HK40013317B - Novel amino-imidazopyridine derivatives as janus kinase inhibitors and pharmaceutical use thereof - Google Patents

Description

Novel amino-imidazopyridine derivatives as Janus kinase inhibitors and their pharmaceutical applications

[Technical Field]

This invention relates to compounds that are Janus kinase inhibitors and their derivatives, intermediates for preparing said compounds, said compounds for treatment, and pharmaceutical compositions comprising said compounds.

[Invention Background]

This invention relates to novel compounds that are inhibitors of protein tyrosine kinases, such as Janus kinases (JAK1, JAK2, JAK3 and TYK2), and particularly Janus kinase 1 (JAK1).

Protein tyrosine kinases are a family of enzymes that catalyze the transfer of the terminal phosphate of adenosine triphosphate (ATP) to tyrosine residues in protein substrates. Phosphorylation of tyrosine residues on protein substrates leads to the transduction of intracellular signaling that regulates numerous processes, such as cell growth, differentiation and activation, metabolism, hematopoiesis, host defense, and immune regulation. Due to the elucidation of the molecular mechanisms in various inflammatory diseases and other immune system disorders (e.g., autoimmune diseases), the crucial role of these intracellular signaling pathways has been highlighted, making the regulation of protein tyrosine kinase activity an attractive approach for controlling inflammatory diseases. A large number of protein tyrosine kinases have been identified, which can be receptor protein tyrosine kinases (e.g., insulin receptor) or non-receptor protein tyrosine kinases.

Protein tyrosine kinases JAK1, JAK2, JAK3, and TYK2 selectively associate with the cytoplasmic domains of various cytokine receptor chains and play crucial roles in cytokine-dependent regulation of tissue homeostasis, initiation of innate immunity, formation of adaptive immune responses, and inflammatory processes. They are particularly important in the signal transduction that responds to their activation via tyrosine phosphorylation of cytokine receptors. (1) Schindler C. et al., JAK-STAT signaling: from interferons to cytokines. J. Biol. Chem 2007; 282(28):20059; (2) O’Shea J.J. Targeting the Jak/STAT pathway for immunosuppression; Ann. Rheum. Dis. 2004; 63Suppl 2:ii67; (3) Schindler C. Series introduction. JAK-STAT signaling in human disease; J. Clin. Invest. 2002; 109(9):1133); (4) O’Shea et al., Cell, Vol. 109, S121-S131, 2002; (5) Schwartz D.M. et al., Nat. Rev. Rheumatol., 2016; 12(1):25-36; (6) O’Shea et al., New. Eng. J. Med. 2013; 368(2):161-170.

Although JAK1, JAK2, and TYK2 are expressed everywhere, JAK3 is mainly expressed in hematopoietic cells.

JAK1 plays a crucial role in mediating biological responses, and it is widely expressed and associates with several major cytokine receptor families. It participates in signal transduction through members of the IL-2 receptor γ subunit family (IL-2, IL-4, IL-7R, IL-9R, IL-15R, and IL-21R), the IL-4 receptor family (IL-4R, IL-13R), the gp130 receptor family, and class II cytokine receptors including the IL-10 receptor family and type I and type II IFN receptor families.

JAK2 is involved in signal transduction through several single-chain receptors (including Epo-R, GHR, and PRL-R), the IL-3 receptor family, the gp130 receptor family, the IL-12 receptor family (IL-12 and IL-23), and some class II receptor cytokine families. Therefore, JAK2 plays a crucial role in transducing signals targeting Epo, IL-3, GM-CSF, IL-5, and IFNγ. JAK2 knockout mice exhibit an embryonic lethal phenotype.

JAK3 participates in signal transduction by using receptors on the common γ chain of the type I cytokine receptor family (also known as the IL-2 receptor family) (e.g., IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21). The XSCID patient population has been identified as having reduced JAK3 protein levels or genetic defects with the common γ chain, suggesting that immunosuppression should be caused by blocking signal transduction via the JAK3 pathway. Animal studies have shown that JAK3 not only plays a crucial role in the maturation of B and T lymphocytes, but is also constitutively essential for maintaining T cell function. This novel mechanism of modulating immune activity may prove useful in the treatment of T-cell proliferative disorders, such as immune system diseases, particularly autoimmune diseases.

TYK2 is involved in the signaling of type I interferons IL-6, IL-10, IL-12, and IL-23. Human patients with TYK2 deficiency and primary immunodeficiency syndromes characterized by high IgE levels and numerous opportunistic infections caused by viruses, bacteria, and fungi have been documented. Given the important role of IL-23 in many chronic inflammatory diseases, it is conceivable that TYK2 inhibitors would be highly effective in treating diseases affected by IL-23.

Anemia and neutropenia may be associated with the inhibition of EPO and GM-CSF, respectively, since the biological effects of these two cytokines apparently depend solely on JAK2 activation. Similarly, IL-12 and IL-23 are involved in innate and adaptive immune defenses against viruses, bacteria, and fungi. Since these cytokines bind to receptors that recruit JAK2 and TYK2 in their signaling cascade, it is conceivable that selective JAK1 inhibitors would not affect their biological activity and would therefore be safer compared to compounds that inhibit JAK1, JAK2, JAK3, and TYK2.

Activation of JAK leads to activation of STAT molecules and thus the initiation of the JAK/STAT signaling pathway, which is highly regulated by phosphorylation events. STAT activation is considered a potent pharmacodynamic marker of JAK activity, and the activity of a specific JAK molecule can be evaluated by the level of preferentially recruited active STAT molecules.

Specifically, the IL-4 receptor expressed by immune cells is composed of two high-affinity ligands and signal transduction molecules, IL-4Ra and a common γ chain. Upon ligand binding, it activates JAK1 and JAK3, respectively, leading to the recruitment and activation of STAT6. Similarly, the IL-6 receptor is a heterodimeric receptor formed by the IL-6 high-affinity receptor chain (IL-6Ra) and the glycoprotein 130 (gp130) signal transduction molecule, which is preferentially associated with JAK1. The gp130 chain activates the JAK1 and STAT3 signaling pathways upon ligand binding. Therefore, to study JAK1 activity, the levels of active STAT6 or STAT3 can be evaluated in immune cells after stimulation with IL-4 or IL-6, respectively.

Furthermore, the erythropoietin receptor (EPOR) is a homodimeric receptor composed of two identical receptor chains. Therefore, the EPOR chain is both a high-affinity ligand-binding and signal transduction molecule chain, and upon ligand binding, it only activates the associated JAK2 molecule, leading to the recruitment and activation of STAT5. The GM-CSF receptor is a heterodimeric receptor composed of a GM-CSF high-affinity receptor chain (GM-CSFRα) and a JAK2-specifically associated signal transduction molecule chain (GM-CSFRβ). Upon ligand binding, the association of the α and β receptor chains leads to the activation of both the JAK2 and STAT5 signaling pathways. Therefore, to study JAK2 activity, the level of active STAT5 can be evaluated in immune cells after stimulation with GM-CSF or erythropoietin (EPO).

Inhibitors of Janus kinases are expected to show efficacy in the treatment of inflammatory and non-infectious autoimmune diseases involving these kinases. Recently, pan-JAK inhibitors tofacitinib and ruxolitinib have been introduced for the treatment of rheumatoid arthritis and myelofibrosis, respectively. The JAK1 inhibitor PF-04965842 is currently in a Phase III clinical trial for the treatment of atopic dermatitis; the JAK1 inhibitor baricitinib has been introduced for the treatment of rheumatoid arthritis and is in a Phase III trial for the treatment of atopic dermatitis; and the JAK1 inhibitor upadacitinib is currently in a Phase III clinical trial for the treatment of rheumatoid arthritis and psoriatic arthritis, and in a Phase II trial for the treatment of atopic dermatitis, Crohn's disease, and ulcerative colitis.

Therefore, JAK inhibitors can also be used to treat diseases associated with Janus kinase activity, including, for example, skin diseases such as psoriasis, atopic dermatitis, scleroderma, rosacea, skin cancer, dermatitis, herpetic dermatitis, dermatomyositis, vitiligo, alopecia areata, contact dermatitis, eczema, xerosis, ichthyosis, urticaria, chronic idiopathic pruritus, pyoderma gangrenosa, cutaneous lupus erythematosus, and lichen planus; respiratory diseases such as asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, cystic fibrosis, rhinitis, bronchiolitis, cotyledonous dermatitis, pneumoconiosis, bronchiectasis, allergic pneumonia, lung cancer, and mesothelioma. And sarcoidosis; gastrointestinal diseases such as inflammatory bowel disease, ulcerative colitis, Crohn's disease, retroperitoneal fibrosis, celiac disease and cancer; eye diseases such as myasthenia gravis, Scholenne's syndrome, conjunctivitis, scleritis, uveitis, dry eye syndrome, keratitis, iritis; systemic indications such as lupus, multiple sclerosis, rheumatoid arthritis, type I diabetes and complications of diabetes, cancer, ankylosing spondylitis and psoriatic arthritis; cancers such as bone and soft tissue tumors, head and neck cancer and other autoimmune diseases and indications, among which immunosuppression is necessary, for example, in organ transplantation.

WO2013007768 discloses tricyclic heterocyclic compounds, compositions, and methods of using them as JAK inhibitors.

WO2013007765 discloses fused tricyclic compounds used as Janus kinase inhibitors.

WO2011086053 discloses tricyclic heterocyclic compounds, compositions and methods of use thereof.

Zak, M. et al., J. Med. Chem., (2013), 56, 4764-85 disclose imidazopyrrolopyridine as a JAK1 inhibitor.

There is still a need for new compounds that can effectively and selectively inhibit specific JAK enzymes, particularly inhibitors that selectively inhibit JAK1 relative to JAK2, in order to reduce adverse effects without affecting overall anti-inflammatory efficacy.

[Summary of the Invention]

The compounds of the present invention exhibit inhibitory activity against Janus kinases; and in particular, they exhibit inhibitory activity against JAK1. Therefore, the compounds of the present invention show JAK kinase inhibitory selectivity; in particular, they show JAK1 inhibitory selectivity relative to JAK2. Thus, the compounds of the present invention may also show inhibitory selectivity relative to STAT5 against STAT6 or STAT3. Some compounds of the present invention possess pharmacokinetic properties particularly advantageous for systemic use, such as high metabolic stability and high water solubility. Some compounds of the present invention possess particularly advantageous toxicological properties, such as high kinase activity and general off-target selectivity, no CYP inhibition, low or no CYP induction, low cytotoxicity; and good tolerability in repeated-dose toxicology studies.

Therefore, this invention relates to compounds of formula (I).

in

A represents _C6 -cycloalkyl, wherein the _C6 -cycloalkyl is optionally substituted with one or more deuterium atoms;

_R1 represents a _C1 -alkyl group, wherein the _C1 -alkyl group is optionally substituted with one or more deuterium groups;

_R2 represents a _C1 -alkyl group, wherein the _C1 -alkyl group is substituted with a substituent selected from _R6 ; and wherein the _C1 -alkyl group is optionally substituted with one or more deuterium groups;

_R3 represents a _C2 -alkyl group, wherein the _C2 -alkyl group is substituted with a substituent selected from _R7 and wherein the _C2 -alkyl group is optionally substituted with one or more deuterium groups;

R ₄ represents hydrogen or deuterium;

R ₅ represents hydrogen or deuterium;

R ₆ represents cyano;

R ₇ represents a hydroxyl group;

Or its pharmaceutically acceptable salt, hydrate or solvate.

In another aspect, the present invention relates to pharmaceutical compositions comprising a compound of general formula (I) as defined herein, and a pharmaceutically acceptable solvent or excipient or a pharmaceutically acceptable carrier, optionally and one or more other therapeutically active compounds.

In another aspect, the present invention relates to compounds of general formula (I) as defined herein, which are used as pharmaceuticals.

In another aspect, the present invention relates to compounds of general formula (I) as defined herein, for the prevention and/or treatment of diseases of the immune system (e.g., autoimmune diseases) or diseases related to immune system dysregulation.

In another aspect, the present invention relates to intermediates that can be used to prepare compounds of general formula (I).

[Detailed Description of the Invention]

definition

The term "C _{_a}-alkyl" is intended to indicate the group obtained when a hydrogen atom is removed from a branched or straight-chain hydrocarbon. The alkyl group contains 1-2 carbon atoms, such as methyl and ethyl. The number of carbon atoms in "alkyl" is indicated by the prefix "C _{_a} ", where a is the number of carbon atoms in the hydrocarbon group. Therefore, C _₁-alkyl indicates an alkyl group containing 1 carbon atom, i.e., methyl. C _₂-alkyl indicates an alkyl group containing 2 carbon atoms, i.e., ethyl.

The term "cyano" is intended to refer to a -CN group that is attached to a portion of the parent molecule via a carbon atom.

The term "C ₆ -cycloalkyl" is intended to refer to a saturated cycloalkane hydrocarbon group containing 6 carbon atoms, namely cyclohexyl.

The term "hydrocarbon" is intended to refer to a group containing only hydrogen and carbon atoms, which may contain one or more carbon-carbon double bonds and/or carbon-carbon triple bonds, and may include a cyclic portion in combination with a branched or straight-chain portion. The hydrocarbon contains 1-10 carbon atoms, preferably 1-6, for example 1-4, 1-3, 1-2, or 6 carbon atoms. This term includes alkyl and cycloalkyl groups as indicated herein.

The term "hydroxyl" or "hydroxyl group" is intended to indicate the -OH group.

BrettPhos intends to indicate 2-(dicyclohexylphosphino)3,6-dimethoxy-2′,4′,6′-triisopropyl-1,1'-biphenyl.

tBuBrettPhos indicates 2-(di-tert-butylphosphino)-2′,4′,6′-triisopropyl-3,6-dimethoxy-1,1′-biphenyl.

tBuXPhos is intended to indicate 2-di-tert-butylphosphino-2′,4′,6′-triisopropylbiphenyl.

BrettPhos Pd G1 is intended to indicate chloro[2-(dicyclohexylphosphino)-3,6-dimethoxy-2',4',6'-triisopropyl-1,1'-biphenyl][2-(2-aminoethyl)phenyl]palladium(II).

BrettPhos Pd G3 is intended to indicate the presence of palladium(II) of mesylate [(2-di-cyclohexylphosphino-3,6-dimethoxy-2',4',6'-triisopropyl-1,1'-biphenyl)-2-(2'-amino-1,1'-biphenyl)].

tBuBrettPhos Pd G3 is intended to indicate the presence of palladium(II) of mesylate [(2-di-tert-butylphosphino-3,6-dimethoxy-2',4',6'-triisopropyl-1,1'-biphenyl)-2-(2'-amino-1,1'-biphenyl)].

tBuXPhos Pd G1 is intended to indicate [2-(di-tert-butylphosphino)-2′,4′,6′-triisopropyl-1,1′-biphenyl][2-(2-aminoethyl)phenyl)]palladium(II) chloride.

tBuXPhos Pd G3 is intended to indicate the presence of palladium(II) of mesylate [(2-di-tert-butylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)-2-(2′-amino-1,1′-biphenyl)].

If substituents are described as being selected independently from the group, then each substituent is chosen independently of the others. Therefore, each substituent may be the same as or different from the other substituents.

The term “optional substitution” means “unsubstituted or substituted”, and therefore the general formulas described herein cover compounds containing the specified optional substituents as well as compounds not containing the optional substituents.

The term "pharmaceutically acceptable salt" is intended to refer to a salt prepared by reacting a compound of formula (I) containing a basic moiety with a suitable inorganic or organic acid, such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, 2,2-dichloroacetic acid, adipic acid, ascorbic acid, L-aspartic acid, L-glutamic acid, mucoic acid, lactic acid, maleic acid, L-malic acid, phthalic acid, citric acid, propionic acid, benzoic acid, glutaric acid, gluconic acid, D-glucuronic acid, methanesulfonic acid, salicylic acid, succinic acid, malonic acid, tartaric acid, benzenesulfonic acid, ethane-1,2-disulfonic acid, 2-hydroxyethanesulfonic acid, toluenesulfonic acid, aminosulfonic acid, fumaric acid, acetic acid, L-lactic acid, glycolic acid, oxalic acid, saccharic acid, DL-mandelic acid, or L-tartaric acid. Other examples of pharmaceutically acceptable salts are listed in Berge, S.M.; J. Pharm. Sci.; (1977), 66(1), 1-19, which are incorporated herein by reference.

The term "solvent" is intended to refer to a substance formed by the interaction between a compound (e.g., compound of formula (I)) and a solvent (e.g., alcohol, glycerol, dioxane, or water), wherein the substance is in crystalline or amorphous form. When water is used as the solvent, the substance is called a hydrate.

As used herein, the term "treatment" refers to the management and care of a patient for the purpose of combating a disease, condition, or ailment. This term is intended to include delaying the progression of the disease, condition, or ailment, improving, alleviating, or relieving symptoms and complications, and/or curing or eliminating the disease, condition, or ailment. The term also includes the prevention of disease, which should be understood as the management and care of a patient for the purpose of combating a disease, condition, or ailment, and includes the administration of active compounds to prevent the onset of symptoms or complications. However, preventive (preventative) and therapeutic (curative) treatment are two separate aspects.

All references cited in this document (including publications, patent applications and patents) are incorporated herein by full reference as if each reference were individually and specifically indicated to be incorporated by reference, regardless of any separate inclusion of a particular document elsewhere in this document.

Embodiments of the present invention

In one embodiment, the present invention provides a compound of general formula (I), wherein formula (I) is general formula (Ia).

_R1 - _R2 and _R4 - _R7 are as defined above, and Ra, Rb, Rc and Rd are each independently selected from hydrogen and deuterium;

Or its pharmaceutically acceptable salt, hydrate or solvate.

In one embodiment, the present invention provides a compound of general formula (I); wherein formula (I) is general formula (Ib).

_R1 - _R2 and _R4 - _R7 are as defined above, and Ra, Rb, Rc and Rd are each independently selected from hydrogen and deuterium;

Or its pharmaceutically acceptable salt, hydrate or solvate.

In one embodiment, the present invention provides a compound of general formula (I); wherein formula (I) is general formula (Ic).

_R1 - _R2 and _R4 - _R7 are as defined above, and Ra, Rb, Rc and Rd are each independently selected from hydrogen and deuterium;

Or its pharmaceutically acceptable salt, hydrate or solvate.

In one embodiment, the present invention provides a compound of general formula (I); wherein formula (I) is general formula (Id).

_R1 - _R2 and _R4 - _R7 are as defined above, and Ra, Rb, Rc and Rd are each independently selected from hydrogen and deuterium;

Or its pharmaceutically acceptable salt, hydrate or solvate.

In one embodiment, the present invention provides a compound of general formula (I) as defined herein; wherein

A represents _C6 -cycloalkyl; _R1 represents _C1 -alkyl; _R2 represents _C1 -alkyl, wherein the _C1 -alkyl is substituted with a substituent selected from _R6 ; _R3 represents _C2 -alkyl, wherein the _C2 -alkyl is substituted with a substituent selected from _R7 ; _R4 represents hydrogen; _R5 represents hydrogen; _R6 represents cyano; _R7 represents hydroxyl.

Or its pharmaceutically acceptable salt, hydrate or solvate.

In one embodiment, the present invention provides a compound of general formula (I) as defined herein; wherein

A represents a _C6 -cycloalkyl group; _R1 represents a _C1 -alkyl group optionally substituted with one or more deuterium groups; _R2 represents a _C1 -alkyl group, wherein the _C1 -alkyl group is substituted with a substituent selected from _R6 ; _R3 represents a _C2 -alkyl group, wherein the _C2 -alkyl group is substituted with a substituent selected from _R7 ; _R4 represents hydrogen; _R5 represents hydrogen; _R6 represents cyano; _R7 represents hydroxyl.

Or its pharmaceutically acceptable salt, hydrate or solvate.

Any combination of two or more embodiments described herein is considered to be within the scope of this invention.

The present invention includes all embodiments in which _R1 , _R2 , _R3 , _R4 , _R5 , _R6 and _R7 are combined in any combination as described elsewhere herein.

In one embodiment, the present invention provides a compound of general formula (I) as defined herein; said compound is selected from:

trans-2-[4-[2-[1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile,

trans-2-[4-[2-[(1S)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile,

trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile,

trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(trideuteriummethylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile,

trans-2-[4-[2-[1,2,2,2-tetradeuter-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile,

cis-2-[4-[2-[1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile and

cis-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile

Or its pharmaceutically acceptable salt, hydrate or solvate.

In one embodiment, the present invention provides compounds selected from those of general formula (I) as defined herein:

trans-2-[4-[2-[1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile,

trans-2-[4-[2-[(1S)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile and

trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile

Or its pharmaceutically acceptable salt, hydrate or solvate.

In one embodiment, the present invention provides compounds of general formula (I) as defined herein, which are selected from the deuterated forms of the following compounds:

trans-2-[4-[2-[1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile,

trans-2-[4-[2-[(1S)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile and

trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile

Or its pharmaceutically acceptable salt, hydrate or solvate.

One embodiment of the present invention provides a compound of formula (I), said compound being trans-2-[4-[2-[1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile.

Or its pharmaceutically acceptable salt, hydrate or solvate.

One embodiment of the present invention provides a compound of formula (I), said compound being trans-2-[4-[2-[(1S)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile or a pharmaceutically acceptable salt, hydrate or solvate thereof.

One embodiment of the present invention provides a compound of formula (I), said compound being trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile or a pharmaceutically acceptable salt, hydrate or solvate thereof.

One embodiment of the present invention provides a compound of formula (I), said compound being trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(trideuterylmethylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile or a pharmaceutically acceptable salt, hydrate or solvate thereof.

One embodiment of the present invention provides a compound of formula (I), said compound being trans-2-[4-[2-[1,2,2,2-tetradeuter-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile or a pharmaceutically acceptable salt, hydrate or solvate thereof.

One embodiment of the present invention provides a compound of formula (I), said compound being cis-2-[4-[2-[1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile or a pharmaceutically acceptable salt, hydrate or solvate thereof.

One embodiment of the present invention provides a compound of formula (I), said compound being cis-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile or a pharmaceutically acceptable salt, hydrate or solvate thereof.

In one embodiment, the present invention provides a compound of general formula (I); wherein the compound is:

trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile,

Or its pharmaceutically acceptable salt.

In one embodiment, the present invention provides a compound of general formula (I); wherein the compound is:

trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile,

Or its hydrates.

In one embodiment, the present invention provides a compound of general formula (I); wherein the compound is:

trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile,

Or its solvates.

In one embodiment, the present invention provides a compound of general formula (I); wherein the compound is a deuterated form of trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile.

In one embodiment, the present invention provides a compound of general formula (I); wherein the compound is:

trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile.

In one embodiment, the present invention provides a compound of general formula (I); wherein the compound is:

trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile malonate.

In one embodiment, the present invention provides a compound of general formula (I); wherein the compound is:

trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile glycolate.

In one embodiment, the present invention provides a compound of general formula (I); wherein the compound is:

trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile L-tartrate.

In one embodiment, the present invention provides a compound of general formula (I); wherein the compound is:

trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile L-malate.

In one embodiment, the present invention provides a compound of general formula (I); wherein the compound is:

trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile sulfate.

In one embodiment, the present invention provides a compound of general formula (I); wherein the compound is:

trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile hydrochloride.

In one embodiment, the present invention provides a compound of general formula (I); wherein the compound is:

trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile succinate.

In one embodiment, the present invention provides a compound of general formula (I); wherein the compound is:

trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile oxalate.

In one embodiment, the present invention provides a compound of general formula (I); wherein the compound is:

trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile fumarate.

In one embodiment, the present invention provides a compound of general formula (I); wherein the compound is:

trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile 1,5-naphthalenedisulfonate.

In one embodiment, the present invention provides a compound of general formula (I); wherein the compound is:

trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile DL-amyric acid salt.

In one or more embodiments, the present invention provides an intermediate of general formula (II).

in

A indicates _C6 -cycloalkyl;

_R2 represents a _C1 -alkyl group, wherein the _C1 -alkyl group is substituted with a substituent selected from _R6 ; and wherein the _C1 -alkyl group is optionally substituted with one or more deuterium groups;

R ₄ represents hydrogen or deuterium;

R ₅ represents hydrogen or deuterium;

R ₆ represents cyano;

R ₈ indicates halogen;

or its salt;

It is used to prepare compounds of general formula (I).

In one embodiment, the present invention provides intermediates selected from the following:

2-[trans-4-[(5-amino-2-chloropyridin-4-yl)amino]cyclohexyl]acetonitrile

And its salt.

In one or more embodiments, the present invention provides an intermediate of general formula (III):

in

_R2 represents a _C1 -alkyl group, wherein the _C1 -alkyl group is substituted with a substituent selected from _R6 ; and wherein the _C1 -alkyl group is optionally substituted with one or more deuterium groups;

R ₄ represents hydrogen or deuterium;

R ₅ represents hydrogen or deuterium;

R ₆ represents cyano;

R ₇ represents a hydroxyl group;

Ra, Rb, Rc, and Rd are each independently selected from hydrogen and deuterium;

R ₈ indicates halogen;

or its salt;

It is used to prepare compounds of general formula (Ia).

In one embodiment, the present invention provides intermediates selected from the following:

2-[trans-4-[6-chloro-2-(1-hydroxyethyl)-1H-imidazo[4,5-c]pyridin-1-yl]cyclohexyl]-acetonitrile,

2-[trans-4-[6-chloro-2-[(1R)-1-hydroxyethyl]-1H-imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile and

trans-2-[4-[6-chloro-2-(1,2,2,2-tetradeuter-1-hydroxy-ethyl)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile

Or its salt.

In one embodiment, the present invention relates to a method for preparing compound (Ia) from compound (III), comprising amination of compound (III) in the presence of a palladium catalyst.

in

_R1 represents a _C1 -alkyl group, wherein the _C1 -alkyl group is optionally substituted with one or more deuterium groups;

_R2 represents a _C1 -alkyl group, wherein the _C1 -alkyl group is substituted with a substituent selected from _R6 ; and wherein the _C1 -alkyl group is optionally substituted with one or more deuterium groups;

R ₄ represents hydrogen or deuterium;

R ₅ represents hydrogen or deuterium;

R ₆ represents cyano;

R ₇ represents a hydroxyl group;

Ra, Rb, Rc, and Rd are each independently selected from hydrogen and deuterium;

R ₈ indicates halogen;

Or its salt.

In one embodiment, the present invention relates to a method for preparing compound (Ia) from compound (III), comprising amination of compound (III) in the presence of a palladium catalyst, wherein the palladium catalyst comprises a ligand of BrettPhos, tBuBrettPhos, or tBuXPhos.

In one embodiment, the present invention relates to a method for preparing compound (Ia) from compound (III), comprising amination of compound (III) in the presence of a palladium catalyst, wherein the palladium catalyst is selected from BrettPhos Pd G1, BrettPhos Pd G3, tBuBrettPhos Pd G3, tBuXPhos Pd G1 or tBuXPhos Pd G3.

In one embodiment, the present invention relates to a method for preparing compound (Ia) from compound (III), comprising amination of compound (III) in the presence of a palladium catalyst, wherein the palladium catalyst is prepared from a combination of a palladium source (e.g., _PdCl₂ , _Pd₂ (dba) _₃ or Pd(OAc) _₂ ) and a ligand of BrettPhos, tBuBrettPhos or tBuXPhos.

Compounds of formula (I) can be obtained directly by concentration from an organic solvent, or by crystallization or recrystallization from an organic solvent or a mixture of said solvent and a cosolvent, which may be organic or inorganic (e.g., water), to obtain crystalline form. Crystals can be separated in substantially solvent-free form or as solvates (e.g., hydrates). This invention covers all crystalline forms (e.g., polymorphs and pseudopolymorphs) and mixtures thereof.

Compounds of formula (I) contain asymmetrically substituted (chiral) carbon atoms, resulting in the presence of isomers (e.g., enantiomers and diastereomers). This invention relates to all such isomers, whether in optically pure form or mixtures thereof (e.g., racemic mixtures or partially purified optical mixtures). Pure stereoisomers of the compounds and intermediates of this invention can be obtained by methods known in the art. Different isomers can be separated by physical separation methods such as selective crystallization and chromatographic techniques (e.g., high-performance liquid chromatography using a chiral stationary phase). Enantiomers can be separated from each other by selective crystallization of their diastereomer salts formed from optically active acids. Optically purified compounds can then be released from said purified diastereomer salts. Enantiomers can also be resolved by forming diastereomer derivatives. Alternatively, enantiomers can be separated by chromatographic techniques using a chiral stationary phase. Pure stereoisomers can also be derived from corresponding pure stereoisomers of suitable starting materials, provided the reaction is carried out in a stereoselective or stereospecific manner. Preferably, if a specific stereoisomer is desired, the compound will be synthesized by a stereoselective or stereospecific preparation method. This method will advantageously employ chiral pure starting materials.

Furthermore, geometric isomers can be formed when double bonds or fully or partially saturated ring systems are present in the molecule. Any geometric isomers, whether isolated, pure, or partially purified, or mixtures thereof, are intended to be included within the scope of this invention.

Disubstituted cycloalkanes (e.g., disubstituted cyclohexane) can form geometric isomers; namely, cis- and trans-isomers. The cis-isomer has two substituents on the same side of the ring, and the trans-isomer has substituents on opposite sides of the ring. Any geometric isomer, whether isolated, pure, or partially purified, or a mixture thereof, is intended to be included within the scope of this invention.

In compounds of formula (I), atoms may exhibit their natural isotopic abundance, or one or more atoms may be artificially enriched with specific isotopes having the same atomic number but different atomic masses or mass numbers from those found in nature. This invention aims to include all suitable isotopic variations of compounds of formula (I). For example, different isotopic forms of hydrogen include ^¹H , ^²H , and ^³H ; different isotopic forms of carbon include ^¹²C , ^¹³C , and ^¹⁴C ; and different isotopic forms of nitrogen include ^¹⁴N and ^¹⁵N . Enrichment of deuterium ( ^²H ) may, for example, increase the in vivo half-life or reduce the dosage regimen, or provide compounds that can be used as standards for characterizing biological samples. Isotopically enriched compounds of formula (I) may be prepared using conventional techniques well known to those skilled in the art or using methods similar to those described herein with appropriate isotope-enriching reagents and/or intermediates.

In one or more embodiments of the present invention, compounds of Formula I as defined above can be used therapeutically and particularly for treating, for example, skin diseases such as proliferative and inflammatory skin conditions, psoriasis, atopic dermatitis, scleroderma, rosacea, skin cancer, dermatitis, herpetic dermatitis, dermatomyositis, vitiligo, alopecia areata, contact dermatitis, eczema, xerosis, ichthyosis, urticaria, chronic idiopathic pruritus, pyoderma gangrenosa, cutaneous lupus erythematosus, and lichen planus; respiratory diseases such as asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, cystic fibrosis, rhinitis, bronchiolitis, cotyledonous dermatitis, pneumoconiosis, and bronchiectasis. Allergic pneumonia, lung cancer, mesothelioma, and sarcoidosis; gastrointestinal diseases such as inflammatory bowel disease, ulcerative colitis, Crohn's disease, retroperitoneal fibrosis, celiac disease, and cancer; eye diseases such as myasthenia gravis, Scholenren's syndrome, conjunctivitis, scleritis, uveitis, dry eye syndrome, keratitis, and iritis; systemic indications such as lupus, multiple sclerosis, rheumatoid arthritis, type 1 diabetes and its complications, cancer, ankylosing spondylitis, and psoriatic arthritis; cancers such as bone and soft tissue tumors, head and neck cancer, and other autoimmune diseases and indications, among which immunosuppression is desirable, for example, in organ transplantation.

In one embodiment, the present invention provides a compound of formula I as defined above for the prevention and/or treatment of psoriasis or atopic dermatitis.

In one embodiment, the present invention provides a compound of formula I as defined above for the prevention and/or treatment of atopic dermatitis.

In one embodiment, the present invention provides a method for preventing, treating, or improving immune system diseases (e.g., autoimmune diseases), the method comprising administering to a person suffering from at least one of the diseases an effective amount of one or more compounds of general formula I above, optionally along with a pharmaceutically acceptable carrier or one or more excipients, optionally in combination with other therapeutically active compounds.

In one embodiment, the present invention provides a method for preventing, treating, or improving psoriasis or atopic dermatitis, the method comprising administering to a person suffering from at least one of the diseases an effective amount of one or more compounds of general formula I above, optionally along with a pharmaceutically acceptable carrier or one or more excipients, optionally in combination with other therapeutically active compounds.

In one embodiment, the present invention provides a method for preventing, treating, or improving atopic dermatitis, the method comprising administering to a person suffering from at least one of the diseases an effective amount of one or more compounds of general formula I above, optionally along with a pharmaceutically acceptable carrier or one or more excipients, optionally in combination with other therapeutically active compounds.

In one embodiment, the present invention provides a compound of formula I for preparing a medicament for preventing and/or treating immune system diseases, such as autoimmune diseases, like psoriasis or atopic dermatitis.

In one embodiment, the present invention provides a compound of formula I for preparing a medicament for preventing and/or treating immune system diseases, such as autoimmune diseases, such as atopic dermatitis.

In one or more embodiments of the present invention, compounds of Formula I as defined above can be used as anti-inflammatory agents that are capable of modulating the activity of protein tyrosine kinases of the JAK family (e.g., JAK1, JAK2, JAK3, or TYK2 protein tyrosine kinases).

In one or more embodiments, the present invention provides a compound of general formula (I) for treating a disease in which the disease responds to an inhibitory response to JAK1 kinase activity.

In addition to their use in human treatment, the compounds of the present invention can also be used in veterinary treatment of animals, including mammals such as horses, cattle, sheep, pigs, dogs and cats.

The pharmaceutical composition of the present invention

For use in therapy, the compounds of the present invention are generally in the form of pharmaceutical compositions. Therefore, the present invention relates to pharmaceutical compositions comprising compounds of formula (I), optionally and one or more other therapeutically active compounds, and pharmaceutically acceptable excipients, solvents, or carriers. The excipients must be "acceptable" in the sense that they are compatible with the other components of the composition and harmless to the recipient.

Suitable, the active ingredient accounts for 0.0001% to 99.9% of the weight of the formulation.

In dosage units, the compound may be administered once or more at appropriate intervals between days; however, this always depends on the patient’s condition and is based on a prescription from a practicing physician. Suitably, the dosage units of the formulation contain a compound of formula (I) between 0.001 mg and 1000 mg, preferably between 0.1 mg and 300 mg, more preferably between 1-150 mg, for example 3-100 mg.

The appropriate dosage of the compounds of this invention will (in particular) depend on the patient's age and condition, the severity of the disease to be treated, and other factors known to the practicing physician. The compounds may be administered orally, non-enterally, topically, transdermally, intradermally, or via other routes according to different dosing schedules (e.g., daily, weekly, or monthly intervals). Generally, a single dose will range from 0.001 mg/kg body weight to 400 mg/kg body weight, for example, 0.1 g to 4 mg/kg. The compounds may be administered as a bolus (i.e., a single administration of the entire daily dose) or as split doses twice or more daily.

In the case of local treatment, it may be more appropriate to refer to the “unit of use”, which refers to a single dose that can be administered to the patient and is easily manipulated and packaged, which remains a physically and chemically stable unit dose, comprising the active material itself or a mixture thereof with solid, semi-solid or liquid pharmaceutical diluents or carriers.

The term “unit of use” in relation to topical application means a unit, i.e. a single dose, which can be applied topically to a patient in the form of 0.001 micrograms to 1 mg, preferably 0.05 micrograms to 0.5 mg of the active ingredient of interest per square centimeter of treatment area.

It is also envisioned that in some treatment regimens, it would be beneficial to administer the medication at longer intervals, such as every other day, every week, or even longer intervals.

If treatment involves the administration of other therapeutically active compounds, it is recommended to consult Goodman & Gilman’s The Pharmacological Basis of Therapeutics, 9th edition, edited by J.G. Hardman and L.E. Limbird, McGraw-Hill 1995 for the available dosages of the compounds.

The compounds of the present invention may be applied simultaneously or sequentially with one or more other active compounds.

Formulations include those suitable for oral, rectal, non-intestinal (including subcutaneous, intraperitoneal, intramuscular, intra-articular, and intravenous), transdermal, intradermal, ocular, topical, nasal, sublingual, or buccal administration.

The formulation may suitably exist in dosage units and may be prepared by, but not limited to, any method well known in the pharmaceutical industry, such as that disclosed in Remington, The Science and Practice of Pharmacy, 21st edition, 2005. All methods include the step of conjugating the active ingredient with a carrier constituting one or more auxiliary ingredients. Generally, the formulation is prepared by homogenizing and adequately conjugating the active ingredient with a liquid carrier, a semi-solid carrier, or a micro-solid carrier, or a combination thereof, and then (if necessary) shaping the product into the desired formulation.

Formulations of the present invention suitable for oral and buccal administration may be in the following forms: discrete units each containing a predetermined amount of the active ingredient, such as capsules, pouches, tablets, chewing gum, or lozenges; powders, granules, or pills; solutions or suspensions in aqueous or non-aqueous liquids; or gels, nanoemulsions or microemulsions, oil-in-water emulsions, water-in-oil emulsions, or other dispersion systems. Suitable dispersants or suspending agents for aqueous suspensions include synthetic or natural surfactants and thickeners. The active ingredient may also be administered as a bolus, electuary, or paste.

Tablets can be prepared by compression, molding, or lyophilization of an active ingredient, optionally with one or more excipients. Compressed tablets can be prepared by compressing an active ingredient (e.g., powder or granules) into a free-flowing form in a suitable machine, said active ingredient optionally mixed with a binder and/or filler; lubricant; disintegrant or dispersant. Molded tablets can be prepared by molding a mixture of a powdered active ingredient wetted with an inert liquid diluent and a suitable carrier in a suitable machine. Lyophilized tablets can be formed from a solution of the active pharmaceutical ingredient in a lyophilizer. Suitable fillers may be included.

Formulations for rectal administration may be in suppository form, wherein the compounds of the present invention are mixed with low-melting-point, water-soluble or insoluble solids.

Formulations suitable for non-enteral administration suitably comprise sterile oily or aqueous formulations of the active ingredient, preferably isotonic with the recipient's blood, such as isotonic saline, isotonic glucose solution, or buffer solution. Furthermore, the formulation may contain co-solvents, solubilizers, and/or complexing agents. The formulation may be suitably sterilized by, for example, filtration through a bacterial retention filter, addition of a sterilizing agent to the formulation, irradiation, or heating. Liposome formulations, such as those disclosed in the Encyclopedia of Pharmaceutical Technology, Volume 9, 1994, are also suitable for non-enteral administration.

Alternatively, the compound of formula (I) may exist in the form of a sterile solid dosage form, such as a lyophilized powder that can be easily dissolved in a sterile solvent just before use.

Transdermal formulations may be in the form of ointments, patches, microneedles, liposomes or nanoparticle delivery systems or other dermal formulations applied to the skin.

Formulations suitable for ocular application may be in the form of a sterile aqueous formulation of the active ingredient (which may be in microcrystalline form), for example, in the form of an aqueous microcrystalline suspension. Liposome formulations or biodegradable polymer systems, such as those disclosed in Encyclopedia of Pharmaceutical Technology, Volume 2, 1989, may also be used to provide the active ingredient for ocular application.

Preparations suitable for topical (e.g., skin, dermis, or eye) application include liquid or semi-solid preparations such as liniments, lotions, gels, applicants, sprays, foams, film-forming systems, microneedles, microemulsions or nanoemulsions, oil-in-water or water-in-oil emulsions such as creams, ointments, or pastes; or solutions or suspensions such as drops.

For topical application, the compound of formula (I) is typically present in an amount from 0.001% to 20% by weight of the composition, for example, from 0.01% to about 10%, but may also be present in an amount up to about 100% of the composition.

Preparations suitable for nasal or buccal administration include powders, self-propelled and spray formulations, such as aerosols and nebulizers. Such preparations are disclosed in more detail in, for example, the following: Modern Pharmaceutics, 2nd edition, G.S. Banker and C.T. Rhodes (eds.), pp. 427-432, Marcel Dekker, New York; Modern Pharmaceutics, 3rd edition, G.S. Banker and C.T. Rhodes (eds.), pp. 618-619 and 718-721, Marcel Dekker, New York; and Encyclopedia of Pharmaceutical Technology, Vol. 10, J. Swarbrick and J.C. Boylan (eds.), pp. 191-221, Marcel Dekker, New York.

In addition to the ingredients mentioned above, formulations of compounds of formula (I) may include one or more additional ingredients, such as diluents, buffers, flavoring agents, colorants, surfactants, thickeners, penetration enhancers, solubilizers, preservatives (e.g., methylparaben (including antioxidants)), emulsifiers, etc.

Preparation method

The compounds of this invention can be prepared in a variety of ways well known to those skilled in the art. For example, compounds of formula (I) can be prepared using the reactions and techniques outlined below, as well as methods known in the field of synthetic organic chemistry or variations thereof as understood by those skilled in the art. Preferred methods include (but are not limited to) those described below. The reactions are carried out in solvents suitable for the reagents and materials used and conducive to achieving the transformation. Furthermore, in the synthetic methods described below, it should be understood that all proposed reaction conditions (including the choice of solvent, reaction atmosphere, reaction temperature, duration of the experiment, and post-treatment procedures) are selected as standard conditions for the reactions, which should be readily apparent to those skilled in the art. Not all compounds falling into a given category are compatible with some of the reaction conditions required in some of the methods described. Such limitations on substituents compatible with the reaction conditions will be readily apparent to those skilled in the art, and alternative methods may be used. If necessary, standard methods well known to synthetic organic chemists can be used to purify the compounds of this invention or any intermediates, such as those described in “Purification of Laboratory Chemicals,” 6th edition, 2009, W. Amarego and C. Chai, Butterworth-Heinemann. The starting materials are commercially available known compounds, or can be prepared by conventional synthetic methods well known to those skilled in the art.

General methods, preparation and examples

Starting materials are commercially available or known in the literature. Reagents and solvents are commercially available and used unpurified unless otherwise specified. Chromatographic purification is performed using a Grace system or Teledyne Isco Rf system with a pre-packed silica rapid column or manually using silica gel 60. ^¹H NMR spectra are recorded at 300 MHz, 400 MHz, or 600 MHz on a Bruker instrument, with tetramethylsilane (δ = 0.00 ppm) as an internal standard. Chemical shift values (δ, in ppm) are relative to the internal tetramethylsilane (δ = 0.00) standard reference. In cases where doublets (d), triplets (t), quartets (q), or undefined (m) are defined, the values for multiplets are approximately taken at the midpoint unless ranges are listed. (br) indicates a broad peak, while (s) indicates a singlet.

The following abbreviations are used throughout:

AcOH Acetic acid

Boc tert-butoxycarbonyl

Cbz Benzyloxycarbonyl

DCM (Dichloromethane)

dba Dibenzylacetone

DIPEA (N,N-diisopropylethylamine)

DMF (dimethylformamide)

DMP (Dess-Martin periodinane)

DMSO (Dimethyl sulfoxide)

DSC (Differential Scanning Calorimetry)

ee Enantiomer excess

Et ethyl

EtOAc Ethyl acetate

EtOH Ethanol

h hours

HATU 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolidine

[4,5-b]pyridinium 3-oxide hexafluorophosphate

HPLC (High Performance Liquid Chromatography)

HRMS (High Resolution Mass Spectrometry)

L liters

m ...

MeCN Acetonitrile

MeOH (Methanol)

min

m.p. Melting point

Ms Methanesulfonyl group

MS (Mass Spectrometry)

NMR (Nuclear Magnetic Resonance Spectroscopy)

rt Room temperature, i.e., 18-30℃, and usually 20℃

RuPhos 2-Dicyclohexylphosphino-2',6'-diisopropoxybiphenyl

SFC (Supercritical Fluid Chromatography)

TBS tert-butyldimethylsilyl

TFA (Trifluoroacetic acid)

THF (Tetrahydrofuran)

Tj Temperature of the heating jacket

TLC (Thin Layer Chromatography)

tr Retention time

Tr Temperature of the reaction mixture

UHPLC (Ultra-High Performance Liquid Chromatography)

UPLC (Ultra-High Performance Liquid Chromatography)

Xantphos 4,5-Bis(diphenylphosphino)-9,9-dimethylxanthanium

Preparative HPLC

acidic method

Apparatus: Gilson HPLC system with Gilson UV/VIS-155 detector

Column: Waters SunFire ^TM Prep C18 5μm OBD 19×250mm

Reagents: (A) 0.1% formic acid-water solution; (B) MeCN

Pump:

- Flow rate: 30 mL/min

Time [min] [％]B 0.0 10 2.0 10 9.0 100 13.0 100

alkaline method

Apparatus: Gilson HPLC system with Gilson UV/VIS-155 detector

Column: WatersPrep C18 5μm OBD 19×250mm

Reagents: (A) 50mM _{NH₄HCO₃ solution} ; (B) MeCN

Pump:

- Flow rate: 30 mL/min

Time [min] [％]B 0.0 0 2.0 0 9.0 60 10.0 100 13.0 100

Analytical LC-MS

Method A

Equipment: Shimadzu UHPLC 2020

Column: Acquity UPLC HSS C18, 1.8μm, 2.1×50mm

Reagent: Formic acid ≥98%, Sigma-Aldrich

- Acetonitrile for HPLC UV/gradient stages, Baker

- Pure dimethyl sulfoxide (DMSO), Chempur

Purified water for HPLC

HPLC conditions: - Wavelength: 214nm±4nm, 254nm±4nm

- Flow rate: 0.5 ml/min

- Column temperature: 25℃

-Automatic sampler temperature: 20℃

Injection volume: 3.0 μl

-Analysis time: 6 minutes

- Elution: Gradient

Time [min] Mobile phase A [%) Mobile phase B [%] Flow rate [ml/min] 0.0 95 5 0.5 4.0 5 95 0.5 5.0 5 95 0.5 5.2 95 5 0.5 6.0 95 5 0.5

Mobile phase A: 0.1% v/v aqueous solution of formic acid

Mobile phase B: acetonitrile solution of 0.1% v/v formic acid

Solution for syringe washing: 20% MeOH

MS conditions:

- Mass range: 100-1000 m/z

-Ionization: Alternating

- Scan time: 0.5s

Method B

UPLC-MS analysis was performed using a Waters Acquity UPLC system with a 2.1 × 50 mm Acquity HSS T3 1.8 μm column and an Acquity SQ detector operating in positive ionization electrospray mode. The mobile phase consisted of 0.1% formic acid in 10 mM ammonium acetate aqueous solution for buffer A and 0.1% formic acid in acetonitrile for buffer B. A binary gradient (A:B 95:5 → 5:95) was used over 1.4 min at a flow rate of 1.2 mL/min and a column temperature of 60 °C.

Method C

High-resolution mass spectrometry (UPLC-MS) analysis was performed using a Waters Acquity UPLC system with UV detection at 254 nm and a Waters LCT Premier XE high-resolution TOF mass spectrometer operating in positive ionization electrospray mode. The same column and mobile phases A and B as in Method B were used, but with a slower gradient (A:B 99:1 → 1:99 over 4.8 min; 0.7 mL/min; column temperature 40 °C).

Method D

LC-MS: Mass spectra were obtained on a Shimadzu LCMS-2010EV spectrometer using electrospray ionization and atmospheric pressure chemical ionization; column: Acquity BEH C18 (50 mm × 2.1 mm, 1.7 μm); mobile phase: A: 0.1% formic acid in water; B: 0.1% formic acid in acetonitrile; gradient: time (min)/%B: 0/2, 0.2/2, 2.3/98, 3.4/98, 3.41/2, 3.5/2; column flow rate: 0.8 mL/min.

Method E

LC-MS: Mass spectra were obtained using electrospray ionization and atmospheric pressure chemical ionization on a Shimadzu LCMS-2010EV spectrometer; Column: Acquity BEH C18 (50 mm × 2.1 mm, 1.7 μm); Mobile phase: A: 0.1% formic acid in water; B: 0.1% formic acid in acetonitrile; Gradient time (min)/%B: 0/3, 0.4/3, 2.5/98, 3.4/98, 3.5/3, 4/3; Column temperature: 35 °C; Flow rate: 0.6 mL/min.

Analytical chiral stationary phase SFC

Chiral stationary phase SFC analysis was performed using a Waters UPC2 SFC instrument equipped with a Phenomenex 3 μm cellulose-4 (150 × 4.6 mm) column. Isocratic conditions were used, with the mobile phase consisting of _CO₂ :MeOH 80:20 and a flow rate of 3 mL/min. Enantiomer ratios of analytes were determined by integrating the UV peak areas.

intermediate

Intermediate 1

2-[trans-4-[(2-chloro-5-nitropyridin-4-yl)amino]cyclohexyl]acetonitrile

A solution of trans-4-(cyanomethyl)cyclohexyl]trifluoroacetate ammonium (Li, Y.-L. et al., US2014/0121198) (26.7 g, 105.8 mmol) and 2,4-dichloro-5-nitropyridine (22.4 g, 116.3 mmol) in anhydrous acetonitrile was frozen in an ice bath, and N,N-diisopropylethylamine (55.3 mL, 317 mmol) was added dropwise. The resulting mixture was stirred at rt for 20 hours. The reaction mixture was diluted with a saturated aqueous solution _{of NaHCO3} and extracted with DCM (3 × 500 mL). The _combined organic layers were dried over _Na2SO4 , filtered, and evaporated under vacuum. The crude product was ground with hexane, diethyl ether, and water to provide the title compound (29.8 g, 95%) as a yellow solid.

UPLC-MS (Method A): t _R = 3.41 minutes, m/z = 294.9 (M+H ⁺ ).

¹ H NMR (400MHz, DMSO-d ₆ )δ8.87(s,1H),8.03(d,J＝8.3Hz,1H),7.30(s,1H),3.73(dtd,J＝11.5,7.7,4.2Hz,1H),2.50(d,J＝4.2Hz,2H)2.04-1.91 (m,2H),1.86-1.75(m,2H),1.64(ddd,J＝11.6,5.7,3.0Hz,1H),1.53-1.39(m,2H),1.31(ddd,J＝25.4,12.9,4.2Hz,2H).

Intermediate 2

2-[trans-4-[(5-amino-2-chloropyridin-4-yl)amino]cyclohexyl]acetonitrile

Iron (1.86 g, 33.2 mmol) and ammonium chloride (1.91 g, 35.6 mmol) were added to a solution of intermediate 1 (3.5 g, 11.9 mmol) in MeOH:water 9:1 (99 mL). The resulting mixture was stirred under reflux for 5 hours. After cooling to rt, the solid was removed by filtration through a diatomaceous earth plug. The filter cake was washed with MeOH and the filtrate was concentrated to remove volatiles. The residue was diluted with a saturated aqueous solution of _NaHCO3 (50 mL) and extracted with EtOAc (3 × 30 mL). _The organic phase was dried over _Na2SO4 and concentrated under vacuum to give the title compound (3.0 g, 95%) as a brown solid.

UPLC-MS (Method A): t _R = 1.85 minutes, m/z = 265 (M + H ⁺ ).

¹ H NMR (400MHz, DMSO-d ₆ )δ7.37(s,1H),6.37(s,1H),5.38(d,J＝7.7Hz,1H),4.74(s,2H),2.48(d,J＝6.4Hz ,2H),2.06-1.92(m,2H),1.87-1.74(m,2H),1.71-1.57(m,1H),1.33-1.16(m,5H).

Intermediate 3

2-[trans-4-[6-chloro-2-(1-hydroxyethyl)-1H-imidazo[4,5-c]pyridin-1-yl]cyclohexyl]-acetonitrile

A mixture of triethyloxonium tetrafluoroborate (9.3 g, 49.1 mmol) and lactamide (4.4 g, 49.1 mmol) in THF (40 mL) was stirred at rt for 2 hours (clear solution). This solution was added to a solution of intermediate 2 (2.6 g, 9.8 mmol) in EtOH (70 mL). The resulting mixture was heated to reflux and held for 18 hours. The reaction mixture was concentrated and the residue was partitioned between water (40 mL) and EtOAc (25 mL). The aqueous phase was extracted with EtOAc (3 × 20 mL). _The organic phase was dried over _Na₂SO₄ and concentrated under vacuum. Column chromatography (20% EtOAc in DCM and 10% MeOH in DCM as eluent) provided a semi-solid, which was ground with diethyl ether to give the title compound (2.3 g, 73%) as a red solid.

UPLC-MS (Method A): t _R = 2.52 minutes, m/z = 319 (M+H ⁺ ).

¹ H NMR (400MHz, DMSO-d ₆ )δ8.70(s,1H),8.02(s,1H),5.81(d,J＝6.8Hz,1H),5.10(p,J＝6.5Hz,1H),4.74-4.60(m,1H),2.55(d,J＝6.1 Hz,2H),2.39-2.17(m,2H),2.16-2.03(m,1H),1.98-1.84(m,4H),1.60(d,J=6.5Hz,3H),1.38-1.22(m,2H).

Example

Example 1

trans-2-[4-[2-[1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile (Compound 1)

Step 1:

2-[trans-4-[6-amino-2-(1-hydroxyethyl)-1H-imidazo[4,5-c]pyridin-1-yl]cyclohexyl]-acetonitrile

Intermediate 3 (0.20 g, 0.63 mmol), tert-butyl carbamate (0.15 g, 1.25 mmol), tris-(dibenzylacetone)dipalladium(0) (0.06 g, 0.06 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthone (0.11 g, 0.19 mmol), and tripotassium phosphate (0.31 g, 1.44 mmol) were mixed in 1,4-dioxane (15 mL). The resulting mixture was degassed with argon for 20 min and then heated at 130 °C under microwave irradiation for 45 min. The mixture was filtered through a diatomaceous earth plug, the filter cake was washed with MeOH/DCM (1:9), and the filtrate was concentrated. Due to the volume limitations of the microwave reactor, this reaction was repeated four times using the same amounts and conditions. The resulting residues were combined. Short column chromatography (2% MeOH in DCM as eluent) provided a crude mixture (0.7 g), which was dissolved in DCM (15 mL). TFA (1.3 mL, 17.5 mmol) was added dropwise to the solution. The resulting mixture was stirred at room temperature for 24 hours. The mixture was concentrated under vacuum. The residue was dissolved in DCM (15 mL) and washed with _NaHCO3 aqueous solution (5 mL). The aqueous phase was extracted with DCM (5 × 15 mL). The _organic phase was dried over _Na2SO4 and concentrated under vacuum. Column chromatography (gradient, from MeOH:DCM 4:96 to 7.5 M _NH3 in MeOH:DCM 5:95) provided the title compound (0.195 g, 21%) as a yellow foam.

UPLC-MS (Method A): t _R = 1.72 minutes, m/z = 300 (M + H ⁺ ).

¹ H NMR (400MHz, DMSO-d ₆ )δ8.26(d,J＝0.9Hz,1H),6.66(d,J＝1.0Hz,1H),5.59(d,J＝6.7Hz,1H),5.39(s,2H),4.96(p,J＝6.6Hz,1H),4.62-4 .47(m,1H),2.57(d,J＝6.4Hz,2H),2.23-2.06(m,2H),2.01-1.72(m,5H),1.54(d,J＝6.5Hz,3H),1.40-1.22(m,2H).

Step 2:

trans-2-[4-[2-[1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile

Paraformaldehyde (0.078 g, 2.61 mmol) and sodium methoxide (0.176 g, 3.26 mmol) were added to a solution of the product from step 1 (0.195 g, 0.65 mmol) in methanol (5 mL). The resulting mixture was heated to reflux. After 2 hours, the mixture was cooled to 0 °C and sodium borohydride (0.099 g, 2.61 mmol) was added. The resulting mixture was heated to reflux for 1 hour. After cooling to rt, the reaction was quenched by carefully adding a saturated aqueous solution _of NaHCO3 (10 mL). The aqueous phase was extracted with EtOAc (3 × 10 mL). _The organic phase was dried over _Na2SO4 and concentrated under vacuum. Column chromatography (4% to 10% MeOH in DCM as eluent) yielded the title compound (0.152 g, 76%) as a white foamy solid.

HPLC-MS (Method C): t _R = 1.67 min, m/z = 314.1939 (M+H ⁺ ).

¹ H NMR (400MHz, DMSO-d ₆ )δ8.34(d,J＝0.9Hz,1H),6.48(d,J＝0.9Hz,1H),5.90(q,J＝4.9Hz,1H),5.59(d,J＝6.6Hz,1H),4.96(p,J＝6.6Hz,1H),4.61-4.50(m, 1H), 2.80 (d, J = 4.9Hz, 3H), 2.56 (d, J = 6.2Hz, 2H), 2.30-2.11 (m, 2H), 1.98-1.82 (m, 5H), 1.55 (d, J = 6.5Hz, 3H), 1.39-1.23 (m, 2H).

Examples 2 and 3

The compounds are trans-2-[4-[2-[(1S)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile (compound 2) and trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile (compound 3).

The enantiomers (166 mg) of Example 1 were separated by chiral stationary phase SFC under the following conditions:

column Amy-C (20×250mm, 5μm) Column temperature 40℃ Flow rate 50 mL/min Back pressure regulator 125 bars detector wavelength 228nm Injection volume/sample mass 300μL/12mg Eluent (isocratic) <![CDATA[30:70MeOH:CO₂+0.1%v/v NH₃]]>

The absolute configuration was determined by comparison with a sample of the (R) enantiomer prepared from (R)-lactamide, see Example 3 below (alternative preparation).

Example 2 (Compound 2)

Yield: 73mg, >98% ee

UPLC-MS (Method C): t _R = 1.67 minutes, m/z = 314.1908 (M+H ⁺ ).

¹ H NMR (300MHz, DMSO-d ₆ )δ8.33(d,J＝0.8Hz,1H),6.47(d,J＝1.0Hz,1H),5.89(q,J＝5.0Hz,1H),5.58(d,J＝6.7Hz,1H),4.96(p,J＝6.5Hz,1H),4.63-4.47(m, 1H), 2.79 (d, J = 5.0Hz, 3H), 2.55 (d, J = 6.1Hz, 2H), 2.30-2.10 (m, 2H), 2.01-1.76 (m, 5H), 1.54 (d, J = 6.4Hz, 3H), 1.40-1.20 (m, 2H).

Example 3 (Compound 3)

Yield: 67mg, >98% ee

UPLC-MS (Method C): t _R = 1.67 minutes, m/z = 314.1975 (M+H ⁺ ).

¹ H NMR (300MHz, DMSO-d ₆ )δ8.33(d,J＝0.9Hz,1H),6.47(d,J＝1.1Hz,1H),5.89(q,J＝4.9Hz,1H),5.58(d,J＝6.5Hz,1H),4.96(p,J＝6.4Hz,1H),4.63-4.46(m, 1H), 2.79 (d, J = 5.0Hz, 3H), 2.55 (d, J = 6.1Hz, 2H), 2.29-2.11 (m, 2H), 2.00-1.77 (m, 5H), 1.54 (d, J = 6.5Hz, 3H), 1.39-1.19 (m, 2H).

¹ H NMR (600MHz, DMSO-d ₆ )δ8.33(d,J＝0.9Hz,1H),6.48(d,J＝1.0Hz,1H),5.90(q,J＝5.0Hz,1H),5.59(d,J＝6.7Hz,1 H),4.96(p,J＝6.6Hz,1H),4.55(tt,J＝12.3,4.0Hz1H),2.79(d,J＝5.0Hz,3H),2.55(d,J＝6 .4Hz,2H),2.20(qdd,J＝12.8,10.4,3.7Hz,2H),1.93(ddd,J＝13.1,6.3,3.2Hz,2H),1.89- 1.86(m,2H),1.86-1.84(m,1H),1.54(d,J＝6.5Hz,3H),1.30(qdt,J＝12.3,7.6,3.6Hz,2H).

¹³ C NMR (151MHz, DMSO-d ₆ ) δ 155.5, 155.4, 141.3, 139.2, 133.3, 119.5, 86.4, 61.9, 54.0, 32.9, 30.7, 30.7, 29.1, 28.8, 28.8, 22.9, 21.5.

Example 3 (Alternative Preparation No. 1)

trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile (compound 3)

Step 1

2-[trans-4-[6-chloro-2-[(1R)-1-hydroxyethyl]-1H-imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile

Triethyloxonium tetrafluoroborate (8.00 g, 42.1 mmol) and anhydrous THF (30 mL) were packed into a 100 mL screw-cap vial under argon pressure. (R)-lactamide (3.87 g, 42.1 mmol) was added to the white suspension in a single addition. The resulting solution was stirred at rt. After approximately 6 minutes, a weak exothermic reaction occurred, and the reaction vessel was cooled in a water bath. After 2 hours at rt, this solution was added to a suspension of intermediate 2 (2.23 g, 8.42 mmol) in anhydrous ethanol (45 mL), and the resulting solution was stirred overnight at 80 °C. The evaporation was distilled off, and the residue was treated with a saturated aqueous solution of sodium bicarbonate (50 mL). The mixture was extracted with EtOAc (3 × 80 mL), and the combined organic phases were washed with brine (50 mL), dried over sodium sulfate, and filtered. The evaporation yielded a residue (13.5 g), which was purified using rapid chromatography (DCM:MeOH 98:2 to 95:5) to give a brown foamy substance (1.92 g). This was ground together with ether (20 mL) to give the title compound as a solid (1.62 g, 57%).

UPLC-MS (Method B): t _R = 0.52 minutes, m/z = 319.2 (M+H ⁺ ).

¹ H NMR (300MHz, DMSO-d ₆ )δ8.69(d,J＝0.8Hz,1H),8.00(d,J＝0.9Hz,1H),5.80(d,J＝6.8Hz,1H),5.10(p,J＝6.5Hz,1H),4.74-4.59(m,1H),2.54 (d,J＝6.4Hz,2H),2.38-2.16(m,2H),2.17-2.00(m,1H),1.99-1.81(m,4H),1.59(d,J＝6.5Hz,3H),1.41-1.18(m,2H).

The absolute configuration of the title compound was confirmed by single-crystal X-ray diffraction.

Step 2

2-[4-[2-[(1R)-1-[tert-butyl(dimethyl)silyl]oxyethyl]-6-chloro-imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile

The product from step 1 (2.47 g, 7.75 mmol) in THF (35 mL) was cooled in an ice bath and imidazole (791 mg, 11.6 mmol) was added. After 5 minutes, TBSCl (1.28 g, 8.52 mmol) in THF (11 mL) was added and the ice bath was removed. After 5 hours at rt, the mixture was heated to 45 °C and stirred overnight at this temperature. To achieve complete conversion, another portion of imidazole (791 mg, 11.6 mmol) and TBSCl (1.28 g, 8.52 mmol) in THF (5 mL) was added. The mixture was stirred at 45 °C for another 24 hours and poured into a mixture of brine (35 mL) and water (35 mL). It was extracted with EtOAc (2 × 80 mL), the organic layer was washed with brine, dried over _Na₂SO₄ , filtered _, and evaporated. The residue was purified by rapid chromatography (DCM:EtOAc 90:10 to 80:20) to provide the title compound (2.95 g, 83%) in solid form.

UPLC-MS (Method B): t _R = 0.92 minutes, m/z = 433.3 (M+H ⁺ ).

¹ H NMR (600MHz, CDCl ₃ )δ8.75(d,J＝0.8Hz,1H),7.42(d,J＝0.9Hz,1H),5.34(q,J＝6.8Hz,1H),5.00(tt,J＝12.5,4.1Hz,1H),2.41(d,J＝6.4Hz,2H),2.31-2.19(m,2H ),2.17-2.09(m,2H),2.09-2.03(m,2H),2.00-1.91(m,1H),1.62(d,J＝ 6.4Hz,3H),1.48-1.33(m,2H),0.91(s,9H),0.14(s,3H),0.03(s,3H).

Step 3

2-[4-[2-[(1R)-1-[tert-butyl(dimethyl)silyl]oxyethyl]-6-[(4-methoxyphenyl)methyl-methyl-amino]imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile

The product of step 2 (46.2 mg, 0.107 mmol) and 4-methoxy-N-methylbenzylamine (32.3 mg, 0.213 mmol) were packed into a 2 mL screw-cap vial. The vial was purged with argon and a mixture of sodium tert-butoxide (12.3 mg, 0.128 mmol), RuPhos (3.0 mg, 0.0064 mmol), and palladium(II) acetate (0.72 mg, 0.0032 mmol) was added. The mixture was stirred under argon at 110 °C for 17 hours. It was cooled to rt and dichloromethane (0.45 _mL ) was added. The mixture was washed with brine:water 2:1 (0.45 mL) and the aqueous layer was extracted with dichloromethane (2 × 0.45 mL). The combined organic layers were dried over _Na₂SO₄ , filtered, and evaporated to provide a crude product containing the title compound and the corresponding TBS-free analog (75.3 mg). This substance was used in the next step without purification.

UPLC-MS (Method B): t _R = 0.95 minutes, m/z = 548.4 (M+H ⁺ ).

Step 4

trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile

At 0°C, the crude product from step 3 was dissolved in TFA (0.25 mL) and the mixture was stirred at rt for 1.5 h. Volatiles were removed under vacuum, and the residue was dissolved in 4M hydrogen chloride (0.25 mL) in dioxane. The mixture was stirred at rt for 18 h, the volatiles were evaporated, and the residue was dissolved in dichloromethane (0.25 mL). 2M ammonia was added to methanol (0.60 mL) to adjust the pH to approximately 10. Volatiles were removed under vacuum, the residue was dissolved in DCM:MeOH 95:5 (2 mL), and the mixture was filtered. The filtrate was evaporated, and the residue was purified by chromatography (4 g pre-packed silica column, eluting with DCM:MeOH 96:4 to 94:6) to provide the title compound (37.7 mg) in a semi-solid state containing approximately 30% 4-methoxy-N-methylbenzylamine.

UPLC-MS (Method B): t _R = 0.38 minutes, m/z = 314.3 (M+H ⁺ ).

Analytical chiral stationary phase SFC: (S) enantiomer (minor) t _R = 5.37 min; (R) enantiomer (major) t _R = 5.78 min.

Example 3 (Alternative Preparation No. 2)

trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile (compound 3)

Step 1

2-[trans-4-[(2-chloro-5-nitropyridin-4-yl)amino]cyclohexyl]acetonitrile

A 20L glass reactor equipped with a mechanical stirrer, reflux condenser, and argon inlet was evacuated and purged twice with argon. 700g of 2,4-dichloro-5-nitropyridine (3.63mol, 1.0 equivalent), 665g of trans-4-(cyanomethyl)cyclohexylammonium hydrochloride (3.81mol, 1.05 equivalent), and 7.00L of 2-propanol were added to the reactor. The resulting slurry was stirred at 25°C, and 1.90L of diisopropylethylamine (1.41kg, 10.9mol, 3.0 equivalent) was added. The addition tube was washed with 0.100L of 2-propanol, and the slurry was heated to reflux (Tj setpoint = 95°C). The mixture was stirred under reflux for 15 hours and then cooled to 25°C. A conversion rate of 99% was observed in the process control (LCMS).

The product was separated by filtration and washed on the filter with 1.75 L of 2-propanol followed by 3.80 L of 2-propanol. The filter cake was transferred to a glass and dried under vacuum at 50 °C until constant weight was achieved; 1.00 kg (94%) of the title compound as a yellow solid was obtained; HPLC purity: 98.8% (area%).

Step 2

2-[trans-4-[(5-amino-2-chloropyridin-4-yl)amino]cyclohexyl]acetonitrile

A 2.0 L Parr shaker flask was purged with argon and filled with 5.00 g of 5% Pt/C (a paste with 50% water, 0.05 g/g, 1.3 mmol), 100 g of 2-[trans-4-[(2-chloro-5-nitropyridin-4-yl)amino]cyclohexyl]acetonitrile (339 mmol), and 1.00 L of ethanol. The Parr shaker flask was placed in the shaker and evacuated and refilled with argon twice. Next, the flask was evacuated and refilled with hydrogen. The pressure was adjusted to 1.5 bar and the shaker was started. Hydrogen was added a few more times, but hydrogen consumption was stopped after 2 hours. A conversion of >99% was shown in the process control (HPLC), and 600 mL of dichloromethane was added to prevent precipitation of the title compound. The resulting mixture was stirred for 10 minutes and filtered through diatomaceous earth. The filter cake was washed with 250 mL of dichloromethane, and the solvent was removed from the combined filtrate by evaporation under reduced pressure at 50 °C. The residue was transferred to a glass beaker and dried under vacuum at 50 °C until constant weight was achieved, yielding 85.1 g (95%) of the title compound as a brown solid; HPLC purity: 94%.

Step 3

2-[trans-4-[6-chloro-2-[(1R)-1-hydroxyethyl]-1H-imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile

A 20L glass reactor equipped with a mechanical stirrer, reflux condenser, and argon inlet was evacuated and purged twice with argon. 409g(R)-lactamide (4.59mol, 2.5 equivalents) and 4.90L tetrahydrofuran were charged into the reactor. The temperature was adjusted to 23°C, and 872g of triethyloxonium tetrafluoroborate (4.59mol, 2.5 equivalents) was added in a single batch. Note! The reaction is exothermic, and the temperature reached 43°C after the addition. The reaction mixture was cooled and stirred at 23°C for 90 minutes. The mixture was transferred to a 10L blue-capped flask and stored under argon. The reactor was rinsed with 2.7L ethanol, and 486g of 2-[trans-4-[(5-amino-2-chloropyridin-4-yl)amino]cyclohexyl]acetonitrile (1.84mol, 1.0 equivalent) and 7.3L ethanol were charged into the clean reactor. The temperature was adjusted to 23°C, and a tetrahydrofuran solution containing (R)-lactamide and triethyloxonium tetrafluoroborate was added over a period of approximately 2–4 minutes. The reaction mixture was heated to reflux (Tr = 70°C, Tj set point = 85°C). A white salt precipitate was observed. The reaction mixture was not clear and was orange. After 6 hours, a conversion of >95% was observed on process control (HPLC), and the reaction mixture was cooled to 23°C and stirred for another 16 hours. The mixture was filtered and the filter cake was washed with 2.0 L of tetrahydrofuran. The filtrate was transferred back to the reactor, and the solvent was removed by distillation under reduced pressure at 50°C. Distillation was stopped after 7 hours when condensation stopped at Tr = 31°C/17 mbar. The residual slurry was diluted with 7.3 L of methyl tert-butyl ether and 7.3 L of 10% sodium carbonate aqueous solution. The slurry was stirred at 23°C for 14 hours, after which the title compound was separated by filtration. The filter cake was washed with 5.0 L of water, dried, and transferred back to the reactor. Next, 5.0 L of water was added to the reactor and the resulting slurry was stirred at 23 °C for 60 minutes and then filtered. The filter cake was washed with 5.0 L of water and blotted dry. The resulting off-white solid was transferred to a glass and dried under vacuum at 50 °C until constant weight was achieved; the typical yield of the title compound as an off-white solid was 70-90%; the HPLC purity of the wet filter cake was 95% (area%).

Step 4

trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile

A 400 mL EasyMax glass reactor equipped with a mechanical stirrer, reflux condenser, and argon inlet was purged with argon. 5.43 g sodium tert-butoxide (56.5 mmol, 1.2 equivalents), 5.54 g phenol (58.8 mmol, 1.25 equivalents), and 195 mL tetrahydrofuran were charged into the reactor. The mixture was stirred at 25 °C for 20 minutes, after which 15.0 g of 2-[trans-4-[6-chloro-2-[(1R)-1-hydroxyethyl]-1H-imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile (47.1 mmol, 1.0 equivalents) was added. The feeding funnel was rinsed with 30 mL tetrahydrofuran and 94.1 mL of 2M methylamine (188 mmol, 4.0 equivalents) in tetrahydrofuran was added. The resulting mixture was heated to 55 °C (Tj setpoint = 55 °C). In a separate reaction flask, 0.210 g ^{of tBuBrettPhosPd} G3 (0.235 mmol, 0.005 equivalent) was dissolved in 4.0 mL of tetrahydrofuran. The ^{tBuBrettPhosPd} G3 solution was added to the reaction mixture at 55 °C. After 23 hours, the dark, homogeneous solution was cooled to 23 °C and transferred to a separatory funnel. The reaction mixture was washed with 2 × 225 mL of 2M sodium hydroxide aqueous solution. The organic phase was concentrated to approximately 50% of its volume by evaporation under reduced pressure at 50 °C and diluted with 125 mL of heptane.

Filtered through a silica stopper; 105 g of silica gel (7.0 g/g) was activated with 250 mL of heptane/ethyl acetate (1:1) and poured into a glass filter (8 cm diameter). The organic phase containing the crude title compound was slowly loaded onto the silica stopper. Impurities were eluted with 2 × 250 mL of heptane/ethyl acetate (1:1) followed by 250 mL of ethyl acetate. Next, the title compound was eluted with 5 × 200 mL of tetrahydrofuran. The fraction containing the title compound was collected and the solvent was removed by evaporation under reduced pressure, yielding 12.3 g (83%) of crude title compound as a brown solid; HPLC purity 95% %.

Pd removal: 3.00 g of crude title compound (9.57 mmol) and 45 mL of methanol were charged into a 100 mL flask. The mixture was stirred until all solids dissolved, and then 0.30 g of DMT (10% w/w dimercaptotriazine, 40-63 μm, 60A) was added. The slurry was heated to 50 °C and stirred for 4 hours, after which the mixture was cooled to 23 °C and filtered through diatomaceous earth. The filter cake was washed with 6.0 mL of methanol, and the solvent was removed by evaporation of the combined filtrate under reduced pressure, yielding 2.75 g (92% recovery) of the title compound.

Recrystallization: 3.00 g (9.57 mmol; HPLC purity 96 area%) of the title compound and 20 mL of ethyl acetate were packed into a 100 mL flask. The mixture was heated to reflux and stirred until all solids dissolved. The mixture was cooled and then 10 mL of methyl tert-butyl ether was added dropwise to the warm solution. The mixture was cooled to room temperature and stirred for 17 hours. The precipitate was separated by filtration, washed on the filter with 2 × 1.0 mL of methyl tert-butyl ether, and dried under vacuum at 50 °C until constant weight, yielding 1.81 g (60%) of the title compound as a grayish-white solid (crystallized, m.p. (DSC onset temperature) 143 ± 2 °C); HPLC purity 98 area%.

Example 4

trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(trideuteriummethylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile (compound 4)

Step 1

1,1,1-Trideuterium-N-[(4-methoxyphenyl)methyl]methylamine

Trideuterium methylamine hydrochloride (282 mg, 4.00 mmol) and TEA (0.558 mL, 4.00 mmol) were added to a mixture of 4-methoxybenzaldehyde (0.243 mL, 2.00 mmol) and anhydrous ethanol (3.0 mL) under rt and argon conditions. Tetraisopropoxytitanium (IV) was added to the suspension over 2 minutes with slight cooling. The reaction mixture was then stirred overnight under rt. After 17 hours under rt, _NaBH4 (113 mg, 3.00 mmol) was added. The suspension was then stirred for another 6.5 hours under rt, and 2M ammonia (6 mL) was carefully added under cooling. The solid fraction was removed by filtration and the filter cake was washed with dichloromethane (10 mL). The organic phase was separated. The filter cake was washed again with dichloromethane (10 mL). The combined filtrates were washed with water and then treated with 1M HCl aqueous solution (5 mL). The acidic aqueous phase was washed with dichloromethane (10 mL), and the pH was adjusted to approximately 11 by adding 2 M NaOH. The aqueous phase was then extracted with dichloromethane (3 × 10 mL). The combined organic phases were washed with brine, dried over sodium sulfate, and filtered. Evaporation under reduced pressure (60 mbar/35 °C) provided the title compound (268 mg, 83%) as a colorless liquid.

¹ H NMR (600MHz, CDCl ₃ ) δ7.25-7.21(m,2H), 6.89-6.84(m,2H), 3.80(s,3H), 3.68(s,2H).

Step 2

trans-2-[4-[2-[(1R)-1-[tert-butyl(dimethyl)silyl]oxyethyl]-6-[(4-methoxyphenyl)methyl-trideuterylmethyl-amino]imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile

A 4 mL screw-cap vial was filled with trans-2-[4-[2-[(1R)-1-[tert-butyl(dimethyl)silyl]oxyethyl]-6-chloro-imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile (150 mg, 0.346 mmol) and 1,1,1-trideuterium-N-[(4-methoxyphenyl)methyl]methylamine (107 mg, 0.693 mmol). The vial was purged with argon and a mixture of sodium tert-butoxide (40 mg, 0.416 mmol), RuPhos (9.7 mg, 0.021 mmol), and palladium(II) acetate (2.3 mg, 0.010 mmol) was added. The mixture was stirred under argon at 110 °C for 18 hours. It was cooled to rt and a 1.5 mL solution of dichloromethane/EtOH 95:5 was added. The mixture was washed with brine:water 2:1 (1.2 mL) and the aqueous layer was extracted with dichloromethane/EtOH 95:5 solution ( ₂ × 1.5 mL). The combined organic layers were dried over _Na₂SO₄ , filtered, and evaporated. Column chromatography (1% to 5% MeOH in DCM as eluent) yielded the title compound as a yellow foam and the corresponding TBS-free analogue (0.109 g). This material was used in the next step without further purification.

Step 3

trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(trideuteriummethylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile

The crude product from step 2 was dissolved in TFA (0.75 mL) at 0 °C, and the mixture was stirred at rt for 1.5 h. Volatiles were removed under vacuum, and the residue was dissolved in 4 M hydrogen chloride in dioxane (0.75 mL) at 0 °C. The mixture was stirred at rt for 17 h, and then volatiles were removed by evaporation. Water (0.5 mL) was added to the residue, and the aqueous phase was washed with EtOAc (2 × 0.5 mL). The organic phase was washed with water (0.5 mL). The pH of the combined aqueous phases was adjusted to approximately 11 with a saturated aqueous solution of sodium carbonate. The aqueous phase was then extracted with DCM:MeOH 95:5 (3 × 0.5 mL) _, and the combined organic phases were dried over _Na₂SO₄ , filtered, and evaporated. Column chromatography (3% to 5% MeOH in DCM as eluent) yielded the title compound (48 mg, 42%) as a grayish-white foam.

UPLC-MS (Method B): t _R = 0.38 minutes, m/z = 317.3 (M+H ⁺ ).

¹ H NMR (600MHz, DMSO-d ₆ ) δ8.33 (br s, 1H), 6.47 (br s,1H),5.87(s,1H),5.59(d,J＝6.8Hz,1H),4.96(p,J＝6.5Hz,1H),4.59-4.49(m,1H),2.55(d, J＝6.3Hz,2H),2.25-2.14(m,2H),1.96-1.80(m,5H),1.54(d,J＝6.5Hz,3H),1.35-1.24(m,2H).

Example 5

trans-2-[4-[2-[1,2,2,2-tetradeuter-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile (compound 5)

Step 1

trans-2-[4-(2-acetyl-6-chloro-imidazo[4,5-c]pyridin-1-yl)cyclohexyl]acetonitrile

DMP (10 g, 23.5 mmol) was added fractionally to a solution of 2-[trans-4-[6-chloro-2-[(1R)-1-hydroxyethyl]-1H-imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile (5.0 g, 15.6 mmol) in DCM (50 mL) at 0 °C to 5 °C to RT. The reaction mixture was stirred at RT for 16 hours. Upon completion, the reaction mixture was filtered through a diatomaceous earth bed and washed with DCM. The filtrate was washed with saturated _NaHCO3 (300 mL) and brine, _dried over anhydrous _Na2SO4 , concentrated under reduced pressure, and purified by silica gel (100-200 mesh) column chromatography (10% to 20% EtOAc in petroleum ether as eluent) to provide the title compound (3.5 g, 71%) as a grayish-white solid.

LC-MS (Method D): t _R = 1.84 minutes, m/z = 316.11 (M+H ⁺ ).

¹ H NMR (400MHz, CDCl ₃ )δ8.98(s,1H),8.21(s,1H),5.23-5.18(m,1H),2.75(s,3H),2.54(d,J＝6.5,2H ),2.31-2.23(m,2H),2.08-2.05(m,1H),1.94-1.89(m,4H),1.30-1.27(m,2H).

Step 2

trans-2-[4-[6-chloro-2-(2,2,2-trideuterylacetyl)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile

_K₂CO₃ (4.5 g, 33.2 mmol) was added to a suspension of trans-2-[4-(2-acetyl-6-chloro-imidazo[4,5-c]pyridin-1-yl)cyclohexyl]acetonitrile (3.5 g, 11.1 mmol) in _DMF (35 mL) at RT, and the mixture was stirred at the same temperature for 16 h. The reaction mixture was quenched with _D₂O (10 mL) and stirred at RT for 4 h, followed by the addition of ice-cold water. Ethyl acetate (70 mL) was added. The phases were separated. The aqueous phase was extracted with ethyl acetate (2 × ₃₅ mL) and the combined organic phases were washed with brine, dried over anhydrous _Na₂SO₄ , concentrated under reduced pressure, and purified by column chromatography using silica gel (100-200 mesh) (10-20% EtOAc in petroleum ether as eluent), yielding 2.5 g of crude product as a grayish-white solid. The material obtained was a mixture of isotopes. Based on ^1H NMR, the isotope distribution is as follows: undeuterated ( _CH3 ): 7.6%, monodeuterated ( _CH2D ): 26.33%, dideuterated ( _CHD2 ): 32.34%, and trideuterated ( _CD3 ): 33.72%.

The above procedure was repeated using the obtained crude product to provide 1.8 g of a new crude product as a grayish-white solid. Based on ^1H NMR, the isotopic distribution was as follows: undeuterated ( _CH3 ): 0.66%, monodeuterated ( _CH2D ): 4.47%, dideuterated ( _CHD2 ): 23.84%, and trideuterated ( _CD3 ): 71.02%.

The procedure was repeated using the crude product to yield 1.2 g (35% gross yield) of the title compound as a grayish-white solid. Isotopic purity: 99.3%. Based on ^1H NMR, the isotopic distribution was: undeuterated ( _CH₃ ): 0.66%, monodeuterated ( _CH₂D ): 3.47%, dideuterated ( _CHD₂ ): 21.85%, and trideuterated ( _CD₃ ): 74.02%.

LC-MS (Method E): t _R = 1.79 minutes, m/z = 320.19 (M+H ⁺ ).

¹ H NMR (400MHz, CDCl ₃ )δ8.98(s,1H),7.54(s,1H),5.40-5.45(m,1H),2.39(d,J＝6.5,2H),2.27 -2.219(m,2H),2.13-2.04(m,4H),1.96-1.90(m,1H),1.45-1.39(m,2H).

Step 3

trans-2-[4-[6-chloro-2-(1,2,2,2-tetradeuter-1-hydroxy-ethyl)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile

NaBD4 (0.29 g, 7.04 mmol) was added to a suspension of trans-2-[4-[6-chloro- _2- (2,2,2-trideuterylacetyl)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile (1.5 g, 4.69 mmol) in _CD3 OD (15 mL) at 0 °C to 5 °C. The reaction mixture was stirred at RT for 1 h. Upon completion, the reaction was quenched with _D2O and the mixture was concentrated under reduced pressure. The crude compound was dissolved in _H2O and extracted with 10% MeOH in DCM (2 × 15 mL). The combined organic layers were washed with brine, _dried over anhydrous _Na2SO4 , concentrated under reduced pressure, and purified by silica gel (100-200 mesh) column chromatography (1% to 3% MeOH in DCM as eluent) to provide the title compound (1.2 g, 80%) as a grayish-white solid. The obtained compound was a mixture of isotopes. Based on ^1H NMR, the isotopic distribution was as follows: monodeuterated ( _CDOHCH₃ ): 0.66%, dideuterated ( _CDOHCH₂D ): 3.47%, trideuterated (CDOHCHD₂): 21.85%, and _{tetradeuterated} ( _CDOHCD₃ ): 74.02%.

LC-MS (Method E): t _R = 1.48 minutes, m/z = 323.23 (M+H ⁺ ).

¹ H NMR (400MHz, CDCl ₃ )δ8.69(s,1H),8.01(s,1H),5.77(s,1H),4.68-4.63(m,1H),2.54(d,J＝6,2H) ,2.27-2.21(m,2H),2.10-2.08(m,1H),1.93-1.86(m,4H),1.32-1.26(m,2H).

Step 4

trans-2-[4-[2-[1,2,2,2-tetradeuter-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile

In a sealed tube, Cs₂CO₃ (0.302 g, 0.927 mmol) and Brettphos Pd G1 (0.037 g, 0.046 mmol) were added to a solution of trans-2-[ _4- [6-chloro- _2- (1,2,2,2-tetradeuter-1-hydroxyethyl)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile (0.10 g, 0.309 mmol) in degassed 1,4-dioxane (5.0 mL), and the mixture was purged with argon for 10 min. Then, 2M _CH₃NH₂ (0.60 mL, 1.24 mmol) was added to _THF . The reaction mixture was stirred at 80 °C for 16 h. Upon completion, the reaction mixture was cooled to RT, filtered through a diatomaceous earth pad, and washed with EtOAc. The filtrate was concentrated under reduced pressure. The resulting residue was dissolved in water and extracted twice with DCM (2 × 10 mL). The combined organic phases were washed with brine, dried over _{anhydrous Na₂SO₄} , and concentrated under reduced pressure. The crude product was purified by silica gel (100-200 mesh) column chromatography (3% MeOH in DCM as eluent) to provide the title compound (0.04 g, 41%) as a light brown solid.

LC-MS (Method E): t _R = 1.14 minutes, m/z = 318 (M + H ⁺ ).

Deuterium isotope distribution estimated in mole percent: _D0 , _D1 , _D2 , _D3 , _D4 = 0, 0, 3, 21, 76.

¹ H NMR (400MHz, DMSO-d ₆ ): δ(ppm)8.32(s,1H),6.47(s,1H),5.93-5.89(m,1H),5.58(s,1H),4.57-4.51(m,1H),2.78(d ,J＝5.2Hz,3H),2.55(d,J＝6.4Hz,2H),2.24-2.17(m,2H),1.94-1.86(m,5H),1.31-1.23(m,2H).

Example 6

cis-2-[4-[2-[1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile (compound 6)

Following a small-scale procedure similar to that outlined in “Alternative Preparation No. 2” of Example 3, cis-2-[4-[2-[1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile was obtained, with the only major difference being that in step 1, trans-4-(cyanomethyl)cyclohexyl]ammonium hydrochloride was replaced with cis-4-(cyanomethyl)cyclohexyl]ammonium hydrochloride (CAS Registry No. 1461718-40-0) and in step 3, (R)-lacticamide was replaced with lactamide.

UPLC-MS (Method C): t _R = 1.62 minutes, m/z = 314.4 (M+H ⁺ ).

¹ H NMR (400MHz, DMSO-d ₆ )δ8.34(s,1H),6.51(s,1H),5.97-5.88(m,1H),5.56(d,J＝6.8Hz,1H),4.95(p,J＝6.5Hz,1H),4. 60-4.46(m,1H),2.85-2.77(m,5H),2.27-2.08(m,3H),1.89-1.62(m,6H),1.54(d,J＝6.5Hz,3H).

Example 7

cis-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile (compound 7)

Following a small-scale procedure similar to that outlined in “Alternative Preparation No. 2” of Example 3, cis-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile was obtained, with the only major difference being that in the first step of the reaction sequence, trans-4-(cyanomethyl)cyclohexyl]ammonium hydrochloride was replaced with cis-4-(cyanomethyl)cyclohexyl]ammonium hydrochloride (CAS Registry No. 1461718-40-0).

UPLC-MS (Method C): t _R = 1.62 minutes, m/z = 314.4 (M+H ⁺ ).

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.35(s,1H),6.53(s,1H),5.98(br s,1H),5.58(d,J＝6.7Hz,1H),4.95(p,J＝6.5Hz,1H),4.60-4.46(m,1H),2.8 6-2.76(m,5H),2.27-2.07(m,3H),1.89-1.62(m,6H),1.54(d,J＝6.5Hz,3H).

Example 8

trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile malonate (compound 8)

At 45 °C, trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile (1.40 g, 4.46 mmol) was dissolved in isopropanol (50 mL). Malonic acid (232 mg, 2.23 mmol) was added to the isopropanol (5.0 mL). The volume of the reaction mixture was reduced by evaporation under reduced pressure at 45 °C (approximately 30 mL of isopropanol was distilled off). A small amount of reference crystals of the title compound was added, and crystallization began. The volume of the reaction mixture was further reduced by evaporation under reduced pressure (approximately 15 mL of isopropanol was distilled off). The resulting suspension was cooled in an ice bath, and shortly thereafter the solids were filtered off and washed with ice-cold isopropanol (3 × 2 mL). Drying under reduced pressure provided the title compound (932 mg, approximately 100%) as off-white crystals.

¹ H NMR (600MHz, DMSO-d ₆ ) δ8.37(s,1H),6.54(s,1H),6.10(br s,1H),5.66(br s,1H),4.98(q,J＝6.5Hz,1H),4.62-4.51(m,1H),3.17(s,2H),2.81(s,3H),2.55(d,J＝6. 3Hz,2H),2.25-2.14(m,2H),1.97-1.81(m,5H),1.55(d,J=6.5Hz,3H),1.36-1.24(m,2H).

M.p. (DSC starting temperature) 109±2℃.

Example 9

trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile glycolate (compound 9)

At 45 °C, trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile (1.80 g, 5.74 mmol) was dissolved in isopropanol (65 mL). Glycolic acid (437 mg, 5.74 mmol) was added to isopropanol (8.0 mL). The volume of the reaction mixture was reduced by evaporation under reduced pressure at 45 °C (approximately 65 mL of isopropanol was distilled off). A small amount of reference crystals of the title compound was added, and crystallization began slowly. EtOAc (15 mL) was added. The resulting suspension was cooled in an ice bath, and shortly thereafter the solids were filtered off and washed with an ice-cold 9:1 mixture of EtOAc and isopropanol (2 × 2 mL). Drying under reduced pressure provided the title compound (1.57 g, 70%) as off-white crystals.

¹ H NMR (600MHz, DMSO-d ₆ ) δ8.33(s,1H),6.47(s,1H),5.90(br s,1H),5.58(d,J＝6.6Hz,1H),4.96(p,J＝5.3Hz,1H),4.59-4.51(m,1H),3.90(s,2H),2.79(s,3H),2. 55(d,J＝6.2Hz,2H),2.25-2.14(m,2H),1.97-1.80(m,5H),1.54(d,J＝6.4Hz,3H),1.36-1.25(m,2H).

M.p. (DSC starting temperature) 100±2℃.

Example 10

trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile L-tartrate (compound 10)

At 45 °C, trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile (1.50 g, 4.79 mmol) was dissolved in methanol (25 mL). L-tartaric acid (360 mg, 2.40 mmol) was added to methanol (10 mL). The volume of the reaction mixture was reduced by evaporation under reduced pressure at 45 °C (approximately 10 mL of methanol was distilled off). A small amount of reference crystals of the title compound was added, and crystallization began. Isopropanol (30 mL) was added, and the volume of the reaction mixture was reduced by evaporation under reduced pressure at 45 °C (approximately 20 mL was distilled off). The resulting suspension was cooled in an ice bath, and shortly thereafter the solids were filtered off and washed with ice-cold isopropanol (4 × 4 mL). Drying under reduced pressure provided the title compound (1.55 g, 83%) as off-white crystals.

¹ H NMR (600MHz, DMSO-d ₆ ) δ8.34(s,1H),6.49(s,1H),5.98(br s,1H),5.60(br s,1H),4.96(q,J＝6.4Hz,1H),4.60-4.49(m,1H),4.28(s,1H),2.80(s,3H),2.55(d,J＝6. 3Hz, 2H), 2.26-2.13 (m, 2H), 1.97-1.80 (m, 5H), 1.54 (d, J = 6.5Hz, 3H), 1.36-1.25 (m, 2H).

M.p. (DSC starting temperature) 98±2℃.

Example 11

trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile L-malate (compound 11)

At 45 °C, trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile (1.50 g, 4.79 mmol) was dissolved in methanol (25 mL). L-malic acid (332 mg, 2.40 mmol) was added to methanol (10 mL). The volume of the reaction mixture was reduced by evaporation under reduced pressure at 45 °C (approximately 20 mL of methanol was distilled off). A small amount of reference crystals of the title compound was added, and crystallization began. Isopropanol (30 mL) was added, and the volume of the reaction mixture was reduced by evaporation under reduced pressure at 45 °C (approximately 10 mL was distilled off). The resulting suspension was cooled in an ice bath, and shortly thereafter the solid was filtered off and washed with ice-cold isopropanol (4 × 4 mL). Drying under reduced pressure provided the title compound (1.51 g, 79%) as off-white crystals.

¹ H NMR (600MHz, DMSO-d ₆ ) δ8.34(s,1H),6.49(s,1H),5.95(br s,1H),5.59(d,J＝6.7Hz,1H),4.96(p,J＝6.5Hz,1H),4.59-4.51(m,1H),4.23(dd,J＝7.5,5.3Hz,0.5H),2.79(s,3H),2.60(dd,J＝15.6,5.3Hz, 0.5H), 2.55 (d, J = 6.4Hz, 2H), 2.43 (dd, J = 15.6, 7.5Hz, 0.5H), 2.25-2.14 (m, 2H), 1.96-1.81 (m, 5H), 1.54 (d, J = 6.5Hz, 3H), 1.36-1.25 (m, 2H).

M.p. (DSC starting temperature) 94℃±2℃.

Example 12

trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile sulfate (compound 12)

At 45 °C, 1.50 g (4.79 mmol) of trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile (1.50 g, 4.79 mmol) was dissolved in methanol (10 mL). Sulfuric acid (1.0 M, 4.79 mL, 4.79 mmol) was added to isopropanol (5.0 mL). The volume of the reaction mixture was reduced by evaporation under reduced pressure at 45 °C (approximately 7 mL was distilled off). A small amount of reference crystals of the title compound was added, and crystallization began. The resulting suspension was cooled in an ice bath, and shortly thereafter the solid was filtered off and washed with ice-cold isopropanol (2 × 2 mL). Drying under reduced pressure yielded the title compound (1.57 g) as off-white crystals.

¹ H NMR (600MHz, DMSO-d ₆ ) δ13.32(br s,1H),8.61(s,1H),7.49(br s,1H),6.95(s,1H),5.91(br s,1H),5.07(q,J＝6.5Hz,1H),4.67-4.58(m,1H),2.96(s,3H),2.56(d,J＝6.0Hz,2H ),2.26-2.14(m,2H),1.99-1.87(m,5H),1.57(d,J＝6.5Hz,3H),1.38-1.27(m,2H).

M.p. (DSC starting temperature) 169±2℃.

Example 13

trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile hydrochloride (compound 13)

At 45 °C, trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile (2.00 g, 6.38 mmol) was dissolved in isopropanol (70 mL). Methanol hydrochloric acid (3.0 M, 6.38 mL, 19.1 mmol) was added under reduced pressure. The volume of the reaction mixture was reduced by evaporation at 45 °C under reduced pressure (approximately 45 mL distilled off). A small amount of reference crystals of the title compound was added, and crystallization began. The volume of the reaction mixture was further reduced by evaporation under reduced pressure (approximately 5 mL distilled off). The resulting suspension was cooled in an ice bath, and shortly thereafter, the solids were filtered off and washed with ice-cold isopropanol (4 × 4 mL). The title compound (1.30 g) was dried under reduced pressure as off-white crystals.

¹ H NMR(600MHz, DMSO-d ₆ )δ14.06(br s,1H),8.60(s,1H),7.84(br s,1H),7.02(s,1H),6.16(br s,1H),5.07(q,J＝6.5Hz,1H),4.69-4.60(m,1H),2.98(s,3H),2.56(d,J＝6.0Hz,2H ),2.27-2.16(m,2H),2.01-1.86(m,5H),1.57(d,J=6.5Hz,3H),1.39-1.28(m,2H).

M.p. (DSC starting temperature) 148±2℃.

Example 14

trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile succinate (compound 14)

At approximately 50 °C, trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile (31.3 mg, 0.100 mmol) was dissolved in ethanol (0.20 mL). Succinic acid (11.8 mg, 0.100 mmol) was added to ethanol (0.25 mL). The volume of the reaction mixture was reduced by evaporation under reduced pressure at 45 °C (approximately 0.14 mL of ethanol was distilled off). After crystallization, ethanol (0.10 mL) was added. The resulting suspension was cooled in an ice bath and filtered shortly thereafter, providing the title compound (16 mg, 33%) as off-white crystals.

¹ H NMR (600MHz, DMSO-d ₆ ) δ12.16(br s,3H),8.33(s,1H),6.48(s,1H),5.91(br s,1H),5.59(d,J＝6.7Hz,1H),4.96(p,J＝6.5Hz,1H),4.59-4.50(m,1H),2.79(s,3H),2.55(d,J＝6.3H z, 2H), 2.42 (s, 6H), 2.25-2.14 (m, 2H), 1.97-1.80 (m, 5H), 1.54 (d, J = 6.5Hz, 3H), 1.35-1.24 (m, 2H).

M.p. (DSC starting temperature) 162±2℃.

Example 15

trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile oxalate (compound 15)

At approximately 50 °C, trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile (31.3 mg, 0.100 mmol) was dissolved in ethanol (0.20 mL). Oxalic acid (4.5 mg, 0.050 mmol) was added to ethanol (0.25 mL). The reaction mixture was cooled in an ice bath and then filtered shortly thereafter, yielding the title compound (27 mg) as off-white crystals.

¹ H NMR (600MHz, DMSO-d ₆ )δ8.39(s,1H),6.57(s,1H),4.98(q,J＝6.5Hz,1H),4.60-4.52(m,1H),2.83(s,3H),2.55(d,J ＝6.3Hz,2H),2.25-2.14(m,2H),1.97-1.82(m,5H),1.55(d,J＝6.4Hz,3H),1.36-1.25(m,2H).

M.p. (DSC starting temperature) 134±2℃.

Example 16

trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile fumarate (compound 16)

Trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile (3.00 g, 9.57 mmol) was dissolved in ethanol (6.0 mL). Fumaric acid (1.11 g, 9.57 mmol), dissolved in ethanol (12 mL) at about 50 °C, was slowly added. Crystallization occurred. The resulting suspension was brought to rt, and the solid was filtered off shortly thereafter and washed with ethanol (2 × 0.5 mL). Drying under reduced pressure provided the title compound (3.26 g, 79%) as off-white crystals.

1H NMR (600MHz, DMSO-d ₆ )δ8.34(s,1H),6.63(s,2H),6.49(s,1H),4.97(q,J＝6.5Hz,1H),4.60-4.51(m,1H),2.80(s,3H),2. 55(d,J＝6.3Hz,2H),2.26-2.14(m,2H),1.97-1.80(m,5H),1.54(d,J＝6.5Hz,3H),1.35-1.24(m,2H).

M.p. (DSC starting temperature) 111±2℃.

Example 17

trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile 1,5-naphthalenedisulfonate (compound 17)

At approximately 50 °C, trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile (11.8 mg, 0.037 mmol) was dissolved in ethyl acetate (1 mL). 1,5-Naphthalenedisulfonic acid (tetrahydrate) (15.0 mg, 0.042 mmol) was added to _H₂O (150 μL). The solution was stirred very slowly on a magnetic stirrer for approximately 3 days, after which grayish-white crystals were separated by filtration.

¹ H NMR (600MHz, DMSO-d ₆ ) δ8.88 (d, J = 8.5 Hz, 2H), 8.60 (s, 1H), 7.95 (dd, J = 7.1, 1.2 Hz, 2H), 7.54 (br s,1H),7.42(dd,J＝8.5,7.1Hz,2H),6.97(s,1H),5.06(q,J＝6.5Hz,1H),4.63-4.56(m,3H),2.95(s,3H) ,2.51(d,J＝5.4Hz,2H),2.28-2.06(m,2H),1.95-1.72(m,5H),1.56(d,J＝6.5Hz,3H),1.38-1.18(m,2H).

M.p. (DSC starting temperature) 110±2℃.

Example 18

trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile DL-amyginate (compound 18)

At approximately 50 °C, trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile (9.92 mg, 0.032 mmol) and DL-mandelic acid (5.3 mg, 0.035 mmol) were dissolved in ethyl acetate (1 mL). The solution was stirred very slowly on a magnetic stirrer for approximately 3 days, after which grayish-white crystals were separated by filtration.

¹ H NMR (600MHz, DMSO-d ₆ ) δ8.33 (d, J = 0.9 Hz, 1H), 7.45-7.37 (m, 2H), 7.37-7.31 (m, 2H), 7.31-7.25 (m, 1H), 6.48 (d, J = 1.0Hz, 1H), 5.92 (br s,1H),5.59(br s,1H),5.00(s,1H),4.96(q,J＝6.8Hz,1H),4.63-4.47(m,1H),2.79(s,3H),2.55(d,J＝6. 4Hz, 2H), 2.27-2.11 (m, 2H), 1.97-1.75 (m, 5H), 1.54 (d, J = 6.5Hz, 3H), 1.38-1.21 (m, 2H).

M.p. (DSC starting temperature) 153±2℃.

Example 19

trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile dioxane solvate (compound 19)

A suspension of approximately 15 mg of trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile in 0.4 mL of a 1,4-dioxane:heptane (1:1) mixture was prepared in a 2.5 mL vial fitted with a small magnetic rod. The vial was placed on a magnetic stirrer and stirred for two weeks at approximately 600 rpm under normal temperature and humidity. The solid material was separated by filtration, dried, and then the melting point was determined.

M.p. (DSC starting temperature) 77±2℃.

JAK kinase analysis

Janus kinases (JAK) 1, 2, 3, and tyrosine kinase (TYK) 2 expressed by human baculovirus were purchased from Carna Biosciences, Inc. (product numbers 08-144, -045, -046, and -147, respectively). All four purified enzymes contained only the catalytic domain. JAK1 (aa 850-1154) and TYK2 (aa 871-1187) were expressed with an N-terminal fused GST-tag, while JAK2 and JAK3 had an N-terminal fused His-tag. Phosphorylation inhibition of the synthetic peptides was measured in an HTRF-based assay (CisBio product number 62TK0PEC). First, 75 nL of the test compound solution (100% DMSO) was added to a white, shallow 384-well plate (NUNC product number 264706) using a Labcyte ECHO 550 liquid dispensing apparatus. Next, add 1 μL of compound dilution buffer (50 mM HEPES, 0.05% bovine serum albumin) and 2 μL of TK solution (TK substrate-biotin in kinase buffer [1× enzyme buffer, 1 mM DTT from the HTRFKinEASE TK kit]). Then, add 5 μL of kinase-ATP mixture (prepared in kinase buffer) to the wells and incubate the plates at RT for 20 min (JAK2, JAK3, and TYK2) to 40 min (JAK1). For all four kinases, use the ATP concentration corresponding to K _{_m} of ATP. The final concentrations of buffer, substrate, kinase, and ATP were as follows: JAK1: 50 mM Hepes buffer (pH 7.0), 0.01% BSA, ₁₀ mM MgCl2, 1 mM DTT, 7 μM ATP, 50 nM SEB, 1 μM TK substrate-biotin, and 5 ng/well JAK1; JAK2: 50 mM Hepes buffer (pH 7.0), 0.01% BSA, 5 mM MgCl2, ₁ mM DTT, 4 μM ATP, 1 μM TK substrate-biotin, and 0.1 ng/well JAK2; JAK3: 50 mM Hepes buffer (pH 7.0), 0.01% BSA, 5 mM MgCl2, ₁ mM DTT, 2 μM ATP, 1 μM TK substrate-biotin, and 0.3 ng/well JAK3; TYK2: 50 mM Hepes buffer (pH 7.0), 0.01% BSA, 10 mM MgCl2, 1 mM DTT, 2 μM ATP, 1 μM TK substrate-biotin, and 0.3 ng/well JAK3; The following solutions were prepared: 7.0), 0.01% BSA, 5mM _MgCl2 , 1mM DTT, 13μM ATP, 50nM SEB, 1μM TK substrate-biotin, and 0.8 ng/well TYK2. The kinase reaction was then stopped by adding 4 μL of the assay mixture (final concentration: 50mM Hepes buffer (pH 7.0), 0.01% BSA, 0.8M KF, 20mM EDTA, 42nM streptavidin-XL665, and 1:400 STK AbCryptate) and the plates were incubated overnight in the dark. The HTRF signal was quantified using a PerkinElmer Envision reader with the following filters: 320nm excitation filter, 665nm emission filter, and 615nm second emission filter. The ratio for each well was calculated ((665/615) × ^10⁴ ).

STAT6 analysis

25 μL of STAT6bla-RA1 (Invitrogen ID K1243) cell suspension was seeded at a density of 30–40,000 cells/well in analytical medium (Opti-MEM (Invitrogen ID 11058-021) + 0.5% heat-inactivated fetal bovine serum (Invitrogen ID 10082-147) + 1% non-essential amino acids (Invitrogen ID 11140-050) + 1% sodium pyruvate (Invitrogen ID 11360-070) + 1% penicillin/streptomycin (Invitrogen ID 15140-122) containing 550 ng/mL CD40 ligand (Invitrogen ID PHP0025) in 384-well Black View plates (PerkinElmer ID 6007460) with clear bottoms. Cell culture plates were incubated overnight in a humidified 37°C air/ _CO2 (95%/5%) incubator. The following day, 125 nL of solutions of the test and reference compounds were transferred to the cell culture plates using a Labcyte Echo 550 liquid handler. The plates were then incubated for 1 hour in a humidified 37°C air/ _CO2 (95%/5%) incubator. Subsequently, recombinant human interleukin-4 (Invitrogen ID: PHC0045) was added to the plates using a Labcyte Echo 550 until a final concentration of 10 ng/mL was achieved. The cells were then incubated for 4 1/2–5 hours in a humidified 37°C air/ _CO2 (95%/5%) incubator. Then, 8 μL of LiveBLAzer substrate mixture (Invitrogen ID: K1095) was added to the analysis plate, and the plate was incubated overnight at RT. Fluorescence was then measured: excitation: 405 nm; emission: 460 nm (green channel); emission: 535 nm (blue channel). Subtract the background from the two emission channels and calculate the ratio of 460/535nm for each aperture.

STAT5 analysis

25 μL of STAT5irf1-bla TF1 (Invitrogen ID K1219) cell suspension was seeded at a density of approximately 10,000 cells/well in analytical medium (Opti-MEM (Invitrogen ID 11058-021) + 0.5% heat-inactivated fetal bovine serum (Invitrogen ID 10082-147) + 1% non-essential amino acids (Invitrogen ID 11140-050) + 1% sodium pyruvate (Invitrogen ID 11360-070) + 1% penicillin/streptomycin (Invitrogen ID 15140-122)) in 384-well Black View plates (PerkinElmer ID 6007460) with clear bottoms. The cell plates were incubated overnight in a humidified 37°C air/ _CO2 (95%/5%) incubator. The following day, 125 nL of solutions containing the test and reference compounds were transferred to cell culture plates using a Labcyte Echo 550 liquid handler. The plates were then incubated for 1 hour in a humidified 37°C air/ _CO2 (95%/5%) incubator. Subsequently, recombinant human erythropoietin (EPO) (Invitrogen catalog number PHC9634) was added to the plates using a Labcyte Echo 550 until a final concentration of 10 ng/mL was achieved. Cells were then incubated for 4 1/2–5 hours in a humidified 37°C air/ _CO2 (95%/5%) incubator. 8 μL of LiveBLAzer substrate mixture (Invitrogen catalog number K1095) was then added to the analysis plate, and the plate was incubated overnight at RT. Fluorescence was then measured: excitation: 405 nm; emission: 460 nm (green channel), emission: 535 nm (blue channel). Background was subtracted from the two emission channels, and the ratio of 460/535 nm per well was calculated.

The compounds of the present invention were tested in JAK1, JAK2, JAK3 and TYK2 kinase assays and STAT6 and STAT5 assays. The results are disclosed in Table 1.

Table 1

*Examples 641 and 644 of WO2011086053 are prepared according to WO2011086053.

JAK1 selectivity relative to JAK2 or JAK3 is calculated as the JAK2:JAK1 or JAK3:JAK1 ratio at their respective _EC50 . Selectivity relative to STAT5 for STAT6 is calculated similarly. As can be seen from Table 1, the compounds of the present invention exhibit high selectivity for JAK1 inhibition relative to JAK2 and JAK3; and high selectivity for STAT6 inhibition (reflecting JAK1 inhibition) relative to STAT5 (reflecting JAK2 inhibition).

Kinase selectivity

The kinase selectivity profiles of Examples 3 and 644 and 641 of WO2011086053 were evaluated by CARNA Biosciences Inc. using a group consisting of: 23 tyrosine kinases (including JAK1 (ABL, CSK, EGFR, EPHA2, EPHB1, EPHB4, FGFR1, FLT3, IGF1R, ITK, JAK1, JAK3, KDR, LCK, MET, PDGFRα, PDGFRβ, PYK2, SRC, TIE2, TRKA, and TYRO3)) and 68 serine and threonine kinases (LCK, MET, AKT1, AMPKα). 1/β1/γ1, AurA, AurB, AurC, BRSK2 ([ATP] = Km value), CaMK1α ([ATP] = Km value), CaMK2α ([ATP] = Km value), CaMK4, CDC2/CycB1, CDC7/ASK, CDK2/CycA2, CDK2/CyE1, CDK3/CyE1 ([ATP] = Km value), CDK4/CyD3, CDK6/CyD3, CDK7/CycH/MAT1, CD K9/CycT1, CHK1, CK1ε, CK2α1/β, CK2α2/β, CLK1, CLK2, DAPK1, DYRK1B, Erk2, GSK3α, GSK3β, HGK, IKKβ, IRAK4( [ATP]=Km value), JNK2, LOK ([ATP]=Km value), MAPKAP2, MLK1, MLK2, MNK2 ([ATP]=Km value), MST1, MST2 ([ATP]=Km value), NEK1, NEK2, NEK6, NEK7, NEK9, p38α, p70S6K, PAK1 ([ATP] = Km value), PAK2, PAK5 ([ATP] = Km value), PBK, PDK1, PIM1, PIM2, PKACα, PKCα, OKD2, PKN1 ([ATP] = Km value), PLK1, PLK2 ([ATP] = Km value), ROCK1, RSK1, SGK, skMLCK ([ATP] = Km value), and TSSK1). Assessments were typically performed at 1 mM ATP concentration; however, for some kinases, ATP concentrations close to the corresponding Km value were used (this is stated in parentheses for each relevant kinase). Inhibition percentages were measured at approximately 1000 times the inhibitor concentration of JAK1 _EC50 . Results are summarized in Table 2.

Table 2

As shown in Table 2, Example 3 of the present invention demonstrates an extremely high degree of selectivity for a large group of kinases; that is, no tested kinase other than JAK1 was inhibited by more than 50%.

Examples 644 and 641 of WO2011086053 inhibit 50% or more of 9 other kinases besides JAK1.

Inhibiting off-target kinases increases the risk of adverse effects; that is, high kinase selectivity can reduce the risk of adverse effects.

CYP inhibition and CYP induction

Reversible CYP inhibition, time-dependent CYP inhibition (TDI), and CYP induction were tested on Cyprotex PLCs according to authoritative guidelines.

For reversible CYP inhibition, IC50 was measured at substrate concentrations up to 50 μM for Example 3 and up to 25 μM for Examples 644 and 641 of WO2011086053.

The results of reversible CYP inhibition are summarized in Table 3.

Table 3

ND = Not measured

As can be seen from Table 3, Example 3 showed no CYP inhibition when measured at substrate concentrations up to 50 μM.

Examples 644 and 641 of WO2011086053 indicate weak CYP3A4 inhibition.

Example 3 indicates time-independent CYP inhibition.

CYP3A4 inhibition can indicate undesirable drug-drug interactions. CYP inhibition can affect plasma levels and increase exposure to co-administered drugs, potentially leading to adverse drug responses or toxicity.

The induction of CYP1A2, CYP2B6, and CYP3A4 gene expression can be used as a sensitive representative endpoint for the activation of aryl hydrocarbon receptor (AhR), pregnane X receptor (PXR), and constitutive androstenedione receptor (CAR), respectively. The induction of these nuclear receptors was evaluated by measuring the increase in the mRNA encoding of AhR, CAR, and PXR at relevant concentrations.

A shift of more than 2 times indicates CYP induction.

The results from the three highest cultivation concentrations are summarized in Table 4.

Table 4: CYP-induced mean fold change compared to solvent

As can be seen from Table 4, Example 3 did not induce CYP3A4 at a concentration of at least 50 μM or CYP1A2 and CYP2B6 at a concentration of at least 10 μM.

Example 644 of WO2011086053 induces CYP3A4 at a concentration of 10 μM and CYP2B6 at a concentration of 10 μM, while Example 641 of WO2011086053 induces CYP3A4 at a concentration of 20 μM and above.

CYP3A4 induction can indicate undesirable drug-drug interactions. CYP induction can reduce exposure to co-administered drugs, leading to decreased efficacy. Additionally, CYP induction can also cause toxicity by increasing the formation of reactive metabolites.

Water solubility of crystalline materials

The water solubility of Crystallization Example 3, Examples 644 and 641 of WO2011086053 at different pH values was evaluated. The solubility (mg/mL) is summarized in Table 5:

Table 5

Generally, water solubility is a key aspect affecting the development of oral formulations, and oral bioavailability is highly dependent on drug solubility. High solubility drives rapid dissolution of compounds in the gamma tract, and the resulting high concentrations drive absorption across the intestinal epithelium. Therefore, high solubility can significantly increase the likelihood of achieving high oral bioavailability and desired systemic exposure at relevant doses.

Claims (14)

1. Compounds of general formula (I), in A represents _C6 -cycloalkyl, wherein the _C6 -cycloalkyl is optionally substituted with one or more deuterium atoms; _R1 represents a _C1 -alkyl group, wherein the _C1 -alkyl group is optionally substituted with one or more deuterium groups; _R2 represents a _C1 -alkyl group, wherein the _C1 -alkyl group is substituted with a substituent selected from _R6 ; and wherein the _C1 -alkyl group is optionally substituted with one or more deuterium groups; _R3 represents a _C2 -alkyl group, wherein the _C2 -alkyl group is substituted with a substituent selected from _R7 , and wherein the _C2 -alkyl group is optionally substituted with one or more deuterium groups; R ₄ represents hydrogen or deuterium; R ₅ represents hydrogen or deuterium; R ₆ represents cyano; R ₇ represents a hydroxyl group; Or its pharmaceutically acceptable salt, hydrate or solvate. 2. The compound according to claim 1, wherein formula (I) is general formula (Ia). Wherein _R1 - _R2 and _R4 - _R7 are as defined in claim 1, and wherein Ra, Rb, Rc and Rd are each independently selected from hydrogen and deuterium. 3. The compound according to claim 1 or 2, wherein formula (I) is general formula (Ib). Wherein _R1 - _R2 , _R4 - _R7 , Ra, Rb, Rc and Rd are as defined in claim 1 or 2. 4. The compound according to claim 1, wherein the compound is selected from... trans-2-[4-[2-[1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile, trans-2-[4-[2-[(1S)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile, trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile, trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(trideuteriummethylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile, trans-2-[4-[2-[1,2,2,2-tetradeuter-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile, cis-2-[4-[2-[1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile and cis-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile Or its pharmaceutically acceptable salt, hydrate or solvate. 5. The compound according to claim 1, wherein the compound is trans-2-[4-[2-[(1R)-1-hydroxyethyl]-6-(methylamino)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile Or its pharmaceutically acceptable salt. 6. Use of the compound according to any one of claims 1 to 5 in the preparation of a medicament for the prevention and/or treatment of immune system diseases or diseases related to immune system disorders. 7. The use according to claim 6, wherein the immune system disease is an autoimmune disease. 8. According to claim 6, the immune system disease is atopic dermatitis. 9. A pharmaceutical composition comprising a compound according to any one of claims 1 to 5 and a pharmaceutically acceptable solvent or excipient or a pharmaceutically acceptable carrier. 10. Use of the compound according to any one of claims 1 to 5 in the preparation of a treatment for a disease in which the disease responds to an inhibitory response of JAK1 kinase activity. 11. Use of the compound according to any one of claims 1 to 5 in a medicament for the preparation of a method for preventing, treating or improving immune system diseases, the method comprising administering to a person suffering from at least one of said diseases an effective amount of one or more of the compounds according to any one of claims 1 to 5, optionally and pharmaceutically acceptable carriers or one or more excipients. 12. The immune system disease according to claim 11 is an autoimmune disease. 13. A compound selected from: 2-[trans-4-[(5-amino-2-chloropyridin-4-yl)amino]cyclohexyl]acetonitrile Or its salt. 14. A compound selected from: 2-[trans-4-[6-chloro-2-(1-hydroxyethyl)-1H-imidazo[4,5-c]pyridin-1-yl]cyclohexyl]-acetonitrile, 2-[trans-4-[6-chloro-2-[(1R)-1-hydroxyethyl]-1H-imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile and trans-2-[4-[6-chloro-2-(1,2,2,2-tetradeuter-1-hydroxy-ethyl)imidazo[4,5-c]pyridin-1-yl]cyclohexyl]acetonitrile Or its salt.