EA040902B1 - 5-7-MEMBERED HETEROCYCLIC AMIDES AS JAK INHIBITORS - Google Patents

Description

The field of technology to which the invention belongs

The invention relates to compounds which are useful as Janus kinase (JAK) inhibitors. The invention also relates to pharmaceutical compositions containing such compounds and methods of using such compounds for the treatment of respiratory diseases.

State of the art

Asthma is a chronic respiratory disease for which there is no prevention or treatment available. The disease is characterized by inflammation, fibrosis, hyperreactivity, and airway remodeling, all of which lead to airflow limitation. An estimated 300 million people worldwide have asthma, and it is expected that by 2025 the number of people with asthma will increase by more than 100 million. In the United States, asthma affects 6 to 8% of the population, making it one of the most the most common chronic diseases in the country. Although most patients can control their asthma symptoms with inhaled corticosteroids, which can be combined with a leukotriene modifier and/or a long-acting beta-agonist, there remains a subset of patients with severe asthma who are not controlled by conventional therapies. Severe persistent asthma is defined as an illness that remains uncontrolled with high doses of inhaled corticosteroids. While severe asthmatics are estimated to account for approximately 5% of all asthma patients, they are at high risk of morbidity and mortality and account for a disproportionate share of asthmatic health care resources. There is still a need for new treatments for these patients.

Cytokines are intercellular signaling molecules that include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factor. Cytokines are critical for normal cell growth and immunoregulation, but they also cause immune disease and promote the growth of malignant cells. The pathology of inflammation in asthma is associated with elevated levels of many cytokines. For example, antibody-based therapy directed at interleukins (IL) 5 and 13 has been shown to be clinically effective in subgroups of patients with severe asthma. Many of the cytokines involved in asthma inflammation act through Janus family tyrosine kinase (JAK) dependent signaling pathways that signal through the signal transducer and transcription activator (STAT) family of transcription factors. Cytokines involved in asthma inflammation signaling 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 (IFNy) and granulocyte and macrophage colony stimulating factor (GM-CSF).

The JAK family consists of four members: JAK1, JAK2, JAK3, and tyrosine kinase 2 (TYK2). Binding of the cytokine to the JAK-dependent cytokine receptor causes dimerization of the receptor, which results in phosphorylation of tyrosine residues on the JAK kinase, influencing JAK activation. Phosphorylated JAKs, in turn, bind and phosphorylate various STAT proteins, which dimerize, internalize in the cell nucleus, and directly modulate gene transcription, leading, among other things, to downstream effects associated with inflammatory disease. JAKs are usually associated with cytokine receptors in pairs as homodimers or heterodimers. Certain cytokines are associated with certain paired JAKs. Each of the four members of the JAK family is involved in the signaling of at least one of the cytokines associated with asthma inflammation. Therefore, a chemical inhibitor with pan activity against all members of the JAK family could modulate a wide range of pro-inflammatory pathways that contribute to the development of severe asthma.

However, the broad anti-inflammatory effect of such inhibitors can suppress normal immune cell function, potentially leading to an increased risk of infection. Evidence of an increased risk of infection has been observed with the oral JAK inhibitor tofacitinib for the treatment of rheumatoid arthritis. In asthma, inflammation is localized in the airways. Inflammation of the airways is also characteristic of other respiratory diseases besides asthma. Chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), pneumonitis, interstitial lung disease (including idiopathic pulmonary fibrosis), acute lung injury, acute respiratory distress syndrome, bronchitis, emphysema, and sarcoidosis are also respiratory diseases whose pathophysiology is thought to be related. with cytokines signaling through JAK. Local administration of a JAK inhibitor to the lungs by inhalation may be therapeutically effective by delivering a potent anti-cytokine agent directly to the site of action, limiting systemic exposure and thus limiting the possibility of adverse systemic immunosuppression. There remains a need for a strong JAK inhibitor suitable for topical administration to the lungs for the treatment of respiratory disease.

Cytokines signaling through JAK also play an important role in the activation of T cells, a subtype of immune cells that are central to many immune processes. Pathological T cell activation is critical in the etiology of numerous respiratory diseases. Autoreactive T cells play a role in bronchiolitis obliterans with organizing pneumonia (also called COS). Like COS, the etiology of lung transplant rejection is associated with aberrant activation of the recipient's T cells by the donor's transplanted lung.

Lung transplant rejection can occur early as primary graft dysfunction (PGD), organizing pneumonia (OP), acute rejection (AR), or lymphocytic bronchiolitis (LB), or it can occur years after lung transplant as chronic lung graft dysfunction (CLAD). CLAD, formerly known as bronchiolitis obliterans (BO), is now considered a syndrome that can present with various pathological manifestations, including VO, restrictive CLAD (rCLAD or RAS), and neutrophilic allograft dysfunction. Chronic lung allograft dysfunction (CLAD) is a major problem in the long-term management of lung transplant recipients as it leads to a gradual loss of functionality of the transplanted lung (Gauthier et al., Curr. Transplant. Rep., 2016, 3(3), 185-191) . CLAD is poorly treatable and therefore there remains a need for effective compounds capable of preventing or treating this condition. Some JAK-dependent cytokines, such as IFNy and IL-5, are upregulated in CLAD and lung transplant rejection (Berastegui et al., Clin. Transplant. 2017, 31, el2898). Moreover, high levels of CXCR3 chemokines in the lungs, such as CXCL9 and CXCL10, which are downstream of JAK-dependent IFN signaling, are associated with worse outcomes in lung transplant patients (Shino et al., PLOS One, 2017, 12(7) , e0180281). Systemic JAK inhibition has been shown to be effective in kidney transplant rejection (Vicenti et al., American Journal of Transplantation, 2012, 12, 2446-56). Therefore, JAK inhibitors may be effective in the treatment or prevention of lung transplant rejection and CLAD. Similar T cell activation events described as a major cause of lung transplant rejection are also considered to be a major cause of lung graft-versus-host disease (GVHD) that can develop after hematopoietic stem cell transplantation. Like CLAD, GVHD of the lung is a chronic progressive disease with extremely poor outcomes and there is currently no approved treatment. In a retrospective multicenter study of 95 patients with acute or chronic steroid-resistant GVHD who received the systemic JAK inhibitor ruxolitinib as adjuvant therapy, the majority of patients showed a complete or partial response to ruxolitinib, including patients with pulmonary GVHD (Zeiser et al., Leukemia, 2015, 29, 10, 2062-68). Because systemic JAK inhibition is associated with severe side effects and a small therapeutic index, there remains a need for an inhaled non-systemic JAK inhibitor targeting the lungs to prevent and/or treat lung transplant rejection or GVHD of the lungs.

The essence of the invention

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

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

Where

A is a 6-7-membered monocyclic heterocyclic ring containing two nitrogen atoms, where A is connected to the carbonyl group in (I) through a nitrogen atom, while A is substituted by 13 R ² groups, and optionally two R ² groups form a ring with A through -(CH2)-bridge connection; or

A is pyrrolidinyl, where the pyrrolidinyl is bonded to the carbonyl group in (I) via a nitrogen atom, and where the pyrrolidinyl is substituted with NR ³ R ⁴ ;

R ¹ is C _1-3 alkyl;

each R ² is independently C _1-3 alkyl optionally substituted with -OH;

R ³ is C _1-3 alkyl;

and R ⁴ is C _1-3 alkyl;

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

The invention also provides a compound of the invention as provided herein for use in medical therapy, as well as the use of a compound of the invention in the manufacture of a formulation or medicament for the treatment of a respiratory disease in a mammal.

Detailed description of the invention

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

Where

A is a 6-7-membered monocyclic heterocyclic ring containing two nitrogen atoms, where A is connected to the carbonyl group in (I) through a nitrogen atom, while A is substituted by 1 3 R ² groups, and optionally two R ² groups form with A ring through -(CH ₂ )- bridging; or

A is pyrrolidinyl, where the pyrrolidinyl is bonded to the carbonyl group in (I) via a nitrogen atom, and where the pyrrolidinyl is substituted with NR ³ R ⁴ ;

R ¹ is C3 alkyl;

each R ² is independently C3 alkyl optionally substituted with —OH;

R ³ is C3 alkyl;

and R ⁴ is C3 alkyl;

or a pharmaceutically acceptable salt thereof.

In some embodiments, compound (I) has the formula (D)

In some embodiments, R ¹ is selected 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]heptanyl, and 1,4-diazacycloheptanyl, each of which is substituted with 1 or 2 R ² groups, where each R ² group is independently C3 alkyl , optionally substituted with -OH, or A is pyrrolidinyl substituted with NR ³ R ⁴ , where R ³ is C3 alkyl and R ⁴ is C3 alkyl.

In some embodiments, A is selected from the group consisting of

NR ³ R ⁴

N

where R ² is Cs alkyl, optionally substituted with -OH, R ³ is Cs alkyl, and R ⁴ is Cs alkyl.

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

or a pharmaceutically acceptable salt thereof.

In another embodiment, the invention provides a compound of formula

-3 040902

or a pharmaceutically acceptable salt thereof.

In another embodiment, the invention provides a compound of formula

or a pharmaceutically acceptable salt thereof.

In another embodiment, the invention provides a compound of formula

or a pharmaceutically acceptable salt thereof.

In another embodiment, the invention provides a compound of formula

or a pharmaceutically acceptable salt thereof.

In another embodiment, the invention provides a compound of formula (II)

Where

R ¹ is C1-3 alkyl;

And ¹ is selected from the group consisting of

where R ² represents C _1-3 alkyl, optionally substituted with -OH;

or a pharmaceutically acceptable salt thereof.

In another embodiment, the compound of formula (II) has the formula (II')

- 4 040902

„ „ 1 < ^P '> .

In some embodiments, R ¹ is selected from the group consisting of methyl and propyl.

In some embodiments, A ¹ is selected from the group consisting of

In some embodiments, A ¹ is selected from the group consisting of

and R ¹ is selected from the group consisting of methyl and propyl.

In some embodiments, the invention provides a compound of formula (III)

Where

R ¹ is C _1-3 alkyl;

And ² are selected from the group consisting of:

where each R ² is independently C1-3 alkyl, optionally substituted with -OH, R ³ is C1-3 alkyl and R ⁴ is C _1-3 alkyl;

or a pharmaceutically acceptable salt thereof.

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

In some embodiments, R ¹ is propyl.

In some embodiments, A ² is selected from the group consisting of methyl, isopropyl, and selected from the group consisting of

NI—N

N

And

In some embodiments, R ¹ is selected from the group consisting of methyl, isopropyl, and propyl, and A ² is selected from the group consisting of

- 5 040902

In some embodiments, the invention provides a compound of formula (IV)

(IV) where

R ¹ is C _1-3 alkyl;

And ³ are selected from the group consisting of

where each R ² is independently C1-3 alkyl, optionally substituted with -OH, R ³ is C1-3 alkyl, and R ⁴ is C _1-3 alkyl;

or a pharmaceutically acceptable salt thereof.

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

In some embodiments, R ¹ is selected from the group consisting of methyl, isopropyl, and propyl.

In some embodiments, A ³ is selected from the group consisting of

In some embodiments, R ¹ is selected from the group consisting of methyl, isopropyl, and propyl, and A ³ is selected from the group consisting of

In addition, the imidazo portion of the tetrahydroimidazopyridine moiety exists in the tautomeric forms illustrated below for a moiety of the compounds of the invention.

According to the IUPAC convention, these representations lead to different numbering of the atoms of the imidazole part: (1H-indazol-3-yl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-с]pyridine (structure A) compared with (1H-indazol-3-yl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine (structure B). It should be understood that while structures are shown or named for a particular form, the invention also includes its tautomer.

- 6 040902

The compounds of the present invention may contain one or more chiral centers, and therefore such compounds (and their intermediates) may exist as racemic mixtures; pure stereoisomers (ie enantiomers or diastereomers); mixtures enriched in stereoisomers, and the like. Chiral compounds shown or named herein without defined stereochemistry at the chiral center include any or all possible variants of stereoisomers at the undefined stereocenter, unless otherwise indicated. The depiction or naming of a particular stereoisomer means that the indicated stereocenter has the indicated stereochemistry, assuming that minor amounts of other stereoisomers may also be present, unless otherwise indicated, provided that the presence of another stereoisomer does not preclude the benefit of the depicted or named compound.

The compounds of the present invention may also contain several basic groups (e.g., amino groups) and, therefore, such compounds may exist as a free base or in various salt forms, such as a monoprotonated salt form, a diprotonated salt form, a triprotonated salt form, or their mixtures. All such forms are included within the scope of the present invention unless otherwise indicated.

The present invention also includes isotopically labeled compounds of formulas (I), (I'), (II), (II'), (III), (III'), (IV) and (IV'), i. compounds of formula (I), (I'), (II), (II'), (III), (III'), (IV) and (IV'), in which one or more atoms are substituted or enriched with an atom having the same atomic number, but an atomic mass different from the atomic mass prevailing in nature. Examples of isotopes that may be included in a compound of formula (I), (I'), (II), (II'), (III), (III'), (IV) and (IV') include, without limitation , ² H, ³ H, ^U C, ¹³ C, ¹⁴ C, ¹³ N, ¹⁵ N, ¹⁵ 0, ¹⁷ O and ¹⁸ O. Compounds of formula (I), (I'), (II), ( II'), (III), (III'), (IV) and (IV') enriched in tritium or carbon-14, which can be used, for example, in tissue distribution studies. Also of particular interest are compounds of formula (I), (I'), (II), (II'), (III), (III'), (IV) and (IV') enriched in deuterium, especially at the site of metabolism, and these compounds are expected to have higher metabolic stability. In addition, of particular interest are compounds of formula (I), (I'), (II), (II'), (III), (III'), (IV) and (IV') enriched in a positron-emitting isotope, such as 11C, ¹⁵ O and ¹³ N, which can be used, for example, in positron emission tomography (PET) studies.

Definitions

In describing the present invention, including its various aspects and embodiments, the following terms have the following meanings, unless otherwise indicated.

The term alkyl means a monovalent saturated hydrocarbon group, which may be linear or branched, or combinations thereof. Unless otherwise indicated, such alkyl groups typically contain from 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, 2,2-dimethylpropyl, 2-methylbutyl, 3methylbutyl, 2-ethylbutyl, 2,2-dimethylpentyl , 2-propylpentyl, and the like.

If a particular term provides for a specific number of carbon atoms, the number of carbon atoms is indicated before that term. For example, the term N-3alkyl means an alkyl group having 1 to 3 carbon atoms, where the carbon atoms are in any chemically acceptable configuration, including linear or branched configurations.

The term cycloalkyl means a monovalent saturated carbocyclic group, which may be monocyclic or polycyclic. Unless otherwise indicated, such cycloalkyl groups typically contain from 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 c-propyl means cyclopropyl.

The term heterocyclyl, heterocycle, heterocyclic or heterocyclic ring means a monovalent saturated or partially unsaturated cyclic non-aromatic group having from 3 to 10 ring atoms in common, where the ring contains from 2 to 9 ring carbon atoms and from 1 to 4 ring heteroatoms selected from nitrogen, oxygen and sulfur. Heterocyclic groups may be monocyclic or polycyclic (ie, fused or bridged). Representative heterocyclic groups include, for example, pyrrolidinyl, piperidinyl, piperazinyl, imidazolidinyl, morpholinyl, thiomorpholyl, indolin-3-yl, 2-imidazolinyl, tetrahydropyranyl, 1,2,3,4-tetrahydroisoquinolin-2-yl, quinuclidinyl, 7-azanorbornanyl , northropanil, and the like, wherein the point of attachment is on any available ring carbon or nitrogen atom. Where the point of attachment of a heterocyclic group is evident from the context, such groups may alternatively be referred to as non-valent species, ie. pyrrolidine, piperidine, piperazine, imidazole, tetrahydropyran, etc.

The term halogen means fluorine, chlorine, bromine or iodine.

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

- 7 040902

The term treating or treating means preventing, alleviating, or suppressing a medical condition, disease, or disorder being treated (eg, a respiratory disease) in a patient (particularly, a human); or alleviating symptoms of a medical condition, disease, or disorder.

The term pharmaceutically acceptable salt means a salt that is acceptable for administration to a patient or a mammal, such as a human (eg, a salt that has acceptable mammalian safety for a given dosage regimen). Typical pharmaceutically acceptable salts include acetic, ascorbic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, edisyl, fumaric, gentisic, gluconic, glucuronic, glutamic, hippuric, hydrobromic, hydrochloric, isethionic, lactic, lactobionic, maleic, malic, mandelic, methanesulfonic, mucinic, naphthalenesulfonic, naphthalene-1,5disulfonic, naphthalene-2,6-disulfonic, nicotinic, nitric, orotic, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic and xinafoic acids, etc.

The term salt thereof means a compound formed when the hydrogen of an acid is replaced by a cation such as a metal cation or an organic cation and the like. For example, the cation may be the protonated form of a compound of formula (I), (I'), (II), (II'), (III), (III'), (IV) and (IV'), i.e., a form in which one or more amino groups are protonated with an acid. Typically, the salt is a pharmaceutically acceptable salt, although this is not a requirement for salts of intermediates that are not intended to be administered to a patient.

General synthesis procedures

The compounds of the present invention and their intermediates can be prepared according to the following general methods and procedures using commercially available or commonly obtained starting materials and reagents. Substituents and variables (eg, A, R ¹ , R ² , R ³ , etc.) used in the schemes below have the same meanings as defined elsewhere in this specification, unless otherwise indicated. In addition, compounds containing an acidic or basic atom or functional group can be used or can be obtained in the form of a salt, unless otherwise indicated (in some cases, the use of a salt in a particular reaction requires, before carrying out this reaction, the conversion of the salt to a non-salt form , such as the free base, using conventional procedures).

While a particular embodiment of the present invention may be shown or described in the procedures below, those skilled in the art will appreciate that other embodiments or aspects of the present invention may also be prepared using such procedures or other methods, reagents, and starting materials. known to those skilled in the art. In particular, it should be understood that the compounds of the present invention can be obtained in various ways in which the reactants are combined in different orders to obtain different intermediates during the preparation of the final products.

The general method for obtaining the final compounds of the invention is illustrated in the following scheme.

K-18 K-19

Compound K-18 is reacted 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 formation conditions to give compound (I). Typically, the carboxylic acid is contacted with about 1-4 equivalents of amine A in the presence of an excess of base. Coupling agents such as N,N,N',N'-tetramethylO-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (HATU) or other amide coupling agents known in the art may be used in the amide bond formation reaction. this area. Hydrazine may be added to remove unwanted by-products. The reaction is generally carried out at room temperature for about 5 minutes to about 24 hours, or until the reaction is substantially complete.

Pharmaceutical compositions

The compounds of the present invention and their pharmaceutically acceptable salts are generally used in the form of a pharmaceutical composition or formulation. Such pharmaceutical compositions may advantageously be administered to a patient by inhalation. In addition, pharmaceutical compositions can be administered by any acceptable route of administration, including, without limitation, oral, rectal, nasal, topical (including transdermal), and parenteral routes of administration.

Accordingly, in one of its compositional aspects, the invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient and a compound of formula (I), (I'), (II), (II'), (III ), (III'), (IV) and (IV'), where, as defined above, the compound of formula (I), (I'), (II), (II'), (III), (III') , (IV) or (IV') means a compound of formula (I), (I'), (II), (II'), (III), (III'), (IV) and (IV') or its pharmaceutical acceptable salt. Optionally, such pharmaceutical compositions may contain, if necessary, other therapeutic agents and/or formulation agents. When discussing compositions and their uses, the compound of the invention may also be referred to herein as the active agent. Used in the present description, the term compound according to the invention includes all compounds covered by formula (I), as well as species covered by formula (I) and their pharmaceutically acceptable salts.

Pharmaceutical compositions of the present invention generally contain a therapeutically effective amount of a compound of the present invention. However, those skilled in the art will recognize that a pharmaceutical composition may contain more than a therapeutically effective amount, i. bulk compositions, or less than a therapeutically effective amount, i. separate unit doses intended for repeated administration to achieve a therapeutically effective amount.

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

Any conventional carrier or excipient may be used in the pharmaceutical compositions of the invention. The choice of a particular carrier or excipient, or combination of carriers or excipients, will depend on the route of administration used to treat a particular patient, or the type of medical condition or severity of the disease. In this regard, the preparation of a suitable pharmaceutical composition for a particular mode of administration is within the skill of the pharmaceutical art. In addition, carriers or excipients used in the pharmaceutical compositions of the present invention are commercially available. By way of further illustration, conventional formulation methods are described in Remington: The Science and Practice of Pharmacy, ^20th Edition, Lippincott Williams & White, Baltimore, Maryland (2000); and H. C. Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Edition, Lippincott Williams & White, Baltimore, Maryland (1999).

Representative examples of materials that can serve as pharmaceutically acceptable carriers include, without limitation, 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 such as 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; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffer solutions; and other non-toxic compatible substances used in pharmaceutical compositions.

Pharmaceutical compositions are usually prepared by intimately mixing the active agent with a pharmaceutically acceptable carrier and one or more optional ingredients. The resulting uniformly mixed mixture can then be shaped or filled into tablets, capsules, pills, and the like. using conventional procedures and equipment.

In one aspect, the pharmaceutical composition is suitable for inhalation administration. Pharmaceutical compositions for inhalation administration are usually in the form of an aerosol or powder. Such compositions are typically administered via inhaled delivery devices such as a dry powder inhaler (DPI), metered dose inhaler (MDI), nebulizer inhaler, or similar delivery device.

In a specific embodiment, the pharmaceutical composition is administered by inhalation using a dry powder inhaler. Such dry powder inhalers typically administer the pharmaceutical composition as a free flowing powder that is dispersed in an air stream during inhalation by the patient. To form a free-flowing powder composition, the therapeutic agent is usually formulated with a suitable excipient such as lactose, starch, mannitol, dextrose, polylactic acid (PLA), polylactide-glycolide copolymer (PLGA), or combinations thereof. Typically, the therapeutic agent is comminuted and combined with a suitable carrier to form a composition suitable for inhalation.

A typical pharmaceutical composition for use in a dry powder inhaler includes lactose and a compound of the present invention in micronized form. Such a dry powder composition can be obtained, for example, by combining dry milled lactose with a therapeutic agent and then dry mixing the components. The composition is then typically loaded into a dry powder dispenser or into inhalation cartridges or capsules for use with a dry powder delivery device.

Inhalation dry powder delivery devices suitable for the administration of therapeutic agents by inhalation are described in the art, and examples of such devices are commercially available. For example, exemplary inhalation dry powder delivery devices or products include the 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); Twisthaler (Schering-Plough); Turbuhaler (AstraZeneca); Ultrahaler (Aventis); and so on.

In another specific embodiment, the pharmaceutical composition is administered by inhalation using a metered dose inhaler. Such metered dose inhalers typically deliver a metered amount of therapeutic agent using a pressurized propellant gas. Accordingly, pharmaceutical compositions administered by metered dose inhaler typically contain a solution or suspension of the therapeutic agent in a liquefied propellant. Any suitable liquefied propellant may be used, 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 such as CCl ₃ F. In a particular embodiment, the propellant is hydrofluoroalkanes. In some embodiments, the hydrofluoroalkane formulation contains a co-solvent such as ethanol or pentane and/or a surfactant such as sorbitan trioleate, oleic acid, lecithin, and glycerin.

A typical pharmaceutical composition for use in a metered dose inhaler comprises from about 0.01% to about 5% by weight of a compound of the invention; from about 0 wt.% to about 20 wt.% ethanol; and from about 0 wt.% to about 5 wt.% surfactant; and the rest is HFA propellant. Such compositions are typically prepared by adding chilled or pressurized hydrofluoroalkane to a suitable container containing the therapeutic agent, ethanol (if present) and surfactant (if present). To prepare a suspension, the therapeutic agent is finely ground and then combined with the propellant. The composition is then loaded into an aerosol can, which is usually part of a metered dose inhaler.

Metered dose inhalers suitable for the administration of therapeutic agents by inhalation are described in the art, and examples of such devices are commercially available. For example, exemplary metered dose inhaler devices or products include the AeroBid inhalation system (Forest Pharmaceuticals); inhalation aerosol Atrovent (Boehringer Ingelheim); Flovent (GlaxoSmithKline); Maxair inhaler (3M); Proventil Inhaler (Schering); inhalation aerosol Serevent (GlaxoSmithKline); and so on.

In another specific aspect, the pharmaceutical composition is administered by inhalation using a nebulizer inhaler. Such nebulizer devices typically provide a high-velocity air stream that nebulizes the pharmaceutical composition as a mist that is carried into the patient's airways. Accordingly, when preparing a composition for use in a nebulizer inhaler, the therapeutic agent may be dissolved in a suitable carrier to form a solution. Alternatively, the therapeutic agent can be ground to microparticle or nanoparticle size and combined with a suitable carrier to form a suspension.

A typical pharmaceutical composition for use in a nebulizer inhaler includes a solution or suspension containing from about 0.05 μg/ml to about 20 mg/ml of a compound of the present invention and excipients compatible with spray formulations. In one embodiment, the solution has a pH of about 3 to about 8.

Nebulizer devices suitable for the administration of therapeutic agents by inhalation are described in the art, and examples of such devices are commercially available. For example, typical nebulizers or products include the Respimat Softmist inhaler (Boehringer Ingelheim); AERx pulmonary delivery system (Aradigm Corp.); reusable nebulizer PARI LC Plus (Pari GmbH); and so on.

In yet another aspect, the pharmaceutical compositions of the invention may alternatively be formulated into a dosage form intended for oral administration. Suitable pharmaceutical compositions for oral administration may be in the form of capsules, tablets, pills, lozenges, cachets, dragees, powders, granules; or as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil liquid emulsion; or as an elixir or syrup; and so on.; each of them contains a given amount of the compound of the present invention as an active ingredient.

When the pharmaceutical compositions of the invention are intended for oral administration in solid dosage form, they usually contain the active agent and one or more pharmaceutically acceptable carriers such as sodium citrate or dicalcium phosphate. Optionally or alternatively, such solid dosage forms may also include: fillers or extenders, binders, humectants, dissolution retardants, absorption accelerators, wetting agents, absorbents, lubricants, coloring agents, and buffering agents. Release agents, wetting agents, coating agents, sweeteners, flavoring and flavoring agents, preservatives and antioxidants may also be present in the pharmaceutical compositions of the present invention.

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

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

Dry powder composition

The micronized compound of formula (I) (1 g) is mixed with milled lactose (25 g). This mixed mixture is then loaded into individual blisters of a peelable blister pack in an amount sufficient to provide from about 0.1 mg to about 4 mg of the compound of formula (I) per dose. The contents of the blisters are administered using a dry powder inhaler.

Dry powder composition

The micronized compound of formula (I) (1 g) is mixed with milled lactose (20 g) to form a bulk composition having a weight ratio of compound to milled lactose of 1:20. The mixed composition is packaged in a dry powder inhalation device capable of delivering from about 0.1 mg to about 4 mg of the compound of formula (I) per dose.

Composition for metered dose inhaler

Micronized compound of formula (I) (10 g) is dispersed in a solution obtained by dissolving lecithin (0.2 g) in demineralized water (200 ml). The resulting slurry is spray dried and then micronized to form a micronized composition containing particles with an average diameter of less than about 1.5 microns. The micronized composition is then loaded into metered dose inhaler cartridges containing pressurized 1,1,1,2-tetrafluoroethane in an amount sufficient to provide from about 0.1 mg to about 4 mg of a compound of formula (I) per dose when administered by a metered dose inhaler.

Composition for nebulizer

The compound of formula (I) (25 mg) is dissolved in a solution containing 1.5-2.5 equivalents of hydrochloric acid, followed by the addition of sodium hydroxide to adjust the pH to 3.55.5 and 3% by weight of glycerol. The solution is well mixed until all components are dissolved. The solution is administered using a nebulizer that provides from about 0.1 mg to about 4 mg of the compound of formula (I) per dose.

Utility

The JAK inhibitors of the present invention have been developed for the treatment of inflammatory and fibrotic diseases of the airways. In particular, the compounds have been designed to allow delivery of a strong anti-cytokine agent directly to the site of exposure to respiratory disease in the lungs to limit systemic exposure.

The compounds of the present invention have been shown to be potent inhibitors of the JAK family of enzymes: JAK1, JAK2, JAK3 and TYK2. In addition, the compounds showed strong inhibition of proinflammatory and profibrotic cytokines. It is recognized that the broad-spectrum anti-inflammatory effect of JAK inhibitors can suppress normal immune cell function, potentially leading to an increased risk of infection. Therefore, the present compounds are optimized to limit lung-to-plasma absorption, thus minimizing the risk of immunosuppression.

As described below in the experimental section, absorption and distribution profiles of representative compounds were obtained in preclinical studies. Compounds 1 to 6 were tested in mice and showed high concentration in lung tissue and low plasma absorption at 5 hours post-dose. Compounds of the invention have been shown to inhibit the action of the pro-inflammatory cytokine IL-13 in mouse lung tissue. In particular, the compounds showed inhibition of IL-13-induced STAT6 phosphorylation in lung tissue, indicative of local JAK targeting in the lung in vivo. This effect was observed when the pro-inflammatory cytokine IL-13 was administered 4 hours after administration of the test compound to provide additional evidence for its preferential retention in the lungs.

The test compounds have been shown to exhibit both strong inhibitory activity at the cellular level and that they are preferentially retained in lung tissue. Extensive research by the present inventors has shown that while it is possible to identify compounds that are potent at the cellular level, or compounds that preferentially linger in the lungs, it is much more difficult to find compounds that have both desirable characteristics at the same time.

The anti-inflammatory activity of JAK inhibitors has been convincingly demonstrated in preclinical models of asthma (Malaviya et al., Int. Immunopharmacol., 2010, 10, 829-836; Matsunaga et al., Biochem. And Biophys. Res. Commun., 2011, 404, 261-267; Kudlacz et al., Eur J. Pharmacol, 2008, 582, 154161). Cytokines 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 (IFNy) and granulocyte and macrophage colony stimulating factor (GM-CSF). Accordingly, the compounds of the invention are expected to be useful in the treatment of inflammatory respiratory diseases, in particular asthma. Pulmonary inflammation and pulmonary fibrosis are characteristic of respiratory diseases other than asthma, such as chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), pneumonitis, interstitial lung disease (including idiopathic pulmonary fibrosis), acute lung injury, acute respiratory distress syndrome , bronchitis, emphysema, bronchiolitis obliterans and sarcoidosis. Therefore, the compounds of the present invention are also expected to be useful in the treatment of chronic obstructive pulmonary disease, cystic fibrosis, pneumonitis, interstitial lung disease (including idiopathic pulmonary fibrosis), acute lung injury, acute respiratory distress syndrome, bronchitis, emphysema, bronchiolitis obliterans and sarcoidosis. .

Compounds 1 to 6 have been shown to have similar JAK activity when compared to their respective fluorine analogs (Compounds C-1, C-2, C-3, C-4, C-5 and C-6). However, they have the advantage that they cause significantly lower levels of metabolic pathway sulfation, as shown in Assay 5. This is important because metabolic pathway sulfation occurs in the lungs, which can lead to a rapid decrease in the concentration of the active parent compound.

The compounds of the invention have shown inhibition of cytokines associated with inflammation. Therefore, the compounds of the present invention are likely to be useful in the treatment of certain specific respiratory diseases, as detailed below.

Eosinophilic airway inflammation is a characteristic feature of diseases collectively referred to as eosinophilic lung diseases (Cottin et al., Clin. Chest. Med., 2016, 37(3), 535-56). Eosinophilic diseases are associated with IL-4, IL-13 and IL5 signaling. Eosinophilic lung diseases include infections (especially helminthic infections), drug pneumonitis (caused, for example, by therapeutic drugs such as antibiotics, phenytoin, or tryptophan), fungal pneumonitis (eg, allergic bronchopulmonary aspergillosis), hypersensitivity pneumonitis, and eosinophilic granulomatosis with polyangiitis (previously known like Churg-Strauss syndrome). Eosinophilic lung diseases of unknown etiology include idiopathic acute eosinophilic pneumonias, idiopathic chronic eosinophilic pneumonias, hypereosinophilic syndrome, and Loeffler's syndrome.

IL-6 gene polymorphism is associated with elevated IL-6 levels and an increased risk of developing pulmonary arterial hypertension (PAH) (Fang et al., J. Am. Soc. Hypertens., 2017, 11(3), 171-177). In support of the role of IL-6 in the development of PAH, inhibition of the gp130 receptor chain of IL-6 ameliorated the course of the disease in a rat model of PAH (Huang et al., Can. J. Cardiol., 2016, 32(11), 1356.e1-1356. e10).

Cytokines such as IFNy, IL-12 and IL-6 are implicated in a number of non-allergic lung diseases such as sarcoidosis and lymphangioleiomyomatosis (El-Hashemite et al., Am. J. Respir. Cell. Mol. Biol., 2005, 33 , 227-230, and El-Hashemite et al., Cancer Res., 2004, 64, 3436-3443). The compounds of the present invention have also been shown to inhibit IFNy signaling.

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

Pathological T cell activation is critical in the etiology of numerous respiratory diseases. Autoreactive T cells play a role in bronchiolitis obliterans with organizing pneumonia (also called COS). Like COS, the etiology of lung transplant rejection is associated with aberrant activation of the recipient's T cells by the donor's transplanted lung. Lung transplant rejection can occur early as primary graft dysfunction (PGD), organizing pneumonia (OP), acute rejection (AR), or lymphocytic bronchiolitis (LB), or it can occur years after lung transplant as chronic lung graft dysfunction (CLAD). CLAD, formerly known as bronchiolitis obliterans (BO), is now considered a syndrome that can present with various pathological manifestations, including VO, restrictive CLAD (rCLAD or RAS), and neutrophilic allograft dysfunction. Chronic lung allograft dysfunction (CLAD) is a major problem in the long-term management of lung transplant recipients as it leads to a gradual loss of functionality of the transplanted lung (Gauthier et al., Curr. Transplant. Rep., 2016, 3(3), 185-191) . CLAD is poorly treatable and therefore there remains a need for effective compounds capable of preventing or treating this condition. Some JAK-dependent cytokines, such as IFNy and IL-5, are upregulated in CLAD and lung transplant rejection (Berastegui et al., Clin. Transplant. 2017, 31, e12898). Moreover, high levels of CXCR3 chemokines in the lungs, such as

- 12 040902

CXCL9 and CXCL10, which are downstream of JAK-dependent IFN signaling, are associated with worse outcomes in lung transplant patients (Shino et al., PLOS One, 2017, 12(7), e0180281). Systemic JAK inhibition has been shown to be effective in kidney transplant rejection (Vicenti et al., American Journal of Transplantation, 2012, 12, 2446-56). Therefore, JAK inhibitors may be effective in the treatment or prevention of lung transplant rejection and CLAD. Similar T cell activation events described as a major cause of lung transplant rejection are also considered to be a major cause of lung graft-versus-host disease (GVHD) that can develop after hematopoietic stem cell transplantation. Like CLAD, GVHD of the lung is a chronic progressive disease with extremely poor outcomes and there is currently no approved treatment. In a retrospective multicenter study of 95 patients with acute or chronic steroid-resistant GVHD who received the systemic JAK inhibitor ruxolitinib as adjuvant therapy, the majority of patients showed a complete or partial response to ruxolitinib, including patients with pulmonary GVHD (Zeiser et al., Leukemia, 2015, 29, 10, 2062-68). Because systemic JAK inhibition is associated with severe side effects and a small therapeutic index, there remains a need for an inhaled non-systemic JAK inhibitor targeting the lungs to prevent and/or treat lung transplant rejection or GVHD of the lungs. The compounds of the invention have the characteristics necessary to meet this need. More recently, with the increased use of immune checkpoint inhibitors, immune checkpoint inhibitor-induced pneumonitis, another T-cell mediated lung disease, has emerged. Cancer patients receiving these T-cell stimulating agents may develop pneumonitis, which is fatal.

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 invention, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of of the invention or a pharmaceutically acceptable salt thereof.

In one aspect, the respiratory disease is asthma, chronic obstructive pulmonary disease, cystic fibrosis, pneumonitis, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), pneumonitis, interstitial lung disease (including idiopathic pulmonary fibrosis), acute lung injury, acute respiratory distress syndrome, bronchitis, emphysema obliterans 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, eosinophilic disease, helminthic infection, pulmonary arterial hypertension, sarcoidosis, lymphangioleiomyomatosis, bronchiectasis, infiltrative lung disease, drug-induced pneumonitis, fungal pneumonitis, allergic bronchopulmonary aspergillosis, hypersensitivity pneumonitis, eosinophilic granulomatosis with polyangiitis, idiopathic acute eosinophilic pneumonia, idiopathic chronic eosinophilic pneumonia, hypereosinophilic syndrome, Loeffler's syndrome, bronchiolitis obliterans with organizing pneumonia, acute and chronic lung transplant rejections (including PGD, OP, LB, AR and CLAD, BO, restrictive CLAD and neutrophilic allograft dysfunction), disease pulmonary graft versus host, bronchiolitis obliterans with organizing pneumonia, pulmonary arterial hypertension, bronchiectasis, or immune checkpoint inhibitor-induced pneumonitis.

The invention also relates to a method for treating asthma in a mammal, comprising administering to the mammal a therapeutically effective amount of a compound of the invention, 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.

When used to treat asthma, the compounds of the present invention are typically administered as a single daily dose or 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 the physician, taking into account relevant circumstances, including the condition being treated, the chosen route of administration, the compound actually administered and its relative potency, the age, weight, and response of the individual patient, the severity of the patient's symptoms; and the like.

The invention also relates to a method for treating a respiratory disease (including, without limitation, the disease disclosed herein) in a mammal, the method comprising administering to the mammal a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing a pharmaceutically acceptable carrier and a compound according to the invention, or a pharmaceutically acceptable salt thereof.

When used to treat a respiratory disease (including, but not limited to, the disease disclosed herein), the compounds of the present invention are typically administered as a single daily dose or 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 the physician, taking into account relevant circumstances, including the condition being treated, the chosen route of administration, the compound actually administered and its relative potency, 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 may also be useful in the treatment of a variety of other diseases. The compounds of the present invention may be useful in various inflammatory diseases of the gastrointestinal tract, which include, without limitation, inflammatory bowel disease, ulcerative colitis (proctosigmoiditis, pancolitis, ulcerative proctitis, and left-sided colitis), Crohn's disease, collagenous colitis, lymphocytic colitis, disease Behçet, celiac disease, immune checkpoint inhibitor-induced colitis, ileitis, eosinophilic esophagitis, graft-versus-host disease-associated colitis, and infectious colitis. Ulcerative colitis (Reimund et al., J. Clin. Immunology, 1996, 16, 144-150), Crohn's disease (Woywodt et al., Eur. J. Gastroenterology Hepatology, 1999, 11, 267-276), collagenous colitis ( Kumawat et al., Mol. Immunology, 2013, 55, 355-364), lymphocytic colitis (Kumawat et al., 2013), eosinophilic esophagitis (Weinbrand-Goichberg et al., Immunol. Res., 2013, 56, 249- 260), graft-versus-host disease-associated colitis (Coghill et al., Blood, 2001, 117, 3268-3276), infectious colitis (Stallmach et al., Int. J. Colorectal Dis., 2004, 19, 308- 315), Behçet's disease (Zhou et al., Autoimmun. Rev., 2012, 11, 699-704), celiac disease (de Nitto et al., World J. Gastroenterol., 2009, 15, 4609-4614), colitis induced by immune checkpoint inhibitors (for example, CTLA-4 inhibitor colitis; (Yano et al., J. Translation. Med., 2014, 12, 191), colitis induced by PD-1 or PD-L1 inhibitor), and ileitis (Yamamoto et al., Dig. Liver Dis., 2008, 40, 253-259) is characterized by increased levels of pro-inflammatory cytokines. Because many pro-inflammatory cytokines signal through JAK activation, the compounds described herein may alleviate inflammation and relieve symptoms. In particular, the compounds of the present invention may be useful in the induction and maintenance of remission of ulcerative colitis, as well as in the treatment of Crohn's disease, immune checkpoint inhibitor-induced colitis, and gastrointestinal side effects of graft versus host disease. Thus, in one aspect, the present invention provides a method of treating an inflammatory disease of the gastrointestinal tract in a mammal (e.g., a human), the method comprising administering to the mammal a compound of the invention, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound according to of the invention or a pharmaceutically acceptable salt thereof.

Atopic dermatitis and other inflammatory skin diseases are associated with increased levels of pro-inflammatory cytokines that are dependent on the JAK-STAT pathway. Therefore, the compounds of the present invention, or pharmaceutically acceptable salts thereof, may be useful against a number of skin inflammatory or pruritic conditions, which include, without limitation, atopic dermatitis, alopecia areata, vitiligo, psoriasis, dermatomyositis, cutaneous T-cell lymphomas (Netchiporouk et al. , Cell Cycle 2014; 13, 3331-3335) and subtypes (Cesari syndrome, mycosis fungoides, pagetoid reticulosis, granulomatous lax skin, lymphomatoid papulosis, chronic lichenoid pityriasis, lichenoid acute parapsoriasis, CD30+ cutaneous T-cell lymphoma, secondary CD30+ cutaneous large cell lymphoma, nonmycotic fungal CD30 ^- cutaneous large T-cell lymphoma, pleomorphic T-cell lymphoma, Lennert's lymphoma, subcutaneous T-cell lymphoma, angiocentric lymphoma, blast NK-cell lymphoma), nodular pruritus, lichen planus, primary localized cutaneous amyloidosis, bullous pemphigoid, skin manifestations of trans disease graft versus host, pemphigoid, discoid lupus, granuloma annulare, lichen simplex, vulvar/scrotal/perianal pruritus, lichen sclerosus, pruritus after herpetic neuralgia, lichen planus, and decalval folliculitis. In particular, atopic dermatitis (Bao et al., JAK-STAT, 2013, 2, e24137), alopecia areata (Xing et al., Nat. Med. 2014, 20, 1043-1049), vitiligo (Craiglow et al., JAMA Dermatol. 2015, 151, 11101112), pruritus nodosa (Sonkoly et al., J. Allergy Clin. Immunol. 2006, 117, 411-417), lichen planus (Welz-Kubiak et al., J. Immunol. Res 2015 , ID: 854747), primary localized cutaneous amyloidosis (Tanaka et al., Br. J. Dermatol. 2009, 161, 1217-1224), bullous pemphigoid (Feliciani et al., Int. J. Immunopathol. Pharmacol. 1999, 12 , 55-61), and cutaneous manifestations of graft-versus-host disease (Okiyama et al., J. Invest. Dermatol. 2014, 134, 992-1000) are characterized by elevated levels of certain cytokines that signal through JAK activation. Accordingly, the compounds of the present invention, or pharmaceutically acceptable salts thereof, are capable of alleviating the associated skin inflammation or pruritus caused by these cytokines. In particular, the compounds of the invention, or pharmaceutically acceptable salts thereof, are expected to be useful in the treatment of atopic dermatitis and other inflammatory skin diseases. Thus, in one aspect of the present invention, there is provided a method for treating an inflammatory skin disease in a mammal (e.g., a human), comprising applying to the skin of the mammal a pharmaceutical composition containing a compound of the present invention, or a pharmaceutically acceptable salt thereof.

- 14 040902 and a pharmaceutical carrier. In one aspect, the inflammatory skin disease is atopic dermatitis.

Many eye diseases have been shown to be associated with an increase in pro-inflammatory cytokines that are dependent on the JAK-STAT pathway. Thus, the compounds of the present invention, or pharmaceutically acceptable salts thereof, may be useful in the treatment of a number of ocular diseases, which include, but are not limited to, uveitis, diabetic retinopathy, diabetic macular edema, dry eye disease, age-related macular degeneration, and atopic keratoconjunctivitis. In particular, uveitis (Horai and Caspi, J. Interferon Cytokine Res., 2011, 31, 733-744), diabetic retinopathy (Abcouwer, J. Clin. Cell. Immunol., 2013, Suppl 1, 1-12), diabetic macular edema (Sohn et al., American Journal of Opthamology, 2011, 152, 686-694), dry eye disease (Stevenson et al., Arch. Ophthalmol., 2012, 130, 90-100) and age-related macular degeneration ( Knickelbein et al., Int. Ophthalmol. Clin., 2015, 55(3), 63-78) are characterized by increased levels of some pro-inflammatory cytokines that signal through the JAK-STAT pathway. Accordingly, the compounds of the present invention, or pharmaceutically acceptable salts thereof, are capable of alleviating associated ocular inflammation and reversing disease progression or providing symptomatic relief. Thus, in one aspect, the present invention provides a method for 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 invention, 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 disease, age-related macular degeneration, or atopic keratoconjunctivitis. In one aspect, the method comprises administering a compound of the invention, or a pharmaceutically acceptable salt thereof, by intravitreal injection. The compounds of the invention, or pharmaceutically acceptable salts thereof, may also be used in combination with one or more compounds useful in ophthalmic conditions.

The compounds of the invention, or pharmaceutically acceptable salts thereof, may also be useful in the treatment of other diseases such as other inflammatory diseases, autoimmune diseases, or cancer. The compounds of the invention, or pharmaceutically acceptable salts thereof, may be useful in the treatment of one or more of the following: arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, transplant rejection, xerophthalmia, psoriatic arthritis, diabetes, insulin dependent diabetes, motor neuron disease, myelodysplastic syndrome, pain, sarcopenia, cachexia, septic shock, systemic lupus erythematosus, leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, acute lymphoblastic leukemia, acute myelogenous leukemia, ankylosing spondylitis, myelofibrosis, B-cell lymphoma, hepatocellular carcinoma, Hodgkin's disease, breast cancer, multiple myeloma , melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian clear cell carcinoma, ovarian tumor, pancreatic tumor, polycythemia vera, Sjögren's syndrome, soft tissue sarcoma, sarcoma, splenomegaly, T-cell lymphoma, and thorax assemia.

Combination Therapy

The compounds of the invention, or pharmaceutically acceptable salts thereof, may be used in combination with one or more agents that act by the same or different mechanisms to treat a disease. Various agents can be administered sequentially or simultaneously, in separate compositions or in one composition. Useful classes of agents for combination therapy include, without limitation, beta2-adrenergic receptor agonist, muscarinic receptor antagonist, glucocorticoid agonist, G protein-coupled receptor-44 antagonist, leukotriene D4 receptor antagonist, muscarinic M3 receptor antagonist, histamine III receptor antagonist , Immunoglobulin E antagonist, PDE 4 inhibitor, IL-4 antagonist, M1 muscarinic receptor antagonist, Histamine receptor antagonist, IL-13 antagonist, IL-5 antagonist, 5-lipoxygenase inhibitor, beta-adrenergic agonist, CCR3 chemokine antagonist, CFTR stimulant, immunoglobulin modulator, interleukin 33 ligand inhibitor, PDE 3 inhibitor, phosphoinositide 3-kinase delta isoform inhibitor, thromboxane A2 antagonist, elastase inhibitor tyrosine kinase inhibitor Kit, leukotriene E4 antagonist, leukotriene antagonist, PGD2 antagonist, TNF-alpha ligand inhibitor, TNF-binding agent, complement cascade inhibitor, eotaxin ligand inhibitor, in glutathione reductase inhibitor, histamine H4 receptor antagonist, IL-6 antagonist, IL2 gene stimulator, immunoglobulin gamma Fc region IIB receptor modulator, interferon gamma ligand, interleukin-13 ligand inhibitor, interleukin-17 ligand inhibitor, L-selectin antagonist, leukocyte elastase inhibitor, leukotriene C4 antagonist, leukotriene-C4 synthase inhibitor, membrane copper amine oxidase inhibitor, metalloprotease-12 inhibitor, metalloprotease-9 inhibitor, mite allergen modulator, muscarinic receptor modulator, nicotinic acetylcholine receptor agonist, nuclear factor kappa B inhibitor, antagonist p-selectin, PDE5 inhibitor, PDGF receptor antagonist, gamma-phosphoinositide 3-kinase inhibitor, TLR-7 agonist, TNF antagonist, Ab1 tyrosine kinase inhibitor, acetylcholine receptor antagonist, mammalian acid chitinase inhibitor, ACTH receptor agonist, modulator of actin polymerization, adenosine antagonist

- 15 040902 A1 receptor, adenylate cyclase stimulator, adrenergic antagonist, adrenocorticotropic hormone ligand, alcohol dehydrogenase 5 inhibitor, alpha-1 antitrypsin stimulator, alpha proteinase inhibitor, androgen receptor modulator, angiotensin-converting enzyme 2 stimulator, ANP agonist, Bcr protein inhibitor, beta-1 antagonist adrenoreceptor, beta-2 adrenoceptor antagonist, beta-2 adrenoceptor modulator, beta-amyloid modulator, BMP10 gene inhibitor, BMP15 gene inhibitor, calcium channel inhibitor, cathepsin G inhibitor, CCL26 gene inhibitor, CCR3 chemokine modulator, CCR4 chemokine antagonist, cell cell molecule inhibitor adhesion stimulator, chitinase inhibitor, collagen I antagonist, complement C3 inhibitor, CSF1 antagonist, CXCR2 chemokine antagonist, cytokine common beta chain receptor modulator, cytotoxic T-lymphocyte protein-4 stimulator, deoxyribonuclease I stimulator, deoxyribonuclease stimulator, dipeptide inhibitor lpeptidase I, DNA gyrase inhibitor, prostanoid DP receptor modulator, E-selectin antagonist, tyrosine kinase ERBB receptor family inhibitor, elastin modulator, ET-A endothelin antagonist, ET-B endothelin antagonist, epoxide hydrolase inhibitor, FGF3 receptor antagonist, Fyn tyrosine kinase inhibitor, transcription factor GATA 3 inhibitor, glucosylceramidase modulator, glutamate receptor modulator, GM-CSF ligand inhibitor, guanylate cyclase stimulator, H ⁺ K ⁺ ATPase inhibitor, hemoglobin modulator, heparin agonist, histone deacetylase inhibitor, histone deacetylase-2 stimulator, HMG CoA reductase inhibitor, 1-kappa inhibitor.

B beta kinase, ICAM1 gene inhibitor, IL-17 antagonist, IL-17 receptor modulator, IL-23 antagonist, IL-4 receptor modulator, immunoglobulin G modulator, immunoglobulin G1 agonist, immunoglobulin G1 modulator, Fc region Ia receptor antagonist epsilon -immunoglobulin, IIB receptor antagonist of the gamma-immunoglobulin Fc region, immunoglobulin kappa chain modulator, insulin sensitizer, interferon beta ligand, interleukin 1 receptor-like protein antagonist, interleukin 18 ligand inhibitor, interleukin 17A receptor antagonist, interleukin-1 beta ligand inhibitor, interleukin 5 ligand inhibitor, interleukin-6 ligand inhibitor, KCNA voltage-gated potassium channel-3 inhibitor, Kit ligand inhibitor, laminin-5 agonist, CysLT1 leukotriene receptor antagonist, CysLT2 leukotriene receptor antagonist, LOXL2 gene inhibitor, Lyn tyrosine kinase inhibitor, protein inhibitor MARCKS, MDR-associated protein 4 inhibitor, metalloprotease-2 modulator, modulator metalloprotease-9, mineralocorticoid receptor antagonist, muscarinic M2 receptor antagonist, muscarinic M4 receptor antagonist, muscarinic M5 receptor antagonist, natriuretic peptide A receptor agonist, natural killer cell receptor modulator, nicotinic ACh receptor alpha-7 subunit stimulator, NK cell receptor modulator , nuclear factor kappa B modulator, opioid growth factor receptor agonist, P-glycoprotein inhibitor, purinoceptor P2X3 antagonist, MAP kinase p38 inhibitor, peptidase 1 modulator, phospholipase A2 inhibitor, phospholipase C inhibitor, plasminogen activator type 1 inhibitor, activation factor receptor antagonist platelet, PPAR gamma agonist, prostacyclin agonist, protein tyrosine kinase inhibitor, inositol phosphatase 1 SH2 domain stimulator, transduction signal inhibitor, sodium channel inhibitor, STAT-3 modulator, stem cell antigen-1 inhibitor, superoxide dismutase modulator, CD28 surface g inhibitor T cell lycoprotein, T cell surface glycoprotein CD8 inhibitor, TGF-beta agonist, TGF-beta antagonist, thromboxane synthetase inhibitor, thymic stromal lymphoprotein ligand inhibitor, thymosin agonist, thymosin beta 4 ligand, TLR-8 agonist, TLR-9 agonist, TLR9 gene stimulator, topoisomerase IV inhibitor, fast skeletal muscle troponin I stimulator, fast skeletal muscle troponin T stimulator, type I IL-1 receptor antagonist, type II TNF receptor modulator, ion channel modulator, uteroglobin stimulator, and VIP agonist.

Specific agents that can be used in combination with the JAK inhibitor compounds of the present invention include, but are not limited to, rosiptor acetate, umeclidinium bromide, secukinumab, methenkephalin acetate, tridecactide acetate, fluticasone propionate, alpha-cyclodextrin-stabilized sulforaphane, tezepelumab, mometasone furoate, BI -1467335, dupilumab, aclidinium, formoterol, AZD-1419, HI-1640V, rivipansel, CMP-001, mannitol, ANB-020, omalizumab, tregalizumab, mitizax, benralizumab, golimumab, roflumilast, imatinib, REGN-3500, apremilast, apremilast , RPL554, astimmun, adalimumab, rupatadine, parogrelil, MK-1029, beclomethasone dipropionate, formoterol fumarate, mogamulizumab, seratrodast, UCB-4144, nemiralisib, CK-2127107, fevipiprant, danitriksin, bosentan, abatacept, EC-18, doparuvelisib , ciprofloxacin, salbutamol HFA, erdosteine, PrEP-001, nedocromil, CDX-0158, salbutamol, enobosarm, R-TPR-022, lenzilumab, fluticasone furoate, vilanterol triphenate, fluticasone propion at, salmeterol, PT-007, PRS-060, remestemcel-L, citrulline, RPC-4046, nitric oxide, DS-102, gerilimzumab, actair, fluticasone furoate, umeclidinium, vilanterol, AG-NPP709, gamunex, infliximab, ampion, acumapimod, canakinumab, INS-1007, CYP001, sirukumab, fluticasone propionate, mepolizumab, pitavastatin, solithromycin, etanercept, ivacaftor, anakinra, MPC-300-IV, glycopyrronium bromide, aclidinium bromide, FP-025, risanquizumab, glycopyrronium, formterol adipocell, YPL-001, tiotropium bromide, glycopyrronium bromide, indacaterol maleate, andecaliximab, olodaterol, esomeprazole, dust mite vaccine, vaccine

- 16 040902 against wormwood pollen allergen, vamorolone, gefapixant, revefenacin, gefitinib, ReJoin, tipelukast, bedoradrin, SCM-CGH, SHP-652, RNS-60, brodalumab, BIO-11006, umeclidinium bromide, vilanterol trifenatate, ipratropium bromide, tralokinumab , PUR-1800, VX-561, VX-371, oloppatadine, tulobuterol, formoterol fumarate, triamcinolone acetonide, reslizumab, salmeterol xinafoate, fluticasone propionate, beclomethasone dipropionate, formoterol fumarate, tiotropium bromide, ligelizumab, RUTI, glycolyzumabromimumab, berylimumab, berylimumab , SENS-111, declomethasone dipropeonate, CHF5992, LT-4001, indacaterol, glycopyrronium bromide, mometasone furoate, fexofenadine, glycoperronium bromide, azithromycin, AZD-7594, formoterol, CHF-6001, batefenterol, OATD-01, olodaterol, CJM11 , salmeterol, setipiprant, inhaled interferon beta, AZD-8871, plecanatide, fluticasone, salmeterol, eicosapentaenoic acid monoglycerides, lebrikizumab, RG-6149, QBKPN, mometasone, indacaterol, AZD-9898, pyruvate on tria, zileuton, CG-201, imidafenacin, CNTO-6785, CLBS-03, mometasone, RGN-137, procaterol, formoterol, CCI-15106, POL-6014, indacaterol, beclomethasone, MV-130, GC-1112, Allergovac depot , MEDI-3506, QBW-251, ZPL-389, udenafil, GSK-3772847, levocetirizine, AChP-1275, ADC-3680, timapiprant, abeditherol, AZD-7594, ipratropium bromide, salbutamol sulfate, tadekinig alfa, AST-774312, dornase alfa, iloprost, batefenterol, fluticasone furoate, alicaforsen, ciclesonide, emeramide, arformoterol, SB-010, ozagrel, VTT-1023, dectrecumab, levalbuterol, pranlukast, hyaluronic acid, GSK-2292767, formoterol, NOV-14, lucinactant, salbutamol , prednisolone, ebastine, dexamethasone cypecylate, GSK-2586881, BI-443651, GSK-2256294, VR-179, VR-096, hdm-ASIT+, budesonide, GSK-2245035, VTX-1463, emedastine, dexpramipexole, levalbuterol, N- 6022, dexamethasone sodium phosphate, PIN-201104, OPK-0018, TEV-4810, suplatast, BI-1060469, gemilukast, interferon gamma, dalazatide, bilastine, fluticasone propionate, salmeterol xy nafoate, RP-3128, bicyclochidia bromide, reslizumab, PBF-680, CRTH2 antagonist, pranlukast, salmeterol xinafoate, fluticasone propionate, tiotropium bromide monohydrate, mazilukast, RG-7990, doxofullin, abeditherol, glycopyrronium bromide, TEV-46024, , fliticasone propionate, glycopyrronium bromide, salmeterol xinofoate, salbutamol, TA-270, flinisolid, sodium chromoglycate, epsi-gam, ZPL-521, salbutamol, aviptadil, TRN-157, zafirlukast, stempeucel, pemirolast sodium, nadolol, fluticasone propionate+salmeterol xinafoate, RV-1729, salbutamol sulfate, carbon dioxide+perfluorooctyl bromide, APL-1, dectrecumab+VAK-694, lysine acetylsalicylate, zileuton, TR-4, human adipocyte-derived allogeneic mesenchymal progenitor cell therapy, MEDI-9314, PL -3994, HMP-301, TD-5471, NKTT-120, pemirolast, beclomethasone dipropionate, trantinterol, monosodium alpha-luminol, IMD-1041, AM-211, TBS-5, ARRY-502, seratrodast, recombinant midismase, ASM -8, deflazacort, bambuterol, RB x-10017609, ipratropium+fenoterol, fluticasone+formoterol, epinastine, WIN-901X, VALERGEN-DS, OligoG-COPD-5/20, tulobuterol, oxis turbuhaler, DSP-3025, ASM-024, mizolastine, budesonide+salmeterol, LH -011, AChR-E, histamine + human immunoglobulin, YHD-001, theophylline, ambroxol + erdosteine, ramatroban, montelukast, pranlukast, AG-1321001, tulobuterol, ipratropium + salbutamol, tranilast, methylprednisolone suleptanate, colforsin daropat, repyrinast and doxophylline.

The present invention also relates to a pharmaceutical composition containing a compound of the invention, or a pharmaceutically acceptable salt thereof, and one or more other therapeutic agents. The therapeutic agent may be selected from the class of agents mentioned above and from the list of specific agents described above. In some embodiments, the implementation of the pharmaceutical composition is suitable for delivery to the lungs. In some embodiments, the pharmaceutical composition is suitable for administration by inhalation or nebulization. In some embodiments, the implementation of the pharmaceutical composition is a dry powder or liquid composition.

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

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

Examples

The following synthetic examples and biological examples are provided to illustrate the invention and should in no way be construed as limiting the scope of the invention. In the examples below, the following abbreviations have the following meanings, unless otherwise noted. Abbreviations not defined below have their conventional meanings.

ACM=acetonitrile.

DCM=dichloromethane.

DIPEA=N,N-diisopropylethylamine.

- 17 040902

DMF=N,N-dimethylformamide.

EtOAc=ethyl acetate.

h=hour(s).

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

1PA = isopropyl alcohol.

1PAc=isopropyl acetate.

MeOH=methanol.

min=minute(s).

Pd(PPh ₃ ) ₄ =tetrakis(triphenylphosphine)palladium (0).

RT=room temperature.

TFA=trifluoroacetic acid.

THF=tetrahydrofuran.

bis(Pinacolato)diborone=4,4,5,5,4',4',5',5'-octamethyl-[2,2']bi[[1,3,2]dioxaborolanyl].

Reagents and solvents were purchased from commercial suppliers (Aldrich, Fluka, Sigma, etc.) and used without further purification. Reaction mixtures were monitored by thin layer chromatography (TLC), analytical high performance liquid chromatography (anal HPLC) and mass spectrometry. The reaction mixtures were worked up as specifically described for each reaction; they were usually purified by extraction and other purification methods such as crystallization, depending on the temperature and solvent, and by precipitation. In addition, reaction mixtures were typically purified by column chromatography or preparative HPLC, typically using C18 or BDS-filled columns and conventional eluents. Typical preparative HPLC conditions are described below.

Characterization of the reaction products was usually obtained by mass spectrometry and ¹ H NMR. For NMR analysis, samples were dissolved in a deuterated solvent (such as CD ₃ OD, CDCl ₃ or d6DMSO) and ¹ H-NMR spectra were obtained using a Varian Gemini 2000 instrument (400 MHz) under standard observation conditions. Mass spectrometric identification of compounds was performed by electrospray ionization (ESMS) using an Applied Biosystems (Foster City, CA) model API 150 EX instrument or a Waters (Milford, MA) 3100 instrument connected to autopurification systems.

Preparative HPLC conditions.

Column: C18, 5 µm, 21.2x150 mm; or C18, 5 µm 21x250; or C14, 5 µm 21x150 mm.

Column temperature: room temperature.

Flow rate: 20.0 ml/min.

Mobile phases: A=water+0.05% TFA.

B=ACN+0.05% TFA.

Injection volume: (100-1500 µl).

Detector wavelength: 214 nm.

The crude compounds were dissolved in 1:1 water:acetic acid at about 50 mg/mL. A 4 minute assay scale test was performed on a C18 2.1 x 50 mm column followed by a 15 or 20 minute run in preparative mode using a 100 µl gradient injection based on % retention time B in the assay mode test run. The exact gradients depended on the sample. Samples with close run-time impurities were tested using a 21x250 mm C18 column and/or a 21x150 mm C14 column for better separation. Fractions containing the desired product were identified by mass spectrometric analysis.

Preparation 1: 4-(benzyloxy)-2-ethylphenyl)trifluoro-λ ⁴ -borane, potassium salt I-5.

(a) 1-(Benzyloxy)-3-ethylbenzene (I-2).

To a stirred solution of 3-ethylphenol (I-1) (25.0 g, 204.0 mmol) in ACN (250 ml, 10 vols) was added potassium carbonate (42.0 g, 306 mmol) at room temperature. The resulting reaction mass was stirred at room temperature for 15 minutes, after which benzyl bromide (24.0 ml, 204 mmol) was added dropwise. The resulting reaction mixture was stirred for 6 hours at room temperature. After completion of the reaction (TLC control), the resulting reaction mass was

- 18 040902 was poured into water (1.0 L) followed by extraction of the compound with EtOAc (2x2 L). The combined organic layers were washed with cold water, brine and dried over sodium sulfate, filtered and evaporated under reduced pressure. The crude product was then purified by silica gel column chromatography (100-200 M) using 2% EtOAc in hexane as eluents to give the desired product (I-2) as a light yellow oily compound (35.0 g, 81%) . ¹ H NMR (400 MHz, chloroform-d) δ 7.46-7.44 (m, 2H), 7.39 (t, J=7.6 Hz, 2H), 7.34-7.31 (m , 1H), 7.21 (t, J=7.6 Hz), 6.86-6.80 (m, 3H), 5.07 (s, 2H), 2.64 (q, J=7, 6 Hz, 2H), 1.24 (t, J=7.6 Hz, 3H).

(b) 4-(Benzyloxy)-1-bromo-2-ethylbenzene (I-3).

To an ice-cold, stirred solution of 1-(benzyloxy)-3-ethylbenzene.

(I-2) (35.0 g, 164 mmol) in ACN (525 mL, 15 vols) was added N-bromosuccinimide (32.0 g, 181 mmol) in portions over 15 min. The resulting reaction mixture was stirred for 1 hour at room temperature. After completion of the reaction (TLC control), the resulting reaction mass was poured into ice water (1.50 L) followed by extraction of the compound with EtOAc (2x1 L). The combined organic layers were washed with water and dried over sodium sulfate, filtered and evaporated under reduced pressure to give the crude product. n-Hexane (250 ml) was added to the crude material to form a suspension which was then filtered through a Schott funnel. The mother liquor was evaporated under reduced pressure to give the desired product I-3 as a light yellow oily compound (42.0 g, 87%). 1H NMR (400 MHz, chloroform-d) δ 7.52-7.29 (m, 7H), 6.88 (s, 1H), 6.68 (d, J=6.0 Hz, 1H), 5 .04 (s, 2H), 2.69 (q, J=7.6 Hz, 2H), 1.20 (t, J=7.5 Hz, 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) (42.0 g, 144 mmol), bis(pinacolato)diborone (44.0 g, 173 mmol) and potassium acetate (28 g , 288 mmol) in dioxane (440 mL) was degassed by purging N2 (gas) for 15 min followed by the addition of PdCl ₂ (dppf)-DCM complex (11.0 g, 15 mmol). The resulting reaction mixture was heated to 80°C for the next 16 hours. After completion of the reaction (TLC monitoring), the reaction mass was filtered through a pad of celite and the mother liquor was evaporated under reduced pressure to obtain a crude product. The crude residue was purified by silica gel column chromatography (100-200 M) using 1% EtOAc in hexane as eluents to give the desired product (I-4) as a light yellow oily compound (32.0 g, 66%). 1H NMR (400 MHz, chloroform-d) δ 7.74 (d, J=8.4 Hz, 1H), 7.45-7.36 (m, 5H), 6.84-6.78 (m, 2H), 5.08 (s, 2H), 2.91 (q, J=7.6 Hz), 1.33 (s, 12H), 1.19 (t, J=7.6 Hz, 3H) .

(d) (4-(Benzyloxy)-2-ethylphenyl)trifluoro-λ ⁴ -borane, potassium salt (I-5).

To a stirred solution of 2-(4-(benzyloxy)-2-ethylphenyl)-4,4,5,5-tetramethyl-1,3,2dioxaborolane (I-4) (20 g, 59.0 mmol) in acetone :methanol (200 mL, 1:1 ratio, 10 vols) was added 3M potassium fluoride (23.0 g, 295 mmol, dissolved in 98.0 mL water). The resulting reaction mixture was stirred at room temperature for 16 hours. After completion of the reaction (TLC monitoring), the resulting reaction mass was evaporated under reduced pressure. The solid thus obtained was taken up in water (100 ml) and stirred at room temperature for 30 minutes. The resulting reaction mass was filtered through a Schott funnel, washed with n-hexane and dried under reduced pressure to give the desired product (I-5) as a white solid (14.0 g, 74%). 1H NMR (400 MHz, chloroform-d) δ 7.43 (d, J=7.2 Hz, 2H), 7.37 (t, J=7.5 Hz, 2H), 7.30 (t, J =7.1 Hz, 1H), 7.22 (d, J=8.0 Hz), 6.58 (s, 1H), 6.53 (d, J=7.9 Hz, 1H), 5, 00 (s, 2H), 2.65 (kv, J=7.5 Hz, 2H), 1.07 (t, J=7.4 Hz, 3H).

Preparation 2: 6-benzyl-5-(tert-butyl)-(S)-3-benzyl-3,4,6,7-tetrahydro-5H-imidazo[4,5-c]niridine5,6-dicarboxylate (I -eleven).

1-10 1-11 (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) (5.0 kg, 32.14 mol) in water (40 L, 8 volumes) was added dropwise concentrated hydrochloric acid (3.93 L, 33.75 mol) followed by the addition of formaldehyde (5.50 L, 67.5 mol, 37% aqueous solution). The resulting solution was stirred for 30 min at the same temperature and then heated at 80° C. for 8 h. The progress of the reaction was monitored by LCMS. The water was removed under reduced pressure to give a crude product and the resulting crude product was stirred for 2 hours in toluene (20 L). The organics were removed under reduced pressure to remove excess water and the compound was azeotropically dried. The resulting material was then taken up in diethyl ether (20 L) and stirred for 2 hours. The solid material was then filtered and air dried to give the desired product (I-7) as an off-white solid (6.50 kg, 85 %). ¹ H NMR (400 MHz, D ₂ O) δ 8.69 (s, 1H), 4.56 (d, J=15.4 Hz, 1H), 4.42 (d,

J=15.5 Hz, 1H), 4.20 (dd, J=5.5, 5.2 Hz, 1H), 3.42 (dd, J=5.0, 17.0 Hz, 1H), 3.11 (dd, J=10.2, 16.8 Hz, 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 an ice-cold stirred solution of (S)-4,5,6,7-tetrahydro-3H-imidazo[4,5c]pyridine-6-carboxylic acid dihydrochloride (I-7) (6.10 kg, 25.40 mol) in 1,4-dioxane (48 L, 8 vols) and water (48 L, 8 vols) triethylamine (12.36 L, 89 mol) was added dropwise followed by di-tert-butyl dicarbonate (18.07 L, 78 .74 mol, dissolved in 5 l of 1,4-dioxane) for 30 min. The resulting reaction mixture was stirred at room temperature for a further 16 hours. After completion of the reaction (monitored by TLC and LCMS), the yellowish reaction mixture was diluted with water (10 L) and washed successively with diethyl ether (2x10 L) and EtOAc (2x7.50 L). The organic phase was discarded. The aqueous layer was cooled and adjusted to pH ~3 with 6N HCl; the aqueous phase was extracted with EtOAc (3x10 L). The combined organic layers were washed with brine, dried over sodium sulfate and concentrated under reduced pressure. The oily residue was crystallized from 30% EtOAc:hexanes to give the desired product (I-8) as an off-white solid (5.1 kg, 55%). (m/z): [M+H]+ calculated for C ₁₇ H ₂₅ N ₃ O ₆ 368.18, found 368.21.

(c) 6-Benzyl-3,5-di-tert-butyl-(S)-6,7-dihydro-3H-imidazo[4,5-c]pyridine-3,5,6(4Н)-tricαcarboxylate ( I-9).

To an ice-cold solution of (S)-3,5-bis(tert-butoxycarbonyl)-4,5,6,7-tetrahydro-3Himidazo[4,5-c]pyridine-6-carboxylic acid (I-8) (5 .1 kg, 13.88 mol) in DCM (51 L, 10 vols) were added successively saturated aqueous sodium bicarbonate (41.0 L, 8 vols), tetrabutylammonium iodide (5.13 kg, 13.88 mol) and benzyl bromide ( 2.47 L, 20.82 mol). The resulting reaction mixture was stirred at room temperature for the next 16 hours. After completion of the reaction (monitoring by TLC and LCMS), the biphasic solution was separated. The aqueous layer was extracted with DCM (3x10 L). The combined organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to give the crude product, which was purified by column chromatography through silica gel (100-200 M) using 40% EtOAc in hexane as eluents to give the desired product ( I-9) as a viscous oil (4.50 kg, 72%). (m/z): [M+H]+ calculated for C ₂₄ H ₃₁ N ₃ O ₆ 458.22, found 458.60.

(d) 6-Benzyl-5-(tert-butyl)-(S)-3,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridine-5,6-dic-carboxylate (I-10 ).

To an ice-cold solution of 6-benzyl-3,5-di-tert-butyl-(S)-6,7-dihydro-3H-imidazo[4,5c]pyridine-3,5,6(4H)-tricarboxylate (I -9) (4.50 kg, 9.84 mol) in IPA (45 L, 10 volumes) was added dropwise ammonium hydroxide (36 L, 8 volumes). The resulting reaction mixture was further stirred at room temperature for a further 16 hours. After completion of the reaction (monitored by TLC and LCMS), the resulting mixture was diluted with water (25 L) followed by extraction with EtOAc (3x20 L). The combined organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to give the crude product, which was purified by column chromatography through silica gel (100-200 M) using 2% MeOH in DCM as eluents to give the desired product ( I-10) as a thick viscous oil (2.70 kg, 77%). (m/z): [M+H]+ calculated for C ₁₉ H ₂₃ N ₃ O ₄ 358.17, found 358.33.

(e) 6-Benzyl-5-(tert-butyl)-(S)-3-benzyl-3,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridine-5,6-dicarboxylate (I-11).

To an ice-cold solution of 6-benzyl-5-(tert-butyl)-(S)-3,4,6,7-tetrahydro-5H-imidazo[4,5c]pyridine-5,6-dicarboxylate (I-10) (2.70 kg, 7.55 mol) in DCM (32.4 L, 12 vols) was added 1 N aqueous sodium hydroxide solution (24.3 L, 9 vols) followed by sequential addition of tetrabutylammonium iodide (2.80 kg, 7.55 mol) and benzyl bromide (0.99 L, 8.31 mol). The resulting reaction mixture was stirred at room temperature for the next 2 hours. After completion of the reaction (monitored by TLC and LCMS), the biphasic solution was separated. The aqueous layer was extracted with DCM (3x10 L). The combined organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to give the crude product, which was purified by column chromatography on silica gel (100-200 M) using 40% EtOAc in hexane as eluents to give the desired product ( I-11) as a viscous oil (1.70 kg, 63%). (m/z): [M+H]+ calculated for C ₂₆ H ₂₉ N ₃ O ₄ 448.22, found 448.20.

Preparation 3: 6-benzyl-5-(tert-butyl)-(S)-3-benzyl-2-(6-(4-(benzyloxy)-2-ethylphenyl)-1H-indazol3-yl)-3.4 ,6,7-tetrahydro-5H-imidazo[4,5-c]pyridine-5,6-dicarboxylate (I-16).

- 20 040902

(a) 4-Bromo-2-fluorobenzoyl chloride (I-13).

Oxalyl chloride (0.98 L, 11.42 mol). The resulting reaction mixture was stirred for 10 minutes at the same temperature. Then DMF (150 ml) was added dropwise to the reaction mixture. The resulting reaction mass was allowed to warm to room temperature and stirred for 2 hours. After completion of the reaction (TLC monitoring), excess oxalyl chloride was removed under reduced pressure under nitrogen to give crude product (I-13) (1.08 kg, 80%) , which was used in the next step without further purification.

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

To a stirred solution of 6-benzyl-5-(tert-butyl)-(S)-3-benzyl-3,4,6,7-tetrahydro-5nimidazo[4,5-c]pyridine-5,6-dicarboxylate (I -11) (1.70 kg, 3.80 mol) in ACN (13.6 L, 8 vols) was added triethylamine (2.11 L, 15.2 mol) followed by 4-bromo-2-fluorobenzoyl chloride (I -13) (1.08 kg, 4.56 mol in 3.4 L ACN, 2 volumes) at room temperature. After completion of the addition, the light yellow color of the resulting reaction mixture turned brown. The resulting reaction mixture was stirred at the same temperature for 30 min, and the progress of the reaction was monitored by TLC. The resulting reaction mixture was quenched with ice water (10 L) followed by extraction with EtOAc (3x5 L) and the combined organic layers washed with brine. The organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure to give the crude product, which was purified by column chromatography on silica gel (100-200M) using 20% EtOAc in hexane as eluents to give the desired product (I-14) (1.74 kg, 71%). (m/z): [M+H]+ calculated for C ₃₃ H ₃₁ BrFN ₃ O ₅ 648.14, found 648.20.

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

To a stirred solution of 6-benzyl-5-(tert-butyl)-(S)-3-benzyl-2-(4-bromo-2-fluorobenzoyl)3,4,6,7-tetrahydro-5H-imidazo[4, 5-c]pyridine-5,6-dicarboxylate (I-14) (1.74 kg, 2.68 mol) in THF (26.0 L, 15 vols) was added hydrazine hydrate (0.705 L, 13.4 mol) at room temperature. The resulting reaction mixture was heated at 60°C for 3 hours. After completion of the reaction (TLC monitoring), the resulting reaction mass was poured into ice water (10 L) followed by extraction of the compound with EtOAc (3x10 L). The combined organic layers 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 (100-200 M) using 20% EtOAc in hexane as eluents to give the desired product ( I-15) as an off-white solid (1.12 kg, 65%). (m/z): [M+H] ⁺ calculated for C ₃₃ H ₃₂ BrFN ₅ O ₄ 642.16, found 642.21.

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

Bis(pinacolato)diborone (250 g, 984 mmol) was charged into a 5 L three-necked single-walled flask pre-etched with fluorides, together with propan-2-ol (1.882 L, 2.46E+04 mmol) and the mixture was stirred until complete. dissolution. Dissolution was endothermic (-4°C). In a 4 L Erlenmeyer flask pre-etched with fluoride compounds, potassium fluoride hydrofluoride (538 g, 6891 mmol) was dissolved in water (2.306 L, 1.28E+05 mmol) to form a 3M solution. Dissolution was endothermic (-12°C). The solution was then filtered to remove a small amount of undissolved material from the potassium fluoride hydrofluoride. Once both solutions were completely dissolved, the contents of the Erlenmeyer flask were batch-loaded into the single-walled flask for 15 min. Observed a moderate exothermic effect (+10°C). During the addition, the solution turned into a thick and translucent translucent gray suspension, and stirring force

- 21 040902 to keep the contents well mixed. The mixture was stirred for 1.5 hours and then filtered through a sintered glass funnel (4 L, 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 (cake cracking observed). The solids were transferred back to a cleaned 5 L three neck single wall flask and resuspended in water (1.33 L, 7.38E+04 mmol). The suspension was stirred for 2 hours, resulting in the formation of a transparent homogeneous hydrogel. The solution was stirred for 1 hour, after which time the solids/gel were filtered off using a 4 L coarse filter glass funnel (pre-etched). The solids were allowed to dry on the filter for 30 minutes. The solids were transferred back to a cleaned 5 L three neck single wall flask and resuspended in acetone (1.084 L, 1.48E+04 mmol). The white-gray suspension was stirred for 1 h, and then filtered onto a 4 L glass coarse filtration funnel (previously pickled). It took 20 minutes to complete the filtration, and then funnel drying was performed for another 1 hour. During this time, the solids were stirred from time to time to ensure uniform drying. After drying, a light white powder remained on the filter. The solids were dried for 20 hours at 55° C. under vacuum under a slow stream of nitrogen to give a fluffy white solid (200.3 g collected).

To a stirred solution of 6-benzyl-5-(tert-butyl)-(S)-3-benzyl-2-(6-bromo-1H-indazol-3-yl)3,4,6,7-tetrahydro-5H- (4-(benzyloxy)-2- ethylphenyl)trifluoro-λ ⁴ -borane, potassium salt (I-5) (8.0 g, 20 mmol) and the fluffy white solid obtained above (0.20 g). The resulting reaction mixture was degassed with nitrogen for 30 min. To this solution was added the prepared aqueous cesium carbonate solution (20.0 g, 62.0 mmol in 60 ml of water, 6 volumes). The resulting reaction mixture was further degassed for 15 min followed by the addition of bis(ditert-butyl(4-dimethylaminophenyl)phosphine)dichloropalladium (II) (0.66 g, 0.93 mmol) and the reaction mixture was evacuated and washed with nitrogen. The resulting reaction mixture was heated at 110°C for 20 h. After completion of the reaction (TLC and LCMS monitoring), the resulting reaction mixture was cooled to room temperature and filtered through a pad of celite, then washed with EtOAc (3x0.5 L). The combined organic layers were washed with 1 N sodium hydroxide solution (3x0.5 L). The combined organic layers were then washed with brine and dried over sodium sulfate, filtered and evaporated under reduced pressure to give a crude product which was purified by column chromatography on silica gel (100-200M) using 20% EtOAc in hexane as eluents to give the desired product. (I-16) (as a mixture of N-benzyl regioisomers) as a light yellow solid (8.0 g, 66%). (m/z): [M+H]+ calculated for C ₄₈ H ₄₇ N ₅ O ₅ 774.36, found 774.59.

Preparation 4: (S)-2-(6-(2-ethyl-4-hydroxyphenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-3H-imidazo[4.5c]pyridine -6-carboxylic acid, hydrochloride (I-18).

VPO VPO (a) Benzyl-(S)-3-benzyl-2-(6-(4-(benzyloxy)-2-ethylphenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro -3Himidazo[4,5-c]pyridine-6-carboxylate, hydrochloride (I-17).

6-Benzyl-5-(tert-butyl)-(S)-3-benzyl-2-(6-(4-(benzyloxy)-2-ethylphenyl)-1H-indazol-3-yl)-3.4, 6,7tetrahydro-5H-imidazo[4,5-c]pyridine-5,6-dicarboxylate (I-16) (1.0 g, 1.292 mmol) was dissolved in dioxane (8 ml) and water (1.5 ml) , then a 4M solution of hydrogen chloride in dioxane (7 ml, 28.0 mmol) was added and the reaction mixture was stirred at room temperature for 3 h (reaction progress monitored by LCMS). The reaction mixture was then frozen and lyophilized, and the crude product (I-17) was used directly in the next reaction (estimated quantitative yield), (m/z): [M+H]+ calculated for C ₄₃ H ₃₉ N ₅ O ₃ 674, 31, found 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).

Benzyl-(S)-3-benzyl-2-(6-(4-(benzyloxy)-2-ethylphenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-3Himidazo[4, 5-c]pyridine-6-carboxylate, hydrochloride (I-17) (0.918 g, 1.292 mmol) was dissolved in 2-propanol (15 ml), 5 M hydrogen chloride in water (0.258 ml, 1.292 mmol) and water (0.292 mmol). 25 ml) at 50° C., then 10 wt% palladium on charcoal, 50% water (0.138 g, 0.065 mmol) was added. The reaction flask was then purged with nitrogen, a hydrogen balloon was attached, and the reaction mixture was stirred at 50° C. for 4 days, replenishing the balloon with hydrogen if necessary (reaction progress monitored by LCMS). Then all solids were removed by filtration, and the resulting solution was concentrated. The residue was dissolved in ACN/water 1:1, frozen and lyophilized. The resulting powder (I-22 040902

18) used without further purification (estimated quantitative yield), (m/z): [M+H]+ calculated for C ₂₂ H ₂₁ N5O ₃ 404.17 found 404.2.

Preparation 5: (S)-2-(6-(2-ethyl-4-hydroxyphenyl)-1H-indazol-3-yl)-5-isopropyl-4,5,6,7-tetrahydro3H-imidazo[4,5 -c] pyridine-6-carboxylic acid (I-19).

(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) (0.25 g, 0.568 mmol) was suspended in DMF (2.5 ml) and acetone (2.5 ml), then acetic acid (0.098 ml, 1.705 mmol) and sodium cyanoborohydride ( 0.179 g, 2.84 mmol) and the reaction mixture was stirred at room temperature for 24 h (reaction progress monitored by LCMS). The reaction mixture was concentrated, then the crude product was purified by reverse phase chromatography (5-70% ACN/water gradient, 50 g C18aq column) to give the TFA salt of the title compound (149 mg, 47% yield). (m/z): [M+H]+ calculated for C ₂₅ H ₂₇ N ₅ O ₃ 446.21, found 446.3.

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

(S)-2-(6-(2-Ethyl-4-hydroxyphenyl)-1H-indazol-3-yl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine6- carboxylic acid, HCl (I-18) (0.160 g, 0.364 mmol) and propionaldehyde (0.039 ml, 0.546 mmol) were dissolved in methanol (3.0 ml), then sodium cyanoborohydride (0.069 g, 1.091 mmol) was added and the reaction mixture was stirred at room temperature for 24 h (the course of the reaction was monitored by LCMS). The reaction mixture was concentrated and the crude product was purified by reverse phase chromatography (5-70% ACN/water gradient, 50 g C18 column) to give the TFA salt of the title compound (78 mg, 38% yield). (m/z): [M+H]+ calculated for C ₂₅ H ₂₇ N ₅ O ₃ 446.21, found 446.3.

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

(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) (0.160 g, 0.364 mmol) and a solution of formaldehyde, 37 wt.% in water (0.032 ml, 0.436 mmol) were dissolved in methanol (3.0 ml), then sodium cyanoborohydride (0.069 g , 1.091 mmol) and the reaction mixture was stirred at room temperature for 4 h (the progress of the reaction was monitored by LCMS). Sodium borohydride (14 mg, 0.364 mmol) was added to quench excess formaldehyde, then the reaction mixture was concentrated. The crude product was purified by reverse phase chromatography (5-70% ACN/water gradient, 50 g C18 column) to give the TFA salt of the title compound (110 mg, 57% yield). (m/z): [M+H]+ calculated for C ₂₃ H ₂₃ N ₅ O ₃ 418.18, found 418.2.

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

- 23 040902

(a) tert-Butyl-(R)-4-(2-hydroxyethyl)-2-methylpiperazine-1-carboxylate (I-23) (R)-1-boc-2-methylpiperazine (0.2 g, 0.999 mmol ), DIPEA (0.698 ml, 3.99 mmol) and 2-bromoethanol (0.085 ml, 1.198 mmol) were dissolved in DMF (5 ml) and the reaction mixture was stirred at 60°C for 16 h (reaction progress monitored by LCMS) . The reaction mixture was concentrated, then 10 ml of diethyl ether was added to the residue. The solution was sonicated until the gelatinous precipitate disappeared and a solid formed. The ether solution was then decanted from the solid. The solid was then used directly in the next reaction (estimated quantitative yield), (m/z): [M+H] ⁺ calculated for C ₁₂ H ₂₄ N ₂ O ₃ 245.18 found 245.4.

(b) (R)-2-(3-methylpiperazin-1-yl)ethan-1-ol, dihydrochloride (I-24) tert-butyl-(R)-4-(2hydroxyethyl)-2-methylpiperazin-1- the carboxylate (0.244 g, 0.999 mmol) was dissolved in dioxane (3.0 ml) and water (0.5 ml), then a 4M solution of hydrogen chloride in dioxane (2.0 ml, 65.8 mmol) was added and the reaction mixture was stirred at room temperature for 3 h (the course of the reaction was monitored by LCMS). The reaction mixture was frozen and lyophilized and the resulting product was used without purification (estimated quantitative yield), (m/z): [M+H] ⁺ calculated for C ₇ H ₁₆ N ₂ O 145.13 found 145.4.

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

(S)-2-(6-(2-Ethyl-4-hydroxyphenyl)-1H-indazol-3-yl)-5-propyl-4,5,6,7-tetrahydro-3H-imidazo[4.5c] pyridine-6-carboxylic acid, TFA (I-20) (40 mg, 0.071 mmol), n-(2-hydroxyethyl)piperazine (0.018 ml, 0.143 mmol) and DIPEA (0.025 ml, 0.143 mmol) were dissolved in DMF (1 .5 ml), then HATU (40.8 mg, 0.107 mmol) was added and the reaction mixture was stirred at room temperature for 3 h (reaction progress monitored by LCMS). Hydrazine (0.011 mL, 0.357 mmol) was added to cleave unwanted 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, column C18) to give the TFA salt of the title compound (31 mg, 56% yield). (m/z): [M+H]+ calculated for C ₃₁ H ₃₉ N ₇ O ₃ 558.31, found 558.3. 1H NMR (400 MHz, DMSOL) δ 13.10 (s, 1H), 12.27 (d, J=48.92 Hz, 1H), 9.40 (s, 1H), 8.28 (t, J =8.10 Hz, 1H), 7.30 (s, 1H), 7.04 (m, 2H), 6.71 (d, J=2.46 Hz, 1H), 6.64 (dd, J =2.50, 8.23 Hz, 1H), 4.44 (t, J=5.31 Hz, 1H), 4.11 (q, J=5.26, 2H), 3.96 (m, 1H), 3.86-3.52 (m, 6H), 3.49 (q, J=6.01 Hz, 2H), 2.95 (m, 2H), 2.48 (q, J=7 .48 Hz, 2H), 2.42-2.21 (m, 4H), 2.37 (t, J=6.20 Hz, 2H), 1.42 (m, 2H), 1.00 (t , J=7.52 Hz, 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-3Himidazo[4, 5-c]pyridin-6-yl)((1S,4S)-5-METHYL-2,5-diazabicyclo[2.2.1]heptan-2-yl)methanone.

(S)-2-(6-(2-Ethyl-4-hydroxyphenyl)-1H-indazol-3-yl)-5-propyl-4,5,6,7-tetrahydro-3H-imidazo[4.5c] pyridine-6-carboxylic acid, TFA (I-20) (40 mg, 0.071 mmol), (1S,4S)-5-methyl-2,5diazabicyclo[2.2.1]heptane dihydrobromide (29.4 mg, 0.107 mmol) and DIPEA (0.062 ml, 0.357 mmol) were dissolved in DMF (1.5 ml), then HATU (40.8 mg, 0.107 mmol) was added and the reaction mixture was stirred at room temperature for 3 h (reaction progress monitored by LCMS) . Hydrazine (0.011 mL, 0.357 mmol) was added to cleave unwanted 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 (gradient 5-70% ACN/water, column

- 24 040902

C18) to give the TFA salt of the title compound (32 mg, 59% yield). (m/z): [M+H]+ calculated for C ₃₁ H ₃₇ N ₇ O ₂ 540.30, found 540.3.

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

(a) tert-Butyl-(S)-4-((S)-2-(6-(2-ethyl-4-hydroxyphenyl)-1H-indazol-3-yl)-5-methyl-4.5, 6,7-tetrahydro3H-imidazo[4,5-c]pyridine-6-carbonyl)-3-methylpiperazine-1-carboxylate (I-25).

(S)-2-(6-(2-ethyl-4-hydroxyphenyl)-1H-indazol-3-yl)-5-methyl-4,5,6,7-tetrahydro-3H-imidazo[4.5c] pyridine-6-carboxylic acid, TFA(I-21) (55 mg, 0.103 mmol), (S)-4-n-boc-2-methylpiperazine (41.5 mg, 0.207 mmol) and DIPEA (0.036 ml, 0.207 mmol) was dissolved in DMF (1.5 ml), then HATU (59.0 mg, 0.155 mmol) was added and the reaction mixture was stirred at room temperature for 16 h (reaction progress monitored by LCMS). Hydrazine (0.016 ml, 0.517 mmol) was added to cleave unwanted 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, 50 g C18 column) to give the TFA salt of the title compound (54 mg, 72% yield). (m/z): [M+H]+ calculated for C ₃₃ H ₄₁ N ₇ O ₄ 600.33, found 600.3.

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

tert-Butyl-(S)-4-((S)-2-(6-(2-ethyl-4-hydroxyphenyl)-1H-indazol-3-yl)-5-methyl-4,5,6,7 -tetrahydro-3Himidazo[4,5-c]pyridine-6-carbonyl)-3-methylpiperazine-1-carboxylate, TFA (I-25) (0.126 g, 0.177 mmol) was dissolved in dioxane (1.5 ml) and water (0.3 ml), then a 4M solution of hydrogen chloride in dioxane (1.5 ml, 6.00 mmol) was added and the reaction mixture was stirred at room temperature for 2 h (reaction progress monitored by LCMS). The reaction mixture was frozen and lyophilized and the resulting powder was used directly in the following reaction (estimated quantitative yield), (m/z): [M+H]+ calculated for C ₂₈ H ₃₃ N ₇ O ₂ 500.27 found 500.3 .

(c) ((S)-2,4-Dimethylpiperazin-1-yl)((S)-2-(6-(2-ethyl-4-hydroxyphenyl)-1H-indazol-3-yl)-5-methyl4 ,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[4.5c ]pyridin-6-yl)((S)-2-methylpiperazin-1-yl)methanone, dihydrochloride (0.101 g, 0.176 mmol) and formaldehyde solution, 37 wt% in water (0.016 ml, 0.212 mmol) were dissolved in methanol (3.0 ml), then sodium cyanoborohydride (0.055 g, 0.882 mmol) was added and the reaction mixture was stirred at room temperature for 16 h (reaction progress monitored by LCMS). Sodium borohydride (7 mg, 0.176 mmol) was added to quench any remaining formaldehyde. The reaction mixture was concentrated, then the crude product was purified by preparative HPLC (5-60% ACN/water gradient, column C18) to give the TFA salt of the title compound (28 mg, 21% yield). (m/z): [M+H]+ calculated for C ₂₉ H ₃₅ N ₇ O ₂ 514.29, found 514.3.

Example 4: (S)-(2-(6-(2-Ethyl-4-hydroxyphenyl)-1H-indazol-3-yl)-5-isopropyl-4,5,6,7-tetrahydro3H-imidazo[4, 5-c]pyridin-6-yl)(4-methyl-1,4-diazepan-1-yl)methanone.

(S)-2-(6-(2-Ethyl-4-hydroxyphenyl)-1H-indazol-3-yl)-5-isopropyl-4,5,6,7-tetrahydro-3H-imidazo[4.5c] pyridine-6-carboxylic acid, TFA (I-19) (50 mg, 0.089 mmol), 1-methylhomopiperazine (0.022 ml, 0.179 mmol) and DIPEA (0.031 ml, 0.179 mmol) were dissolved in DMF (1.5 ml), then HATU (51.0 mg, 0.134 mmol) was added and the reaction mixture was stirred at room temperature for 3 h (reaction progress monitored by LCMS). Hydrazine (0.014 mL, 0.447 mmol) was added to remove unwanted by-products and 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

- 25 040902 (gradient 2-70% ACN/water, column C18) to give the TFA salt of the title compound (29 mg, 42% yield). (m/z): [M+H]+ calculated for C ₃₁ H ₃₉ N ₇ O ₂ 542.32, found 542.3. 1H NMR (400 MHz,

DMSO^) δ 13.08 (s, 1H), 12.21 (d, J=29.9 Hz, 1H), 9.40 (s, 1H), 8.27 (d, J=8.33 Hz , 1H), 7.30 (s, 1H), 7.04 (t, J=8.05, 2H), 6.71 (d, J=2.53 Hz, 1H), 6.64 (dd, J=2.54, 8.23 Hz, 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=13.9 Hz, 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-3Himidazo[4, 5-c]pyridin-6-yl)((R)-4-(2-hydroxyethyl)-2-methylpiperazin-1-yl)methanone.

(S)-2-(6-(2-Ethyl-4-hydroxyphenyl)-1H-indazol-3-yl)-5-methyl-4,5,6,7-tetrahydro-3H-imidazo[4.5c] pyridine-6-carboxylic acid, TFA (I-21) (55 mg, 0.103 mmol), (R)-2-(3-methylpiperazin-1-yl)ethan-1ol, dihydrochloride (I-24) (33.7 mg, 0.155 mmol) and DIPEA (0.090 ml, 0.517 mmol) were dissolved in DMF (1.5 ml), then HATU (59.0 mg, 0.155 mmol) was added and the reaction mixture was stirred at room temperature for 16 h (stroke reactions were monitored by LCMS). Hydrazine (0.016 ml, 0.517 mmol) was added to cleave unwanted 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, column C18) to give the TFA salt of the title compound (22 mg, 28% yield). (m/z): [M+H]+ calculated for C ₃₀ H ₃₇ N ₇ O ₃ 544.30, found 544.3.

Example 6: ((S)-3-(dimethylamino)pyrrolidin-1-yl)((S)-2-(6-(2-ethyl-4-hydroxyphenyl)-1H-indazol3-yl)-5-isopropyl- 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-isopropyl-4,5,6,7-tetrahydro-3H-imidazo[4.5c] pyridine-6-carboxylic acid, TFA (I-19) (50 mg, 0.089 mmol), (S)-(-)-3-(dimethylamino)pyrrolidine (0.023 ml, 0.179 mmol) and DIPEA (0.031 ml, 0.179 mmol ) was dissolved in DMF (1.5 ml), then HATU (51.0 mg, 0.134 mmol) was added and the reaction mixture was stirred at room temperature for 3 h (reaction progress monitored by LCMS). Hydrazine (0.014 mL, 0.447 mmol) was added to cleave unwanted by-products and 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, column C18) to give the TFA salt of the title compound (37 mg, 53% yield). (m/z): [M+H]+ calculated for C ₃₁ H ₃₉ N ₇ O ₂ 542.32, found 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 (20 g, 910.32 mmol) in ACN (250 ml) was added K ₂ CO ₃ (31.55 g, 228.3 mmol) and then added dropwise benzyl bromide (13.10 ml, 109.58 mmol). The resulting reaction mixture was stirred at 80° C. for 2 hours. The aqueous layer was extracted with EtOAc (three times), combined and washed with brine. The organic layer was dried over Na ₂ SO ₄ and evaporated under reduced pressure to give the title intermediate as a pale yellow oil (25 g, 89% yield). 1H NMR (400 MHz, chloroform-d) δ 7.48-7.30 (m, 5H), 7.27 (d, J=10.5 Hz, 1H), 6.87 (d, J=8, 7 Hz, 1H), 5.12 (s, 2H), 2.66 (kv, J=7.5 Hz, 2H), 1.16 (t, J=7.5 Hz, 3H).

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

To a solution of the product from the previous step (12.5 g, 40.45 mmol) in dioxane (100 ml) was added bis(pinacolato)diborone (15.40 g, 60.67 mmol) and KOAc (11.9 g , 121.35 mmol). reactionary

- 26 040902 the mixture was purged with nitrogen for 15 min followed by the addition of [1,1'bis(diphenylphosphino)ferrocene]dichloropalladium (II) complex with dichloromethane (1.65 g, 2.023 mmol). The resulting reaction mixture was stirred and heated at 110°C for 3 h, filtered through celite, and the residue was washed with EtOAc. The filtrate was diluted with excess EtOAc (200 ml), and washed with water (100 ml) then brine (100 ml), dried over sodium sulfate and concentrated in vacuo to give the crude product, which was purified by column chromatography on (100-200 ml) silica gel eluting 35% EtOAc:hexane to give the desired product as an off-white solid (9.50 g, 66% yield). ¹ H NMR (400 MHz, chloroform-d) δ 7.54-7.27 (m, 6H), 6.81 (d, J=7.9 Hz, 1H), 5.16 (s, 2H), 2.84 (q, J=7.5 Hz, 2H), 1.32 (s, 12H), 1.14 (t, J=7.5 Hz, 3H).

Biological analyzes

Compounds of the present invention were characterized using one or more of the following biological assays.

Analysis 1. Biochemical analyzes of JAK kinases.

A LanthaScreen panel for biochemical analyzes of four JAKs (JAK1, 2, 3 and Tyk2) was used in a common kinase reaction buffer (50 mM HEPES, pH 7.5, 0.01% Brij-35, 10 mM MgCl ₂ and 1 mM EGTA ). Recombinant GST-tagged JAK enzymes and GFP-tagged STAT1 peptide substrate were obtained from Life Technologies.

Serially diluted compounds were pre-incubated with each of the four JAK enzymes and substrate in white 384-well microplates (Corning) at ambient temperature for 1 hour. ATP was then added to initiate kinase reactions in a total volume of 10 μl with 1% DMSO. Final enzyme concentrations for JAK1, 2, 3, and Tyk2 were 4.2 nM, 0.1 nM, 1 nM, and 0.25 nM, respectively; the respective ATP Km concentrations used were 25 μM, 3 μM, 1.6 μM and 10 μM; while the substrate concentration was 200 nM in all four assays. Kinase reactions were performed for 1 h at ambient temperature before adding 10 µl of EDTA preparation (final concentration 10 mM) and Tb-anti-pSTAT1 (pTyr701) antibody (Life Technologies, final concentration 2 nM) in TR-FRET dilution buffer ( Life Technologies). The plates were incubated at ambient temperature for 1 hour before reading on an EnVision reader (Perkin Elmer).

The emissivity signals (520 nm/495 nm) were recorded and then used to calculate the percent inhibition considering the DMSO control and background controls.

For dose-response analysis, percentage inhibition was plotted against compound concentrations, and IC ₅₀ values were determined by fitting a 4-parameter logistic model using Prism software (GraphPad Software). The results were expressed as pIC ₅₀ (negative logarithm of IC ₅₀ ) and converted to pK, (negative logarithm of the dissociation constant Ki) using the Cheng-Prusov equation.

Test compounds having a lower Kj value or a higher pKj value in each of the four JAK assays showed greater inhibition of JAK activity.

Assay 2 Inhibition of IL-2-stimulated pSTAT5 in Tall-1 T cells.

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

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

Human T cells from the Tall-1 cell line were cultured in a humidified incubator at 37°C and 5% CO ₂ in RPMI (Life Technologies) supplemented with 15% heat-inactivated fetal bovine serum (FBS, Life Technologies), 2 mM glutamax (Life Technologies), 25 mM HEPES (Life Technologies), and 1X Pen/Strep (Life Technologies). Compounds were serially diluted in DMSO and sonicated into empty wells. Assay medium (DMEM without phenol red (Life Technologies) supplemented with 10% FBS (ATCC)) (4 μl/well) was added and the plates were shaken at 900 rpm for 10 minutes. Cells were seeded in assay medium (4 µl/well) at a rate of 45,000 cells/well and incubated at 37°C and 5% CO2 for 1 h followed by the addition of IL-2 (R&D Systems; final concentration 300 ng/ml) in prewarmed assay medium (4 µl) for 30 min. After cytokine stimulation, cells were lysed with 6 μl of 3x AlphaLisa lysis buffer (PerkinElmer) containing 1x PhosStop and Complete tablets (Roche). The lysate was shaken at 900 rpm for 10 minutes at room temperature (RT). Phosphorylated STAT5 was measured using the pSTAT5 AlphaLisa kit (PerkinElmer). A freshly prepared mixture of acceptor beads was added to the lysate (5 μl) by irradiation with light passing through a green filter and providing an illumination of <100 lux. The plates were shaken at 900 rpm for 2 minutes, centrifuged for a short period of time and incubated for 2 hours at room temperature in the dark. Donor granules (5 μl) were dosed by irradiating with light passing through a green filter and providing

- 27 040902 baking illumination <100 lux.

The plates were shaken at 900 rpm for 2 minutes, centrifuged for a short period of time and incubated overnight at room temperature in the dark. Luminescence was measured at an excitation wavelength of 689 nm and an emission wavelength of 570 nm using an EnVision plate reader (PerkinElmer), irradiating with light passing through a green filter and providing an illumination of <100 lux.

To determine the inhibitory ability of the test compounds in response to IL-2, the average emission intensity of pSTAT5-bound beads in a human T cell line was measured. IC ₅₀ values were determined from the analysis of curves of signal intensity inhibition versus compound concentration. Data were expressed as pIC ₅₀ values (negative decimal logarithm of IC ₅₀ ) (mean ± standard deviation).

Results of in vitro analysis.

Table 1

Example No. JAK1 pK JAK2 pK JAK3 pK, Tyk2 pK Tall-1 pIC50 1 10.2 10.3 9.8 9.0 8.8 C-1 10.2 10.5 9.7 9.1 8.7 2 10.3 10.2 9.8 8.9 8.4 C-2 10.3 10.8 9.9 9.5 8.6 3 10.2 10.4 10.2 9.3 8.8 C-3 10.2 10.5 9.7 9.3 8.7 4 Yu,1 10.4 ιο, ι 9.0 8.6 C-4 10.0 10.6 9.8 9.3 8.7 5 10.3 10.5 ιο, ι 9.2 8.8 C-5 Yu,1 10.7 9.9 9.5 8.6 6 10.2 10.4 10.0 9.0 8.7 C-6 10.2 10.7 9.8 9.3 8.6

Analysis 3 Mouse model of IL-13-induced pSTAT6 induction in lung tissue.

IL-13 is an important cytokine underlying the pathophysiology of asthma (Kudlacz et al., Eur. J. Pharmacol, 2008, 582, 154-161). IL-13 binds to cell surface receptors, activating members of the Janus kinase (JAK) family, which then phosphorylate STAT6 and subsequently activate further transcriptional pathways. In the model described, a dose of IL-13 was delivered locally to the lungs of mice to induce STAT6 (pSTAT6) phosphorylation, which was then measured as an endpoint.

Balb/c adult mice from Harlan were used in the assay. On the day of the study, animals were lightly anesthetized with isoflurane and administered with either vehicle or test compound (1 mg/ml, total volume 50 μl over several breaths) by oral aspiration. After dosing, the animals were left in the lateral position and observed for their full recovery from anesthesia before returning to the home cage. Four hours later, the animals were again briefly anesthetized and sensitized with either vehicle or IL-13 (total delivered dose 0.03 μg, total volume 50 μl) by oral aspiration, after which they were observed for recovery from anesthesia and returned to the home cage. One hour after vehicle or IL-13 administration, whole blood and lung samples were taken to detect pSTAT6 in lung homogenates using the Perkin Elmer AlphaLISA® SureFire® Ultra™ HV p-STAT6 assay kit (Tyr641) and to analyze total drug concentration in lungs and plasma. Blood samples were centrifuged (Eppendorf centrifuge, 5804R) for 4 minutes at approximately 12,000 rpm at 4° C. to collect plasma. Lungs were washed with DPBS (Dulbecco's Phosphate Buffered Saline), dried, flash frozen, weighed and homogenized at a 1:3 dilution in 0.1% formic acid in HPLC water. Plasma and lung test compound levels were determined by LC-MS analysis against analytical standards from which a standard curve was generated in the test matrix. The lung-to-plasma ratio was defined as the ratio of lung concentration in ng/g to plasma concentration in ng/ml after 5 hours.

The activity of the model was confirmed by the decrease in the level of pSTAT6 present in the lungs of the treated animals after 5 hours compared to control IL-13-sensitized animals treated with vehicle. In each experiment, the difference between IL-13-sensitized vehicle-treated controls and vehicle-sensitized controls

- 28 040902 animals treated with the carrier, was determined equal to 0% and 100% inhibitory effect, respectively. Compounds tested in this assay demonstrated inhibition of STAT6 phosphorylation 5 hours after IL-13 sensitization, as indicated below.

Table 2 Observed pSTAT6 Inhibition and Plasma/Lung Concentrations

The observed high concentration of test compound in mouse lungs confirms that the observed inhibition of IL-13-induced pSTAT6 induction is a result of test compound activity. The lung-to-plasma ratio at 5 h showed that the concentration of compounds 1 to 6 in the lung was significantly higher than that in the plasma of mice.

Assay 4: Inhibition of TSLP-induced TARC release in human peripheral blood mononuclear cells.

Thymic stromal lymphopoietin (TSLP) and thymus and activation-regulated chemokine (TARC) are overexpressed in the airways in asthma and correlate with disease severity. In the lungs, TSLP can be released by bronchial epithelial cells in response to allergens and viral infections. TSLP signals via the IL-7Ra/TSLPR heterodimer are found in a wide range of tissues and cell types, including epithelial cells, endothelial cells, neutrophils, macrophages, and mast cells. Binding of TSLP to the receptor causes a conformational change that activates JAK1 and JAK2 to phosphorylate various transcription factors, including STAT3 and STAT5. In immune cells, this triggers a cascade of intracellular events that lead to cell proliferation, anti-apoptosis, dendritic cell migration, and production of cytokines and Th2 chemokines. In peripheral blood mononuclear cells (PBMCs), TSLP has a pro-inflammatory effect by activating myeloid dendritic cells to recruit and stimulate T cells, a process mediated by the TARC chemoattractant.

In this assay, it was shown that TSLP stimulation induces the release of TARC from PBMC and that this response is attenuated in a dose-dependent manner upon treatment with the compound. The efficacy of test compounds was measured by inhibition of TARC release.

PBMC aliquots (previously isolated from whole blood and frozen in aliquots at -80°C) obtained from 3 to 5 donors were thawed at 37°C and added dropwise to 40 ml of pre-warmed, sterile-filtered, complete RPMI medium in 50 ml Falcon tubes. Cells were pelleted and resuspended in complete medium at a density of 2.24x106 cells/ml. Cells were seeded at 85 μl (190,000 cells) per well in a 96 well tissue cultured flat bottom plate. Cells were left for 1 h at 37°C and 5% CO ₂ .

Compounds were obtained as 10 mM stock solutions in DMSO. Performed 3.7-fold serial dilution to obtain 9 concentrations of the test compound in DMSO at 300X the final test concentration for analysis. 150-fold intermediate dilutions were performed in complete medium to give a compound concentration greater than 2X the final test concentration with 0.2% DMSO. After a 1 hour rest period, 95 μl of 2X compound was added to each well of PBMC, with a final assay concentration range of 33.33 μM to 0.95 μM. 95 μl of 0.2% DMSO in complete medium was added to untreated control wells. Prior to stimulation, cells were pre-treated with the compound for 1 h at 37° C. and 5% CO ₂ .

Recombinant human TSLP protein was reconstituted at 10 μg/ml in sterile DPBS with 0.1% BSA and stored in aliquots at -20°C. Immediately prior to use, an aliquot was thawed and prepared at 20X final concentration for analysis in complete medium. 10 μl of 20X TSLP was added to each well of PBMC to give a final assay concentration of 10 ng/ml. 10 μl of complete medium was added to unstimulated control wells. Cells were stimulated in the presence of the compound for 48 h at 37°C and 5% CO ₂ .

After stimulation, cell culture supernatants were harvested and TARC levels were determined by enzyme immunoassay (ELISA) using the Human CCL17/TARC Quantikine ELISA kit.

- 29 040902 (R&D Systems # DDN00) in accordance with the manufacturer's instructions.

For dose response analysis, the logarithm (test compound (M)) was plotted against the percent response of each donor, and IC ₅₀ values were determined by non-linear regression analysis using GraphPad Prism software using a 4-parameter sigmoidal dose response algorithm. with variable slope. Data were expressed as mean pIC ₅₀ values (negative decimal logarithm of IC ₅₀ ) calculated from individual donor pIC ₅₀ values and rounded to one decimal place. The inhibition efficiency values for the starting compounds and their defluoro-modified analogs are given in Table 1. 3.

Table 3 Efficacy values of test compounds for inhibition of TSLP-induced TARC release in human peripheral blood mononuclear cells

Compound р!С50 ± 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. Metabolism of S9 in the lungs.

The in vitro metabolic stability of compounds 1 to 6 and C-1 to C-6 was assessed in the S9 human lung fraction (1 μM compound; 1 mg/ml S9 protein). Samples obtained at 0, 15, 30 and 60 minutes were analyzed for the parent compound by high resolution LC-MS/MS. Human lung fraction S9 (Lot 1410245) was purchased from XenoTech LLC (Lenexa, KS). NADPH (Sigma Aldrich, N1630) and 3-phosphoadenosine-5-phosphosulfate (PAPS) (Sigma Aldrich, A1651) were purchased from Sigma Aldrich (St. Louis, MO). Acetonitrile and water were obtained from VWR (Radnor, PA) in HPLC purity or higher. Raloxifene and formic acid were purchased from Sigma Aldrich (St. Louis, MO). S9 lung incubation was performed in a water bath at 37° C. in a 96-well polypropylene plate. S9 lung solutions consisted of 100 mM potassium phosphate buffered to pH 7.4 (BD Biosciences, Woburn, MA) supplemented with 1 mM NADPH (Sigma-Aldrich, St. Louis, MO), 3 mM magnesium chloride (Sigma Aldrich, M1028 ), in the presence of 100 μM PAPS cofactor (Sigma-Aldrich, St. Louis, MO) with a final protein concentration at 1 mg/mL incubation. Stock solutions of raloxifene in 10 mM DMSO (n=1) and compounds (n=1) were diluted in buffer and incubated to give substrate concentrations of 1 μM (0.001% DMSO by volume). The incubation volumes were 400 μl, at the time points of 0, 15, 30 and 60 min, 70 μl aliquots were taken, which were diluted with 140 μl of acetonitrile (0% formic acid). All samples were centrifuged at 2250 g for 10 min at 5°C. Supernatant (50 μl) was obtained from centrifuged samples and diluted with 100 μl of HPLC water containing an internal standard. Samples were processed on a Dionex Ultimate 3000 Auto sampler and analyzed using a Thermo Q-Exactive high resolution mass spectrometer (Thermo, Waltham, MA) in full scan mode in combination with an Atlantis T3 3 μM - 2.1x50 mm column (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 obtained using Gubbs GMSU software (Gubbs Inc., Alpharetta, GA). Peak area ratios were calculated for each sample by dividing the analyte peak area by the internal standard peak area. For each incubation, the peak area ratios of the analytes at each t ₀ point were set to 100%, and the peak area ratios for the 60 minute samples were converted to percentages remaining relative to the corresponding t ₀ . Qualitative determination of sulfate metabolite formation was performed by observing an early elution peak in the parent ion channel, which, based on previous laboratory data, was consistent with the O-sulfate metabolite of each parent compound. The results of the analysis are shown in table 4 (n=2 repetitions).

- 30 040902

Table 4. Metabolic stability in S9 human lung fraction

Compound Clearance (µl/min/mg) Compound remaining after 60 min (%) The presence of sulfate 1 4.2 78 Yes C-1 20.8 29 Yes 2 3.5 81 Yes C-2 61.0 3 Yes 3 2.6 86 Yes C-3 33.2 14 Yes 4 2.1 88 Yes C-4 53.4 4 Yes 5 3.7 80 Yes C-5 42.2 8 Yes 6 3.4 82 Yes C-6 51.4 5 Yes

Compounds 1 to 6, compared to their corresponding fluoro-containing counterparts (compounds C-1 to C-6), cause significantly lower levels of sulfation in the metabolic pathway in the S9 fraction of the lungs.

Analysis 6 Plasma and lung pharmacokinetics in mice.

Plasma and lung concentrations of test compounds and their ratios were determined as follows. BALB/c mice from Charles River Laboratories were used in the assay. Each test compound was separately formulated at 0.2 mg/ml in 20% propylene glycol in citrate buffer, pH 4, and 50 µl of the dosing solution was injected into the mouse trachea by oral aspiration. At various time points (typically 0.167, 2, 6, 24 hours) post-dose, blood samples were taken from mice by cardiac puncture and intact lungs were dissected. Blood samples were centrifuged (Eppendorf centrifuge, 5804R) for 4 min at approximately 12,000 rpm at 4° C. to collect plasma. The lungs were dried, weighed and homogenized at a 1:3 dilution in sterile water. Plasma and lung test compound concentrations were determined by LC-MS analysis against analytical standards from which a standard curve was generated in the test matrix. The lung-to-plasma ratio was defined as the ratio of lung AUC in µg h/g to plasma AUC in µg h/mL, where AUC is usually defined as the area under the test compound concentration versus time curve.

Table 5 Plasma and Lung Tissue Concentrations Following Single Oral Administration of Test Compounds

Compound Plasma AUC(o-24) (µg h/mL) AUC(o-24) in lung tissue (µg h/g) AUC lung tissue:plasma ratio 1 0.581 64.6 111 5 0.599 29.9 49.9

Assay 7. IL-5 mediated eosinophil survival assay.

The efficacy of test compounds on IL-5 mediated eosinophil survival can be measured in human eosinophils isolated from human whole blood (AllCells). Because IL-5 signals through the JAK, this assay measures JAK cellular activity.

Human eosinophils are isolated from fresh human whole blood (AllCells) from healthy donors. The blood is mixed with 4.5% dextran (Sigma-Aldrich) in 0.9% sodium chloride solution (Sigma-Aldrich). The erythrocytes are left for 35 minutes to precipitate. The leukocyte-rich top layer is removed, applied to Ficoll-Paque (GE Healthcare) and centrifuged at 600 g for 30 minutes. The plasma and mononuclear cell layers are removed before the granulocyte layer is lysed with water to remove any contaminating erythrocytes. Eosinophils were further purified using a human eosinophil isolation kit (Miltenyi Biotec). A fraction of purified eosinophils was incubated with anti-CD16 FITC (Miltenyi Biotec) for 10 min at 4° C. in the dark. Purity is checked with an LSRII flow cytometer (BD Biosciences).

Cells are cultured in a humidified incubator at 37°C and 5% CO ₂ in RPMI 1640 (Life Technolo

- 31 040902 gies) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Life Technologies), 2 mM Glutamax (Life Technologies), 25 mM HEPES (Life Technologies) and 1X Pen-Strap (Life Technologies). Cells are seeded at the rate of 10,000 cells/well in medium (50 μl). The plate is centrifuged at 300 g for 5 minutes and the supernatants are removed. Compounds are subjected to serial dilution in DMSO, and then 500-fold dilution to 2x the final analytical concentration in the medium. Test compounds (50 µl/well) are added to the cells and incubated at 37°C and 5% CO ₂ for 1 h, followed by the addition of IL-5 (R&D Systems; final concentrations 1 ng/ml and 10 pg/ml) in pre- heated analytical medium (50 µl) for 72 hours.

After cytokine stimulation, cells are centrifuged at 300 g for 5 min and washed twice with cold DPBS (Life Technologies). To determine viability and apoptosis, cells were incubated with propidium iodide (Thermo Fisher Scientific) and APC Annexin V (BD Biosciences) and analyzed with an LSRII flow cytometer (BD Biosciences). IC ₅₀ values are determined from analysis of percentage cell viability versus compound concentration curves. Data are expressed as pIC ₅₀ values (negative decimal logarithm of IC ₅₀ ).

Assay 8 Inhibition of IFNy and IL-27 induced chemokines CXCL9 and CXCL10 in 3D human airway cultures.

EpiAirway tissue cultures are available from Mattek (AIR-100). Cultures are obtained from asthmatic donors. In a cell culture insert, human tracheal/bronchial epithelial cells were grown and differentiated on a porous membrane support to create an air-liquid interface with a heated culture medium below the cells and a gaseous test atmosphere above them. The tissues are cultured in maintenance medium (Mattek, AIR-100-MM) in a humidified incubator at 37°C and 5% CO ₂ . Four donors can be tested. On day 0 tissue cultures are treated at a concentration of 10 μM, 1 μM and/or 0.1 μM. Compounds are diluted with dimethyl sulfoxide (DMSO, Sigma) to a final concentration of 0.1%. As a control carrier, DMSO can be used at a concentration of 0.1%. Test compounds are incubated with cultures for 1 hour at 37° C. and 5% CO ₂ , after which prewarmed medium containing IFNy (R&D Systems) or IL-27 (R&D Systems) at a final concentration of 100 ng/mL is added. Tissue cultures are maintained for 8 days. Medium is replaced every 2 days with fresh medium containing compounds and IFNy or IL-27. On day 8, tissue cultures and supernatants are collected for analysis. Supernatant samples are analyzed for CXCL10 (IP-10) and CXCL9 (MIG) using the Luminex assay platform (EMD Millipore). Data are expressed as % inhibition ± standard deviation (± STDV). Percent inhibition is determined by the ability of the compound to inhibit IFNy or IL-27 induced CXCL10 or CXCL9 secretion compared to vehicle-treated cells. Data are averages for 3 or 4 donors.

Assay 9 Cellular JAK Activity Assay: Inhibition of IL-2/anti-CD3-stimulated IFN-y in human PBMCs.

The potency of test compounds to inhibit interleukin-2 (IL-2)/anti-CD3 stimulated interferon gamma (IFNy) can be measured in human peripheral blood mononuclear cells (PBMCs) isolated from human whole blood (Stanford Blood Center). Because IL-2 signals through the JAK, this assay measures JAK cellular activity.

(1) Human peripheral blood mononuclear cells (PBMC) are isolated from whole blood of healthy human donors using a ficoll gradient. Cells are cultured in a humidified incubator at 37°C and 5% CO ₂ in RPMI (Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Life Technologies), 2 mM glutamax (Life Technologies), 25 mM HEPES (Life Technologies ) and 1X Pen-Strap (Life Technologies). Cells are plated at 200,000 cells/well in medium (50 μl) and cultured for 1 hour. Compounds are serially diluted in DMSO and then 500-fold diluted (to 2x the final assay concentration) in medium. Test compounds (100 µl/well) are added to cells and incubated for 24 h at 37°C and 5% CO2 for 1 h followed by addition of IL-2 (R&D Systems; final concentration 100 ng/mL) and anti-CD3 (BD Biosciences; final concentration 1 µg/ml) in pre-warmed assay medium (50 µl).

(2) After cytokine stimulation, the cells are centrifuged at 500 g for 5 min, the supernatants are removed and frozen at -80°C. To determine the inhibitory ability of a test compound in response to IL-2/anti-CD3, IFNy concentrations in the supernatant are measured using ELISA (R&D Systems). IC ₅₀ values are determined from analysis of IFNy concentration versus compound concentration curves. Data are expressed as pIC ₅₀ values (negative decimal logarithm of IC ₅₀ ).

Assay 10 Cellular JAK Activity Assay: Inhibition of IL-2-stimulated pSTAT5 in CD4+ T cells.

The efficacy of test compounds in inhibiting interleukin-2 (IL-2)/anti-CD3 stimulated STAT5 phosphorylation can be measured by flow cytometry in CD4-positive (CD4+) T cells in peripheral blood mononuclear cells (PBMCs). ) isolated from human whole blood (Stanford Blood Center). Because IL-2 signals through

JAK, this assay measures the cellular activity of JAK.

CD4+ T cells are identified using a phycoerythrobilin (PE) conjugated anti-CD4 antibody (Clone RPA-T4, BD Biosciences), while an Alexa Fluor 647 conjugated anti-pSTATS antibody (pY694, Clone 47, BD Biosciences) is used to detect phosphorylation of STAT5.

(1) Follow assay protocol 9, paragraph (1), except that cytokine stimulation with anti-CD3 antibody is performed for 30 minutes instead of 24 hours.

(2) After cytokine stimulation, cells are fixed with pre-warmed fixative solution (200 µl; BD Biosciences) for 10 min at 37°C and 5% CO ₂ , washed twice with DPBS buffer (1 ml, Life Technologies) and resuspended in ice-cold Perm buffer Buffer III (1000 µl, BD Biosciences) for 30 min at 4°C. Cells are washed twice with 2% FBS in DPBS (FACS buffer) and then resuspended in FACS buffer (100 µl) containing anti-CD4 PE (1:50 dilution) and anti-CD3 antiCD3Alexa Fluor 647 (1:5 dilution) for 60 min at room temperature in the dark. After incubation, cells are washed twice with FACS buffer before analysis performed with an LSRII flow cytometer (BD Biosciences). To determine the inhibitory ability of test compounds in response to IL-2/anti-CD3, the mean fluorescence intensity (MFI) of pSTAT5 in CD4+ T cells is measured. IC ₅₀ values are determined from analysis of MFI inhibition versus compound concentration curves. Data are expressed as pIC ₅₀ values (negative decimal logarithm of IC ₅₀ ).

Assay 11 Cellular JAK Activity Assay: Inhibition of IL-4-stimulated pSTAT6 in CD3+ T cells.

The efficacy of test compounds in inhibiting interleukin-4 (IL-4) stimulated STAT6 phosphorylation can be measured by flow cytometry in CD3 positive (CD3+) T cells in peripheral blood mononuclear cells (PBMCs) isolated from human whole blood (Stanford Blood Center ). Because IL-4 signals through the JAK, this assay measures JAK cellular activity.

CD3+ T cells are identified with a phycoerythrobilin (PE)-conjugated anti-CD3 antibody (Clone UCHT1, BD Biosciences), while an Alexa Fluor 647-conjugated anti-pSTAT6 antibody (pY641, Clone 18/P, BD Biosciences) is used to detect STAT6 phosphorylation.

Human peripheral blood mononuclear cells (PBMCs) are isolated from whole blood of healthy donors as in assays 9 and 10. Cells are plated at 250,000 cells/well in medium (200 μl), cultured for 1 h, and then resuspended in assay medium ( 50 µl) (RPMI supplemented with 0.1% bovine serum albumin (Sigma), 2 mM Glutamax, 25 mM HEPES and 1X Pen-Strap) containing various concentrations of test compounds. Compounds are serially diluted in DMSO and then 500-fold diluted (to 2x final assay concentration) in assay medium. Test compounds (50 µl) are incubated with cells at 37°C and 5% CO2 for 1 h followed by the addition of IL-4 (50 µl) (R&D Systems; final concentration 20 ng/ml) in prewarmed assay medium for 30 min. After cytokine stimulation, cells are fixed with pre-warmed fixative solution (100 μl) (BD Biosciences) for 10 min at 37°C and 5% CO ₂ , washed twice with FACS buffer (1 ml) (2% FBS in DPBS) and resuspended in ice-cold Perm Buffer III (1000 µl) (BD Biosciences) for 30 min at 4°C. Cells are washed twice with FACS buffer and then resuspended in FACS buffer (100 µl) containing anti-CD3 PE (1:50 dilution) and anti-pSTAT6 Alexa Fluor 647 (1:5 dilution) for 60 min at room temperature in the dark . After incubation, cells were washed twice in FACS buffer before analysis performed using an LSRII flow cytometer (BD Biosciences).

To determine the inhibitory ability of a test compound in response to IL-4, measure the mean fluorescence intensity (MFI) of pSTAT6 in CD3+ T cells. IC ₅₀ values are determined from analysis of MFI inhibition versus compound concentration curves. Data are expressed as pIC ₅₀ (negative base 10 logarithm of IC ₅₀ ).

Assay 12 Cellular JAK Activity Assay: Inhibition of IL-6-stimulated pSTAT3 in CD3+ T cells.

To determine the efficacy of a test compound in inhibiting interleuken-6 (IL-6) stimulated STAT3 phosphorylation, a protocol similar to assay protocol 11 can be used. Alexa Fluor 647 conjugated anti-pSTAT3 antibodies (pY705, Clone 4/) can be used to detect STAT3 phosphorylation. P, BD Biosciences).

Analysis 13. Mouse model of eosinophilic lung inflammation induced by Alternaria alternata.

Airway eosinophilia is a hallmark of asthma in humans. Alternaria alternata is a fungal aeroallergen that can exacerbate asthma in humans and cause eosinophilic inflammation in the lungs of mice (Havaux et al., Clin Exp Immunol. 2005 Feb; 139(2): 179-88). It has been demonstrated in mice that alternariosis indirectly activates resident lymphoid

- 33 040902 Type 2 innate immunity cells in the lung that respond (to eg IL-2 and IL-7) and release JAK-dependent cytokines (eg IL-5 and IL-13) and coordinate eosinophilic inflammation ( Bartemes et al., J Immunol 2012 Feb 1; 188(3): 1503-13).

Male Taconic C57 mice between seven and nine weeks of age can be used in the study. On the day of the study, animals are lightly anesthetized with isoflurane and either vehicle or test compound (0.03-1.0 mg/mL, total volume 50 µl over several breaths) is administered by oral aspiration. After dosing, the animals are left to lie in a horizontal position and their full recovery from anesthesia is observed before returning to the home cage. One hour later, the animals are again lightly anesthetized and sensitized with either vehicle or Alternaria extract (total extract delivered 200 μg, total volume 50 μl) by oral aspiration, after which they are observed for recovery from anesthesia and returned to the home cage. 48 hours after Alternariosis administration, bronchoalveolar lavage fluid (BALF) was collected and eosinophils were counted in BALF using an Advia 120 hematology system (Siemens). The activity of the model is confirmed by the decrease in the level of eosinophils present in BALF treated animals after 48 hours compared to control animals treated with vehicle and sensitized with Alternaria. Data are expressed as percent inhibition of eosinophil response in BALF in vehicle treated animals sensitized with Alternaria. To calculate percent inhibition, the eosinophil count in BALF for each condition is converted to a percentage of the mean eosinophil count in BALF of vehicle-treated, Alternaria-sensitized animals and subtracted from one hundred percent.

Assay 14 Cellular JAK Activity Assay: Inhibition of IFNy-induced pSTAT1.

The potency of test compounds in inhibiting interferon gamma (IFNy) stimulated STAT1 phosphorylation was measured by flow cytometry in CD14 positive (CD14+) monocytes derived from human whole blood (Stanford Blood Center). Since IFNy signals through JAK, this assay allows the measurement of JAK cellular activity.

Monocytes were identified with a fluorescein isothiocyanate (FITC) conjugated anti-CD14 antibody (clone RM052, Beckman Coulter), while an Alexa Fluor 647 conjugated anti-pSTAT1 antibody (pY701, Clone 4a, BD Biosciences) was used to detect STAT1 phosphorylation.

Human peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of healthy donors using a ficoll gradient. Cells were cultured in a humidified incubator at 37°C and 5% CO ₂ in RPMI (Life Technologies) supplemented with 10% fetal bovine serum (FBS, Life Technologies), 2 mM glutamax (Life Technologies), 25 mM HEPES (Life Technologies) and 1X Pen-Strap (Life Technologies). Cells were plated at 250,000 cells/well in medium (200 µl), cultured for 2 h, and resuspended in assay medium (50 µl) (RPMI supplemented with 0.1% bovine serum albumin (Sigma), 2 mM glutamax, 25 mM HEPES and 1X Pen-Strap) containing various concentrations of the test compound. Compounds were subjected to serial dilution in DMSO, and then 1000-fold dilution to obtain a final concentration of DMSO equal to 0.1%. Test compound dilutions were incubated with cells at 37°C and 5% CO ₂ for 1 h, followed by the addition of prewarmed IFNy (R&D Systems) to the medium (50 µl) to a final concentration of 0.6 ng/ml for 30 min. After cytokine stimulation, cells were fixed with pre-warmed fixative solution (100 μl) (BD Biosciences) for 10 min at 37°C and 5% CO ₂ , washed twice with FACS buffer (1 ml) (1% BSA in PBS), resuspended in the ratio 1:10 in anti-CD14 FITC:FACS buffer (100 µl) and incubated at 4°C for 15 min. Cells were washed once and then resuspended in ice-cold Perm Buffer III (BD Biosciences) (100 μl) for 30 min at 4°C. Cells were washed twice with FACS buffer and then resuspended at a ratio of 1:10 in anti-pSTAT1 Alexa Fluor 647:FACS buffer (100 µl) for 30 min at room temperature in the dark, washed twice in FACS buffer and analyzed using a MACSQuant flow cytometer. (Miltenyi).

To determine the inhibitory ability of a test compound, the mean fluorescence intensity (MFI) of pSTAT1 in CD14+ monocytes was measured. IC ₅₀ values were determined from analysis of MFI inhibition versus compound concentration curves. Data were expressed as pIC ₅₀ values (negative decimal logarithm of IC ₅₀ ). In this assay, Compound 1 had a pIC ₅₀ value of 7.5. In this assay, compound 4 had a pIC ₅₀ value of 7.3.

Assay 15. JAK cell activity assay: inhibition of GM-CSF-induced pSTAT5.

The potency of test compounds in inhibiting granulocyte and macrophage colony stimulating factor (GM-CSF) stimulated STAT5 phosphorylation was measured by flow cytometry in CD14-positive (CD14+) monocytes derived from human whole blood (Stanford Blood Center). Because GM-CSF signals through JAK, this assay allows the measurement of JAK cellular activity.

Monocytes were identified using fluorescein isothiocyanate-conjugated

- 34 040902 (FITC) aHTu-CD14 antibody (clone RM052, Beckman Coulter), while an Alexa Fluor 647 conjugated aHTu-pSTAT5 antibody (pY694, BD Biosciences) was used to detect phosphorylation

STAT5.

Human peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of healthy donors using a ficoll gradient. Cells were cultured in a humidified incubator at 37°C and 5% CO ₂ and in RPMI (Life Technologies) supplemented with 10% fetal bovine serum (FBS, Life Technologies), 2 mM glutamax (Life Technologies), 25 mM HEPES (Life Technologies) and 1X Pen-Strap (Life Technologies). Cells were plated at 250,000 cells per well in medium (200 µl), cultured for 2 h, and resuspended in assay medium (50 µl) (RPMI supplemented with 0.1% bovine serum albumin (Sigma), 2 mM glutamax, 25 mM HEPES and 1X Pen-Strap) containing various concentrations of test compounds. Compounds were subjected to serial dilution in DMSO, and then 1000-fold dilution in medium to obtain a final concentration of DMSO equal to 0.1%. Test compound dilutions were incubated with cells at 37°C and 5% CO ₂ for 1 h, followed by the addition of prewarmed GM-CSF (R&D Systems) to the medium (50 µl) to a final concentration of 0.3 ng/ml for 15 min . After cytokine stimulation, cells were fixed with pre-warmed fixative solution (100 μl) (BD Biosciences) for 10 min at 37°C and 5% CO ₂ , washed twice with FACS buffer (1 ml) (1% BSA in PBS), resuspended in the ratio 1:10 in anti-CD14 FITC:FACS buffer (100 µl) and incubated at 4°C for 15 min. Cells were washed once and then resuspended in ice-cold Perm Buffer III (BD Biosciences) (100 μl) for 30 min at 4°C. Cells were washed twice with FACS buffer and then resuspended at a ratio of 1:10 in anti-pSTAT1 Alexa Fluor 647:FACS buffer (100 µl) for 30 min at room temperature in the dark, washed twice in FACS buffer and analyzed using a MACSQuant flow cytometer. (Miltenyi).

To determine the inhibitory ability of a test compound, the mean fluorescence intensity (MFI) of pSTAT5 in CD14+ monocytes was measured. IC ₅₀ values were determined from analysis of MFI inhibition versus compound concentration curves. Data were expressed as pIC50 values (negative decimal logarithm of IC ₅₀ ). In this assay, Compound 1 had a pIC ₅₀ value of 6.7. In this assay, compound 4 had a pIC ₅₀ value of 6.7.

Assay 16. JAK cell activity assay: inhibition of IL-12-induced pSTAT4.

The potency of test compounds in inhibiting interleukin-12 (IL-12) stimulated STAT4 phosphorylation was measured by flow cytometry in CD3 positive (CD3+) T cells derived from human whole blood (Stanford Blood Center). Since IL-12 signals through the JAK, this assay allows the measurement of JAK cellular activity.

CD3+ T cells were identified with a phycoerythrin (PE) conjugated anti-CD3 antibody (clone UCHT1, BD Biosciences), while an Alexa Fluor 647 conjugated anti-pSTAT4 antibody (clone 38/p-Stat4, BD Biosciences) was used to detect phosphorylation of STAT4.

Human peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of healthy donors using a ficoll gradient. Cells were cultured in RPMI (Life Technologies) supplemented with 10% fetal bovine serum (FBS, Life Technologies), 2 mM Glutamax (Life Technologies), 25 mM HEPES (Life Technologies), 1X Pen-Strap (Life Technologies) bound to the plate purified anti-CD3 antibody (5 μg/ml, clone UCHT1, BD Biosciences) and dissolved anti-CD28 antibody (1 μg/ml, clone CD28.2, BD Biosciences) for 3 days in a humidified incubator at 37°C and 5 % CO ₂ . Cells were collected, washed with medium, and then resuspended in medium containing interleukin-2 (IL-2, 10 ng/ml, R&D Systems). Cells were cultured for 3 days in a humidified incubator at 37°C and 5% CO ₂ . Cells were harvested, washed with RPMI and seeded at 250,000 cells/well in medium (200 µl), cultured for 2 h and resuspended in assay medium (50 µl) (RPMI supplemented with 0.1% bovine serum albumin (Sigma), 2 mM Glutamax, 25 mM HEPES and 1X PenStrepa) containing various concentrations of test compounds. Compounds were subjected to serial dilution in DMSO, and then 1000-fold dilution in medium to obtain a final concentration of DMSO equal to 0.1%. Test compound dilutions were incubated with cells at 37°C and 5% CO ₂ for 1 h followed by the addition of prewarmed IL-12 (R&D Systems) to the medium (50 µl) to a final concentration of 10 ng/ml for 30 min. After cytokine stimulation, cells were fixed with pre-warmed fixative solution (100 μl) (BD Biosciences) for 10 min at 37°C and 5% CO ₂ , washed twice with FACS buffer (1 ml) (1% BSA in PBS) and resuspended in chilled ice-cold Perm Buffer III (1000 µl) (BD Biosciences) for 30 min at 4°C. Cells were washed twice with FACS buffer and then resuspended in FACS buffer (100 µl) containing anti-CD3 PE (1:50 dilution) and anti-pSTAT4 Alexa Fluor 647 (1:10 dilution) for 45 min at room temperature in the dark . After incubation, cells were washed twice with FACS buffer before analysis using a MACSQuant flow cytometer (Miltenyi). To determine the inhibitory ability of a test compound, the mean fluorescence intensity (MFI) of pSTAT4 in CD3+ T cells was measured. IC ₅₀ values were determined from analysis of MFI inhibition curves as a function of compound concentration. Data are expressed as pIC ₅₀ values (negative decimal

Claims (36)

exact logarithm of IC5o). Compound 1 had a pIC5o value of 6.2 in this assay. In this assay, compound 4 had a pIC ₅₀ value of 6.0. Structure of crystals. A co-crystal structure of compound D-1 bound to human JAK1 was obtained with a resolution of 2.28 A. The ligand was found to bind at the ATP binding site. Seven specific interactions were identified based on hydrogen bonds over a distance of 3.5 A or less between donor and acceptor atoms. Of particular note, a hydrogen bond interaction has been identified between the carbonyl of the exocyclic amide group of compound D-1 and the Arg879 side chain of JAK1. A similar interaction can be expected for the compounds of the invention. In earlier modeling studies, this interaction was proposed as a way to provide selectivity for JAK1 over other tyrosine kinases because, other things being equal, closely related kinases (eg, TRKA, VEGFR, ABL1) did not have an arginine residue at the equivalent site. The observed results of the interaction based on hydrogen bonds in the crystal structure and the improved selectivity of kinome compared to species that do not contain an exocyclic amide group support the hypothesis of the presence of this structure. While the present invention has been described with reference to specific aspects or embodiments thereof, those skilled in the art will appreciate that various changes or equivalent substitutions may be made without departing from the spirit and scope of the invention. In addition, to the extent permitted by applicable patent laws and regulations, all publications, patents and patent applications cited in the present description are hereby incorporated by reference in their entirety to the same extent as if each document has been incorporated herein separately by reference. CLAIM 1. Compound of formula (I): (I) where A is a 6-7-membered monocyclic heterocyclic ring containing two nitrogen atoms, where A is connected to the carbonyl group in (I) through a nitrogen atom, while A is substituted by 13 R ² groups, and optionally two R ² groups form a ring with A through -(CH2)-bridge connection; or A is pyrrolidinyl, wherein the pyrrolidinyl is bonded to the carbonyl group in (I) via a nitrogen atom, and wherein the pyrrolidinyl is substituted with NR3R ⁴ ; R ¹ is C _1-3 alkyl; each R ² is independently C _1-3 alkyl optionally substituted with -OH; R ³ is C _1-3 alkyl; and R ⁴ is C _1-3 alkyl; or a pharmaceutically acceptable salt thereof. 2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, having the formula (I') 3. A compound according to any one of claims 1 or 2, or a pharmaceutically acceptable salt thereof, wherein R ¹ is selected from the group consisting of methyl, propyl or 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]heptanyl, and 1,4-diazacycloheptanyl, each of which is substituted with 1 or 2 R ² groups, where each R ² group is independently C 1-3 alkyl optionally substituted with -OH, or A is pyrrolidinyl substituted with NR 3 R ⁴ , where R ³ is C 1-3 alkyl and R ⁴ is C _{1 -3} alkyl. - 36 040902 5. A compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, wherein A is selected from the group consisting of where R ² is C1-3 alkyl, optionally substituted with -OH, R ³ is C1-3 alkyl and R ⁴ is C _1-3 alkyl. 6. Compound Formula or a pharmaceutically acceptable salt thereof. 7. A compound of formula or a pharmaceutically acceptable salt thereof. 8. A compound of formula or a pharmaceutically acceptable salt thereof. 9. A compound of formula or a pharmaceutically acceptable salt thereof. 10. A compound of formula or a pharmaceutically acceptable salt thereof. 11. Compound formula - 37 040902 or a pharmaceutically acceptable salt thereof. 12. A pharmaceutical composition containing a compound according to any one of claims 1 to 11 and a pharmaceutically acceptable carrier. 13. The use of a compound according to any one of claims 1 to 11 in the treatment of a respiratory disease in a mammal. 14. Use according to claim 13, wherein the respiratory disease is selected from the group consisting of asthma, chronic obstructive pulmonary disease, cystic fibrosis, pneumonitis, idiopathic pulmonary fibrosis, acute lung injury, acute respiratory distress syndrome, bronchitis, emphysema, bronchiolitis obliterans, sarcoidosis eosinophilic disease, helminthic infection, pulmonary arterial hypertension, lymphangioleiomyomatosis, bronchiectasis, infiltrative lung disease, drug-induced pneumonitis, fungal pneumonitis, allergic bronchopulmonary aspergillosis, hypersensitivity pneumonitis, eosinophilic granulomatosis with polyangiitis, idiopathic acute eosinophilic pneumonia, idiopathic chronic eosinophilic pneumonia, hypereosinophilic syndrome, Loeffler's syndrome, bronchiolitis obliterans with organizing pneumonia, lung graft versus host disease, and immune checkpoint inhibitor-induced pneumonitis. 15. Use according to claim 13, wherein the respiratory disease is asthma or chronic obstructive pulmonary disease. 16. The use of a compound according to any one of claims 1 to 11 in the treatment of lung transplant rejection in a mammal. 17. The use of claim 16, wherein the lung graft rejection is selected from the group consisting of primary graft dysfunction, organizing pneumonia, acute rejection, lymphocytic bronchiolitis, and chronic lung allograft dysfunction. 18. The use of claim 16 wherein the lung transplant rejection is acute lung transplant rejection. 19. The use of claim 16, wherein the lung graft rejection is chronic lung allograft dysfunction. 20. The use of claim 16, wherein lung graft rejection is selected from the group consisting of bronchiolitis obliterans, restrictive chronic lung allograft dysfunction, and neutrophilic allograft dysfunction. 21. The use of a compound according to any one of claims 1 to 11 for the manufacture of a medicament for the treatment of a respiratory disease in a mammal. 22. Use according to claim 21, wherein the respiratory disease is selected from the group consisting of asthma, chronic obstructive pulmonary disease, cystic fibrosis, pneumonitis, idiopathic pulmonary fibrosis, acute lung injury, acute respiratory distress syndrome, bronchitis, emphysema, bronchiolitis obliterans, sarcoidosis eosinophilic disease, helminthic infection, pulmonary arterial hypertension, lymphangioleiomyomatosis, bronchiectasis, infiltrative lung disease, drug-induced pneumonitis, fungal pneumonitis, allergic bronchopulmonary aspergillosis, hypersensitivity pneumonitis, eosinophilic granulomatosis with polyangiitis, idiopathic acute eosinophilic pneumonia, idiopathic chronic eosinophilic pneumonia, hypereosinophilic syndrome, Loeffler's syndrome, bronchiolitis obliterans with organizing pneumonia, lung graft versus host disease, and immune checkpoint inhibitor-induced pneumonitis. 23. Use according to claim 21, wherein the respiratory disease is asthma or chronic obstructive pulmonary disease. 24. The use of a compound according to any one of claims 1 to 11 for the manufacture of a medicament for the treatment of lung transplant rejection in a mammal. 25. The use of claim 24, wherein the lung graft rejection is selected from the group consisting of primary graft dysfunction, organizing 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 graft rejection is chronic lung allograft dysfunction. 28. Use according to claim 24, wherein lung transplant rejection is selected from the group - 38 040902 consisting of obliterating bronchiolitis, restrictive chronic pulmonary allograft dysfunction and neutrophilic allograft dysfunction. 29. A method of treating a respiratory disease in a mammal, comprising administering to the mammal a pharmaceutical composition containing 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, pneumonitis, idiopathic pulmonary fibrosis, acute lung injury, acute respiratory distress syndrome, bronchitis, emphysema, bronchiolitis obliterans, sarcoidosis, eosinophilic disease, helminthic infection, pulmonary arterial hypertension, lymphangioleiomyomatosis, bronchiectasis, infiltrative lung disease, drug-induced pneumonitis, fungal pneumonitis, allergic bronchopulmonary aspergillosis, hypersensitivity pneumonitis, eosinophilic granulomatosis with polyangiitis, idiopathic acute eosinophilic pneumonia, idiopathic chronic eosinophilic pneumonia , Loeffler's syndrome, bronchiolitis obliterans with organizing pneumonia, lung graft versus host disease, and immune checkpoint inhibitor-induced pneumonitis. 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, 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. 33. The method of claim 32, wherein the lung transplant rejection is selected from the group consisting of primary graft dysfunction, organizing pneumonia, acute rejection, lymphocytic bronchiolitis, and chronic lung allograft dysfunction. 34. The method of claim 32, wherein the lung graft rejection is acute lung graft rejection. 35. The method of claim 32, wherein the lung graft rejection is chronic lung allograft dysfunction. 36. The method of claim 32, wherein lung graft rejection is selected from the group consisting of bronchiolitis obliterans, restrictive chronic lung allograft dysfunction, and neutrophilic allograft dysfunction. ^gj) Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2