agonists, actin polymerization modulators, adenosine A1 receptor antagonists, adenylate cyclase stimulators, adrenoreceptor antagonists, corticotropin ligands, alcohol dehydrogenase 5 inhibitors, alpha 1 antitrypsin stimulators, alpha 1 protease inhibitors, androgen receptor modulators, vasoconstriction convertase 2 stimulators, ANP agonists, Bcr protein inhibitors, beta 1 adrenoreceptor antagonists, beta 2 adrenoreceptor modulators, beta amyloid modulators, BMP10 gene inhibitors, BMP15 gene inhibitors, calcium channel inhibitors, cathepsin G inhibitors, CCL26 gene inhibitors, CCR3 chemokine modulators, CCR4 chemokine antagonists, cell adhesion molecule inhibitors, chaperone stimulators, chitinase inhibitors, collagen I antagonists, complement C3 inhibitors, CSF-1 antagonists, CXCR2 chemokine antagonists, interleukin receptor-sharing beta chain modulators, cytotoxic T-lymphoglobulin-4 stimulators, DNAse I stimulators, DNAse stimulators, dipeptidyl peptidase I inhibitors, DNA gyrase inhibitors, DP prostaglandin receptor modulators, E-selectin antagonists, EGFR family tyrosine kinase receptor inhibitors, elastin modulators, endothelin ET-A antagonists, endothelin ET-B antagonists, epoxide hydrolase inhibitors, FGF3 receptor antagonists, Fyn tyrosine kinase inhibitors, GATA 3 transcription factor inhibitors, Glucosceramidase modulators, glutamate receptor modulators, GM-CSF ligand inhibitors, guanylate cyclase stimulators, H + K + ATPase inhibitors, heme modulators, heparin agonists, histone deacetylase inhibitors, histone deacetylase-2 stimulators, HMG CoA reductase inhibitors, I- κ B kinase β inhibitors, ICAM1 gene inhibitors, IL-17 antagonists, IL-17 receptor modulators, IL-23 antagonists, IL-4 receptor modulators, immunoglobulin G1 agonists, immunoglobulin G1 modulators, immunoglobulin ε Fc receptor IA antagonists, immunoglobulin γ Fc receptor IIB antagonists, immunoglobulin κ modulators, insulin sensitizers, interferon β ligands, interleukin 1-like receptor antagonists, interleukin 18 ligand inhibitors, An interleukin receptor 17A antagonist, an interleukin-1 beta ligand inhibitor, an interleukin-5 ligand inhibitor, an interleukin-6 ligand inhibitor, a KCNA voltage-gated potassium channel-3 inhibitor, a Kit ligand inhibitor, a laminin-5 agonist, a leukotriene CysLT1 receptor antagonist, a leukotriene CysLT2 receptor antagonist, a LOXL2 gene inhibitor, a Lyn tyrosine kinase inhibitor, a MARCKS protein inhibitor, an MDR-related protein 4 inhibitor, a metalloproteinase-2 modulator, a metalloproteinase-9 modulator, a mineralocorticoid receptor antagonist, a muscarinic M2 receptor antagonist, a muscarinic M4 receptor antagonist, a muscarinic M5 receptor antagonist, a natriuretic peptide receptor A agonist, a natural killer cell receptor modulator, a nicotinic ACh receptor alpha 7 subunit stimulator, an NK cell receptor modulator, a cytokine antagonist, a cytokine such as a cytokine antagonist, a human interferon-1 beta ligand inhibitor, a human factor receptor antagonist, a human factor, Nuclear factor kappa B modulators, opioid growth factor receptor agonists, P-glycoprotein inhibitors, P2X3 purine receptor antagonists, P38 MAP kinase inhibitors, peptidase 1 modulators, phospholipase a2 inhibitors, phospholipase C inhibitors, plasminogen activator inhibitor 1 inhibitors, platelet activating factor receptor antagonists, PPAR γ agonists, prostacyclin agonists, protein tyrosine kinase inhibitors, SH2 domain inositol phosphatase 1 stimulators, signal transduction inhibitors, sodium channel inhibitors, STAT-3 modulators, stem cell antigen-1 inhibitors, superoxide dismutase modulators, T cell surface glycoprotein CD28 inhibitors, T cell surface glycoprotein CD8 inhibitors, TGF β agonists, TGF β antagonists, thromboxane synthase inhibitors, thymic matrix lymphokine ligand inhibitors, thymosin agonists, Thymosin beta 4 ligands, TLR-8 agonists, TLR-9 agonists, TLR9 gene stimulators, topoisomerase IV inhibitors, troponin I fast skeletal muscle stimulators, troponin T fast skeletal muscle stimulators, type I IL-1 receptor antagonists, type II TNF receptor modulators, ion channel modulators, uteroglobin stimulators, and VIP agonists.

Specific agents that may be used in combination with the present JAK inhibitor compounds include (but are not limited to): rosemamectin acetate (rosiptor acetate), umeclidinium bromide (umeclidinium bromide), secukinumab (secukinumab), mithradine acetate (metekefalin acetate), terfenac acetate (tridececatide acetate), fluticasone propionate (fluticasone propionate), alpha-cyclodextrin stabilized sulforaphane, tezephalumab (tezephalumab), mometasone furoate (mometasone furoate), BI-1467335, doluzumab (dupilumab), aclidinium (aclidinium), formoterol (formoterol), AZD-1419, HI-1640V, rivastigmine (rivansel), CMP-001, mannitol, ANB-020, omalizumab (omalizumab), tremelimumab (force application), mitralizumab (mitrializumab), mitrazurix (mitakazacil), mithramycin (3500), troglib-554, troglib (rpjirimycin), troglib-554, troglitinib (rpjikurtib), troglizumab (rpjikurtib), mithradine acetate (tricitabine acetate), mithradine acetate (metilin acetate (metiramate), mithradine acetate (rpjikum acetate), mithradine acetate (rpb (rpjikutib), mithramide (rpb), mithramide (rppib) and mithramide (rppib) as-554) Achrome (Actimmune), adalimumab (adalimumab), rupatadine (rupatadine), Paroglelil (parogrelil), MK-1029, beclomethasone dipropionate (beclometasone diproprionate), formoterol fumarate (formoterol fumarate), mogrolizumab (mogamulizumab), seratrodast (seratrodast), UCB-4144, nanoxib (nemalisib), CK-2127107, novilant (fevipiprant), danirixin (danixin), bosentan (bosentan), arbasicept (abatacept), EC-18, duvexib (duvistate), doxyprolimus (docaprepint), ciprofloxacin (ciprofloxacin), probucol (HFA), domethamine (HFA), dolastacin (HFR), furazolidone (CDR), tprex (berromazine), TPR (mogroside), TPR (triglucrolimus), TPR (berroside), TPR (glibenralin (berroside), TPR (ber, Fluticasone propionate, salmeterol (salmeterol), PT-007, PRS-060, Remidt Meile-L (remestemcel-L), citrulline, RPC-4046, nitric oxide, DS-102, Geilimizumab (gerilimizumab), Actarat (Actair), Fluticasone furoate, umeclidinium (umeclidinium), Willandiole (vilaterol), AG-NPP709, Cammins (Gamunex), Inexiximab (infliximab), Amipin (Ampion), Arkumamimod (Acumaimod), Cannabumemamod (Canavamod), Canavamab (Canavamab), INS-1007, CYP-001, Sirukumab (Sirukulimumab), Fluticasone propionate, Melitumomab (merlizumab), Pipitan, Soxhlinomycin (sorethrin), Poncirus (MPC), Aceramicin (beramide), Icarinicamid-300, Iridamycin IV), Iridamycin (berella), Flutica (berella IV), Flutica (berella), Actarativudine), Actarlandium (Acinetoramin, Aclina (Aclina, Aclinagliomab), Aclinagliomab (Aclinagliomab), Aclinagliomab (Aclinagli, FP-025, Rinsengumab (risankizumab), glycopyrronium (glycopyrronium), formoterol fumarate, Addisopressin (Adipocell), YPL-001, tiotropium bromide (tiotropium bromide), glycopyrronium bromide, indacaterol maleate (indaterol maleate), Andeximab (andecaiximab), ondterol (olodaterol), esomeprazole (esomeprazole), dust mite vaccine, Artemisia pollen allergen vaccine, Vamololone (Vamorolone), Gefaxax (gefapixant), resifanazine (revafenac), gefitinib (gefitinib), resilin (Rejouin), Tilauster (tiphaskast), Bedoramine (bedoradine), SCM-1800, SHP-652, RNS-60, R-60, Rumalbradine (Rejodine), VXylotab-561, VXylazine (VXylotab-R-561), ipratropium bromide (VXylotab-371), hydramine (VXylotab-371), VX-R-III, VX-A-III, Ipomona (valprozin-R-III), VX-III, Xylotab, VX-R-III, VX-III, R-III, Tobuterol (tulobutenol), formoterol fumarate, triamcinolone acetonide (triamcinolone acetonide), Raylelizumab (resizumab), salmeterol xinafoate (salmeterol xinafoate), fluticasone propionate, beclomethasone dipropionate, formoterol fumarate, tiotropium bromide, lidelizumab (ligelizumab), RUTI, batelimumab (bertilimumab), omalizumab, glycopyrrolate, SENS-111, beclomethasone dipropionate, CHF-5992, LT-4001, indacaterol (indacaterol), canobromide, mometasone furoate, fexofenadine (fexofenadine), glycopyrrolate, azithromycin (AZazythromycin), AZD-7594, formoterol, CHF-6001, tefeletrol (tefentefelol), seiflodine (TD-01, beta-112), olazine (AZAPIREPIROMETIN), SALT-71, SALT-D (AZE), SALT-D, CIT-D (IRE-D), CIT-6001, CIT-E (SALT-D, CIT-D (SALT-D, CIT-D-E (SALT-D, CIT-D, Fluticasone, salmeterol, eicosapentaenoic acid monoglyceride (eicosapentaenoic acid monogylceride), rebeccrine (lebrikizumab), RG-6149, QBKPN, Mometasone (Mometasone), indacaterol, AZD-9898, sodium pyruvate, zileuton (zileuton), CG-201, imidafenacin (imidafenacin), CNTO-6785, CLBS-03, Mometasone, RGN-137, procaterol (procaterol), formoterol, CCI-15106, POL-6014, indacaterol, beclomethasone (beclomethasone), MV-130, GC-1112, Resogluca (allegroc depot), MEDI-3506, QBW-251, ZPL-389, udenafil (udenafacil), GSK-3772847, levocetirizine (axilizine), azurite-94, Azaditopine-3680, Azadipamoate (Aceramide), Azadipinol-D-9880, Azadipinol-1275, Azadipinol (azurite-D-12780), propineb (zizanide), Cetrolene, Clinolide (benzalkonium, Clinolide), Cleotide (Clinolide), Clinolide-D-, Salbutamol sulfate (salbutamol sulfate), tadecanib (tadekingalfa), ACT-774312, deoxyribonuclease a, iloprost (iloprost), benifetrobol (batefentol), fluticasone furoate, Aliskiren (alicafensen), ciclesonide (ciclesonide), emery amine (emeramid), arformoterol (arformotel), SB-010, Ozagrel (Ozagrel), BTT-1023, daclizumab (Dectrekumab), levalbuterol (levalbuterol), Pravalukast (pranlukast), hyaluronic acid, GSK-2292767, formoterol, NOV-14, Lucinatan (Lucinactant), salbutamol, prednisone (prednisolone), ebastine (dexamethasone), dexamethasone (VTK-539), dexamethasone (ASV-2245035, GSK-3667179, GSK-2245035, GSH-363682, GSK-3636363682, GSH-2245035, GSK-3695, dexamethasone, and dexamethasone, Emedastine (Emedastine), dexpramipexole (dexpramipexole), levalbuterol, N-6022, dexamethasone sodium phosphate (dexamethasone sodium phosphate), PIN-201104, OPK-0018, TEV-48107, sertraline (suplatast), BI-1060469, gemiukast (Gemilukast), interferon gamma, dalazatide (dalazatide), bilastine (bilastine), fluticasone propionate, salmeterol xinafoate, RP-3128, benconolaquine bromide (benconloquidium bromide), Raylelizumab, PBF-680, CRTH2 antagonist, prasterone, salmeterol xinafoate, fluticasone propionate, tiotropium bromide monohydrate (trongylolimumab), Marukalukast (Arthrokast), theophylline 7990, doxolone (RG), doxolone propionate, 02417, bromate, salmeterol propionate, salmeterol, Salbutamol, TA-270, Flunisolide (Flunisolide), cromolyn sodium, Epsi-gam, ZPL-521, salbutamol, aviptadil (aviptadil), TRN-157, Zafirlukast (Zafirlukast), Spdiaplasl (Stempelucel), Perrolase sodium (pemirolast sodium), nadolol (nadolol), fluticasone propionate + salmeterol xinafoate, RV-1729, salbutamol sulphate, carbon dioxide + perfluorooctylbromide, APL-1, Dekemab + VAK-694, lysine acetyl salicylate (lysine acetylsalicylate), zileuton, TR-4, human allogeneic fat progenitor derived mesenchymal cell therapy, MEDI-9314, PL-3994, HMP-301, TD-5471, NKTT-120, pimentol (pimentol), alpha-salmonel (alpha-acetate), alpha-butol, alpha-salmonel, alpha-salmonel, IMD-1041, AM-211, TBS-5, ARRY-502, seratrodast, recombinant Maydimes (recombinant midismase), ASM-8, deflazacort (deflazacort), bambuterol (bambuterol), RBx-10017609, ipratropium (ipratropium) + fenoterol (fenoterol), fluticasone + formoterol, epinastine (epinastine), WIN-901X, VALERGEN-DS, OligoG-COPD-5/20, tobuterol, oxepan (oxidas Turbuhaler), DSP-3025, ASM-024, mizolastine (mizolastine), budesonide + salmeterol, AXP-011, histamin human immunoglobulin, YHD-001, base (theophylline), bromamine (ambroxol, triptan) + -502, montelukast (amantalin), montelukast AG + sarkokast, amantadine (AG + 24), montelukast (formoterol + albuterol), montelukast + 1321001, montelukast (isoxatilin), montelukast + g-24, montelukast, montelukas, Tranilast (tranilast), methylprednisolone suleptate (methylprednisolone sultanate), colestipol (colforsin daropate), repirast (repirinast) and doxofylline.

Also provided herein is a pharmaceutical composition comprising a compound of the disclosure, or a pharmaceutically acceptable salt thereof, and one or more other therapeutic agents. The therapeutic agent may be selected from the classes of agents specified above and the list of specific agents described above. In some embodiments, the pharmaceutical composition is suitable for delivery to the lung. In some embodiments, the pharmaceutical composition is suitable for inhalation or spray administration. In some embodiments, the pharmaceutical composition is a dry powder or a liquid composition.

In addition, in a method aspect, the present disclosure provides a method of treating a disease or disorder in a mammal comprising administering to the mammal a compound of the present disclosure, or a pharmaceutically acceptable salt thereof, and one or more other therapeutic agents.

When used in combination therapy, the agents may be formulated as a single pharmaceutical composition, or the agents may be provided in separate compositions for administration by the same or different routes of administration, either simultaneously or at different times. These compositions may be packaged separately or may be packaged together as a kit. The two or more therapeutic agents in the kit can be administered by the same route of administration or by different routes of administration.

Examples of the invention

The following synthetic and biological examples are provided to illustrate the invention and are not to be construed in any way as limiting the scope of the invention. In the examples below, the following abbreviations have the following meanings, unless otherwise indicated. Abbreviations not defined below have their generally acceptable meaning.

ACN ═ acetonitrile

DCM ═ dichloromethane

DIPEA ═ N, N-diisopropylethylamine

DMF ═ N, N-dimethylformamide

EtOAc ═ ethyl acetate

h is hour

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

IPA (isopropyl alcohol)

IPAc ═ isopropyl acetate

MeOH ═ methanol

min is minutes

Pd(PPh₃)₄Tetra (triphenylphosphine) palladium (0)

RT ═ room temperature

TFA ═ trifluoroacetic acid

THF ═ tetrahydrofuran

Bis (pinacolato) diboron ═ 4,4,5,5,4',4',5',5' -octamethyl- [2,2' ] bis [ [1,3,2] dioxaborolane ]

Reagents and solvents were purchased from commercial suppliers (Aldrich, Fluka, Sigma, etc.) and used without further purification. The progress of the reaction mixture was monitored by Thin Layer Chromatography (TLC), analytical high performance liquid chromatography (analytical high performance liquid chromatography; and HPLC), and mass spectrometry. The reaction mixture was treated as specifically described in each reaction; the reaction mixture is usually purified by extraction and other purification methods, such as temperature-dependent and solvent-dependent crystallization and precipitation. Furthermore, the reaction mixture is conventionally purified by column chromatography or by preparative HPLC, usually using C18 or BDS column packing and customary eluents. Typical preparative HPLC conditions are described below.

By mass spectrometry and¹H-NMR spectroscopy is routinely performed for the characterization of the reaction product. For NMR analysis, the sample is dissolved in a deuterated solvent (e.g., CD)₃OD、CDCl₃Or d₆-DMSO) and obtained with a Varian Gemini2000 instrument (400MHz) under standard observation conditions¹H-NMR spectrum. Mass spectral identification of compounds was performed by electrospray ionization method (ESMS) using an Applied Biosystems (Foster City, CA) model API150EX instrument or Waters (Milford, MA)3100 instrument coupled to an automated purification system.

Preparative HPLC conditions

Pipe column: c18,5 μm.21.2X 150mm or C18,5 μm 21X 250 or

C14,5μm 21×150mm

Temperature of the pipe column: at room temperature

Flow rate: 20.0mL/min

Mobile phase: a ═ water + 0.05% TFA

B＝ACN+0.05％TFA，

Injection volume: (100-

Detector wavelength: 214nm

The crude compound was dissolved at about 50mg/mL in 1:1 water acetic acid. The 4 minute analytical scale testing run was performed using a 2.1X 50mm C18 column followed by 15 or 20 minute preparative scale runs using 100 μ L of injection with% B retention based on the analytical scale testing run. The exact gradient depends on the sample. Samples with tight handling impurities were examined with a 21 x 250mm C18 column and/or a 21 x 150mm C14 column for optimal separation. Fractions containing the desired product were identified by mass spectrometry.

Preparation 1: 4- (Phenylmethoxy) -2-ethylphenyl) trifluoro-lambda⁴Borane, potassium salt I-5

(a)1- (Phenylmethoxy) -3-ethylbenzene (I-2)

To a stirred solution of 3-ethylphenol (I-1) (25.0g, 204.0mmol) in ACN (250mL, 10 volumes) was added potassium carbonate (42.0g, 306mmol) at room temperature. The resulting reaction mass was stirred at room temperature for 15 minutes, followed by the addition of bromotoluene (24.0mL, 204mmol) in a dropwise manner. The resulting reaction mixture was stirred at room temperature for 6 hours. After completion of the reaction (TLC monitoring), the resulting reaction cake was poured into water (1.0L) followed by extraction of the compound with EtOAc (2 × 2L). The combined organics were washed with cold water, brine solution and dried over sodium sulfate, filtered and evaporated under reduced pressure. The crude product was then purified by column chromatography on silica gel (100-.¹H NMR (400MHz, chloroform-d) δ 7.46-7.44(m,2H),7.39(t, J ═ 7.6Hz,2H),7.34-7.31(m,1H),7.21(t, J ═ 7.6Hz),6.86-6.80(m,3H),5.07(s,2H),2.64(q, J ═ 7.6Hz,2H),1.24(t, J ═ 7.6Hz, 3H).

(b)4- (Phenylmethoxy) -1-bromo-2-ethylbenzene (I-3)

To an ice-cooled stirred solution of 1- (benzyloxy) -3-ethylbenzene (I-2) (35.0g, 164mmol) in ACN (525mL, 15 volumes) was added N-bromosuccinimide (32.0g, 181mmol) portionwise over a period of 15 minutes. The resulting reaction mixture was stirred at room temperature for another 1 hour. After completion of the reaction (TLC monitoring), the resulting reaction cake was poured into ice-cold water (1.50L) followed by extraction of the compound with EtOAc (2 × 1L). The combined organics were washed with water and dried over sodium sulfate, filtered and evaporated under reduced pressure to give the crude product. N-hexane (250mL) was added to the crude material, resulting in a mill slurry, which was then filtered through a sintered funnel. The mother liquor was evaporated under reduced pressure to obtain the desired product I-3(42.0g, 87%) as a pale yellow oily compound.¹H NMR (400MHz, chloroform-d) δ 7.52-7.29(m,7H),6.88(s,1H),6.68(d, J ═ 6.0Hz,1H),5.04(s,2H),2.69(q, J ═ 7.6Hz,2H),1.20(t, J ═ 7.5Hz, 3H).

(c)2- (4- (benzyloxy) -2-ethylphenyl) -4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolane (I-4)

A stirred solution of 4- (benzyloxy) -1-bromo-2-ethylbenzene (I-3) (42.0g, 144mmol), bis (pinacolato) diboron (44.0g, 173mmol) and potassium acetate (28g, 288mmol) in dioxane (440mL) was degassed by purging N2(g) for 15min, followed by addition of PdCl2(dppf). DCM complex (11.0g, 15 mmol). The resulting reaction mixture was heated to 80 ℃ for an additional 16 h. After completion of the reaction (TLC monitoring), the reaction cake was filtered through celite bed and the mother liquor was evaporated under reduced pressure to obtain crude product. The crude residue was purified by column chromatography on silica gel (100-200M) using an eluent of 1% EtOAc/hexane to give the desired product (I-4) (32.0g, 66%) as a pale yellow oily compound.¹H NMR (400MHz, chloroform-d) δ 7.74(d, J ═ 8.4Hz,1H),7.45-7.36(m,5H),6.84-6.78(m,2H),5.08(s,2H),2.91(q, J ═ 7.6Hz),1.33(s,12H),1.19(t, J ═ 7.6Hz, 3H).

(d) (4- (Phenylmethoxy) -2-ethylphenyl) trifluoro-lambda⁴Borane, Potassium salt (I-5)

To a stirred solution of the compound 2- (4- (benzyloxy) -2-ethylphenyl) -4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolane (I-4) (20g, 59.0mmol) in acetone: methanol (200mL, 1:1 ratio, 10 volumes) was added a 3M potassium hydrogen fluoride solution (23.0g, 295mmol, dissolved in 98.0mL water). The resulting reaction mixture was stirred at room temperature for 16 hours. After completion of the reaction (TLC monitoring), the resulting reaction cake was evaporated under reduced pressure. The solid thus obtained was dissolved in water (100mL) and stirred at room temperature for 30 min. The resulting reaction cake was filtered through a sintered funnel, washed with n-hexane and dried under reduced pressure to give the desired product (I-5) (14.0g, 74%) as a white solid.¹H NMR (400MHz, chloroform-d) δ 7.43(d, J ═ 7.2Hz,2H),7.37(t, J ═ 7.5Hz,2H),7.30(t, J ═ 7.1Hz,1H),7.22(d, J ═ 8.0Hz),6.58(s,1H),6.53(d, J ═ 7.9Hz,1H),5.00(s,2H),2.65(q, J ═ 7.5Hz,2H),1.07(t, J ═ 7.4Hz, 3H).

Preparation 2: (S) -3-benzyl-3, 4,6, 7-tetrahydro-5H-imidazo [4,5-c ] pyridine-5, 6-dicarboxylic acid 6-benzyl ester 5- (tert-butyl ester) (I-11)

(a) (S) -4,5,6, 7-tetrahydro-3H-imidazo [4,5-c ] pyridine-6-carboxylic acid, hydrochloride salt (I-7)

To an ice-cold stirred suspension of L-histidine (I-6) (5.0kg, 32.14mol) in water (40L, 8 vol) was added concentrated hydrochloric acid (3.93L, 33.75mol), followed by the addition of formaldehyde (5.50L, 67.5mol, 37% aqueous solution) in a dropwise manner. The resulting solution was stirred at the same temperature for 30 minutes and then heated at 80 ℃ for 8 hours. Reaction progress was monitored by LCMS. Water was removed under reduced pressure to obtain a crude product, and the resulting crude material was stirred in toluene (20L) for 2 hours. The organics were removed under reduced pressure to remove excess water and the compound azeotropically dried. The resulting material was then taken up in diethyl ether (20L) and stirred for 2 hours. The solid material was then filtered and air dried to obtain a white solidDesired product (I-7) (6.50Kg, 85%).¹H NMR(400MHz,D2O)δ8.69(s,1H),4.56(d,J＝15.4Hz,1H),4.42(d,J＝15.5Hz,1H),4.20(dd,J＝5.5,5.2Hz,1H),3.42(dd,J＝5.0,17.0Hz,1H),3.11(dd,J＝10.2,16.8Hz,1H)。

(b) (S) -3, 5-bis (tert-butoxycarbonyl) -4,5,6, 7-tetrahydro-3H-imidazo [4,5-c ] pyridine-6-carboxylic acid (I-8)

To (S) -4,5,6, 7-tetrahydro-3H-imidazo [4,5-c]Pyridine-6-carboxylic acid di-hydrochloride (I-7) (6.10Kg, 25.40mol) was added dropwise to an ice-cold stirred solution of 1, 4-dioxane (48L, 8 vol) and water (48L, 8 vol) triethylamine (12.36L, 89mol), followed by the addition of di-tert-butyl dicarbonate (18.07L, 78.74mol, dissolved in 5L of 1, 4-dioxane) over a period of 30 min. The resulting reaction mixture was stirred at room temperature for a further 16 hours. After completion of the reaction (TLC)&LCMS monitoring), the yellowish reaction mixture was diluted with water (10L) and washed successively with diethyl ether (2 × 10L) and EtOAc (2 × 7.50L). The organic phase was discarded. The aqueous layer was cooled and brought to pH about 3 with 6N HCl solution; the aqueous phase was extracted with EtOAc (3X 10L). The combined organics were washed with brine solution, dried over sodium sulfate, and concentrated under reduced pressure. The oily residue was crystallized from 30% EtOAc in hexanes to give the desired product (I-8) as an off-white solid (5.1Kg, 55%). (m/z): c₁₇H₂₅N₃O₆Of [ M + H]+ calculated 368.18, experimental 368.21.

(c) (S) -6, 7-dihydro-3H-imidazo [4,5-c ] pyridine-3, 5,6(4H) -tricarboxylic acid 6-benzyl ester 3, 5-di-tert-butyl ester (I-9)

To (S) -3, 5-bis (tert-butoxycarbonyl) -4,5,6, 7-tetrahydro-3H-imidazo [4, 5-c)]Pyridine-6-carboxylic acid (I-8) (5.1Kg, 13.88mol) in an ice-cold solution of DCM (51L, 10 vol) was added in sequence saturated aqueous sodium bicarbonate (41.0L, 8 vol), tetrabutylammonium iodide (5.13Kg, 13.88mol) and bromotoluene (2.47L, 20.82 mol). The resulting reaction mixture was stirred at room temperature for a further 16 hours. After completion of the reaction (TLC)&LCMS monitoring), the biphasic solution was separated. The aqueous layer was extracted with DCM (3X 10L). The combined organics were washed with brine solution, dried over sodium sulfate, filtered and concentrated under reduced pressure to obtain the crude product which was passed through a silica gel (100-Column chromatography, purified by using eluent 40% EtOAc in hexanes to give the desired product (I-9) as a viscous oil (4.50Kg, 72%). (m/z): c₂₄H₃₁N₃O₆Of [ M + H]+ calculated 458.22, experimental 458.60.

(d) (S) -3,4,6, 7-tetrahydro-5H-imidazo [4,5-c ] pyridine-5, 6-dicarboxylic acid 6-benzyl ester 5- (tert-butyl ester) (I-10)

To (S) -6, 7-dihydro-3H-imidazo [4, 5-c)]Pyridine-3, 5,6(4H) -tricarboxylic acid 6-benzyl ester 3, 5-di-tert-butyl ester (I-9) (4.50Kg, 9.84mol) to an ice-cold solution of IPA (45L, 10 vol) was added ammonium hydroxide (36L, 8 vol) dropwise. The resulting reaction mixture was further stirred at room temperature for 16 hours. After completion of the reaction (TLC)&LCMS monitoring), the resulting mixture was diluted with water (25L) followed by extraction with EtOAc (3 × 20L). The combined organics were washed with brine solution, dried over sodium sulfate, filtered and concentrated under reduced pressure to give the crude product which was purified by column chromatography on silica gel (100-. (m/z): c₁₉H₂₃N₃O₄Of [ M + H]+ calculated 358.17, experimental 358.33.

(e) (S) -3-benzyl-3, 4,6, 7-tetrahydro-5H-imidazo [4,5-c ] pyridine-5, 6-dicarboxylic acid 6-benzyl ester 5- (tert-butyl ester) (I-11)

To (S) -3,4,6, 7-tetrahydro-5H-imidazo [4,5-c]6-benzyl pyridine-5, 6-dicarboxylate 5- (tert-butyl ester) (I-10) (2.70Kg, 7.55mol) in DCM (32.4L, 12 vol) was added with 1N aqueous sodium hydroxide (24.3L, 9 vol), followed by tetrabutylammonium iodide (2.80Kg, 7.55mol) and bromotoluene (0.99L, 8.31mol) in that order. The resulting reaction mixture was stirred at room temperature for an additional 2 hours. After completion of the reaction (TLC)&LCMS monitoring), the biphasic solution was separated. The aqueous layer was extracted with DCM (3X 10L). The combined organics were washed with brine solution, dried over sodium sulfate, filtered and concentrated under reduced pressure to give the crude product which was purified by column chromatography on silica gel (100-. (m/z))：C₂₆H₂₉N₃O₄Of [ M + H]+ calculated 448.22, experimental 448.20.

Preparation 3: (S) -3-benzyl-2- (6- (4- (benzyloxy) -2-ethylphenyl) -1H-indazol-3-yl) -3,4,6, 7-tetrahydro-5H-imidazo [4,5-c ] pyridine-5, 6-dicarboxylic acid 6-benzyl ester 5- (tert-butyl ester) (I-16)

(a) 4-bromo-2-fluorobenzoyl chloride (I-13)

To an ice-cooled stirred solution of 4-bromo-2-fluorobenzoic acid (I-12) (1.25Kg, 5.71mol) in DCM (12.5L, 15 vol) was added oxalyl chloride (0.98L, 11.42mol) in a dropwise fashion. The resulting reaction mixture was stirred at the same temperature for 10 min. DMF (150mL) was then added to the reaction mixture in a dropwise manner. The resulting reaction mass was allowed to warm to room temperature and stirred for 2 hours. After completion of the reaction (monitored by TLC), excess oxalyl chloride was removed under reduced pressure under nitrogen atmosphere to obtain the crude product (I-13) (1.08Kg, 80%) which was used in the next step without further purification.

(b) (S) -3-benzyl-2- (4-bromo-2-fluorobenzoyl) -3,4,6, 7-tetrahydro-5H-imidazo [4,5-c ] pyridine-5, 6-dicarboxylic acid 6-benzyl ester 5- (tert-butyl ester) (I-14)

To (S) -3-benzyl-3, 4,6, 7-tetrahydro-5H-imidazo [4, 5-c) at room temperature]To a stirred solution of pyridine-5, 6-dicarboxylic acid 6-benzyl ester 5- (tert-butyl ester) (I-11) (1.70Kg, 3.80mol) in ACN (13.6L, 8 vol) was added triethylamine (2.11L, 15.2mol), followed by 4-bromo-2-fluorobenzoyl chloride (I-13) (1.08Kg, 4.56mol in 3.4L ACN, 2 vol). After the addition was complete, the resulting reaction mixture changed color from light yellow to brown. The resulting reaction mixture was stirred at the same temperature for 30min, and the reaction progress was monitored by TLC. The resulting reaction mixture was quenched with ice-cold water (10L), then extracted with EtOAc (3 × 5L) and the combined organics washed with brine solution. The organics were dried over sodium sulfate, filtered and concentrated under reduced pressure to give the crude product which was passed through a silica gel (100-200M) column chromatographySpectrum, purified by using eluant 20% EtOAc/hexanes to obtain the desired product (I-14) (1.74Kg, 71%). (m/z): c₃₃H₃₁BrFN₃O₅Of [ M + H]+ calculated 648.14, experimental 648.20.

(c) (S) -3-benzyl-2- (6-bromo-1H-indazol-3-yl) -3,4,6, 7-tetrahydro-5H-imidazo [4,5-c ] pyridine-5, 6-dicarboxylic acid 6-benzyl ester 5- (tert-butyl ester) (I-15)

To (S) -3-benzyl-2- (4-bromo-2-fluorobenzoyl) -3,4,6, 7-tetrahydro-5H-imidazo [4,5-c ] at room temperature]To a stirred solution of pyridine-5, 6-dicarboxylic acid 6-benzyl 5- (tert-butyl) ester (I-14) (1.74Kg, 2.68mol) in THF (26.0L, 15 vol) was added hydrazine hydrate (0.705L, 13.4 mol). The resulting reaction mixture was heated at 60 ℃ for 3 hours. After completion of the reaction (TLC monitoring), the resulting reaction cake was poured into ice-cold water (10L) followed by extraction of the compound with EtOAc (3 × 10L). The combined organics were washed with brine and dried over sodium sulfate, filtered, and evaporated under reduced pressure to give the crude product which was purified by column chromatography on silica gel (100-. (m/z): c₃₃H₃₂BrN₅O₄Of [ M + H]+ calculated 642.16, experimental 642.21.

(d) (S) -3-benzyl-2- (6- (4- (benzyloxy) -2-ethylphenyl) -1H-indazol-3-yl) -3,4,6, 7-tetrahydro-5H-imidazo [4,5-c ] pyridine-5, 6-dicarboxylic acid 6-benzyl ester 5- (tert-butyl ester) (I-16)

Bis (pinacolato) diboron (250g, 984mmol) was charged with propan-2-ol (1.882L, 2.46E +04mmol) to a 5L 3 neck single wall flask previously etched with fluorochemical and the mixture was stirred until fully dissolved. The dissolution was endothermic (-4 ℃). In a 4L Erlenmeyer flask, previously etched with a fluorine chemistry, potassium fluoride hydrofluoride (538g, 6891mmol) was dissolved in water (2.306L, 1.28E +05mmol) to form a 3M solution. The dissolution was endothermic (-12 ℃). The solution was then filtered to remove small amounts of insoluble material from the potassium fluoride hydrofluoride. Once both solutions were sufficiently dissolved, the contents of the erlenmeyer flask were filled portion by portion into a single wall flask over 15 minutes. Moderate heating (+10 ℃) was observed. During the addition, the solution became a thick, translucent, semi-opaque gray slurry and stirring was extended to keep the contents well mixed. The mixture was stirred for 1.5h and then filtered through a coarse glass fritted funnel (4L, pre-etched). It took 30-45 minutes to complete the filtration. The clear biphasic filtrate was discarded. The white solid was dried on the filter for 10 minutes (cleavage of the cake was observed). The solid was transferred back to a clean 5L 3-neck single-walled flask and reslurried with water (1.33L, 7.38E +04 mmol). The slurry was stirred for 2h, after which it formed a clear, homogeneous hydrogel. The solution was stirred for an additional 1h, whereupon the solids/gel were filtered off using a 4L coarse glass funnel (pre-etched). The solid was dried on the filter for 30 minutes. The solid was transferred back to a clean 5L 3-neck single-walled flask and reslurried with acetone (1.084L, 1.48E +04 mmol). The white/grey slurry was stirred for 1h and then filtered on a 4L coarse glass funnel (pre-etched). It took 20 minutes to complete the filtration and then dried on the funnel for another 1 h. During this time, the solids were occasionally agitated to ensure uniform drying. The pale white powder remained on the filter after drying. The solid was dried under vacuum at 55 ℃ for 20h with slow nitrogen bleed to obtain a loose white solid (collected to 200.3 g).

To (S) -3-benzyl-2- (6-bromo-1H-indazol-3-yl) -3,4,6, 7-tetrahydro-5H-imidazo [4, 5-c)]To a stirred solution of pyridine-5, 6-dicarboxylic acid 6-benzyl ester 5- (tert-butyl ester) (I-15) (10.0g, 16.0mmol) in 2-methyltetrahydrofuran (100mL, 10 volumes) was added (4- (benzyloxy) -2-ethylphenyl) trifluoro-lambda. (R)⁴Borane, potassium salt (I-5) (8.0g, 20mmol) and the loose white solid obtained above (0.20 g). The resulting reaction mixture was degassed with nitrogen for 30 minutes. To this solution was added the prepared aqueous cesium carbonate solution (20.0g, 62.0mmol in 60mL of water, 6 volumes). The resulting reaction mixture was further degassed for 15 minutes, then bis (di-tert-butyl (4-dimethylaminophenyl) phosphine) palladium (II) dichloride (0.66g, 0.93mmol) was added and the reaction mixture evacuated under vacuum and flushed with nitrogen. The resulting reaction mixture was heated at 110 ℃ for 20 hours. After completion of the reaction (TLC)&LCMS monitoring), the resulting reaction was mixedThe mixture was cooled to room temperature and filtered through a bed of celite, followed by further washing with EtOAc (3 × 0.5L). The combined organics were washed with 1N sodium hydroxide solution (3X 0.5L). The combined organics were then washed with brine and dried over sodium sulfate, filtered, and evaporated under reduced pressure to give the crude product which was purified by column chromatography on silica gel (100-. (m/z): c₄₈H₄₇N₅O₅Of [ M + H]+ calculated 774.36, experimental 774.59.

Preparation 4: (S) -2- (6- (2-ethyl-4-hydroxyphenyl) -1H-indazol-3-yl) -4,5,6, 7-tetrahydro-3H-imidazo [4,5-c ] pyridine-6-carboxylic acid, hydrochloride (I-18)

(a) (S) -3-benzyl-2- (6- (4- (benzyloxy) -2-ethylphenyl) -1H-indazol-3-yl) -4,5,6, 7-tetrahydro-3H-imidazo [4,5-c ] pyridine-6-carboxylic acid benzyl ester, hydrochloride salt (I-17)

Mixing (S) -3-benzyl-2- (6- (4- (benzyloxy) -2-ethylphenyl) -1H-indazol-3-yl) -3,4,6, 7-tetrahydro-5H-imidazo [4, 5-c)]Pyridine-5, 6-dicarboxylic acid 6-benzyl ester 5- (tert-butyl ester) (I-16) (1.0g, 1.292mmol) was dissolved in dioxane (8mL) and water (1.5mL), followed by the addition of hydrogen chloride solution (4M in dioxane) (7mL, 28.0mmol) and the reaction mixture stirred at room temperature for 3 hours (monitoring of reaction progress by LCMS). The reaction mixture was then frozen and lyophilized, and the crude product (I-17) was used directly in the next reaction (assuming quantitative yield). (m/z): c₄₃H₃₉N₅O₃Of [ M + H]+ calculated 674.31, experimental 674.3.

(b) (S) -2- (6- (2-ethyl-4-hydroxyphenyl) -1H-indazol-3-yl) -4,5,6, 7-tetrahydro-3H-imidazo [4,5-c ] pyridine-6-carboxylic acid, hydrochloride (I-18)

Mixing (S) -3-benzyl-2- (6- (4- (benzyloxy) -2-ethylphenyl) -1H-indazol-3-yl) -4,5,6, 7-tetrahydro-3H-imidazo [4, 5-c)]Pyridine compound-6-Carboxylic acid benzyl ester, hydrochloride salt (I-17) (0.918g, 1.292mmol) was dissolved in 2-propanol (15mL), hydrogen chloride solution (5M in water) (0.258mL, 1.292mmol) and water (0.25mL) at 50 ℃ followed by addition of palladium (10% wt on carbon), 50% water (0.138g, 0.065 mmol). The reaction flask was then purged with nitrogen, a hydrogen balloon attached and the reaction mixture stirred at 50 ℃ for 4 days, with the hydrogen balloon replenished as needed (reaction progress monitored by LCMS). All solids were then removed by filtration and the resulting solution was concentrated. The residue was dissolved in 1:1 ACN/water, frozen and lyophilized. The resulting powder (I-18) was used without further purification (assuming quantitative yield). (m/z): c₂₂H₂₁N₅O₃Of [ M + H]+ calculated 404.17, experimental 404.2.

Preparation 5: (S) -2- (6- (2-ethyl-4-hydroxyphenyl) -1H-indazol-3-yl) -5-isopropyl-4, 5,6, 7-tetrahydro-3H-imidazo [4,5-c ] pyridine-6-carboxylic acid (I-19)

Mixing (S) -2- (6- (2-ethyl-4-hydroxyphenyl) -1H-indazol-3-yl) -4,5,6, 7-tetrahydro-3H-imidazo [4, 5-c)]Pyridine-6-carboxylic acid, HCl (I-18) (0.25g, 0.568mmol) was suspended in DMF (2.5mL) and acetone (2.5mL), followed by the addition of acetic acid (0.098mL, 1.705mmol) and sodium cyanoborohydride (0.179g, 2.84mmol) and the reaction mixture was stirred at room temperature for 24h (monitoring of reaction progress by LCMS). The reaction mixture was concentrated, and the crude product was purified by reverse phase chromatography (5-70% ACN/water gradient, 50g C18aq column) to give the TFA salt of the title compound (149mg, 47% yield). (m/z): c₂₅H₂₇N₅O₃Of [ M + H]+ calculated 446.21, experimental 446.3.

Preparation 6: (S) -2- (6- (2-ethyl-4-hydroxyphenyl) -1H-indazol-3-yl) -5-propyl-4, 5,6, 7-tetrahydro-3H-imidazo [4,5-c ] pyridine-6-carboxylic acid (I-20)

Mixing (S) -2- (6- (2-ethyl-4-hydroxyphenyl) -1H-indazol-3-yl) -4,5,6, 7-tetrahydro-3H-imidazo [4, 5-c)]Pyridine-6-carboxylic acid, HCl (I-18) (0.160g, 0.364mmol) and propionaldehyde (0.039mL, 0.546mmol) were dissolved in methanol (3.0mL), followed by addition of sodium cyanoborohydride (0.069g, 1.091mmol) and stirring of the reaction mixture at room temperature for 24 hours (monitoring of reaction progress by LCMS). The reaction mixture was concentrated and the crude product was purified by reverse phase chromatography (5-70% ACN/water gradient, 50g C18 column) to give the TFA salt of the title compound (78mg, 38% yield). (m/z): c₂₅H₂₇N₅O₃Of [ M + H]+ calculated 446.21, experimental 446.3.

Preparation 7: (S) -2- (6- (2-ethyl-4-hydroxyphenyl) -1H-indazol-3-yl) -5-methyl-4, 5,6, 7-tetrahydro-3H-imidazo [4,5-c ] pyridine-6-carboxylic acid (I-21)

Mixing (S) -2- (6- (2-ethyl-4-hydroxyphenyl) -1H-indazol-3-yl) -4,5,6, 7-tetrahydro-3H-imidazo [4, 5-c)]Pyridine-6-carboxylic acid, HCl (I-18) (0.160g, 0.364mmol) and formaldehyde solution (37 wt% in water) (0.032mL, 0.436mmol) were dissolved in methanol (3.0mL), followed by addition of sodium cyanoborohydride (0.069g, 1.091mmol) and the reaction mixture stirred at room temperature for 4 hours (monitoring the reaction progress by LCMS). Sodium borohydride (14mg, 0.364mmol) was added to quench any excess formaldehyde, followed by concentration of the reaction mixture. The crude product was purified by reverse phase chromatography (5-70% ACN/water gradient, 50g C18 column) to give the TFA salt of the title compound (110mg, 57% yield). (m/z): c₂₃H₂₃N₅O₃Of [ M + H]+ calculated 418.18, experimental 418.2.

Preparation 8: (R) -2- (3-methylpiperazin-1-yl) ethan-1-ol, dihydrochloride (I-24)

(a) (R) -4- (2-hydroxyethyl) -2-methylpiperazine-1-carboxylic acid tert-butyl ester (I-23)

(R) -1-boc-2-methyl-piperazine (0.2g, 0.999mmol), DIPEA (0.698mL, 3.99mmol) and 2-bromoethanol (0.085mL, 1.198mmol) were dissolved in DMF (5mL) and the reaction mixture was stirred at 60 ℃ for 16h (monitoring the reaction progress by LCMS). The reaction mixture was concentrated, and then 10mL of diethyl ether was added to the residue. The solution was sonicated until the residue gel disappeared and a solid formed. The ether solution was then poured from the solid. The solid was then used directly in the next reaction (assuming quantitative yield). (m/z): c₁₂H₂₄N₂O₃Of [ M + H]+ calculated 245.18, experimental 245.4.

(b) (R) -2- (3-methylpiperazin-1-yl) ethan-1-ol, dihydrochloride (I-24)

Tert-butyl (R) -4- (2-hydroxyethyl) -2-methylpiperazine-1-carboxylate (0.244g, 0.999mmol) was dissolved in dioxane (3.0mL) and water (0.5mL), followed by the addition of a solution of hydrogen chloride (4M in dioxane) (2.0mL, 65.8mmol) and the reaction mixture was stirred at room temperature for 3 hours (monitoring of reaction progress by LCMS). The reaction mixture was frozen and lyophilized, and the resulting product was used without purification (assuming quantitative yield). (m/z): c₇H₁₆N₂O of [ M + H]+ calculated 145.13, experimental 145.4.

Example 1: (S) - (2- (6- (2-ethyl-4-hydroxyphenyl) -1H-indazol-3-yl) -5-propyl-4, 5,6, 7-tetrahydro-3H-imidazo [4,5-c ] pyridin-6-yl) (4- (2-hydroxyethyl) piperazin-1-yl) methanone

Mixing (S) -2- (6- (2-ethyl-4-hydroxyphenyl) -1H-indazol-3-yl) -5-propyl-4, 5,6, 7-tetrahydro-3H-imidazo [4, 5-c)]Pyridine-6-carboxylic acid, TFA (I-20) (40mg, 0.071mmol), n- (2-hydroxyethyl) piperazine (0.018mL, 0.143mmol) and DIPEA (0.025mL, 0.143mmol) were dissolved in DMF (1.5mL), followed by HATU (40.8mg, 0.107mmol) and the reaction mixture was stirred at room temperature for 3 hours (monitoring the reaction progress by LCMS). Hydrazine (0.011mL, 0.357mmol) was added to cleave the undesired by-products, then the solution was stirred at room temperature for 10 minutes. The reaction mixture was then concentrated and the crude product was purified by preparative HPLC (5-70% ACN/water gradient, C18 column) to give the TFA salt of the title compound (31mg, 56% yield). (m/z): c₃₁H₃₉N₇O₃Of [ M + H]+ calculated 558.31, experimental 558.3.¹H NMR(400MHz,DMSO-d6)δ13.10(s,1H),12.27(d,J＝48.92Hz,1H),9.40(s,1H),8.28(t,J＝8.10Hz,1H),7.30(s,1H),7.04(m,2H),6.71(d,J＝2.46Hz,1H),6.64(dd,J＝2.50,8.23Hz,1H),4.44(t,J＝5.31Hz,1H),4.11(q,J＝5.26,2H),3.96(m,1H),3.86-3.52(m,6H),3.49(q,J＝6.01Hz,2H),2.95(m,2H),2.48(q,J＝7.48Hz,2H),2.42-2.21(m,4H),2.37(t,J＝6.20Hz,2H),1.42(m,2H),1.00(t,J＝7.52Hz,3H),0.81(m,3H)。

Example 2: ((S) -2- (6- (2-ethyl-4-hydroxyphenyl) -1H-indazol-3-yl) -5-propyl-4, 5,6, 7-tetrahydro-3H-imidazo [4,5-c ] pyridin-6-yl) ((1S,4S) -5-methyl-2, 5-diazabicyclo [2.2.1] hept-2-yl) methanone

Mixing (S) -2- (6- (2-ethyl-4-hydroxyphenyl) -1H-indazol-3-yl) -5-propyl-4, 5,6, 7-tetrahydro-3H-imidazo [4, 5-c)]Pyridine-6-carboxylic acid, TFA (I-20) (40mg, 0.071mmol), (1S,4S) -5-methyl-2, 5-diazabicyclo [2.2.1]Heptane hydrobromide (29.4mg, 0.107mmol) and DIPEA (0.062mL, 0.357mmol) were dissolved in DMF (1.5mL), followed by HATU (40.8mg, 0.107mmol) and the reaction mixture stirred at room temperature for 3 hours (monitoring reaction progress by LCMS). Hydrazine (0.011mL, 0.357mmol) was added to cleave the undesired by-products, then the solution was stirred at room temperature for 10 minutes. The reaction mixture was then concentrated and the crude product was purified by preparative HPLC (5-70% ACN/water gradient, C18 column) to give the TFA salt of the title compound (32mg, 59% yield). (m/z): c₃₁H₃₇N₇O₂Of [ M + H]+ calculated 540.30, experimental 540.3.

Example 3: ((S) -2, 4-dimethylpiperazin-1-yl) ((S) -2- (6- (2-ethyl-4-hydroxyphenyl) -1H-indazol-3-yl) -5-methyl-4, 5,6, 7-tetrahydro-3H-imidazo [4,5-c ] pyridin-6-yl) methanone

(a) (S) -4- ((S) -2- (6- (2-ethyl-4-hydroxyphenyl) -1H-indazol-3-yl) -5-methyl-4, 5,6, 7-tetrahydro-3H-imidazo [4,5-c ] pyridine-6-carbonyl) -3-methylpiperazine-1-carboxylic acid tert-butyl ester (I-25)

Mixing (S) -2- (6- (2-ethyl-4-hydroxyphenyl) -1H-indazol-3-yl) -5-methyl-4, 5,6, 7-tetrahydro-3H-imidazo [4, 5-c)]Pyridine-6-carboxylic acid, TFA (I-21) (55mg, 0.103mmol), (S) -4-n-boc-2-methylpiperazine (41.5mg, 0.207mmol) and DIPEA (0.036mL, 0.207mmol) were dissolved in DMF (1.5mL), followed by addition of HATU (59.0mg, 0.155mmol) and stirring of the reaction mixture at room temperature for 16h (monitoring of reaction progress by LCMS). Hydrazine (0.016mL, 0.517mmol) was added to cleave the undesired by-products, then the reaction mixture was stirred at room temperature for 10 minutes. The reaction mixture was then concentrated and the crude product was purified by reverse phase chromatography (5-70% ACN/water gradient, 50g C18 column) to give the TFA salt of the title compound (54mg, 72% yield). (m/z): c₃₃H₄₁N₇O₄Of [ M + H]+ calculated 600.33, experimental 600.3.

(b) ((S) -2- (6- (2-ethyl-4-hydroxyphenyl) -1H-indazol-3-yl) -5-methyl-4, 5,6, 7-tetrahydro-3H-imidazo [4,5-c ] pyridin-6-yl) ((S) -2-methylpiperazin-1-yl) methanone (I-26)

Mixing (S) -4- ((S) -2- (6- (2-ethyl-4-hydroxyphenyl) -1H-indazol-3-yl) -5-methyl-4, 5,6, 7-tetrahydro-3H-imidazo [4, 5-c)]Pyridine-6-carbonyl) -3-methylpiperazine-1-carboxylic acid tert-butyl ester, TFA (I-25) (0.126g, 0.177mmol) were dissolved in dioxane (1.5mL) and water (0.3mL), followed by addition of a solution of hydrogen chloride (4M in dioxane) (1.5mL, 6.00mmol) and stirring of the reaction mixture at room temperature for 2h (monitoring of reaction progress by LCMS). The reaction mixture was frozen and lyophilized, and the resulting powder was used directly in the next reaction (assuming quantitative yield). (m/z): c₂₈H₃₃N₇O₂Of [ M + H]+ calculated 500.27, experimental 500.3.

(c) ((S) -2, 4-dimethylpiperazin-1-yl) ((S) -2- (6- (2-ethyl-4-hydroxyphenyl) -1H-indazol-3-yl) -5-methyl-4, 5,6, 7-tetrahydro-3H-imidazo [4,5-c ] pyridin-6-yl) methanone

((S) -2- (6- (2-ethyl-4-hydroxyphenyl) -1H-indazol-3-yl) -5-methyl-4, 5,6, 7-tetrahydro-3H-imidazo [4, 5-c)]Pyridin-6-yl) ((S) -2-methylpiperazin-1-yl) methanone, dihydrochloride (0.101g, 0.176mmol) and formaldehyde solution (37 wt% in water) (0.016mL, 0.212mmol) were dissolved in methanol (3.0mL), followed by addition of sodium cyanoborohydride (0.055g, 0.882mmol) and the reaction mixture was stirred at room temperature for 16 hours (monitoring the reaction progress by LCMS). Sodium borohydride (7mg, 0.176mmol) was added to quench any remaining formaldehyde. The reaction mixture was concentrated, and the crude product was purified by preparative HPLC (5-60% ACN/water gradient, C18 column) to give the TFA salt of the title compound (28mg, 21% yield). (m/z): c₂₉H₃₅N₇O₂Of [ M + H]+ calculated 514.29, experimental 514.3.

Example 4: (S) - (2- (6- (2-ethyl-4-hydroxyphenyl) -1H-indazol-3-yl) -5-isopropyl-4, 5,6, 7-tetrahydro-3H-imidazo [4,5-c ] pyridin-6-yl) (4-methyl-1, 4-diazepan-1-yl) methanone

Mixing (S) -2- (6- (2-ethyl-4-hydroxyphenyl) -1H-indazol-3-yl) -5-isopropyl-4, 5,6, 7-tetrahydro-3H-imidazo [4, 5-c)]Pyridine-6-carboxylic acid, TFA (I-19) (50mg, 0.089mmol), 1-methyl homopiperazine (0.022mL, 0.179mmol) and DIPEA (0.031mL, 0.179mmol) were dissolved in DMF (1.5mL), followed by the addition of HATU (51.0mg, 0.134mmol) and the reaction mixture was stirred at room temperature for 3 hours (monitoring of reaction progress by LCMS). Hydrazine (0.014mL, 0.447mmol) was added to cleave the undesired by-products and the solution was stirred at room temperature for 10 min. The reaction mixture was then concentrated and the crude product was purified by preparative HPLC (2-70% ACN/water gradient, C18 column) to give the TFA salt of the title compound (29mg, 42% yield). (m/z): c₃₁H₃₉N₇O₂Of [ M + H]+ calculated 542.32, experimental 542.3.¹H NMR(400MHz,DMSO-d6)δ13.08(s,1H),12.21(d,J＝29.9Hz,1H),9.40(s,1H),8.27(d,J＝8.33Hz,1H),7.30(s,1H),7.04(t,J＝8.05,2H),6.71(d,J＝2.53Hz,1H),6.64(dd,J＝2.54,8.23Hz,1H),4.11(m,3H),3.91-3.52(m,6H),2.97(m,1H),2.91-2.53(m,4H),2.49(q,J＝7.46,2H),2.23(d,J＝13.9Hz,3H),1.76(m,2H),1.0(m,9H)。

Example 5: ((S) -2- (6- (2-ethyl-4-hydroxyphenyl) -1H-indazol-3-yl) -5-methyl-4, 5,6, 7-tetrahydro-3H-imidazo [4,5-c ] pyridin-6-yl) ((R) -4- (2-hydroxyethyl) -2-methylpiperazin-1-yl) methanone

Mixing (S) -2- (6- (2-ethyl-4-hydroxyphenyl) -1H-indazol-3-yl) -5-methyl-4, 5,6, 7-tetrahydro-3H-imidazo [4, 5-c)]Pyridine-6-carboxylic acid, TFA (I-21) (55mg, 0.103mmol), (R) -2- (3-methylpiperazin-1-yl) ethan-1-ol, dihydrochloride (I-24) (33.7mg, 0.155mmol) and DIPEA (0.090mL, 0.517mmol) were dissolved in DMF (1.5mL), followed by addition of HATU (59.0mg, 0.155mmol) and stirring of the reaction mixture at room temperature for 16h (monitoring of reaction progress by LCMS). Hydrazine (0.016mL, 0.517mmol) was added to cleave the undesired by-products, then the reaction mixture was stirred at room temperature for 10 minutes. The reaction mixture was then concentrated, and the crude product was purified by preparative HPLC (5-70% ACN/water gradient, C18 column) to give the TFA salt of the title compound (22mg, 28% yield). (m/z): c₃₀H₃₇N₇O₃Of [ M + H]+ calculated 544.30, experimental 544.3.

Example 6: ((S) -3- (dimethylamino) pyrrolidin-1-yl) ((S) -2- (6- (2-ethyl-4-hydroxyphenyl) -1H-indazol-3-yl) -5-isopropyl-4, 5,6, 7-tetrahydro-3H-imidazo [4,5-c ] pyridin-6-yl) methanone

Mixing (S) -2- (6- (2-ethyl-4-hydroxyphenyl) -1H-indazol-3-yl) -5-isopropyl-4, 5,6, 7-tetrahydro-3H-imidazo [4, 5-c)]Pyridine-6-carboxylic acid, TFA (I-19) (50mg, 0.089mmol), (S) - (-) -3- (dimethylamino) pyrrolidine (0)023mL, 0.179mmol) and DIPEA (0.031mL, 0.179mmol) were dissolved in DMF (1.5mL) followed by the addition of HATU (51.0mg, 0.134mmol) and the reaction mixture was stirred at room temperature for 3 hours (monitoring the reaction progress by LCMS). Hydrazine (0.014mL, 0.447mmol) was added to cleave the undesired by-products and the solution was stirred at room temperature for 10 min. The reaction mixture was then concentrated and the crude product was purified by preparative HPLC (5-70% ACN/water gradient, C18 column) to give the TFA salt of the title compound (37mg, 53% yield). (m/z): c₃₁H₃₉N₇O₂Of [ M + H]+ calculated 542.32, experimental 542.3.

Preparation 9: 2- (4- (benzyloxy) -2-ethyl-5-fluorophenyl) -4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolane

(a)1- (benzyloxy) -4-bromo-5-ethyl-2-fluorobenzene

To a solution of 4-bromo-5-ethyl-2-fluorophenol (20g, 910.32mmol) in ACN (250mL) was added K₂CO₃(31.55g, 228.3mmol), followed by dropwise addition of bromotoluene (13.10mL, 109.58 mmol). The resulting reaction mixture was stirred at 80 ℃ for 2 h. The aqueous layer was extracted with EtOAc (three times), combined and washed with brine. The organic layer was washed with Na₂SO₄Dried and evaporated under reduced pressure to give the title intermediate as a pale yellow oily liquid (25g, 89% yield).¹H NMR (400MHz, chloroform-d) δ 7.48-7.30(m,5H),7.27(d, J ═ 10.5Hz,1H),6.87(d, J ═ 8.7Hz,1H),5.12(s,2H),2.66(q, J ═ 7.5Hz,2H),1.16(t, J ═ 7.5Hz, 3H).

(b)2- (4- (benzyloxy) -2-ethyl-5-fluorophenyl) -4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolane

To a solution of the product of the previous step (12.5g, 40.45mmol) in dioxane (100mL) was added bis (pinacolato) diboron (15.40g, 60.67mmol) and KOAc (11.9g, 121.35 mmol). The reaction mixture was purged with nitrogen for 15min, followed by addition of [1,1' -bis (diphenylphosphino) ferrocene complexed with dichloromethane]Palladium (II) dichloride (1.65g, 2.02)3 mmol). The resulting reaction mixture was stirred and heated at 110 ℃ for 3h, filtered through celite and the residue washed with EtOAc. The filtrate was diluted with excess EtOAc (200mL) and washed with water (100mL), followed by brine (100mL), dried over sodium sulfate and concentrated in vacuo to afford the crude product which was purified by column chromatography on (100-200) silica gel eluting with 3-5% EtOAc: hexanes to afford the desired product as an off-white solid (9.50g, 66% yield).¹H NMR (400MHz, chloroform-d) δ 7.54-7.27(m,6H),6.81(d, J ═ 7.9Hz,1H),5.16(s,2H),2.84(q, J ═ 7.5Hz,2H),1.32(s,12H),1.14(t, J ═ 7.5Hz, 3H).

Preparation 10: 6- (4- (benzyloxy) -2-ethyl-5-fluorophenyl) -1- (tetrahydro-2H-pyran-2-yl) -3- (trimethylstannanyl) -1H-indazole

(a)6- (4- (benzyloxy) -2-ethyl-5-fluorophenyl) -1- (tetrahydro-2H-pyran-2-yl) -1H-indazole

To 6-bromo-1- (tetrahydro-2H-pyran-2-yl) -1H-indazole (50g, 178.57mmol) and 2- (4- (benzyloxy) -2-ethyl-5-fluorophenyl) -4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolane (76.3g, 214.29mmol) in DMF H₂K was added to a solution in O (480:120mL)₃PO₄(94.64g, 446.86 mmol). The reaction mixture was degassed with nitrogen for 15min, followed by addition of Pd (PPh)₃)₂Cl₂Catalyst (6.26g, 8.93mmol) and the mixture was degassed again with nitrogen for 5min, stirred and heated at 100 to 110 ℃ for 5 h. The reaction mixture was filtered through celite and the residue was washed with EtOAc. The filtrate was diluted with EtOAc, washed with cold water and brine, dried over sodium sulfate and concentrated in vacuo to give the crude product, which was purified by flash column chromatography to afford the title intermediate as a white solid (65g, 86% yield). (m/z): c₂₇H₂₇FN₂O₂Of [ M + H]⁺Calculated 431.21, experimental 431.46.¹H NMR (400MHz, chloroform-d) δ 8.06-7.98(m,2H),7.70(d, J ═ 8.2Hz,1H),7.51-7.32(m,5H),7.08(dd, J ═ 809.6,8.3Hz,1H),7.03(d,J＝11.9Hz,1H),6.95(d,J＝8.5Hz,1H),5.76-5.64(m,1H),5.20(s,2H),4.04(d,J＝10.1Hz,1H),3.72(t,J＝9.7Hz,1H),2.52(q,J＝7.5Hz,2H),2.22-2.02(m,3H),1.80-1.71(m,3H),1.06(t,J＝7.5Hz,3H)。

(b)6- (4- (benzyloxy) -2-ethyl-5-fluorophenyl) -1H-indazole

To a solution of the product of the previous step (65g, 151.16mmol) in methanol (700mL) was added concentrated HCl (120mL) and the resulting solution was heated at 60-65 ℃ for 3h, cooled to room temperature, and concentrated in vacuo. The residue was dissolved in EtOAc and taken up with NaHCO₃Saturated aqueous solution and water washing. The organic layer was passed over anhydrous Na₂SO₄Dried and concentrated in vacuo to afford the title intermediate (52g, 99% (crude material)) as a white solid.¹H NMR (400MHz, chloroform-d) δ 8.13(s,1H),7.77(d, J ═ 8.3Hz,1H),7.59-7.30(m,6H),7.10(d, J ═ 8.3Hz,1H),7.01(d, J ═ 11.8Hz,1H),6.96(d, J ═ 8.4Hz,1H),5.21(s,2H),2.53(q, J ═ 7.5Hz,2H),1.05(t, J ═ 7.5Hz, 3H).

(c)6- (4- (benzyloxy) -2-ethyl-5-fluorophenyl) -3-iodo-1H-indazole

To a solution of 6- (4- (benzyloxy) -2-ethyl-5-fluorophenyl) -1H-indazole (56g, 161.18mmol) in DMF (400mL) was added KOH (36.2g, 647.39mmol) and the mixture was stirred for 5 min. A solution of iodine (82.2g, 323.69mmol) in DMF (100mL) was added slowly at 0 ℃ and stirred at room temperature for 30min, diluted with water (3X 150mL) and extracted with EtOAc (3X 200 mL). The organic layer was washed with saturated aqueous sodium metabisulfite (3X 200mL) and water (400mL) over anhydrous Na₂SO₄Dried and concentrated under reduced pressure to give the crude product which was purified by flash column chromatography to give the title intermediate as a light brown semi-solid (64g, 84% yield).¹H NMR (400MHz, chloroform-d) δ 10.49(s,1H),7.57-7.32(m,7H),7.16(d, J ═ 8.3Hz,1H),7.04-6.91(m,2H),5.20(s,2H),2.51(q, J ═ 7.4Hz,2H),1.04(t, J ═ 7.5Hz, 3H).

(d)6- (4- (benzyloxy) -2-ethyl-5-fluorophenyl) -3-iodo-1- (tetrahydro-2H-pyran-2-yl) -1H-indazole

To an ice-cold solution of the product of the previous step (60g, 127.12mmol) in DCM (700mL)P-toluenesulfonic acid (4.84g, 25.423mmol) was added followed by dropwise addition of 3, 4-dihydro-2H-pyran (17.43mL, 190.68 mmol). The reaction mixture was stirred at room temperature overnight, diluted with DCM and with NaHCO₃Saturated aqueous solution and brine. The organic layer was passed over anhydrous Na₂SO₄Dried and concentrated under reduced pressure to give the crude product which was purified by flash chromatography (silica gel) to give the title intermediate as an off-white solid (64g, 91% yield). (m/z): c₂₇H₂₆FIN₂O₂Of [ M + H]⁺Calculated 557.10, experimental 557.30.¹H NMR (400MHz, chloroform-d) δ 7.56-7.31(m,7H),7.14(d, J ═ 8.3Hz,1H),7.01(d, J ═ 11.8Hz,1H),6.95(d, J ═ 8.5Hz,1H),5.68(d, J ═ 9.3Hz,1H),5.20(s,2H),4.08-3.99(m,1H),3.77-3.64(m,1H),2.50(q, J ═ 7.2Hz,2H),2.23-1.97(m,3H),1.81-1.68(m,3H),1.06(t, J ═ 7.4Hz, 3H).

(e)6- (4- (benzyloxy) -2-ethyl-5-fluorophenyl) -1- (tetrahydro-2H-pyran-2-yl) -3- (trimethylstannanyl) -1H-indazole

To a solution of 6- (4- (benzyloxy) -2-ethyl-5-fluorophenyl) -3-iodo-1- (tetrahydro-2H-pyran-2-yl) -1H-indazole (20g, 35.97mmol) in toluene (150mL) was added hexamethylditin (9.2mL, 43.17 mmol). The reaction mixture was degassed with nitrogen for 20min, then four (2.0g, 1.80mmol) was added and then stirred at 100 ℃ for 2h, cooled to room temperature, filtered through celite and the residue washed with EtOAc. The filtrate was concentrated and purified by column chromatography (over neutral alumina) eluting with 2-5% EtOAc: hexanes to give the title compound (17.50g, 82% yield). (m/z): c₃₀H₃₅FN₂O₂[ M + H ] of Sn]⁺Calculated values 595.17, 593.17; experimental values 595.49, 593.55.¹H NMR (400MHz, chloroform-d) δ 7.68(d, J ═ 8.0Hz,1H),7.57-7.29(m,6H),7.13-7.00(m,2H),6.96(d, J ═ 8.4Hz,1H),5.81-5.68(m,1H),5.21(s,2H),4.13-4.00(m,1H),3.81-3.66(m,1H),2.54(q, J ═ 7.3Hz,2H),2.23-2.00(m,2H),1.87-1.59(m,4H),1.08(t, J ═ 7.5Hz,3H),0.47(s, 9H).

Preparation 11: (S) -2-iodo-3- ((2-trimethylsilyl) ethoxy) methyl) -3,4,6, 7-tetrahydro-5H-imidazo [4,5-c ] pyridine-5, 6-dicarboxylic acid 5- (tert-butyl) 6-methyl ester

(a) (S) -4,5,6, 7-tetrahydro-3H-imidazo [4,5-c ] pyridine-6-carboxylic acid

To a stirred suspension of L-histidine (50g, 322.24mmol) in water (420mL) at 0 deg.C was added concentrated HCl (29mL) dropwise, followed by a one-shot addition of formaldehyde (55mL, 676.72mmol) at 0 deg.C. The resulting reaction mixture was stirred for 30min and then heated at 75 ℃ for 6h and concentrated. The resulting crude material was stirred with ether for 2h, filtered and washed with IPA: THF (100:300mL) to give the HCl salt of the title intermediate as an off-white solid (75g, 99% yield (crude material)). (m/z): c₇H₉N₃O₂Of [ M + H]⁺Calculated 168.07, experimental 168.17.

(b) (S) -4,5,6, 7-tetrahydro-3H-imidazo [4,5-c ] pyridine-6-carboxylic acid methyl ester

To a stirred solution of the product of the previous step (75.0g, 312.5mmol) in methanol (1500mL) at 0 deg.C was added SOCl dropwise₂(45.6mL, 625mmol) and stirred at room temperature for 16h, then heated until reflux (70 ℃ C.) for 1 h. The solvent was removed by distillation and the crude product was triturated with methanol followed by diethyl ether to give the crude HCl salt of the title intermediate as an off-white solid (80g of crude material).¹H NMR(400MHz,DMSO-d₆)δ9.05(s,1H),4.71(dd,J＝9.4,5.2Hz,1H),4.36(d,J＝15.5Hz,1H),4.30(d,J＝15.6Hz,1H),3.82(s,3H),3.44-3.21(m,2H)。

(c) (S) -3,4,6, 7-tetrahydro-5H-imidazo [4,5-c ] pyridine-5, 6-dicarboxylic acid 5- (tert-butyl) 6-methyl ester

To a stirred solution of the product of the previous step (80.0g, 314.96mmol) in methanol (1000mL) at 0 deg.C was added DIPEA (282mL, 1574mmol) followed by di-tert-butyl dicarbonate (172mL, 787.48 mmol). The reaction mixture was stirred at room temperature for 16h and then liquid NH was added₃(150mL, 25% in water), and the reaction mixture was stirred again at room temperature for 16h, methanol was removed by distillation and the residue was extracted with DCM (3X 200 mL). Is combined withThe organic extract is processed by anhydrous Na₂SO₄Dried, concentrated and purified by flash chromatography (100-200 mesh silica gel) eluting with 5% MeOH: DCM to afford the title intermediate (41g, 46% yield). (m/z): c₁₃H₁₉N₃O₄Of [ M + H]⁺Calculated 282.14, experimental 282.21.¹H NMR(400MHz,DMSO-d₆)δ11.85(s,1H),7.50(s,1H),5.18(dd,J＝49.3,5.1Hz,1H),4.51(t,J＝14.2Hz,1H),4.09(dd,J＝43.9,16.1Hz,1H),3.59(s,3H),3.08(d,J＝15.5Hz,1H),2.94(d,J＝15.1Hz,1H),1.45(s,9H)。

(d) (S) -2-iodo-3, 4,6, 7-tetrahydro-5H-imidazo [4,5-c ] pyridine-5, 6-dicarboxylic acid 5- (tert-butyl) 6-methyl ester

To a solution of the product of the previous step (41.0g, 145.9mmol) in THF (500mL) at 0 deg.C was added N-iodosuccinimide (66.0g, 291.8mmol) and the resulting solution was stirred at room temperature for 4h, diluted with water and extracted with ethyl acetate. The organic portion was washed with 10% sodium thiosulfate solution (3X 200 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated to give 60g of the title compound (crude material), which was used in the next step without further purification. (m/z): c₁₃H₁₈IN₃O₄Of [ M + H]⁺Calculated 408.03, experimental 408.31.¹H NMR(400MHz,DMSO-d₆)δ12.48(s,1H),5.34-4.97(m,1H),4.67-4.35(m,1H),4.12-3.95(m,1H),3.60(s,3H),3.14-2.82(m,2H),1.44(s,9H)。

(e) (S) -2-iodo-3- ((2-trimethylsilyl) ethoxy) methyl) -3,4,6, 7-tetrahydro-5H-imidazo [4,5-c ] pyridine-5, 6-dicarboxylic acid 5- (tert-butyl) 6-methyl ester

To (S) -2-iodo-3, 4,6, 7-tetrahydro-5H-imidazo [4,5-c at 0 deg.C]To a stirred solution of 5- (tert-butyl) 6-methyl pyridine-5, 6-dicarboxylate (40g, 0.098mol) in DMF (150mL) was added DIPEA (35.1mL, 0.19 mol). The reaction mixture was stirred for 10min, followed by dropwise addition of 2- (trimethylsilyl) -ethoxymethyl chloride (19.1mL, 0.10mol) at 0 ℃. The resulting reaction mixture was stirred at room temperature for 3 h. After 4h, chilled water was added and the reaction mixture was extracted with EtOAc (2X 200 mL). The organic layer was dried over anhydrous sodium sulfate, concentrated and passed through a flashColumn chromatography, eluting with 20-35% EtOAc in hexanes, afforded the title product (27g) as a pale yellow, viscous liquid. (m/z): c₁₉H₃₂IN₃O₅[ M + H ] of Si]⁺Calculated 538.12, experimental 538.42.¹H NMR(400MHz,DMSO-d₆)δ5.33-5.04(m,3H),4.79-4.56(m,1H),4.54-4.14(m,1H),3.60(s,3H),3.47(t,J＝7.8Hz,2H),3.31-3.16(m,1H),2.97(t,J＝18.9Hz,1H),1.44(s,9H),0.92-0.74(m,2H),-0.03(s,9H)。

Preparation 12: (6S) -5- (tert-butoxycarbonyl) -2- (6- (2-ethyl-5-fluoro-4-hydroxyphenyl) -1- (tetrahydro-2H-pipran-2-yl) -1H-indazol-3-yl) -3- ((2- (trimethylsilyl) ethoxy) methyl) -4,5,6, 7-tetrahydro-3H-imidazo [4,5-c ] pyridine-6-carboxylic acid

(a) (6S) -2- (6- (4- (benzyloxy) -2-ethyl-5-fluorophenyl) -1- (tetrahydro-2H-pyran-2-yl) -1H-indazol-3-yl) -3- ((2- (trimethylsilyl) ethoxy) methyl) -3,4,6, 7-tetrahydro-5H-imidazo [4,5-c ] pyridine-5, 6-dicarboxylic acid 5- (tert-butyl) 6-methyl ester

To (S) -2-iodo-3- ((2-trimethylsilyl) ethoxy) methyl) -3,4,6, 7-tetrahydro-5H-imidazo [4, 5-c-]To a stirred solution of 5- (tert-butyl) 6-methyl pyridine-5, 6-dicarboxylate (17.0g, 31.65mmol) in toluene (500mL) was added 6- (4- (benzyloxy) -2-ethyl-5-fluorophenyl) -1- (tetrahydro-2H-pyran-2-yl) -3- (trimethylstannyl) -1H-indazole (20g, 34.82 mmol). The reaction mixture was purged with argon for 15min and Pd (PPh) was added₃)₄(3.6g, 3.16mmol) and copper iodide (1.20g,6.33mmol) and the reaction mixture was stirred at 120 ℃ for 16 h. The reaction mixture was filtered through celite, the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (Redisep 80g column, eluting with DCM for 10min and then 15-20% EtOAc/hexanes) to give the title intermediate as a yellow solid (15.10g, 58% yield). (m/z): c₄₆H₅₈FN₅O₇[ M + H ] of Si]⁺Calculated 840.41, experimental 840.54.¹H NMR (400MHz, chloroform-d) delta 8.43(s,1H),7.54-7.33(m,6H),7.20(s,1H),7.05(d,J＝11.4Hz,1H),6.95(d,J＝8.5Hz,1H),6.09-5.69(m,3H),5.59-5.36(m,1H),5.20(s,2H),4.97-4.80(m,1H),4.12-3.90(m,1H),3.68(s,3H),3.57-3.47(m,2H),3.40(d,1H),3.21-3.05(m,1H),2.74-2.34(m,4H),2.25-2.07(m,2H),1.94-1.65(m,4H),1.54(s,9H),1.12-0.99(m,3H),0.91-0.75(m,2H),-0.12(s,9H)。

(b) (6S) -2- (6- (4- (benzyloxy) -2-ethyl-5-fluorophenyl) -1- (tetrahydro-2H-pyran-2-yl) -1H-indazol-3-yl) -3- ((2- (trimethylsilyl) ethoxy) methyl) -3,4,6, 7-tetrahydro-5H-imidazo [4,5-c ] pyridine-5, 6-dicarboxylic acid 6-benzyl ester 5- (tert-butyl ester)

To a round bottom flask was added toluene (400mL) containing the product of the previous step (15.0g, 17.85mmol), benzyl alcohol (46.3mL) and Ti (OEt)₄(7.15mL, 35.70mmol) and the reaction mixture was refluxed vigorously (140 ℃ C.) for 48h, diluted with water and extracted with DCM. The suspension is filtered and the filtrate is taken over Na₂SO₄Dry, concentrate under reduced pressure and purify by silica gel column chromatography (Redisep 80g column, 0-5% EtOAc/hexanes) for 20min to remove excess benzyl alcohol, followed by elution with 10-15% EtOAc/hexanes to give the title intermediate.¹H NMR was consistent with the structure. (m/z): c₅₂H₆₂FN₅O₇[ M + H ] of Si]⁺Calculated 916.44, experimental 916.86.

(c) (6S) -5- (tert-butoxycarbonyl) -2- (6- (2-ethyl-5-fluoro-4-hydroxyphenyl) -1- (tetrahydro-2H-pipran-2-yl) -1H-indazol-3-yl) -3- ((2- (trimethylsilyl) ethoxy) methyl) -4,5,6, 7-tetrahydro-3H-imidazo [4,5-c ] pyridine-6-carboxylic acid

To a stirred solution of the product of the previous step (21.0g, 22.92mmol) in 1:1IPA: THF (400mL) was added Pd (OH)₂(5.0 g). The reaction mixture was stirred at room temperature under a balloon of hydrogen for 16h, filtered through celite, concentrated under reduced pressure and purified by silica gel column chromatography (Redisep 80g column, eluting with 25-40% EtOAc in hexanes) to give the title compound as an off-white solid (6.1g, 8.29 mmol). (m/z): c₃₈H₅₀FN₅O₇[ M + H ] of Si]⁺Calculated 736.35, experimental 736.5.¹H NMR was consistent with the structure. (m/z):C₃₈H₅₀FN₅O₇[ M + H ] of Si]⁺Calculated 736.35, experimental 736.5.¹H NMR(400MHz,DMSO-d₆)δ12.94(s,1H),9.86(s,1H),8.34(t,J＝7.6Hz,1H),7.66(s,1H),7.20(d,J＝8.7Hz,1H),7.03(d,J＝11.8Hz,1H),6.93(d,J＝9.1Hz,1H),6.11-5.77(m,3H),5.33-5.06(m,1H),4.87-4.56(m,1H),4.52-4.14(m,1H),3.97-3.69(m,2H),3.53-3.40(m,2H),3.23-3.11(m,1H),3.11-2.93(m,1H),2.47-2.44(m,2H),2.13-1.96(m,2H),1.68(d,J＝70.9Hz,4H),1.48(s,9H),1.02(t,J＝7.5Hz,3H),0.86-0.68(m,2H),-0.17(s,9H)。

Preparation 13: (S) -2- (6- (2-ethyl-5-fluoro-4-hydroxyphenyl) -1H-indazol-3-yl) -4,5,6, 7-tetrahydro-3H-imidazo [4,5-c ] pyridine-6-carboxylic acid

To (6S) -5- (tert-butoxycarbonyl) -2- (6- (2-ethyl-5-fluoro-4-hydroxyphenyl) -1- (tetrahydro-2H-pyran-2-yl) -1H-indazol-3-yl) -3- ((2- (trimethylsilyl) ethoxy) -methyl) -4,5,6, 7-tetrahydro-3H-imidazo [4, 5-c) at 0 deg.C]To a stirred solution of pyridine-6-carboxylic acid (5.7g, 7.75mmol) in 5:1 dioxane: water (60mL) was added concentrated HCl (20mL) dropwise. The reaction mixture was allowed to warm and stirred at 90 ℃ for 16h and distilled under vacuum to give a crude residue which was triturated sequentially with chilled diethyl ether and acetonitrile to give the HCl salt of the title compound as a light brown solid (3.6g, 95% yield). (m/z): c₂₂H₂₀FN₅O₃Of [ M + H]⁺Calculated 422.16, experimental 422.24.¹H NMR(400MHz,D₂O/DMSO-d₆)δ8.22(d,J＝8.4Hz,1H),7.49(s,1H),7.19(d,J＝8.1Hz,1H),6.99(d,J＝11.9Hz,1H),6.91(d,J＝9.0Hz,1H),4.56-4.51(m,1H),4.36(d,J＝15.5Hz,1H),4.30(d,J＝15.5Hz,1H),3.35-3.25(m,1H),3.15-3.05(m,1H),2.4-2.55(m,2H),0.97(t,J＝7.5Hz,3H)。

Preparation 14: (S) -2- (6- (2-ethyl-5-fluoro-4-hydroxyphenyl) -1H-indazol-3-yl) -5-propyl-4, 5,6, 7-tetrahydro-3H-imidazo [4,5-c ] pyridine-6-carboxylic acid

To (S) -2- (6- (2-ethyl-5-fluoro-4-hydroxyphenyl) -1H-indazol-3-yl) -4,5,6, 7-tetrahydro-3H-imidazo [4, 5-c)]Pyridine-6-carboxylic acid, HCl (400mg, 0.874mmol) and propionaldehyde (0.095mL, 1.310mmol) in DMF (7mL) was added sodium cyanoborohydride (165mg, 2.62mmol) and the reaction mixture was stirred at room temperature overnight. Sodium borohydride (33mg, 0.874mmol) was added, the solution was concentrated and purified by preparative HPLC to give a TFA salt of the title compound (179mg, 37% yield) (m/z): c₂₅H₂₆FN₅O₃Of [ M + H]⁺Calculated 464.20, experimental 464.5.

Preparation 15: (S) -2- (6- (2-ethyl-5-fluoro-4-hydroxyphenyl) -1H-indazol-3-yl) -5-isopropyl-4, 5,6, 7-tetrahydro-3H-imidazo [4,5-c ] pyridine-6-carboxylic acid

To (S) -2- (6- (2-ethyl-5-fluoro-4-hydroxyphenyl) -1H-indazol-3-yl) -4,5,6, 7-tetrahydro-3H-imidazo [4, 5-c)]To a solution of pyridine-6-carboxylic acid, HCl (400mg, 0.874mmol), acetone (0.192mL, 2.62mmol) and acetic acid (0.150mL, 2.62mmol) in DMF (7mL) was added sodium cyanoborohydride (274mg, 4.37mmol) and the reaction mixture was stirred at room temperature overnight. Sodium borohydride (33mg, 0.874mmol) was added, the solution was concentrated and purified by preparative HPLC to give the TFA salt of the title compound (115mg, 23% yield). (m/z): c₂₅H₂₆FN₅O₃Of [ M + H]⁺Calculated 464.20, experimental 464.5.

Preparation 16: (S) -2- (6- (2-ethyl-5-fluoro-4-hydroxyphenyl) -1H-indazol-3-yl) -5-methyl-4, 5,6, 7-tetrahydro-3H-imidazo [4,5-c ] pyridine-6-carboxylic acid

To (S) -2- (6- (2-ethyl-5-fluoro-4-hydroxyphenyl) -1H-indazol-3-yl) -4,5,6, 7-tetrahydro-3H-imidazo [4, 5-c)]To a solution of pyridine-6-carboxylic acid, HCl (8') (300mg, 0.655mmol) and (37 wt% formaldehyde in water) (0.059mL, 0.786mmol) DMF (5mL) was added sodium cyanoborohydride (165mg, 2.62mmol) and the reaction mixture was stirred at room temperature overnight. Sodium borohydride (25mg, 0.655mmol) was added and the solution was concentrated and purified by flash chromatography (100g column, 5-75% ACN/water) to give the TFA salt of the title compound (85mg, 24% yield). (m/z): c₂₃H₂₂FN₅O₃Of [ M + H]⁺Calculated 436.17, experimental 436.45.

Example 7: (S) - (2- (6- (2-ethyl-5-fluoro-4-hydroxyphenyl) -1H-indazol-3-yl) -5-propyl-4, 5,6, 7-tetrahydro-3H-imidazo [4,5-C ] pyridin-6-yl) (4- (2-hydroxyethyl) piperazin-1-yl) methanone C-1

To (S) -2- (6- (2-ethyl-5-fluoro-4-hydroxyphenyl) -1H-indazol-3-yl) -5-propyl-4, 5,6, 7-tetrahydro-3H-imidazo [4, 5-c)]Pyridine-6-carboxylic acid, TFA (30mg, 0.052mmol), 2- (piperazin-1-yl) ethanol, 2HCl (0.19mL, 0.156mmol) and DIPEA (0.027mL, 0.156mmol) in DMF (1.5mL) was added HATU (29.6mg, 0.078mmol) and the reaction mixture was stirred at room temperature overnight. Hydrazine (5 equivalents) was added and the reaction mixture was concentrated and purified by preparative HPLC to give the TFA salt of the title compound (15.4mg, 37% yield). (m/z): c₃₁H₃₈FN₇O₃Of [ M + H]⁺Calculated 576.30, experimental 576.2.

Example 8: ((S) -2- (6- (2-ethyl-5-fluoro-4-hydroxyphenyl) -1H-indazol-3-yl) -5-propyl-4, 5,6, 7-tetrahydro-3H-imidazo [4,5-C ] pyridin-6-yl) ((1S,4S) -5-methyl-2, 5-diazabicyclo- [2.2.1] hept-2-yl) methanone C-2

To (S) -2- (6- (2-ethyl-5-fluoro-4-hydroxyphenyl) -1H-indazol-3-yl) -5-propyl-4, 5,6, 7-tetrahydro-3H-imidazo [4, 5-c)]Pyridine-6-carboxylic acid, TFA (30mg, 0.052mmol), (1S,4S) -5-methyl-2, 5-diazabicyclo [2.2.1]To a solution of heptane dihydrobromide (42.7mg, 0.156mmol) and DIPEA (0.064mL, 0.364mmol) in DMF (1.5mL) was added HATU (29.6mg, 0.078mmol) and the reaction mixture was stirred at room temperature overnight. Hydrazine (5 equivalents) was added and the reaction mixture was concentrated and purified by preparative HPLC to give the TFA salt of the title compound (27mg, 66% yield). (m/z): c₃₁H₃₆FN₇O₂Of [ M + H]⁺Calculated 558.29, experimental 558.3.¹H NMR (400MHz, methanol-d)₄)δ8.17(dt,1H),7.59-7.50(m,1H),7.32(dd,1H),6.95(d,1H),6.90(d,1H),5.03-4.91(m,2H),4.56-4.34(m,2H),4.30-3.88(m,4H),3.76-3.55(m,1H),3.28-3.10(m,1H),3.10-2.96(m,4H),2.81-2.62(m,2H),2.53(q,2H),2.47-2.33(m,1H),2.31-2.14(m,1H),1.79-1.57(m,2H),1.07(t,3H),0.97(td,3H)。

Example 9: ((S) -2, 4-dimethylpiperazin-1-yl) ((S) -2- (6- (2-ethyl-5-fluoro-4-hydroxyphenyl) -1H-indazol-3-yl) -5-methyl-4, 5,6, 7-tetrahydro-3H-imidazo [4,5-C ] pyridin-6-yl) methanone C-3

(a) (S) -4- ((S) -2- (6- (2-ethyl-5-fluoro-4-hydroxyphenyl) -1H-indazol-3-yl) -5-methyl-4, 5,6, 7-tetrahydro-3H-imidazo [4,5-c ] pyridine-6-carbonyl) -3-methylpiperazine-1-carboxylic acid tert-butyl ester

Mixing (S) -2- (6- (2-ethyl-5-fluoro-4-hydroxyphenyl) -1H-indazol-3-yl) -5-methyl-4, 5,6, 7-tetrahydro-3H-imidazo [4, 5-c)]Pyridine-6-carboxylic acid (0.120g, 0.276mmol), (S) -4-n-boc-2-methylpiperazine (0.110g, 0.551mmol) and DIPEA (0.096mL, 0.551mmol) were dissolved in DMF (3.0mL), followed by the addition of HATU (0.157g, 0.413mmol) and the reaction mixture was stirred at room temperature for 16 hours (monitoring of reaction progress by LCMS). Hydrazine (0.043mL, 1.378mmol) was added to cleave the undesired by-products, then the reaction mixture was stirred at room temperature for 10 minutes. The reaction mixture was then concentrated and the crude product was purified byPurification by reverse phase chromatography (5-70% ACN/water gradient, 50g C18 column) gave the TFA salt of the title compound (126mg, 62% yield). (m/z): c₃₃H₄₀FN₇O₄Of [ M + H]+ calculated 618.32, experimental 618.3.

(b) ((S) -2- (6- (2-ethyl-5-fluoro-4-hydroxyphenyl) -1H-indazol-3-yl) -5-methyl-4, 5,6, 7-tetrahydro-3H-imidazo [4,5-c ] pyridin-6-yl) ((S) -2-methylpiperazin-1-yl) methanone

Mixing (S) -4- ((S) -2- (6- (2-ethyl-5-fluoro-4-hydroxyphenyl) -1H-indazol-3-yl) -5-methyl-4, 5,6, 7-tetrahydro-3H-imidazo [4, 5-c)]Pyridine-6-carbonyl) -3-methylpiperazine-1-carboxylic acid tert-butyl ester, TFA (0.126g, 0.172mmol) were dissolved in dioxane (1.5mL) and water (0.3mL), followed by addition of hydrochloric acid (4M in dioxane) (1.5mL, 49.4mmol) and stirring of the reaction mixture at room temperature for 1 hour (monitoring of reaction progress by LCMS). The reaction mixture was frozen and lyophilized, and the resulting powder was used directly in the next reaction (assuming quantitative yield). (m/z): c₂₈H₃₂FN₇O₂Of [ M + H]+ calculated 518.26, experimental 518.3.

(c) ((S) -2, 4-dimethylpiperazin-1-yl) ((S) -2- (6- (2-ethyl-5-fluoro-4-hydroxyphenyl) -1H-indazol-3-yl) -5-methyl-4, 5,6, 7-tetrahydro-3H-imidazo [4,5-c ] pyridin-6-yl) methanone

((S) -2- (6- (2-ethyl-5-fluoro-4-hydroxyphenyl) -1H-indazol-3-yl) -5-methyl-4, 5,6, 7-tetrahydro-3H-imidazo [4, 5-c)]Pyridin-6-yl) ((S) -2-methylpiperazin-1-yl) methanone, dihydrochloride (0.102g, 0.173mmol) and formaldehyde solution (37 wt% in water) (0.015mL, 0.207mmol) were dissolved in methanol (3.0mL), followed by addition of sodium cyanoborohydride (0.054g, 0.864mmol) and stirring of the reaction mixture at room temperature for 20 hours (monitoring of reaction progress by LCMS). Sodium borohydride (7mg, 0.173mmol) was added to quench any remaining formaldehyde. The reaction mixture was concentrated, and the crude product was purified by preparative HPLC (5-60% ACN/water gradient, C18 column) to give the title compound as a TFA salt (59mg, 45% yield). (m/z): c₂₉H₃₄FN₇O₂Of [ M + H]+ calculated 532.28, experimental 532.3.

Example 10: (S) - (2- (6- (2-ethyl-5-fluoro-4-hydroxyphenyl) -1H-indazol-3-yl) -5-isopropyl-4, 5,6, 7-tetrahydro-3H-imidazo [4,5-C ] pyridin-6-yl) (4-methyl-1, 4-diazepan-1-yl) methanone C-4

To (S) -2- (6- (2-ethyl-5-fluoro-4-hydroxyphenyl) -1H-indazol-3-yl) -5-isopropyl-4, 5,6, 7-tetrahydro-3H-imidazo [4, 5-c)]Pyridine-6-carboxylic acid, TFA (30mg, 0.052mmol), 1-methyl homopiperazine (0.019mL, 0.156mmol) and DIPEA (0.036mL, 0.208mmol) in DMF (1mL) was added HATU (29.6mg, 0.078mmol) and the reaction mixture was stirred at room temperature for 3 h. Hydrazine (5 equivalents) was added and the reaction mixture was stirred at room temperature for 10min, concentrated and purified by preparative HPLC to give the TFA salt of the title compound (26.9mg, 66% yield). (m/z): c₃₁H₃₈FN₇O₂Of [ M + H]⁺Calculated 560.31, experimental 560.2.

Example 11: ((S) -2- (6- (2-ethyl-5-fluoro-4-hydroxyphenyl) -1H-indazol-3-yl) -5-methyl-4, 5,6, 7-tetrahydro-3H-imidazo [4,5-C ] pyridin-6-yl) ((R) -4- (2-hydroxyethyl) -2-methyl-piperazin-1-yl) methanone C-5

To (S) -2- (6- (2-ethyl-5-fluoro-4-hydroxyphenyl) -1H-indazol-3-yl) -5-methyl-4, 5,6, 7-tetrahydro-3H-imidazo [4, 5-c)]Pyridine-6-carboxylic acid, TFA (30mg, 0.052mmol), (R) -2- (3-methylpiperazin-1-yl) ethanol, 2HCl (35.6mg, 0.164mmol) and DIPEA (0.057mL, 0.328mmol) in DMF (1mL) were added HATU (31.1mg, 0.082mmol) and the reaction mixture was stirred at room temperature overnight. Hydrazine (8.57 μ L, 0.273mmol) was added and the reaction mixture was concentrated and purified by preparative HPLC to give the TFA salt of the title compound (15.6mg, 36% yield). (m/z): c₃₀H₃₆FN₇O₃Of [ M + H]⁺Calculated 562.29, experimental 562.2.

Example 12: ((S) -3- (dimethylamino) pyrrolidin-1-yl) ((S) -5-ethyl-2- (6- (2-ethyl-5-fluoro-4-hydroxyphenyl) -1H-indazol-3-yl) -4,5,6, 7-tetrahydro-3H-imidazo [4,5-C ] pyridin-6-yl) methanone C-6

To (S) -2- (6- (2-ethyl-5-fluoro-4-hydroxyphenyl) -1H-indazol-3-yl) -5-isopropyl-4, 5,6, 7-tetrahydro-3H-imidazo [4, 5-c)]Pyridine-6-carboxylic acid, TFA (179mg, 0.310mmol), (S) -N, N-dimethylpyrrolidin-3-amine (0.079mL, 0.620mmol) and DIPEA (0.162mL, 0.930mmol) in DMF (4mL) was added HATU (177mg, 0.465mmol) and the reaction mixture was stirred at room temperature overnight. Hydrazine (5 equivalents) was added and the reaction mixture was concentrated and purified by preparative HPLC to give the TFA salt of the title compound (107mg, 44% yield). (m/z): c₃₁H₃₈FN₇O₂Of [ M + H]⁺Calculated 560.31, experimental 560.2.¹H NMR (400MHz, methanol-d)₄)δ8.21(d,1H),7.50(s,1H),7.26(d,1H),6.94(d,1H),6.90(d,1H),4.83-4.66(m,1H),4.48-4.25(m,2H),4.23-4.12(m,1H),4.12-3.93(m,2H),3.93-3.63(m,3H),3.62-3.48(m,1H),3.26-3.09(m,1H),2.98(d,6H),2.67-2.57(m,1H),2.53(q,2H),2.44-2.12(m,1H),1.41(t,3H),1.31(d,3H),1.05(t,3H)。

Example 13: (S) - (3- (dimethylamino) azetidin-1-yl) (2- (6- (2-ethyl-5-fluoro-4-hydroxyphenyl) -1H-indazol-3-yl) -5-isopropyl-4, 5,6, 7-tetrahydro-3H-imidazo [4,5-c ] pyridin-6-yl) methanone D-1

To (S) -2- (6- (2-ethyl-5-fluoro-4-hydroxyphenyl) -1H-indazol-3-yl) -5-isopropyl-4, 5,6, 7-tetrahydro-3H-imidazo [4, 5-c)]Pyridine-6-carboxylic acid, TFA (179mg, 0.310mmol), N-dimethylazetidin-3-amine, 2HCl (107mg, 0.465mmol) and DIPEA (0.162mL, 0.930mmol) in DMF (4mL) was added HATU (177mg, 0.465mmol) and the reaction mixture was stirred at room temperature overnight. Add hydrazine (5 eq) and concentrateThe reaction mixture was condensed and purified by preparative HPLC to give a TFA salt of the title compound (63mg, 26% yield). (m/z): c₃₀H₃₆FN₇O₂Of [ M + H]⁺Calculated 546.29, experimental 546.7.¹H NMR(400MHz,DMSO-d₆)δ9.90(s,1H),8.29(dd,1H),7.34(s,1H),7.07(d,1H),7.01(d,1H),6.89(d,1H),4.35-4.18(m,1H),4.11-3.94(m,1H),3.94-3.73(m,3H),3.70-3.57(m,2H),3.06-2.94(m,2H),2.87-2.66(m,2H),2.48-2.40(m,2H),2.13-2.00(m,6H),1.07(t,3H),1.03-0.93(m,6H)。

Biological analysis

The compounds of the invention have been characterized in one or more of the following bioassays.

Analysis 1: biochemical JAK kinase assay

Four LanthaScreen JAK biochemical analysis groups (JAK1, 2,3, and Tyk2) were loaded in common kinase reaction buffer (50mM HEPES, pH 7.5, 0.01% Brij-35, 10mM MgCl)₂And 1mM EGTA). Recombinant GST-tagged JAK enzymes and GFP-tagged STAT1 peptide substrates were obtained from Life Technologies.

Serial dilutions of the compound were preincubated with each of the four JAK enzymes and substrate for 1h in white 384-well microtiter plates (Corning) at ambient temperature. ATP was then added in a total volume of 10 μ Ι _ with 1% DMSO to initiate the kinase reaction. Final enzyme concentrations of JAK1, 2,3 and Tyk2 were 4.2nM, 0.1nM, 1nM and 0.25nM, respectively; the corresponding Km ATP concentrations used were 25. mu.M, 3. mu.M, 1.6. mu.M and 10. mu.M; whereas for all four assays the substrate concentration was 200 nM. The kinase reaction was allowed to proceed for 1 hour at ambient temperature before addition of 10 μ l of a formulation of EDTA (10mM final concentration) and Tb anti-pSTAT 1(pTyr701) antibody (Life Technologies, 2nM final concentration) in TR-FRET dilution buffer (Life Technologies). The discs were incubated at ambient temperature for 1h before reading on an EnVision reader (Perkin Elmer). The emissivity signal (520nm/495nm) was recorded and used to calculate percent inhibition values based on DMSO and background control.

For dose response analysis, percent inhibition data were plotted against compound concentration and with Prism software (Graph)Pad Software) determination of IC according to the 4 parameter robust fitting model₅₀The value is obtained. The results are expressed as pIC₅₀(IC₅₀Negative logarithm of) and then converted to pK using the Cheng-Prusoff equation_i(negative logarithm of dissociation constant Ki).

With Low K in each of the four JAK assays_iValue or high pK_iThe test compounds of value showed greater inhibition of JAK activity.

Analysis 2: inhibition of IL-2 stimulated pSTAT5 in Tall-1T cells

The potency of test compounds to inhibit interleukin-2 (IL-2) -stimulated STAT5 phosphorylation was measured in the Tall-1 human T cell line (DSMZ) using AlphaLisa. Because IL-2 signals through JAK1/3, this assay provides a measure of JAK1/3 cellular potency.

Phosphorylated STAT5 was measured by the AlphaLISA SureFire Ultra pSTAT5(Tyr694/699) kit (PerkinElmer).

Human T cells from the Tall-1 cell line were cultured at 37 ℃ with 5% CO₂Incubate in a humidified incubator with RPMI (Life Technologies) supplemented with 15% heat-inactivated fetal bovine serum (FBS, Life Technologies), 2mM Glutamax (Life Technologies), 25mM HEPES (Life Technologies), and 1 XPen/strep (Life Technologies). Compounds were serially diluted in DMSO and acoustically dispensed into empty wells. Assay medium (phenol red free dmem (life technologies) supplemented with 10% fbs (atcc)) was dispensed (4 μ l/well) and the plates were shaken at 900rpm for 10 minutes. Cells were seeded at 45,000 cells/well in assay medium (4. mu.l/well) and 5% CO at 37 ℃₂Incubation for 1 hour followed by addition of IL-2 (R) -containing&D Systems; final concentration 300ng/mL) was allowed to warm up the assay medium (4 μ L) for 30 minutes. After the cytokine stimulation, cells were lysed with 6 μ l of 3 × AlphaLisa Lysis Buffer (PerkinElmer) containing 1 × PhosStop and Complete tablets (Roche). The lysate was shaken at 900rpm for 10 minutes at Room Temperature (RT). Phosphorylated STAT5 was measured by pSTAT5 AlphaLisa kit (PerkinElmer). In filtering green light<The freshly prepared acceptor bead mixture was dispensed onto the lysate (5. mu.L) under 100lux light. The pan was shaken for 2 minutes at 900rpm, simply quicklyCentrifuge rapidly and incubate at room temperature for 2 hours in the dark. In filtering green light<Donor beads (5. mu.L) were dispensed under 100lux light. The plates were shaken at 900rpm for 2 minutes, briefly centrifuged quickly and incubated overnight at room temperature in the dark. In filtering green light<Luminescence was measured at 689nm excitation and 570nm emission using an EnVision disk reader (PerkinElmer) under 100lux light.

To determine the inhibitory potency of test compounds in response to IL-2, the mean emission intensity of beads bound to pSTAT5 was measured in human T cell lines. Determination of IC from inhibition curves of assay Signal intensity versus Compound concentration₅₀The value is obtained. Data are expressed as pIC₅₀(negative decimal logarithm IC)₅₀) Values (mean ± standard deviation).

Results of in vitro analysis

TABLE 1

Analysis 3: IL-13 induced pSTAT6 induced murine (mouse) model in pulmonary tissue

IL-13 is an important cytokine potential in the pathophysiology of asthma (Kudlacz et al, J.Eur. Pharmacol, 2008,582, 154-161). IL-13 binds to cell surface receptors, activating members of the janus family of kinases (JAKs), which in turn phosphorylate STAT6 and subsequently further activate the transcriptional pathway. In the described model, doses of IL-13 were delivered locally into the mouse lungs to induce phosphorylation of STAT6 (pSTAT6), which was then measured as an endpoint.

Adult Balb/c mice from Harlan were used in the assay. On the day of the study, animals were lightly anesthetized with isoflurane and vehicle or test compound (1mg/mL, 50 μ L total volume, over several breaths) was administered by oral suction. After dosing, animals were placed on their side and monitored for complete recovery from anesthesia before returning to their home cages. Four hours later, the animals were briefly anesthetized again and vehicle or IL was administered by oral suction before monitoring recovery from anesthesia and returning to their home cages13 (total delivered dose of 0.03 μ g, total volume of 50 μ L). One hour after vehicle or IL-13 administration, Perkin Elmer was used for two pSTAT6 assays in lung homogenateUltra^TMHV p-STAT6(Tyr641) assay kit and whole blood and lungs were collected for total drug concentration analysis in both lung and plasma. Blood samples were centrifuged (Eppendorf centrifuge, 5804R) at about 12,000rpm for 4 minutes at4 ℃ to collect plasma. The lungs were rinsed with Dulbecco's Phosphate-Buffered Saline (DPBS), dried on pad, flash-frozen, weighed and homogenized in 1:3 dilution in HPLC water containing 0.1% formic acid. Plasma and lung levels of test compounds were determined by LC-MS analysis, against analytical standards constructed as standard curves in a test matrix. The lung to plasma ratio was determined as the ratio of lung concentration (in ng/g) to plasma concentration (in ng/mL) at5 hours.

Activity in the model was demonstrated by a reduction in pSTAT6 levels present in the lungs of treated animals at5 hours compared to vehicle-treated, IL-13-stimulated control animals. Differences between vehicle-treated, IL-13-stimulated control animals and vehicle-treated, vehicle-stimulated control animals indicate 0% and 100% inhibition, respectively, in any given experiment. The compounds tested in the assay exhibited inhibition of STAT6 phosphorylation at5 hours after IL-13 stimulation as noted below.

Table 2: observed inhibition of pSTAT6 and plasma/lung exposure

Observation of significant concentrations of the tested compounds in the mouse lungs confirmed that IL-13-induced observed inhibition of pSTAT 6-induced was the result of the activity of the test compounds. The lung to plasma ratio at5 hours showed that compounds 1 to 6 exhibited significantly more exposure in the lung than plasma in the mice.

Analysis 4: inhibition of TSLP-induced TARC release in human peripheral blood mononuclear cells

Thymic Stromal Lymphopoietin (TSLP) and thymus and activation-regulated chemokines (TARC) are over-expressed in asthmatic airways and are associated with disease severity. In the lung, TSLP can be released by bronchial epithelial cells in response to allergen and viral infections. TSLP signaling through the IL-7R α/TSLPR heterodimer is found in a wide range of tissues and cell types, including epithelial cells, endothelial cells, neutrophils, macrophages, and mast cells. Binding of TSLP to its receptor induces conformational changes that activate JAK1 and JAK2 to phosphorylate a variety of transcription factors, including STAT3 and STAT 5. In immune cells, this triggers a cascade of intracellular events that lead to cell proliferation, anti-apoptosis, dendritic cell migration, and the production of Th2 interleukins and chemokines. In Peripheral Blood Mononuclear Cells (PBMC), TSLP has a pro-inflammatory effect by activating myeloid dendritic cells to attract and stimulate T cells (a method mediated by the chemoattractant TARC).

In this assay, it was shown that TSLP stimulation elicits the release of TARC from PBMCs, and that this response decays in a dose-dependent manner when treated with compounds. The potency of the test compound was measured against inhibition of TARC release.

Aliquots of PBMCs from 3 to 5 donors (previously isolated from whole blood and frozen into aliquots at-80 ℃) were thawed at 37 ℃ and added dropwise to 50mL Falcon tubes containing 40mL of pre-warmed, sterile-filtered, intact RPMI medium. The cells were pelleted and pelleted at 2.24X 10⁶Individual cells/ml were resuspended in complete medium. Cells were seeded at 85 μ L (190,000 cells) per well in tissue culture-treated 96-well flat-bottom microtiter plates. Cells were incubated at 37 ℃ with 5% CO₂The mixture was left standing for 1 hour.

Compounds were received as 10mM stock solutions in DMSO. Serial dilutions 3.7-fold were performed to generate 9 concentrations of test compound in DMSO at 300 x final assay test concentration. 150-fold intermediate dilution in complete medium to yield a final dilution at 2 ×Compounds were assayed at the test concentration (0.2% DMSO). After a1 hour resting period, 95 μ L of 2 × compound was added to each well of PBMCs, with final assay concentrations ranging from 33.33 μ M to 0.95 μ M. 95 μ L of complete medium containing 0.2% DMSO was added to untreated control wells. Cells were stimulated at 37 ℃ with 5% CO₂Next, the mixture was pretreated with a compound for 1 hour.

Recombinant human TSLP protein was reconstituted at 10 μ g/mL in sterile DPBS with 0.1% BSA and stored as aliquots at-20 ℃. Aliquots were thawed immediately prior to use and prepared at 20 × final assay concentration in complete media. mu.L of 20 XTSLP was added to each well of PBMC at a final assay concentration of 10 ng/mL. 10 μ L of complete medium was added to unstimulated control wells. At 37 ℃ with 5% CO₂Next, cells were stimulated in the presence of the compound for 48 hours.

After stimulation, cell culture supernatants were harvested and TARC levels were detected by enzyme-linked immunosorbent assay (ELISA) using the human CCL17/TARC Quantikine ELISA Kit (R & D Systems # DDN00) according to the manufacturer's instructions.

For dose response analysis, log is plotted against the percent response value for each donor [ test compound (M)]And IC was determined using nonlinear regression analysis using a 4-parameter sigmoidal dose-response algorithm with variable slope using GraphPad Prism software₅₀The value is obtained. Data are expressed as pIC from individual donors₅₀Average pIC of value calculation₅₀(negative decimal logarithm IC)₅₀) Value and rounded to a decimal place. The inhibitory potency values using the original compound and its analogs modified with anti-fluoro are summarized in table 3.

Table 3: potency of test Compounds to inhibit TSLP-induced TARC Release in human peripheral blood mononuclear cells

Compound (I)	pIC 50. + -. standard deviation
		1	7.1±0.1
C-1	7.0±0.3
		2	6.8±0.2
C-2	6.6±0.2
		3	7.3±0.2
C-3	7.0±0.1
		4	6.8±0.1
C-4	7.0±0.4
		5	7.5±0.6
C-5	7.3±0.1
		6	6.8±0.2
C-6	6.8±0.2

Analysis 5: lung S9 metabolism

The in vitro metabolic stability of compounds 1 to 6 and C-1 to C-6 was evaluated in a human lung S9 fragment (1. mu.M compound; 1mg/mL S9 protein). Samples at time 0, 15, 30 and 60min were analyzed by high resolution LC-MS/MS for the parent compound. Lung S9 fragment from human (batch 1410245) was purchased from XenoTech LLC (Lenexa, KS). NADPH (Sigma Aldrich, N1630) and adenosine 3-phosphate 5-phosphosulfate (PAPS) (Sigma Aldrich, a1651) were purchased from Sigma Aldrich (st. Acetonitrile and water were obtained from VWR (Radnor, PA) and had HPLC grade or better. Ranoxiphenol (Raloxifene) and formic acid were purchased from Sigma Aldrich (St. Louis, Mo.). Lung S9 incubations were performed in a water bath at 37 ℃ in 96-well polypropylene plates. Lung S9 solution consisted of 100mM potassium phosphate supplemented with 1mM NADPH (Sigma-Aldrich, st. louis, MO), 3mM magnesium chloride (Sigma Aldrich, M1028) buffered to pH 7.4(BD Biosciences, Woburn, MA) and with a final incubation protein concentration of 1mg/mL in the presence of 100 μ M PAPS (Sigma-Aldrich, st. louis, MO) cofactor. Stock solutions of raloxifene (n ═ 1) and compound (n ═ 1) in 10mM DMSO were diluted in buffer and added to the incubation to give a1 μ M substrate concentration (0.001% DMSO v/v). The incubation volume consisted of 400 μ Ι _ and time points were obtained at 0, 15, 30 and 60 minutes by removing 70 μ Ι _ aliquots and diluting into 140 μ Ι _, acetonitrile (0% formic acid). All samples were centrifuged at 2250g for 10min at5 ℃. The supernatant (50 μ L) was taken from the centrifuged sample and diluted into 100 μ L HPLC water containing the internal standard. Samples were run on a Dionex Ultimate 3000 autosampler and analyzed in full scan mode using a Thermo Q-active High Resolution mass spectrometer (Thermo, Waltham, MA) in conjunction with an Atlantis T3 column 3 μ M-2.1X 50mm (Waters Inc., 186003717). Mobile phase a consisted of water + 0.2% formic acid and mobile phase B consisted of acetonitrile + 0.2% formic acid. Peak integration was performed using Gubbs GMSU software (Gubbs inc., Alpharetta, GA). For each sample, the peak area ratio was calculated by dividing the analyte peak area by the internal standard peak area. For each incubation, the peak area ratio of the analyte in each t0 was set to 100%, and the peak area ratio from the 60min sample was converted to the remaining percentage relative to the corresponding t 0. The determination of sulfate metabolite formation is performed qualitatively by observing early elution peaks in the parent ion channels corresponding to the O-sulfate metabolites of each parent compound based on historical internal data. The results of the analysis are summarized in table 4 (n ═ 2 replicates).

Table 4: metabolic stability in human lung fragment S9

Compounds 1 to 6 caused a significant reduction in sulfated metabolism in the lung fragment S9 when compared to their corresponding fluoro analogs (compounds C-1 to C-6).

Analysis 6: pharmacokinetics in plasma and lung in mice

Plasma and lung concentrations of test compounds and ratios thereof were determined in the following manner. BALB/c mice from Charles River Laboratories were used in the analysis. Test compounds were individually formulated at a concentration of 0.2mg/mL in pH 4 citrate buffer containing 20% propylene glycol, and 50 μ Ι _ of dosing solution was introduced into the trachea of mice by oral aspiration. At various time points (typically 0.167, 2,6, 24 hours) post-dose, blood samples were removed by cardiac puncture and the intact lungs were excised from the mice. Blood samples were centrifuged (Eppendorf centrifuge, 5804R) at about 12,000rpm for 4 minutes at4 ℃ to collect plasma. The lungs were plug dried, weighed and homogenized in sterile water at a 1:3 dilution. Plasma and lung concentrations of test compounds were determined by LC-MS analysis, against analytical standards constructed as standard curves in a test matrix. The lung to plasma ratio is determined as the ratio of the lung AUC (in μ g hr/g) to the plasma AUC (in μ g hr/mL), where AUC is routinely defined as the area under the curve of test compound concentration versus time.

Table 5: plasma and lung tissue exposure following single oral suction administration of test compounds

Analysis 7: IL-5 mediated eosinophil survival assay

The potency of test compounds on IL-5 mediated eosinophil survival was measured in human eosinophils isolated from human whole blood (AllCells). Because IL-5 signals through JAKs, this assay provides a measure of JAK cell potency.

Human eosinophils were isolated from fresh human whole blood (AllCells) from healthy donors. Blood was mixed with 4.5% polydextrose (Sigma-Aldrich) in 0.9% sodium chloride solution (Sigma-Aldrich). The red blood cells were left to settle for 35 minutes. The upper layer, which is rich in leukocytes, is removed and layered on a Ficoll-Paque (GE healthcare) and centrifuged at 600g for 30 min. The plasma and monocyte layers were removed prior to lysing the granulocyte layer with water to remove any contaminating red blood cells. Eosinophils were further purified using a human eosinophil isolation kit (Miltenyi Biotec). A portion of the purified eosinophils was incubated with anti-CD 16 FITC (Miltenyi Biotec) in the dark at4 ℃ for 10 minutes. Purity was analyzed using a LSRII flow cytometer (BD Biosciences).

Cells were incubated at 37 ℃ with 5% CO₂Incubate in a humidified incubator with RPMI 1640(Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Life Technologies), 2mM Glutamax (Life Technologies), 25mM HEPES (Life Technologies), and 1 XPen/strep (Life Technologies). Cells were seeded at 10,000 cells/well in culture medium (50 μ L). The dish was centrifuged at 300g for 5 minutes and the supernatant removed. Compounds were serially diluted in DMSO and then diluted an additional 500-fold in culture medium to reach 2x final assay concentration. Test compounds (50. mu.l/well) were added to the cells and 5% CO at 37 ℃₂Incubation for 1 hour followed by addition of IL-5 (R) -containing&D Systems; final concentrations 1ng/mL and 10pg/mL) of pre-warmed assay medium (50 μ L) for 72 hours.

After the interleukin stimulation, cells were centrifuged at 300g for 5min and washed twice with cold dpbs (life technologies). To obtain viability and apoptosis, cells were incubated with propidium iodide (Thermo Fisher Scientific) and APC phospholipid binding protein v (BD Biosciences) and analyzed using an LSRII flow cytometer (BD Biosciences). Determination of IC from analysis of the viability Curve of% cell viability vs. Compound concentration₅₀The value is obtained. Data are expressed as pIC₅₀(negative decimal logarithm IC)₅₀) The value is obtained.

Analysis 8: inhibition of IFN γ and IL-27 induced chemokines CXCL9 and CXCL10 in human 3D tracheal cultures

EpiAirway tissue cultures can be obtained from Mattek (AIR-100). Cultures were derived from asthma donors. In the cell culture insert, human-derived tracheal/bronchial epithelial cells are grown and differentiated on a porous membrane support such that the warmed medium beneath the cells has a gas-liquid interface with the gaseous test atmosphere described above. At 37 ℃ 5% CO₂Tissues were cultured in maintenance medium (Mattek, AIR-100-MM) in a humidified incubator. Four donors can be tested. On day 0, the tissue culture is treated with test compound at 10 μ M,1 μ M and/or 0.1 μ M. Compounds were diluted in dimethyl sulfoxide (DMSO, Sigma) to a final concentration of 0.1%. 0.1% DMSO can be used as vehicle control. Test compounds were incubated with the cultures at 37 ℃ with 5% CO₂Incubation for 1 hour followed by addition of IFN γ (R) at a final concentration of 100ng/mL&D Systems) or IL-27 (R)&D Systems). The tissue culture was maintained for 8 days. The medium was replaced every 2 days with fresh medium containing the compound and either IFN γ or IL-27. On day 8, tissue cultures and supernatants were collected for analysis. Supernatant samples were analyzed using flow fluorometry analysis (EMD Millipore) for CXCL10(IP-10) and CXCL9 (MIG). Data are expressed as% inhibition +/-standard deviation (± STDV). Percent inhibition was determined using the inhibitory potency of the compounds on IFN γ or IL-27 induced CXCL10 or CXCL9 secretion compared to vehicle treated cells. Data are mean values from 3 or 4 donors.

Analysis 9: cellular JAK potency assay: inhibition of IL-2/anti-CD 3 stimulated IFN γ in human PBMC

The potency of a test compound to inhibit interleukin-2 (IL-2)/anti-CD 3-stimulated interferon gamma (IFN γ) can be measured in human Peripheral Blood Mononuclear Cells (PBMCs) isolated from human whole Blood (Stanford Blood Center). Because IL-2 signals through JAKs, this assay provides a measure of JAK cell potency.

(1) Human Peripheral Blood Mononuclear Cells (PBMCs) were isolated from human whole blood of healthy donors using ficoll gradients. Cells were incubated at 37 ℃ with 5% CO₂Incubate in a humidified incubator with RPMI (Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Life Technologies), 2mM Glutamax (Life Technologies), 25mM HEPES (Life Technologies), and 1 XPen/strep (Life Technologies). Cells were seeded at 200,000 cells/well in medium (50 μ L) and cultured for 1 hour. Compounds were serially diluted in DMSO and then diluted an additional 500-fold in culture medium (to 2x final assay concentration). Test compounds (100. mu.l/well) were added to the cells and 5% CO at 37 ℃₂Incubation for 1h followed by addition of IL-2 (R) -containing&D Systems; final concentration 100ng/mL) and anti-CD 3(BD Biosciences; final concentration 1 μ g/mL) of pre-warmed assay medium (50 μ L) for 24 h.

(2) After the cytokine stimulation, the cells were centrifuged at 500g for 5min and the supernatant was removed and frozen at-80 ℃. To determine the inhibitory potency of test compounds in response to IL-2/anti-CD 3, ELISA (R) was performed&D Systems) the supernatant IFN γ concentration was measured. Determination of IC based on analysis of inhibition curves of IFN γ concentration versus Compound concentration₅₀The value is obtained. Data are expressed as pIC₅₀(negative decimal logarithm IC)₅₀) The value is obtained.

Analysis 10: cellular JAK potency assay: inhibition of IL-2 stimulated pSTAT5 in CD4+ T cells

The efficacy of test compounds to inhibit interleukin-2 (IL-2)/anti-CD 3-stimulated STAT5 phosphorylation was measured in CD4 positive (CD4+) T cells in human Peripheral Blood Mononuclear Cells (PBMCs) isolated from human whole Blood (Stanford Blood Center) using flow cytometry. Because IL-2 signals through JAKs, this assay provides a measure of JAK cell potency.

CD4+ T cells were identified using Phycoerythrin (PE) -conjugated anti-CD 4 antibody (inbred RPA-T4, BD Biosciences), while Alexa Fluor 647-conjugated anti-pSTAT 5 antibody (pY694, inbred 47, BD Biosciences) was used to detect STAT5 phosphorylation.

(1) The protocol of paragraph 9 (1) was followed except that the interleukin stimulation with anti-CD 3 was performed for 30min instead of 24 h.

(2) After the cytokine stimulation, the cells were treated with a pre-warmed fixative solution (200. mu.L; BD Biosciences) at 37 ℃ with 5% CO₂Following fixation for 10min, washed twice with DPBS Buffer (1mL, Life Technologies) and resuspended in ice-cold Perm Buffer III (1000. mu.L, BD Biosciences) for 30min at4 ℃. Cells were washed twice with DPBS (FACS buffer) containing 2% FBS and then resuspended in FACS buffer (100 μ L) containing anti-CD 4 PE (1:50 fold dilution) and anti-CD 3 anti-CD 3 Alexa Fluor 647(1:5 fold dilution) at room temperature in the dark for 60 min. After incubation, cells were washed twice with FACS buffer before analysis using a LSRII flow cytometer (BD Biosciences). To determine the inhibitory potency of test compounds in response to IL-2/anti-CD 3, the Median Fluorescence Intensity (MFI) of pSTAT5 was measured in CD4+ T cells. Determination of IC from analysis of inhibition curves of MFI vs. Compound concentration₅₀The value is obtained. Data are expressed as pIC₅₀(negative decimal logarithm IC)₅₀) The value is obtained.

Analysis 11: cellular JAK potency assay: inhibition of IL-4 stimulated pSTAT6 in CD3+ T cells

The efficacy of test compounds to inhibit interleukin-4 (IL-4) -stimulated STAT6 phosphorylation was measured in CD3 positive (CD3+) T cells in human Peripheral Blood Mononuclear Cells (PBMCs) isolated from human whole Blood (Stanford Blood Center) using flow cytometry. Because IL-4 signals through JAKs, this assay provides a measure of JAK cell potency.

CD3+ T cells were identified using Phycoerythrin (PE) -conjugated anti-CD 3 antibodies (pure line UCHT1, BD Biosciences), while Alexa Fluor 647-conjugated anti-pSTAT 6 antibodies (pY641, pure line 18/P, BD Biosciences) were used to detect STAT6 phosphorylation.

Human Peripheral Blood Mononuclear Cells (PBMCs) were isolated from human whole blood of healthy donors as in assays 9 and 10. Cells were seeded at 250,000 cells/well in culture medium (200 μ L), cultured for 1h and then resuspended in assay medium (50 μ L) containing various concentrations of test compound (RPMI supplemented with 0.1% bovine serum albumin (Sigma), 2mM Glutamax, 25mM HEPES and 1 × penstrup). Compounds were serially diluted in DMSO and then diluted an additional 500-fold in assay medium (to 2 × final assay concentration). Test compounds (50. mu.L) were mixed with cells at 37 ℃ with 5% CO₂Incubate for 1h, followed by addition of IL-4 (50. mu.L) (R) in pre-warmed assay medium&D Systems; final concentration 20ng/mL) for 30 min. After interleukin stimulation, cells were plated with pre-warmed fixative solution (100 μ L) (BD Biosciences) at 37 ℃ with 5% CO₂Following fixation for 10min, washed twice with DPBS Buffer (1mL) (DPBS containing 2% FBS) and resuspended in ice-cold Perm Buffer III (1000. mu.L) (BD Biosciences) for 30min at4 ℃. Cells were washed twice with FACS buffer and then resuspended in FACS buffer (100 μ L) containing anti-CD 3 PE (1:50 dilution) and anti-pSTAT 6 Alexa Fluor 647(1:5 dilution) for 60min at room temperature in the dark. After incubation, cells were washed twice with FACS buffer before analysis using a LSRII flow cytometer (BD Biosciences).

To determine the inhibitory potency of test compounds in response to IL-4, Median Fluorescence Intensity (MFI) of pSTAT6 was measured in CD3+ T cells. Determination of IC from analysis of inhibition curves of MFI vs. Compound concentration₅₀The value is obtained. Data are expressed as pIC₅₀(negative decimal logarithm IC)₅₀)。

Analysis 12: cellular JAK potency assay: inhibition of IL-6 stimulated pSTAT3 in CD3+ T cells

A protocol similar to assay 11 was used to determine the potency of test compounds to inhibit interleukin-6 (IL-6) -stimulated STAT3 phosphorylation. Alexa Fluor 647 conjugated anti-pSTAT 3 antibody (pY705, inbred 4/P, BD Biosciences) was used to detect STAT3 phosphorylation.

Analysis 13: mouse model of Alternaria alternata (Alternaria alternata) induced eosinophilic globular inflammation of the lung

Hypereosinophilic tracheitis is the hallmark of human asthma. Alternaria alternata is a fungal aeroallergen that can aggravate asthma in humans and induce eosinophilic coccal inflammation in the lungs of mice (Harvard (Havaux), et al, "clinical and Experimental immunology (Clin Exp Immunol.) -2005-2; 139(2): 179-88). In mice, it has been demonstrated that alternaria indirectly activates tissue-resident type 2 innate lymphocytes in the lung, which react (e.g., IL-2 and IL-7) and release JAK-dependent interleukins (e.g., IL-5 and IL-13) and mediate eosinophilic inflammation (battemes et al, journal of immunology (J Immunol.) 2012, 2 months 1, 188(3): 1503-13).

Seven to nine week old male C57 mice from Taconic were used in the study. On the day of the study, animals were lightly anesthetized with isoflurane and vehicle or test compound (0.03-1.0mg/mL, 50 μ L total volume, over several breaths) was administered by oropharyngeal suction. After dosing, animals were placed on their side and monitored for complete recovery from anesthesia before returning to their home cages. One hour later, the animals were briefly anesthetized again and stimulated by oropharyngeal suction with vehicle or alternaria extract (200 μ g total delivered extract, 50 μ L total volume) before monitoring recovery from anesthesia and returning to their home cages. Forty-eight hours after alternaria administration, bronchoalveolar lavage fluid (BALF) was collected and eosinophils were counted in BALF using the Advia 120Hematology System (Siemens). Activity in the model is evidenced by a decreased level of eosinophils present in BALF of treated animals at forty-eight hours as compared to vehicle-treated, alternaria-stimulated control animals. Data are expressed as percent inhibition of BALF eosinophil response by vehicle treatment, alternaria stimulation. To calculate percent inhibition, the number of BALF eosinophils for each condition was converted to the percent of BALF eosinophils that were averaged vehicle-treated, alternaria-stimulated and subtracted by one hundred percent.

Analysis 14: cellular JAK potency assay: inhibition of IFN gamma-induced pSTAT1

The efficacy of test compounds to inhibit interferon gamma (IFN γ) -stimulated STAT1 phosphorylation was measured in CD14 positive (CD14+) mononuclear spheres derived from human whole Blood (Stanford Blood Center) using flow cytometry. Because IFN γ signals through JAKs, this assay provides a measure of JAK cell potency.

Monoribosomes were identified using Fluorescein Isothiocyanate (FITC) -conjugated anti-CD 14 antibody (inbred RM052, Beckman Coulter) and Alexa Fluor 647-conjugated anti-pSTAT 1 antibody (pY701, inbred 4a, BD Biosciences) for detection of STAT1 phosphorylation.

Human Peripheral Blood Mononuclear Cells (PBMCs) were isolated from human whole blood of healthy donors using a ficoll gradient. Cells were incubated at 37 ℃ with 5% CO₂Incubate in wet air incubators in RPMI (Life Technologies) supplemented with 10% fetal bovine serum (FBS, Life Technologies), 2mM Glutamax (Life Technologies), 25mM HEPES (Life Technologies), and 1 XPen/strep (Life Technologies). Cells were seeded at 250,000 cells/well in culture medium (200 μ L), cultured for 2h and resuspended in assay medium (50 μ L) containing various concentrations of test compound (RPMI supplemented with 0.1% bovine serum albumin (Sigma), 2mM Glutamax, 25mM HEPES and 1 × penstrup). Compounds were serially diluted in DMSO and then diluted an additional 1000-fold in culture medium to bring the final DMSO concentration to 0.1%. Test compound dilutions were incubated with cells at 37 ℃ in 5% CO₂Incubation for 1h followed by addition of IFN γ (R) with pre-warmed heat at a final concentration of 0.6ng/mL&D Systems) for 30 min. After interleukin stimulation, cells were plated with pre-warmed fixative solution (100 μ L) (BD Biosciences) at 37 ℃ with 5% CO₂Following fixation for 10min, washed twice with FACS buffer (1mL) (1% BSA in PBS), resuspended in 1:10 anti-CD 14 FITC: FACS buffer (100. mu.L), and incubated at4 ℃ for 15 min. Cells were washed once and then resuspended in ice-cold Perm Buffer III (BD Biosciences) (100 μ L) for 30min at4 ℃. Cells were washed twice with FACS buffer and then resuspended in 1:10 anti-pSTAT 1 Alexa Fluor 647 FACS buffer (100 μ L) for 30min at room temperature in the dark, washed twice with FACS buffer, and run using a MACSQuuant flow cytometer (BD Biosci)ens) analysis.

To determine the inhibitory potency of the test compounds, Median Fluorescence Intensity (MFI) of pSTAT1 was measured in CD14+ monocytes. Determination of IC from analysis of inhibition curves of MFI vs. Compound concentration₅₀The value is obtained. Data are expressed as pIC₅₀(negative decimal logarithm IC)₅₀) The value is obtained. Compound 1 exhibited a pIC of 7.5 in this assay₅₀The value is obtained. Compound 4 exhibited a pIC of 7.3 in this assay₅₀The value is obtained.

Analysis 15: cellular JAK potency assay: GM-CSF-induced inhibition of pSTAT5

The efficacy of test compounds to inhibit granulocyte-macrophage colony stimulating factor (GM-CSF) -stimulated STAT5 phosphorylation was measured in CD14 positive (CD14+) mononuclear spheres derived from human whole Blood (Stanford Blood Center) using flow cytometry. Because GM-CSF signals through JAKs, this assay provides a measure of JAK cell potency.

The monocytes were identified using Fluorescein Isothiocyanate (FITC) -conjugated anti-CD 14 antibody (pure line RM052, Beckman Coulter) and Alexa Fluor 647-conjugated anti-pSTAT 5 antibody (pY694, BD Biosciences) for detection of STAT5 phosphorylation.

Human Peripheral Blood Mononuclear Cells (PBMCs) were isolated from human whole blood of healthy donors using a ficoll gradient. Cells were incubated at 37 ℃ with 5% CO₂Incubate in wet air incubators in RPMI (Life Technologies) supplemented with 10% fetal bovine serum (FBS, Life Technologies), 2mM Glutamax (Life Technologies), 25mM HEPES (Life Technologies), and 1 XPen/strep (Life Technologies). Cells were seeded at 250,000 cells/well in culture medium (200 μ L), cultured for 2h and resuspended in assay medium (50 μ L) containing various concentrations of test compound (RPMI supplemented with 0.1% bovine serum albumin (Sigma), 2mM Glutamax, 25mM HEPES and 1 × penstrup). Compounds were serially diluted in DMSO and then diluted an additional 1000-fold in culture medium to bring the final DMSO concentration to 0.1%. Test compound dilutions were incubated with cells at 37 ℃ in 5% CO₂Incubate for 1h, followed by addition of pre-warmed GM-CSF (R) at a final concentration of 0.3ng/mL&D Systems) for 15 min. In cellsFollowing interleukin stimulation, cells were plated with pre-warmed fixative solution (100 μ L) (BD Biosciences) at 37 ℃ with 5% CO₂Following fixation for 10min, washed twice with FACS buffer (1mL) (1% BSA in PBS), resuspended in 1:10 anti-CD 14 FITC: FACS buffer (100. mu.L), and incubated at4 ℃ for 15 min. Cells were washed once and then resuspended in ice-cold Perm Buffer III (BD Biosciences) (100 μ L) for 30min at4 ℃. Cells were washed twice with FACS buffer and then resuspended in 1:10 anti-pSTAT 1 Alexa Fluor 647 FACS buffer (100 μ L) for 30min at room temperature in the dark, washed twice with FACS buffer, and analyzed using a macSQurant flow cytometer (Miltenyi).

To determine the inhibitory potency of the test compounds, Median Fluorescence Intensity (MFI) of pSTAT5 was measured in CD14+ monocytes. Determination of IC from analysis of inhibition curves of MFI vs. Compound concentration₅₀The value is obtained. Data are expressed as pIC₅₀(negative decimal logarithm IC)₅₀) The value is obtained. Compound 1 exhibited a pIC of 6.7 in this assay₅₀The value is obtained. Compound 4 exhibited a pIC of 6.7 in this assay₅₀The value is obtained.

Analysis 16: cellular JAK potency assay: IL-12 induced inhibition of pSTAT4

The potency of test compounds to inhibit interleukin-12 (IL-12) -stimulated STAT4 phosphorylation was measured in CD3 positive (CD3+) T cells derived from human whole Blood (Stanford Blood Center) using flow cytometry. Because IL-12 signals through JAKs, this assay provides a measure of JAK cell potency.

CD3+ T cells were identified using Phycoerythrin (PE) -conjugated anti-CD 3 antibodies (inbred UCHT1, BD Biosciences) and Alexa Fluor 647-conjugated anti-pSTAT 4 antibodies (inbred 38/p-Stat4, BD Biosciences) for detection of Stat4 phosphorylation.

Human Peripheral Blood Mononuclear Cells (PBMCs) were isolated from human whole blood of healthy donors using a ficoll gradient. Cells were incubated at 37 ℃ with 5% CO₂Purified anti-CD 3 disc-bound antibody in a humidified incubator supplemented with 10% fetal bovine serum (FBS, Life Technologies), 2mM Glutamax (Life Technologies), 25mM HEPES (Life Technologies), 1 XPen/strep (Life Technologies), disc-bound antibody(5. mu.g/ml, inbred UCHT1, BD Biosciences) and soluble anti-CD 28 antibody (1. mu.g/ml, inbred CD28.2, BD Biosciences) in RPMI (Life technologies) for 3 days. Cells were harvested, washed with medium and then resuspended in a medium containing interleukin-2 (IL-2, 10ng/ml, R)&D Systems). Cells were incubated at 37 ℃ with 5% CO₂Incubate in a humidified incubator for 3 days. Cells were harvested, washed with RPMI and seeded at 250,000 cells/well in culture medium (200 μ L), cultured for 2h and resuspended in assay medium (50 μ L) containing various concentrations of test compound (RPMI supplemented with 0.1% bovine serum albumin (Sigma), 2mM Glutamax, 25mM HEPES and 1 × penstrup). Compounds were serially diluted in DMSO and then diluted an additional 1000-fold in culture medium to bring the final DMSO concentration to 0.1%. Test compound dilutions were incubated with cells at 37 ℃ in 5% CO₂Next, incubation was carried out for 1h, followed by addition of IL-12 (R) containing prewarming heat at a final concentration of 10ng/mL&D Systems) for 30 min. After interleukin stimulation, cells were plated with pre-warmed fixative solution (100 μ L) (BD Biosciences) at 37 ℃ with 5% CO₂Following fixation for 10min, washed twice with FACS Buffer (1mL) (PBS containing 1% BSA) and resuspended in ice-cold Perm Buffer III (1000. mu.L) (BD Biosciences) for 30min at4 ℃. Cells were washed twice with FACS buffer and then resuspended in FACS buffer (100 μ L) containing anti-CD 3 PE (1:50 fold dilution) and anti-pSTAT 4 Alexa Fluor 647(1:10 fold dilution) for 45min at room temperature in the dark. After incubation, cells were washed twice with FACS buffer before analysis using a macSQurant flow cytometer (Miltenyi). To determine the inhibitory potency of the test compounds, Median Fluorescence Intensity (MFI) of pSTAT4 was measured in CD3+ T cells. Determination of IC from analysis of inhibition curves of MFI vs. Compound concentration₅₀The value is obtained. Data are expressed as pIC₅₀(negative decimal logarithm IC)₅₀) The value is obtained. Compound 1 exhibited a pIC of 6.2 in this assay₅₀The value is obtained. Compound 4 exhibited a pIC of 6.0 in this assay₅₀The value is obtained.

Crystal structure

To be provided withThe co-crystal structure of compound D-1 bound to human JAK1 was obtained. Binding of the ligand to the ATP binding site was observed. Based on between donor and acceptor atomsOr smaller distances, identify seven specific hydrogen bonding interactions. It should be especially noted that hydrogen bonding interactions were identified between the carbonyl group of the exocyclic amide of the compound of D-1 and the side chain of Arg879 of JAK 1. Similar interactions can be expected for the compounds of the invention. In early modeling studies, this interaction has been proposed as a way to provide selectivity for JAK1 over other tyrosine kinases, since otherwise closely related kinases (e.g., TRKA, VEGFR, ABL1) do not have an arginine residue at equivalent positions. The observation of hydrogen bonding interactions in the crystal structure and the selectivity of the modified kinase group compared to the series without the cyclic exoamide confirm this design hypothesis.

While the invention has been described with reference to specific aspects or embodiments thereof, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. Moreover, to the extent permitted by applicable patent statutes and laws, all publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety as if each document were individually incorporated by reference.