KR102511209B1 - Benzimidazole derivatives and pharmaceutical compositions thereof for the treatment of inflammatory disorders - Google Patents

Abstract

상기 식에서, Cy, R¹, L₁, R³, R⁴, R⁵, L_a 및 R^a는 본 발명에 정의된 바와 같다. JAK를 억제할 수 있는, 화학식 I에 따른 신규의 벤즈이미다졸을 개시하며, 상기 화합물을 약학 조성물로서 제조할 수 있고, 인간을 포함한 포유동물에서 다양한 상태, 예를 들어 비제한적인 예로서 알러지성 질병, 염증 질병, 자가면역 질병, 증식성 질병, 이식 거부, 연골 턴오버의 장애를 수반하는 질병, 선천성 연골 기형, 및/또는 IL6의 분비과잉 또는 인터페론의 분비과잉과 관련된 질병의 예방 및 치료에 사용할 수 있다.The present invention discloses compounds according to Formula I:
Formula I

In the above formula, Cy, R ¹ , L ₁ , R ³ , R ⁴ , R ⁵ , L _a and R ^a are as defined herein. Disclosed are novel benzimidazoles according to Formula I, which are capable of inhibiting JAK, which compounds can be prepared as pharmaceutical compositions, which can treat various conditions in mammals, including humans, such as allergic as a non-limiting example. For the prevention and treatment of diseases, inflammatory diseases, autoimmune diseases, proliferative diseases, graft rejection, diseases involving disorders of cartilage turnover, congenital cartilage malformations, and/or diseases associated with hypersecretion of IL6 or hypersecretion of interferon. can be used

Description

Benzimidazole derivatives and their pharmaceutical compositions for the treatment of inflammatory diseases

The present invention relates to allergic diseases, inflammatory diseases, autoimmune diseases, proliferative diseases, transplant rejection, diseases involving impaired cartilage turnover, congenital cartilage malformations, and/or diseases associated with hypersecretion of IL6 or hypersecretion of interferon. It relates to compounds that are inhibitors of the tyrosine kinase family, JAK. In particular, the compounds of the present invention inhibit JAK1 and/or TYK2. The present invention also provides methods for preparing the compounds of the present invention, pharmaceutical compositions containing the compounds of the present invention, allergic diseases, inflammatory diseases, autoimmune diseases, proliferative diseases, transplant rejection, cartilage turn by administering the compounds of the present invention. Provided is a method for preventing and/or treating a disease accompanying an over's disorder, a congenital cartilage deformity, and/or a disease associated with hypersecretion of IL6 or hypersecretion of interferon.

Janus kinase (JAK) is a cytoplasmic tyrosine kinase that transmits cytokine signals from membrane receptors to STAT transcription factors. Four JAK family members have been disclosed, JAK1, JAK2, JAK3 and TYK2. When the cytokine binds to its receptor, JAK and its members autophosphorylate and/or transphosphorylate each other, then phosphorylate STATs, which then translocate to the nucleus to regulate transcription. JAK-STAT intracellular signaling provides interferons, most interleukins, as well as various cytokines and endocrine factors such as EPO, TPO, GH, OSM, LIF, CNTF, GM-CSF and PRL (Vainchenker W. et al. al. (2008)).

Combination studies of genetic models and small molecule JAK inhibitors have revealed the therapeutic potential of a number of JAKs. JAK3 has been identified by mouse and human genetics as an immunosuppressive target (O'Shea et al., 2004). JAK3 inhibitors have been successfully clinically developed, initially for organ transplant rejection, but later also for other immune-inflammatory indications, such as rheumatoid arthritis (RA), psoriasis and Crohn's disease (http://clinicaltrials. .gov/).

TYK2 is a potential target for immuno-inflammatory diseases and has been confirmed by human genetics and mouse knock-out studies (Levy and Loomis, 2007).

JAK1 is a target in the field of immune-inflammatory diseases. JAK1 heterodimerizes with other JAKs to transmit cytokine-driven pro-inflammatory signals. Thus, inhibition of JAK1 is associated with pathology-associated cytokines that utilize JAK1 signaling, such as IL-6, IL-4, IL-5, IL-13, or IFNgamma, as well as immune-inflammatory diseases with JAK-mediated signaling. It is important for other diseases driven by transmission.

Degeneration of cartilage is a hallmark of various diseases, of which rheumatoid arthritis and osteoarthritis are the most prominent. Rheumatoid arthritis (RA) is a chronic joint degenerative disease, characterized by inflammation and destruction of joint structures. If the disease is not managed, it leads to substantial disability and pain and even premature death due to loss of joint function. Thus, the goal of RA therapy is to obtain remission in order to stop joint destruction as well as to slow the disease. In addition to the severity of these disease outcomes, the high prevalence of RA (affecting approximately 0.8% of adults worldwide) implies a high socio-economic impact (Choy and Panayi, 2001; Firestein, 2003; Lee and Weinblatt, 2001; O? Dell, 2004; Smolen and Steiner, 2003).

JAK1 and JAK2 are involved in intracellular signaling for a number of cytokines and hormones. Pathologies associated with any of these cytokines and hormones can be ameliorated by JAK1 and JAK2 inhibitors. Thus, a number of allergic or inflammatory conditions and autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, juvenile idiopathic arthritis, osteoarthritis, asthma, chronic obstructive pulmonary disease (COPD), tissue fibrosis, eosinophilic inflammation, esophagitis , inflammatory bowel disease (e.g. Crohn's disease, ulcerative colitis), transplantation, graft-versus-host disease, psoriasis, myositis, multiple sclerosis may benefit from treatment with the compounds disclosed herein (Kopf et al., 2010 ).

Osteoarthritis (also referred to as OA, or abrasive arthritis) is the most common form of arthritis and is characterized by loss of articular cartilage, often associated with bone enlargement and pain (Wieland et al., 2005).

Osteoarthritis is difficult to treat. Currently, a cure cannot be obtained and treatment is focused on relieving pain and preventing deformity of the diseased joint. Conventional treatment involves the use of non-steroidal anti-inflammatory drugs (NSAIDs). Although dietary supplements such as chondroitin and glucosamine sulfate have been claimed as safe and effective treatment options for osteoarthritis, recent clinical trials have shown that neither treatment is capable of reducing pain associated with osteoarthritis (Clegg et al. , 2006).

Stimulation of anabolic processes, blocking of catabolic processes, or a combination of the two may result in stabilization of the cartilage, and possibly even reversal of the damage, thus preventing further progression of the disease. Treatment methods for the correction of articular cartilage lesions appearing in the above osteoarthritis have been developed, but none of them have been able to mediate the regeneration of articular cartilage in situ and in vivo. Taken together, disease modifying osteoarthritis drugs are not available.

Vandeghinste et al. (Vandeghinste et al., 2005) discovered JAK1 as a target whose inhibition may have therapeutic relevance in a number of diseases, including OA. Knockout of the JAK1 gene in mice demonstrated that JAK1 plays an essential and not ineffective role during development: JAK1-/- mice died within 24 hours of birth and lymphocyte development was severely impaired. Moreover, JAK1 −/− cells were not or less responsive to cytokines using class II cytokine receptors, cytokine receptors using the gamma-c subunit for signaling and cytokine receptors using the gp130 subunit for signaling (Rodig et al., 1998).

Various groups have been implicated in JAK-STAT signaling in chondrocyte biology. Li et al. (Li et al., 2001) showed that oncostatin M induces MMP and TIMP3 gene expression by activating the JAK/STAT and MAPK signaling pathways in primary chondrocytes. Osaki et al. (2003) showed that interferon-gamma mediated inhibition of collagen II in chondrocytes involved JAK-STAT signaling. IL1-beta induces cartilage catabolism by reducing the expression of matrix components and inducing the expression of collagenase and inducible nitric oxide synthase (NOS2) (which mediates the production of nitric oxide). Otero et al. (2005) showed that leptin and IL1-beta synergistically induce NO production or NOS2 mRNA expression in chondrocytes and were blocked by JAK inhibitors. Legendre et al. (2003) reported that the IL6/IL6 receptor induces downregulation of the cartilage-specific matrix genes collagen II, aggrecan core and link protein in bovine articular chondrocytes, resulting in JAK/ It was shown to be mediated by STAT signaling. Thus, these observations suggest a role for JAK kinase activity in cartilage homeostasis and therapeutic opportunities for JAK kinase inhibitors.

JAK family members have been implicated in additional conditions including myeloproliferative disorders (O'Sullivan et al, 2007) (where mutations in JAK2 were identified). This indicates that inhibitors of JAK, particularly JAK2, may also be used for the treatment of myeloproliferative disorders. In addition, the JAK family, particularly JAK1, JAK2, and JAK3, is associated with cancer, particularly leukemia, such as acute myelogenous leukemia (O'Sullivan et al, 2007; Xiang et al., 2008) and acute lymphoblastic leukemia (Mullighan et al. , 2009), or solid tumors such as uterine leiomyosarcoma (Constantinescu et al., 2008), prostate cancer (Tam et al., 2007). These results indicate that inhibitors of JAK, particularly JAK1 and/or JAK2, may also have utility in the treatment of cancer (leukemia and solid tumors such as uterine leiomyosarcoma, prostate cancer).

In addition, Castleman's disease, multiple myeloma, mexangiocyte proliferative glomerulonephritis, psoriasis and Kaposi's sarcoma appear to be due to hypersecretion of the cytokine IL-6, whose biological effects are mediated by intracellular JAK-STAT signaling ( Naka et al., 2002). The results show that inhibitors of JAK may also have utility in the treatment of these diseases.

Current therapies are unsatisfactory and therefore allergic diseases, inflammatory diseases, autoimmune diseases, proliferative diseases, graft rejection, diseases involving impaired cartilage turnover, congenital cartilage malformations, and/or hypersecretion of IL6 or interferon secretion. There is still a need to identify additional compounds that can be used in the treatment of excess-related diseases. Accordingly, the present invention provides compounds, methods for their preparation, and pharmaceutical compositions comprising a compound of the present invention together with a suitable pharmaceutical carrier. The present invention also relates to allergic diseases, inflammatory diseases, autoimmune diseases, proliferative diseases, graft rejection, diseases involving disorders of cartilage turnover, congenital cartilage malformations, and/or hypersecretion of IL6 or hypersecretion of interferon. Provided is the use of a compound of the present invention in the manufacture of a medicament for the treatment of a disease.

The present invention provides that the compounds of the present invention can act as inhibitors of JAK and are used to treat allergic diseases, inflammatory diseases, autoimmune diseases, proliferative diseases, graft rejection, diseases involving disorders of cartilage turnover, congenital cartilage malformations, and/or It is based on the discovery that it is useful for the treatment of diseases associated with hypersecretion of IL6 or hypersecretion of interferon. In a specific embodiment the compounds of the invention are inhibitors of JAK1 and/or TYK2. The present invention also provides methods for preparing these compounds, pharmaceutical compositions comprising these compounds, and disorders of allergic diseases, inflammatory diseases, autoimmune diseases, proliferative diseases, transplant rejection, cartilage turnover by administration of the compounds of the present invention. Provided is a method for treating a disease associated with a congenital cartilage deformity, and/or a disease associated with hypersecretion of IL6 or hypersecretion of interferon.

Accordingly, in a first aspect of the present invention there is provided a compound of the present invention according to formula I

[Formula I]

In the above formula,

R ¹ is H or Me;

L ₁ is -NR ² -; -O- or -CH ₂ -;

Cy is phenyl or a 5 to 9 membered monocyclic or fused bicyclic heteroaryl containing 1 to 4 heteroatoms independently selected from N, O and S;

R ² is H or C _1-4 alkyl;

R ³ is H, halo, C _1-4 alkyl optionally substituted with one or more halo, or C _1-4 alkoxy optionally substituted with one or more halo;

R ⁴ is H or halo;

R ⁵ is -CN, halo, or -L ₂ -R ⁶ ;

-L ₂ is absent, or -C(=O)-, -C(=O)NR ⁷ -, -NR ⁷ C(=O)-, -SO ₂ -, -SO ₂ NR ⁷ -, or -NR ⁷ SO ₂ -;

R ⁶ is H or C _1-6 alkyl optionally substituted with one or more independently selected R ⁸ groups;

R ⁷ is H or C _1-4 alkyl;

R ⁸ is OH, CN, halo or C _1-4 alkoxy;

L _a is absent or -C(=0)-, -C(=0)0- or -C(=0)NH-;

R ^a is

-H,

- C _1-4 alkyl optionally substituted with one or more independently selected R ^b ,

- C _3-7 monocyclic cycloalkyl optionally substituted with one or more independently selected R ^c , or

- 4 to 7 membered monocyclic heterocycloalkyl containing one or more heteroatoms independently selected from O, N and S, or

- a 5 to 6 membered monocyclic heteroaryl containing at least one heteroatom independently selected from O, N and S;

^Rb is

- halo,

-CN,

-OH,

- C _1-4 alkoxy;

- C _3-7 cycloalkyl,

- 4 to 7 membered monocyclic heterocycloalkyl containing one or more heteroatoms independently selected from O, N and S, wherein said heterocycloalkyl is optionally substituted with one or more independently selected halo or oxo;

- -SO ₂ -C _1-4 alkyl, or

- -C(=O)NR ^b1 R ^b2 and

R ^c is

- halo,

-CN,

-OH,

- C _1-4 alkyl;

- -C(=O)OH, or

- -C(=0)NR ^c1 R ^c2 ;

Each of R ^bi , R ^b2 , R ^c1 and R ^c2 is independently selected from H and C _1-4 alkyl.

In certain embodiments, the compounds of the invention are inhibitors of JAK1 and/or TYK2.

Surprisingly, it has now been discovered that the compounds of the present invention can exhibit improved in vitro activity compared to closely related analogs.

Moreover, in certain embodiments, the compounds of the present invention may exhibit improved stability in vitro compared to closely related analogs. Such improvements may lower the dosage of drug required in vivo, thereby resulting in reduced toxicity and/or drug-drug interactions.

In a further aspect, the invention provides a pharmaceutical composition comprising a compound of the invention, and a pharmaceutical carrier, excipient or diluent. Moreover, compounds of the present invention useful in the pharmaceutical compositions and methods of treatment disclosed herein are pharmaceutically acceptable when prepared and used. In this aspect of the present invention, the pharmaceutical composition may further comprise additional active ingredients suitable for use with the compounds of the present invention.

In a further aspect, the present invention provides a compound of the present invention or a pharmaceutical composition comprising a compound of the present invention for use as a medicament. In a specific embodiment, the pharmaceutical composition further comprises an additional active ingredient.

In a further aspect of the invention, the invention relates to conditions from those listed herein, and in particular to aberrant JAK activity, e.g. allergic diseases, inflammatory diseases, autoimmune diseases, proliferative diseases, graft rejection, cartilage turnover Provided are methods for the treatment of mammals susceptible to or suffering from conditions as may be associated with diseases involving disorders of the brain, congenital cartilage malformations, and/or diseases associated with hypersecretion of IL6 or hypersecretion of interferon. and the method comprises administering an effective amount of a pharmaceutical composition or compound as disclosed herein. In a specific embodiment, the condition is associated with aberrant JAK1 and/or TYK2 activity.

In a further aspect, the present invention relates to a condition selected from those listed herein, particularly involving aberrant JAK activity, e.g., allergic disease, inflammatory disease, autoimmune disease, proliferative disease, graft rejection, disorder of cartilage turnover. , congenital cartilage malformations, and/or conditions associated with hypersecretion of IL6 or hypersecretion of interferon.

In yet another method of treatment aspect, the present invention provides a method of treating a mammal susceptible to or suffering from a condition causally related to aberrant JAK activity as disclosed herein, and an effective condition-treatment or condition. - administering a prophylactic amount of a pharmaceutical composition or compound of the invention disclosed herein. In a specific embodiment, the condition is causally related to aberrant JAK1 and/or TYK2 activity.

In a further aspect, the present invention provides a compound of the present invention, or a pharmaceutical composition comprising a compound of the present invention, for use as a medicament.

In a further aspect, the invention provides a compound of the invention for use in the treatment or prevention of a condition causally associated with aberrant JAK activity.

In a further aspect, the present invention provides methods for synthesizing the compounds of the present invention by typical synthetic protocols and routes hereinafter disclosed herein.

Accordingly, the main object of the present invention is to provide novel compounds capable of modifying the activity of JAK and thus preventing or treating any condition which may be causally related therewith. In a specific embodiment, the compounds of the invention modulate the activity of JAK1 and/or TYK2.

A further object of the present invention is to treat conditions or symptoms of said conditions which may be causally related to the activity of JAK, in particular JAK1 and/or TYK2, such as allergic diseases, inflammatory diseases, autoimmune diseases, proliferative diseases. , transplant rejection, diseases accompanying disorders of cartilage turnover, congenital cartilage deformities, and/or diseases associated with hypersecretion of IL6 or hypersecretion of interferon are provided.

A still further object of the present invention is a variety of conditions including diseases associated with JAK activity, such as allergic diseases, inflammatory diseases, autoimmune diseases, proliferative diseases, transplant rejection, diseases involving disorders of cartilage turnover, It is to provide a pharmaceutical composition that can be used for the treatment or prevention of congenital cartilage deformities and/or diseases associated with hypersecretion of IL6 or hypersecretion of interferon. In a specific embodiment, the disease is associated with JAK1 and/or TYK2 activity.

Other objects and advantages will be apparent to those skilled in the art upon consideration of the detailed description that follows.

Justice

The following terms are intended to have the meanings given together below and are useful in understanding the disclosure and intended scope of the present invention.

When disclosing the present invention, which may include compounds, pharmaceutical compositions containing such compounds, and methods of using such compounds and compositions, the following terms (where present) have the following meanings unless otherwise indicated: have When disclosed herein, any of the moieties defined below may be substituted with various substituents, and it is intended to include such substituted moieties within their scope as each definition is set forth below. is also of course. Unless stated otherwise, the term "substituted" shall be defined as set forth below. It should also be understood that the terms "group" and "radical" may be considered interchangeable when used herein.

The article 'one' may be used in the present invention to refer to one or more than one (ie at least one) of the grammatical objects of the article. By way of example, 'an analogue' means one analogue or more than one analogue.

'Alkyl' means a straight-chain or branched aliphatic hydrocarbon having the number of carbon atoms specified. Certain alkyl groups have 1 to 8 carbon atoms. More particularly lower alkyl has 1 to 6 carbon atoms. Additional specific groups have 1 to 4 carbon atoms. Exemplary straight chain groups include methyl, ethyl, n-propyl and n-butyl. Branched means that one or more lower alkyl groups, such as methyl, ethyl, propyl or butyl, are attached to the linear alkyl chain; exemplary branched groups include isopropyl, iso-butyl, t-butyl and isoamyl.

'Alkoxy' refers to the group -OR ²⁶ , where R ²⁶ is an alkyl having the specified number of carbon atoms. Particular alkoxy groups are methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, and 1,2- It is dimethylbutoxy. A specific alkoxy group is a lower alkoxy, i.e. an alkoxy having from 1 to 6 carbon atoms. Further specific alkoxy groups have from 1 to 4 carbon atoms.

'Alkenyl' refers to a monovalent olefinic (unsaturated) hydrocarbon group having the specified number of carbon atoms. Certain alkenyls have 2 to 8 carbon atoms, and more particularly 2 to 6 carbon atoms, which may be straight chain or branched, and have one or more and especially 1 to 2 sites of olefinic unsaturation. Particular alkenyl groups include ethenyl (-CH=CH ₂ ), n-propenyl (-CH ₂ CH=CH ₂ ), isopropenyl (-C(CH ₃ )=CH ₂ ), and the like.

'Amino' refers to the radical -NH ₂ .

'Aryl' refers to a monovalent aromatic hydrocarbon group derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system. Aryl in particular refers to a monocyclic or fused polycyclic aromatic ring structure having the specified number of ring atoms. Specifically, the term includes groups containing from 6 to 10 ring members. If the aryl group is a monocyclic ring system, it preferentially contains 6 carbon atoms. Aryl groups in particular include phenyl and naphthyl.

'Cycloalkyl' refers to a monocyclic, condensed polycyclic, bridged polycyclic, or spirocyclic non-aromatic hydrocarbyl ring structure having the specified number of ring atoms. Cycloalkyl may have 3 to 12 carbon atoms, particularly 3 to 10 carbon atoms, and more particularly 3 to 7 carbon atoms. Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.

'Cyano' refers to the radical -CN.

'Halo' or 'halogen' refers to fluoro (F), chloro (Cl), bromo (Br) and iodo (I). Particular halo groups are fluoro or chloro.

'Hetero', when used to describe a compound or group present on a compound, means that one or more carbon atoms in the compound or group have been replaced by a nitrogen, oxygen or sulfur heteroatom. Hetero is any of the aforementioned hydrocarbyl groups, for example an alkyl having 1 to 4, and especially 1 to 3 heteroatoms, more typically 1 or 2 heteroatoms, eg a single heteroatom, for example For example, heteroalkyl, cycloalkyl such as heterocycloalkyl, aryl such as heteroaryl, and the like.

'Heteroaryl' means a monocyclic or fused polycyclic aromatic ring structure comprising one or more heteroatoms independently selected from O, N and S and the number of ring atoms specified. In particular, the aromatic ring structure may have 5 to 9 ring members. Such heteroaryl groups are, for example, condensed 5- and 6-membered rings or two condensed 6-membered rings, or as a further example a 5- or 6-membered monocyclic ring formed from two condensed 5-membered rings or a condensed one. may have a bicyclic structure. Each ring may contain up to 4 heteroatoms, typically selected from nitrogen, sulfur and oxygen. Typically the heteroaryl ring will contain no more than 4, more typically no more than 3, more often no more than 2 heteroatoms, eg a single heteroatom. In one embodiment, the heteroaryl ring contains one or more ring nitrogen atoms. The nitrogen atom in the heteroaryl ring may be basic, as in the case of an imidazole or pyridine, or essentially non-basic, as in the case of an indole or pyrrole nitrogen. Generally the number of basic nitrogen atoms present in the heteroaryl group will be less than 5, including any amino group substituents on the ring.

Examples of 5-membered monocyclic heteroaryl groups include, but are not limited to, pyrrolyl, furanyl, thiophenyl, imidazolyl, furazanyl, oxazolyl, oxadiazolyl, oxatriazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, triazolyl and tetrazolyl groups.

Examples of 6-membered monocyclic heteroaryl groups include, but are not limited to, pyridinyl, pyrazinyl, pyridazinyl, pyrimidinyl and triazinyl. Specific examples of bicyclic heteroaryl groups containing a 5-membered ring fused to another 5-membered ring include, but are not limited to, imidazothiazolyl and imidazoimidazolyl. Specific examples of bicyclic heteroaryl groups containing a 6-membered ring fused to a 5-membered ring include, but are not limited to, benzofuranyl, benzothiophenyl, benzimidazolyl, benzoxazolyl, isobenzoxazolyl, benzisoxa zolyl, benzothiazolyl, benzisothiazolyl, isobenzofuranyl, indolyl, isoindolyl, indolizinyl, purinyl (e.g. adenine, guanine), indazolyl, pyrazolopyrimidinyl, triazolo pyrimidinyl, and pyrazolopyridinyl groups. Specific examples of bicyclic heteroaryl groups containing two condensed six-membered rings include, but are not limited to, quinolinyl, isoquinolinyl, pyridopyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, fr It includes talazinyl, naphthyridinyl and pteridinyl groups. Particular heteroaryl groups are those derived from thiophenyl, pyrrolyl, benzothiophenyl, benzofuranyl, indolyl, pyridinyl, quinolinyl, imidazolyl, oxazolyl and pyrazinyl. Examples of typical heteroaryls include:

In the above, each Y is selected from >C(=0), NH, O and S.

The term 'heterocycloalkyl' refers to a monocyclic, condensed polycyclic, spirocyclic or bridged polycyclic compound containing one or more heteroatoms independently selected from O, N and S and the number of ring atoms specified. It means a fully saturated non-aromatic ring structure of The heterocycloalkyl ring structure may have 4 to 12 ring members, particularly 4 to 10 ring members, and more particularly 4 to 7 ring members. Each ring may contain up to 4 heteroatoms, typically selected from nitrogen, sulfur and oxygen. Typically a heterocycloalkyl ring will contain no more than 4 heteroatoms, more typically no more than 3 heteroatoms, more often no more than 2, eg a single heteroatom. Examples of heterocyclic rings include, but are not limited to, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl (eg 1-pyrrolidinyl, 2-pyrrolidinyl and 3-pyrrolidinyl), morpholinyl , thiomorpholinyl, tetrahydrofuranyl, tetrahydrothiophenyl, imidazolinyl, oxazolinyl, thiazolinyl, piperidinyl (e.g. 1-piperidinyl, 2-piperidinyl, 3- piperidinyl and 4-piperidinyl), pyranyl, dioxanyl, tetrahydropyranyl (e.g. 4-tetrahydropyranyl), 2-pyrazolinyl, pyrazolidinyl or piperazinyl. .

Specific examples of monocyclic heterocycloalkyl groups are shown in the following illustrative examples:

In the above, each W and Y is independently selected from CH ₂ , NH, O and S.

Specific examples of condensed bicyclic heterocycloalkyl groups are shown in the following illustrative examples:

In the above, each W and Y is independently selected from CH ₂ , NH, O and S.

Specific examples of bridged bicyclic heterocycloalkyl groups are shown in the following illustrative examples:

In the above, each of W and Y is independently selected from CH ₂ , NH, O and S, and Z is N or CH.

Specific examples of spirocyclic heterocycloalkyl groups are shown in the following illustrative examples:

In the above, each Y is selected from NH, O and S.

'Hydroxyl' refers to the radical -OH.

'Oxo' refers to the radical ═O.

'Substituted' refers to groups in which one or more hydrogen atoms are each independently substituted with the same or different substituent(s).

'Sulfo' or 'sulfonic acid' refers to a radical such as -SO ₃ H.

A 'thiol' refers to a -SH group.

As used herein, the term 'substituted with one or more' refers to 1 to 4 substituents. In one embodiment this refers to 1 to 3 substituents. In a further embodiment the above refers to 1 or 2 substituents. In a still further embodiment the above refers to one substituent.

'Thioalkoxy' refers to the group -SR ²⁶ , where R ²⁶ is an alkyl having the specified number of carbon atoms and in particular C ₁ -C ₈ alkyl. Specific thioalkoxy groups include thiomethoxy, thioethoxy, n-thiopropoxy, isothiopropoxy, n-thiobutoxy, tert-thiobutoxy, sec-thiobutoxy, n-thiopentoxy, n -thiohexoxy, and 1,2-dimethylthiobutoxy. A more specific thioalkoxy group is a lower thioalkoxy, i.e., a thioalkoxy having 1 to 6 carbon atoms. Further specific alkoxy groups have from 1 to 4 carbon atoms.

Those skilled in the art of organic synthesis know that the maximum number of heteroatoms in a stable, chemically possible heterocyclic ring, whether it is aromatic or non-aromatic, is determined by the size of the ring, the degree of unsaturation and the valency of the heteroatoms. will know In general, heterocyclic rings can have from 1 to 4 heteroatoms as long as the heteroaromatic ring is chemically viable and stable.

'Pharmaceutically acceptable' means approved or approvable by a regulatory agency of the Federal or State governments, or by an appropriate authority outside the United States, or approved by the United States Pharmacopoeia or other generally recognized authorities for use in animals, and more particularly in humans. Means listed in pharmacopeia.

A 'pharmaceutically acceptable salt' refers to a salt of a compound that is pharmaceutically acceptable and possesses the desired pharmacological activity of the parent compound. In particular, such salts are non-toxic and may be inorganic or organic acid addition salts and base addition salts. Specifically, such salts include (1) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or an organic acid such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydride) hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2- Naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary acid addition salts formed with butylacetic acid, lauryl sulfate, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or (2) an acidic proton present in the parent compound is replaced by a metal ion, such as an alkali metal ion, alkaline earth ion, or aluminum ion; or salts formed when coordinated with organic bases such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, and the like. Salts are, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and salts of non-toxic organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate, and the like, when the compound contains a basic functional group. The term 'pharmaceutically acceptable cation' refers to an acceptable cationic counter ion of an acidic functional group. Such cations are exemplified by sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium cations and the like.

'Pharmaceutically acceptable vehicle' refers to a diluent, adjuvant, excipient or carrier with which a compound of the present invention is administered.

A 'prodrug' refers to a compound, including derivatives of a compound of the present invention, which has a cleavable group and which, by solvolysis or under physiological conditions, results in a compound of the present invention that is pharmacologically active in vivo. Such examples include, but are not limited to, choline ester derivatives and the like, N-alkylmorpholine esters and the like.

'Solvate' refers to a form of compound that associates with a solvent, usually by a solvolysis reaction. These physical associations include hydrogen bonds. Common solvents include water, EtOH, acetic acid, and the like. The compounds of the present invention may be prepared, for example, in crystalline form and may be solvated or hydrated. Suitable solvates include pharmaceutically acceptable solvates, such as hydrates, and further include both stoichiometric and non-stoichiometric solvates. In some instances, the solvate may be isolated, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. 'Solvate' includes both solution-phase and isolateable solvates. Typical solvates include hydrates, ethanolates and methanolates.

A 'patient' includes a human being. The terms 'human', 'patient' and 'subject' are used interchangeably in the present invention.

'Effective amount' means an amount of the compound of the present invention sufficient to effect such treatment for the disease when administered to a patient for the treatment of the disease. The 'effective amount' may vary depending on the compound, the disease and its severity, and the age, weight, and the like of the patient to be treated.

'Preventing' or 'prevention' means reducing the risk of acquiring or developing a disease or disorder (i.e., the clinical evaluation of a disease in a patient who may be exposed to a disease-causing agent or predisposed to the disease prior to onset of the disease). one or more of the symptoms do not occur).

The term 'prophylaxis' relates to 'prevention' and refers to measures or procedures aimed at preventing rather than treating or curing disease. Non-limiting examples of prophylactic measures include administration of vaccines; administration of low molecular weight heparin to hospitalized patients at risk of thrombosis, eg due to immobilization; and administration of an anti-malarial agent, such as chloroquine, prior to visiting a geographic area where malaria is endemic or where there is a high risk of contact with malaria.

In one embodiment, 'treating' or 'treatment' of any disease or condition is ameliorating the disease or condition (i.e., inhibiting the disease or reducing the sign, extent or severity of one or more of the clinical symptoms of the disease). decreases). In another embodiment 'treating' or 'treatment' refers to an improvement in one or more physical parameters that is imperceptible to the patient. In yet another embodiment, 'treating' or 'treatment' refers to the disease or disorder physically (eg, stabilization of perceptible symptoms), physiologically (eg, stabilization of a physical parameter), or It refers to mitigating by both of these. In a further embodiment, "treating" or "treatment" relates to slowing the progression of the disease.

As used herein, the term 'allergic disease(s)' refers to diseases of hypersensitivity of the immune system, including allergic airway disease (eg asthma, rhinitis), sinusitis, eczema and urticaria, as well as food allergies or insect poisons. refers to a group of conditions characterized by an allergy to

As used herein, the term 'asthma' refers to any condition of the lungs characterized by changes in pulmonary airflow associated with constriction of the airways, whatever the cause (intrinsic, exogenous, or both; allergic or non-allergic). refers to a disease The term asthma may be used with one or more adjectives indicating a cause.

As used herein, the term 'inflammatory disease(s)' includes rheumatoid arthritis, osteoarthritis, juvenile idiopathic arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, allergic airway disease (eg asthma, rhinitis), chronic Obstructive pulmonary disease (COPD), inflammatory bowel disease (eg Crohn's disease, ulcerative colitis), endotoxin-driven disease conditions (eg post bypass surgery complications or chronic endotoxin conditions contributing to eg chronic heart failure) , and cartilage-related diseases, such as joint diseases. In particular, the term refers to rheumatoid arthritis, osteoarthritis, allergic airway disease (eg asthma), chronic obstructive pulmonary disease (COPD) and inflammatory bowel disease. More particularly the term refers to rheumatoid arthritis, chronic obstructive pulmonary disease (COPD) and inflammatory bowel disease.

As used herein, 'autoimmune disease(s)' refers to obstructive airway disease, including conditions such as COPD, asthma (eg intrinsic asthma, extrinsic asthma, dust asthma, pediatric asthma), particularly chronic or chronic asthma (e.g. late stage asthma and airway hyperresponsiveness), bronchitis including bronchial asthma, systemic lupus erythematosus (SLE), cutaneous lupus erythematosus, lupus nephritis, dermatomyositis, Sjogren's syndrome, multiple sclerosis, psoriasis, dry eye syndrome, type I diabetes and related complications, atopic eczema (atopic dermatitis), thyroiditis (Hashimoto and autoimmune thyroiditis), contact dermatitis and further eczematous dermatitis, inflammatory bowel disease (eg Crohn's disease and ulcerative colitis), atherosclerosis and amyotrophic lateral sclerosis. In particular the term refers to COPD, asthma, systemic lupus erythematosus, type I diabetes and inflammatory bowel disease. As used herein, the term 'proliferative disease(s)' includes cancer (eg uterine leiomyosarcoma or prostate cancer), myeloproliferative diseases (eg polycythemia vera, essential thrombocytosis and bone marrow) fibrosis), leukemia (eg acute myelogenous leukemia, acute and chronic lymphoblastic leukemia), multiple myeloma, psoriasis, restenosis, scleroderma or fibrosis. In particular the term refers to cancer, leukemia, multiple myeloma and psoriasis.

As used herein, the term 'cancer' refers to a malignant or benign proliferation of cells in the skin or body organ, such as but not limited to breast, prostate, lung, kidney, pancreas, stomach or intestine. Cancer tends to invade into adjacent tissue and spread (metastasize) to distant organs such as bone, liver, lungs or brain. As used herein, the term cancer includes the metastatic tumor cell type (eg, but not limited to melanoma, lymphoma, leukemia, fibrosarcoma, rhabdomyosarcoma, and mastocytoma) and the type of tissue carcinoma (eg, non-limiting). colorectal cancer, prostate cancer, small cell lung cancer and non-small cell lung cancer, breast cancer, pancreatic cancer, bladder cancer, kidney cancer, gastric cancer, glioblastoma, primary liver cancer, ovarian cancer, prostate cancer and uterine leiomyosarcoma). In particular, the term 'cancer' refers to acute lymphoblastic leukemia, acute myelogenous leukemia, adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, atypical organoid/rhab tumor, basal cell carcinoma, biliary tract cancer, bladder cancer, and bone cancer (osteosarcoma). and malignant fibrous histiocytosis), brainstem glioma, brain tumor, brain and spinal cord tumor, breast cancer, bronchial tumor, Burkitt's lymphoma, neck cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, cranial carcinoma, cutaneous T- Cell lymphoma, embryonic tumor, endometrial cancer, ependymblastic tumor, ependymoma, esophageal cancer, Ewing sarcoma series tumor, eye cancer, retinoblastoma, gallbladder cancer, gastrointestinal (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), gastrointestinal stromal cell Tumors, germ cell tumors, glioma, hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor (endocrine pancreas), Kaposi's sarcoma, renal cancer, Langerhans cell histiocytosis, Laryngeal cancer, leukemia, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, liver cancer, non-small cell lung cancer, small cell lung cancer, Burkitt's lymphoma, cutaneous T-cell lymphoma, Hodgkin's lymphoma , non-Hodgkin's lymphoma, lymphoma, Waldenstrom's hyperglobulinemia, medulloblastoma, medullary epithelioma, melanoma, mesothelioma, oral cancer, chronic myelogenous leukemia, myelogenous leukemia, multiple myeloma, pharyngeal cancer, neuroblastoma, non-small cell lung cancer , oral cancer, oropharyngeal cancer, osteosarcoma, malignant fibrous histiocytosis of bone, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, papilloma, parathyroid cancer, penile cancer, pharyngeal cancer, intermediate Differentiated Pineal Parenchymal Tumor, Pineal Blastoma and Supratentorial Primitive Neuroectodermal Tumor, Pituitary Tumor, Plasma Cell Neoplasia/Multiple Myeloma, Pleuropulmonary Blastoma, Primary Central Nervous System Lymphoma, Prostate Cancer, Rectal Cancer, Renal Cell (Kidney) Cancer, Retina Blastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, tumors of the Ewing sarcoma family, sarcoma, Kaposi, Sezary syndrome, skin cancer, small cell lung cancer, small bowel cancer, soft tissue sarcoma, squamous cell carcinoma, gastric (gastrointestinal) cancer, Supratentorial primitive neuroectodermal tumors, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's hyperglobulinemia, and Wilms' tumor. In another specific embodiment, the term cancer refers to pancreatic cancer, liver cancer, hepatocellular carcinoma (HCC), breast or colon cancer.

The term 'leukemia' as used herein refers to a neoplastic disease of the blood and blood-forming organs. Such diseases can lead to bone marrow and immune system dysfunction, which makes the host highly susceptible to infection and hemorrhage. In particular, the term leukemia refers to acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL). In another specific embodiment, the term leukemia refers to T-cell acute lymphoblastic leukemia (T-ALL), chronic lymphoblastic leukemia (CLL), or diffuse large B-cell lymphoma (DLBCL).

As used herein, the term 'transplant rejection' includes, for example, cells, tissues or solid organ allo- or xenografts of pancreatic islets, stem cells, bone marrow, skin, muscle, corneal tissue, nerve tissue, heart, lung. , complex heart-lung, kidney, liver, intestine, pancreas, trachea, or esophagus acute or chronic rejection, or graft-versus-host disease.

As used herein, the term 'diseases involving disorders of cartilage turnover' includes osteoarthritis, psoriatic arthritis, juvenile rheumatoid arthritis, gouty arthritis, septic or infectious arthritis, reactive arthritis, sympathetic dystrophy, dystrophy abnormalities, Tietze syndrome or costochondritis, fibromyalgia, osteochondritis, nerve or neuropathic arthritis, arthrosis, endemic forms of arthritis such as endemic deforming osteoarthritis, Mselenie's disease and cold sore; conditions such as degeneration resulting from fibromyalgia, systemic lupus erythematosus, scleroderma, and ankylosing spondylitis.

As used herein, the term 'congenital cartilage malformation(s)' refers to conditions such as hereditary chondrolysis, chondrodysplasia and pseudochondrodysplasia, especially but not limited to microtia, adystrophy, metaphyseal chondrodysplasia, and related diseases. include

As used herein, the term 'disease(s) associated with hypersecretion of IL6' includes conditions such as Castleman's disease, multiple myeloma, psoriasis, Kaposi's sarcoma, and/or mesenchymal proliferative glomerulonephritis.

As used herein, the term 'disease(s) associated with hypersecretion of interferon' includes conditions such as systemic and cutaneous lupus erythematosus, lupus nephritis, dermatomyositis, Sjogren's syndrome, psoriasis, and rheumatoid arthritis.

'Compound(s) of the present invention' and equivalent expressions are meant to include compounds of the formula(s) as disclosed herein, which expressions include pharmaceutically acceptable salts and solvates such as hydrates, and solvates of the above pharmaceutically acceptable salts (where the context permits so). Similarly, reference to an intermediate is meant to include salts, solvates (where the context so permits) of the intermediate, whether or not the intermediate itself is claimed.

Where ranges are recited herein, such as but not limited to C _1-8 alkyl, the recitation of a range is to be regarded as indicating each member of the range.

Other derivatives of the compounds of the present invention have activity in both their acid and acid derivative forms, but in acid sensitive forms often offer the advantages of solubility, tissue compatibility or delayed release to mammalian organisms (Bundgard, 1985). Prodrugs are acid derivatives well known to those skilled in the art, such as esters prepared by the reaction of the parent acid with a suitable alcohol, or amides prepared by the reaction of the parent acid compound with a substituted or unsubstituted amine; or acid anhydrides or mixed anhydrides. Simple aliphatic or aromatic esters, amides and anhydrides derived from acidic groups pendent on the compounds of this invention are particularly useful prodrugs. In some cases it may be desirable to prepare prodrugs of the double ester type, eg, (acyloxy)alkyl esters or ((alkoxycarbonyl)oxy)alkylesters. Certain such prodrugs are C ₁ to C ₈ alkyl, C ₂ to C ₈ alkenyl, C _6-10 optionally substituted aryl, and (C _6-10 aryl)-(C _1-4 alkyl of the compounds of the present invention. ) is an ester.

As used herein, the term 'isotopic variant' refers to such compounds that contain unnatural proportions of isotopes at one or more of the atoms that make up the compound. For example, an 'isotopic variant' of a compound may contain one or more non-radioactive isotopes, such as deuterium ( ² H or D), carbon-13 ( ¹³ C), nitro ( ¹⁵ N), etc. there is. In compounds where such isotopic substitutions are made, the following atoms (if present) can vary, so for example any hydrogen can be ² H/D or any carbon can be ¹³ C It will be appreciated that, or any nitrogen can be 15N, the presence and location of such atoms can be determined within the skill of the art. Likewise, the present invention may include the preparation of isotopic variants with radioisotopes, for example where the resulting compounds can be used in drug and/or substrate tissue distribution studies. The radioactive isotopes, tritium, ³ H, and carbon-14, ¹⁴ C, are particularly useful for this purpose in view of their ease of integration and convenient means of detection. Furthermore, compounds substituted with positron emitting isotopes such as ¹¹ C, ¹⁸ F, ¹⁵ O and ¹³ N can be prepared, which will be useful in positron emission tomography (PET) studies to investigate substrate receptor occupancy. will be.

All isotopic variations of the compounds provided herein, whether radioactive or not, are included within the scope of the present invention.

It should also be noted that compounds that have the same molecular formula but differ in the nature or order of bonding of their atoms or the spatial arrangement of said atoms are termed 'isomers'. Isomers that differ in the arrangement of their atoms in space are termed 'stereoisomers'.

Stereoisomers that are not mirror images of each other are called 'diastereomers', and isomers that are non-superimposable mirror images are called 'enantiomers'. A pair of enantiomers is possible if a compound has an asymmetric center, for example bonded to four different groups. Enantiomers can be characterized by the absolute arrangement of their asymmetric centers, which are initiated by the R- and S-sequencing laws of Cahn and Prelog or by the way the molecule rotates the plane of polarized light and rotates clockwise. It is indicated as either sex or levorotatory (i.e., (+) or (-)-isomer, respectively). Chiral compounds can exist as individual enantiomers or as mixtures thereof. A mixture containing equal proportions of enantiomers is called a 'racemic mixture'.

A 'tautomer' refers to a compound that is an interconvertable form of a particular compound structure and has a variety of substitutions of hydrogen atoms and electrons. Thus, the two structures can exist in equilibrium through the movement of π electrons and atoms (usually H). For example, enols and ketones are tautomers because they are rapidly interconverted by treatment with acids or bases. Another example of tautomerization is the acid- and nitrogen-forms of phenylnitromethane, which are likewise formed by treatment with acids or bases.

Tautomeric forms may be suitable for obtaining optimal chemical reactivity and biological activity of a compound of interest.

The compounds of the present invention may have one or more asymmetric centers; Accordingly, such compounds may be produced as individual (R)- or (S)-stereoisomers or as mixtures thereof.

Unless otherwise indicated, the description or designation of a particular compound in the specification and claims is meant to include both individual enantiomers and racemic or other mixtures thereof. Methods for the determination and separation of the stereochemistry of stereoisomers are well known in the art.

It will be appreciated that the compounds of the present invention may be metabolized to provide biologically active metabolites.

the present invention

The present invention provides that the compounds of the present invention are inhibitors of JAK, allergic diseases, inflammatory diseases, autoimmune diseases, proliferative diseases, graft rejection, diseases involving disorders of cartilage turnover, congenital cartilage malformations, and/or secretion of IL6. It is based on confirmation that it is useful in the treatment of diseases associated with excess or excessive secretion of interferon. In a specific embodiment, the compounds of the invention are inhibitors of JAK1 and/or TYK2.

The present invention also provides methods for preparing the compounds of the present invention, pharmaceutical compositions comprising the compounds of the present invention, and allergic diseases, inflammatory diseases, autoimmune diseases, proliferative diseases, transplant rejection, cartilage by administration of the compounds of the present invention. Provided is a method for treating a disease accompanying a disorder of turnover, a congenital cartilage deformity, and/or a disease associated with hypersecretion of IL6 or hypersecretion of interferon. In a specific embodiment, the compounds of the invention are inhibitors of JAK1 and/or TYK2.

Accordingly, in a first aspect of the present invention there is provided a compound of the present invention according to formula I

Formula I

In the above formula,

R ¹ is H or Me;

L ₁ is -NR ² -; -O- or -CH ₂ -;

Cy is phenyl or a 5 to 9 membered monocyclic or fused bicyclic heteroaryl containing 1 to 4 heteroatoms independently selected from N, O and S;

R ² is H or C _1-4 alkyl;

R ³ is H, halo, C _1-4 alkyl optionally substituted with one or more halo, or C _1-4 alkoxy optionally substituted with one or more halo;

R ⁴ is H or halo;

R ⁵ is -CN, halo, or -L ₂ -R ⁶ ;

-L ₂ is absent, or -C(=O)-, -C(=O)NR ⁷ -, -NR ⁷ C(=O)-, -SO ₂ -, -SO ₂ NR ⁷ -, or -NR ⁷ SO ₂ -;

R ⁶ is H or C _1-6 alkyl optionally substituted with one or more independently selected R ⁸ groups;

R ⁷ is H or C _1-4 alkyl;

R ⁸ is OH, CN, halo or C _1-4 alkoxy;

L _a is absent or -C(=0)-, -C(=0)0- or -C(=0)NH-;

R ^a is

-H,

- C _1-4 alkyl optionally substituted with one or more independently selected R ^b ,

- C _3-7 monocyclic cycloalkyl optionally substituted with one or more independently selected R ^c , or

- 4 to 7 membered monocyclic heterocycloalkyl containing one or more heteroatoms independently selected from O, N and S, or

- a 5 to 6 membered monocyclic heteroaryl containing at least one heteroatom independently selected from O, N and S;

^Rb is

- halo,

-CN,

-OH,

- C _1-4 alkoxy;

- C _3-7 cycloalkyl,

- 4 to 7 membered monocyclic heterocycloalkyl containing one or more heteroatoms independently selected from O, N and S, wherein said heterocycloalkyl is optionally substituted with one or more independently selected halo or oxo;

- -SO ₂ -C _1-4 alkyl, or

- -C(=O)NR ^b1 R ^b2 and

R ^c is

- halo,

-CN,

-OH,

- C _1-4 alkyl;

- -C(=O)OH, or

- -C(=0)NR ^c1 R ^c2 ;

Each of R ^bi , R ^b2 , R ^c1 and R ^c2 is independently selected from H and C _1-4 alkyl.

In one embodiment, a compound of the invention conforms to Formula I wherein R ¹ is Me.

In another embodiment, a compound of the invention conforms to Formula I wherein Cy is phenyl.

In one embodiment, compounds of the invention are of Formula I, wherein Cy is a 5 to 9 membered monocyclic or fused bicyclic heteroaryl containing 1 to 4 heteroatoms independently selected from N, O and S. follow In another embodiment, compounds of the invention are of Formula I, wherein Cy is a 5 to 9 membered monocyclic or fused bicyclic heteroaryl containing 1 or 2 heteroatoms independently selected from N, O and S. follow In certain embodiments, Cy is pyrazolyl, pyrrolyl, imidazolyl, triazolyl, thiophenyl, thiazolyl, furanyl, pyridyl, pyrazinyl, pyrimidyl, pyridazinyl, benzofuranyl, benzothiophenyl , benzothiazolyl, benzothiadiazolyl, benzoxazolyl, indolyl or indazolyl.

In another embodiment, compounds of the invention are according to Formula I, wherein Cy is a 5-6 membered monocyclic heteroaryl containing 1-4 heteroatoms independently selected from N, O and S. In yet another embodiment, compounds of the present invention are according to Formula I, wherein Cy is a 5 to 6 membered monocyclic heteroaryl containing 1 or 2 heteroatoms independently selected from N, O and S. In certain embodiments, Cy is pyrazolyl, pyrrolyl, imidazolyl, furanyl, thiophenyl, triazolyl, tetrazolyl, thiazolyl, oxazolyl, thiadiazolyl or oxadiazolyl. In another specific embodiment, Cy is pyridinyl, pyrazinyl, pyrimidinyl, or pyridazolyl. In a more specific embodiment, Cy is pyridyl.

In one embodiment, the compound of the present invention conforms to Formula IIa, IIb or IIc:

[Formula IIa] [Formula IIb] [Formula IIc]

In the formulas above, L ₁ , R ³ , R ⁴ , L _a , R ^a and R ⁵ are as described in any one of the above embodiments.

In one embodiment, the compound of the present invention conforms to Formula IIIa, IIIb or IIIc:

[Formula IIIa] [Formula IIIb] [Formula IIIc]

In the formulas above, L ₁ , R ³ , R ⁴ , L _a , R ^a and R ⁵ are as described in any one of the above embodiments.

In one embodiment, a compound of the present invention conforms to any of Formulas I to IIIc, wherein L ₁ is —CH ₂ —.

In one embodiment, a compound of the invention conforms to any of Formulas I-IIIc, wherein L ₁ is -O-.

In one embodiment, a compound of the invention is according to any of Formulas I-IIIc wherein L ₁ is -NR ² - and R ² is as disclosed in any of the embodiments above.

In one embodiment, a compound of the invention is according to any of Formulas I-IIIc wherein L ₁ is -NR ² -, wherein R ² is H.

In one embodiment, a compound of the invention is according to any of Formulas I to IIIc, wherein L ₁ is -NR ² -, wherein R ² is C _1-4 alkyl. In certain embodiments, R ² is Me, Et, or iPr. In a more specific embodiment, R ² is Me. In another more specific embodiment, R ² is Et.

In one embodiment, the compounds of the present invention conform to formulas IVa, IVb, IVc, IVd, IVe or IVf:

[Formula IVa] [Formula IVb] [Formula IVc]

[Formula IVd] [Formula IVe] [Formula IVf]

In the above formulas, R ³ , R ⁴ , R ⁵ , L _a and R ^a are as disclosed in any one of the above embodiments.

In one embodiment, a compound of the invention conforms to any one of Formulas I to IVf wherein L _a is absent.

In one embodiment, a compound of the invention is according to any one of formulas I to IVf, wherein L _a is -C(=0)-, -C(=0)0- or -C(=0)NH-.

In one embodiment, the compounds of the present invention conform to the formulas Va, Vb, Vc, Vd, Ve or Vf:

[Formula Va] [Formula Vb] [Formula Vc]

[Formula Vd] [Formula Ve] [Formula Vf]

In the formulas above, R ³ , R ⁴ , R ⁵ and R ^a are as disclosed in any one of the above embodiments.

In one embodiment, a compound of the invention is according to any of Formulas I-Vf, wherein R ⁴ is H or halo. In certain embodiments, R ⁴ is F or Cl. In another specific embodiment, R ⁴ is H.

In one embodiment, a compound of the invention conforms to any of Formulas I-Vf wherein R ³ is H.

In one embodiment, a compound of the invention is according to any of Formulas I-Vf, wherein R ³ is halo. In certain embodiments, R ³ is F or Cl. In a more specific embodiment, R ³ is Cl.

In one embodiment, _a compound of the invention is according to any of Formulas I-Vf, wherein R ³ is C _1-4 alkyl. In certain embodiments, R ³ is Me, Et or n-Pr. In a more specific embodiment, R ³ is Me or Et. In a most specific embodiment, R ³ is Et. In another most specific embodiment R ³ is Me.

In one embodiment, a compound of the invention is according to any _of Formulas I-Vf, wherein R ³ is C _1-4 alkyl substituted with one or more independently selected halo. In certain embodiments, R ³ is -CHF ₂ , -CF ₃ , -CH ₂ -CHF ₂ or -CH ₂ -CF ₃ . In a more specific embodiment, R ³ is -CF ₃ or -CH ₂ -CF ₃ .

In one embodiment, a compound _of the invention is according to any of Formulas I to Vf wherein R ³ is C _1-4 alkoxy. In certain embodiments, R ³ is -OMe, -OEt or -On-Pr. In a more specific embodiment, R ³ is -OMe or -OEt.

In another embodiment, a compound of the invention is according to _any of Formulas I-Vf, wherein R ³ is C _1-4 alkoxy substituted with one or more independently selected halo. In certain embodiments, R ³ is -OCHF ₂ , -OCF ₃ , or -OCH ₂ -CHF ₂ . In a more specific embodiment, R ³ is -OCHF ₂ .

In one embodiment, a compound of the invention conforms to any of Formulas I-Vf wherein R ⁵ is CN.

In one embodiment, a compound of the invention is according to any of Formulas I-Vf, wherein R ⁵ is halo. In certain embodiments, R ⁵ is F or Cl. In a more specific embodiment, R ⁵ is F.

In one embodiment, a compound of the invention is wherein R ⁵ is -L ₂ -R ⁶ , R ⁶ is as described above, and L ₂ is -C(=O)NR ⁷ -, -NR ⁷ C(=O )-, -SO ₂ NR ⁷ - or -NR ⁷ SO ₂ -, wherein R ⁷ is according to any one of formulas I to Vf as defined in any one of the preceding embodiments. In certain embodiments, R ⁷ is H. _In another specific embodiment, R ⁷ is C _1-4 alkyl. In a more specific embodiment, R ⁷ is Me or Et. In a most specific embodiment R ⁷ is Me.

In one embodiment, a compound of the invention is according to any of Formulas I to Vf wherein R ⁵ is -L ₂ -R ⁶ , L ₂ is as defined above, and R ⁶ is H.

In one embodiment, a compound of the invention is according to any of _Formulas I-Vf, wherein R ⁵ is -L ₂ -R ⁶ , L ₂ is as defined above, and R ⁶ is C _1-6 alkyl. In certain embodiments, R ⁶ is Me, Et, iPr or tBu. In a more specific embodiment, R ⁶ is Me or Et.

In one embodiment, a compound of _the invention is of the formula wherein R ⁵ is -L ₂ -R ⁶ , L ₂ is as described above, and R ⁶ is C _1-6 alkyl substituted with one or more independently selected R ⁸ groups. According to any one of I to Vf. In another embodiment, R ⁶ is Me, Et, iPr or tBu, each of which is substituted with one or more independently selected R ⁸ groups. In certain embodiments, R ⁶ is C _1-6 alkyl substituted with 1, 2 or 3 independently selected _R ⁸ groups. In another specific embodiment, R ⁶ is Me, Et, iPr or tBu, each of which is substituted with 1, 2 or 3 independently selected R ⁸ groups. In a more specific embodiment _, R ⁶ is C _1-6 alkyl substituted with one R ⁸ group. In another specific embodiment, R ⁶ is Me, Et, iPr or tBu, each of which is substituted with one R ⁸ group.

In one embodiment, a compound of the invention is according to any of Formulas I to Vf _, wherein R ⁸ is OH, CN, halo or C _1-4 alkoxy. In certain embodiments, R ⁸ is OH, CN, F, Cl, -OMe or -OEt.

In certain embodiments, R ⁵ is -L ₂ -R ⁶ , L ₂ is -C(=O)- or -SO ₂ -, and R ⁶ is C _1-6 alkyl. In a more specific embodiment, R ⁵ is -L ₂ -R ⁶ , L ₂ is -C(=0)- or -SO ₂ , and R ⁶ is Me, Et, iPr or tBu. In the most particular embodiment, R ⁵ is -C(=0)Me, -C(=0)Et, -SO ₂ Me or -SO ₂ Et.

In one embodiment, a compound of the invention wherein R ⁵ is -L ₂ -R ⁶ and L ₂ is -C(=O)NR ⁷ -, -NR ⁷ C(=O)-, -SO ₂ NR ⁷ - or -NR ⁷ SO ₂ -, wherein R ⁶ and R ⁷ are according to any one of Formulas I to Vf as defined in any one of the preceding embodiments. In certain embodiments, R ⁷ is H, Me or Et, and R ⁶ is as defined in any of the preceding embodiments. In another specific embodiment, R ⁷ is as defined in any one of the preceding embodiments and R ⁶ is H. In yet another specific embodiment, R ⁷ is as defined in any one of the preceding embodiments, and R ⁶ is C _1-6 optionally substituted with one or more independently _selected OH, CN, halo or C _1-4 alkoxy _. is an alkyl In a more specific embodiment, R ⁷ is H, Me or Et and R ⁶ is H. In another more specific embodiment, R ⁷ is H, Me or Et and R ⁶ is Me, Et, iPr or tBu, each optionally selected from one or more independently selected OH, CN, halo or C _1-4 alkoxy is replaced In yet another more specific embodiment, R ⁷ is H, Me or Et and R ⁶ is Me, Et, iPr or tBu, each of which is one or more independently selected OH, CN, F, Cl, OMe or OEt randomly substituted. In the most particular embodiment, R ⁵ is -C(=0)NH ₂ or -SO ₂ NH ₂ .

In one embodiment, a compound of the invention conforms to any of Formulas I-Vf wherein R ^a is H.

In one embodiment, a compound of the invention is according to any of Formulas I-Vf _, wherein R ^a is C _1-4 alkyl. In certain embodiments, R ^a is Me, Et, iPr or tBu. In a more specific embodiment, R ^a is Me or Et. In a most specific embodiment, R ^a is Me.

In one embodiment, a compound of the invention is according to any of Formulas I _- Vf, wherein R ^a is C _1-4 alkyl substituted with one or more independently selected R ^b . In another embodiment, R ^a is Me, Et, iPr or tBu, each of which is substituted with one or more independently selected R ^b . In certain embodiments, R ^a is C _1-4 alkyl substituted with 1, 2 or 3 independently selected _R ^b . In another specific embodiment, R ^a is Me, Et, iPr or tBu, each of which is substituted with 1, 2 or 3 independently selected R ^b . In a more specific embodiment _, R ^a is C _1-4 alkyl substituted with one R ^b . In another more specific embodiment, R ^a is Me, Et, iPr or tBu, each of which is substituted with one R ^b .

In one embodiment, a compound of the invention is according to any of Formulas I to Vf, wherein R ^b is halo, CN or OH. In certain embodiments, R ^b is F, Cl, CN or OH.

In one embodiment, a compound _of the invention is according to any of Formulas I to Vf, wherein R ^b is C _1-4 alkoxy. In certain embodiments, R ^b is OMe, OEt or OiPr. In a more specific embodiment, R ^b is OMe.

In one embodiment, a compound of _the invention is according to any of Formulas I to Vf, wherein R ^b is C _3-7 cycloalkyl. In certain embodiments, R ^b is cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. In a more specific embodiment, R ^b is cyclopropyl.

In one embodiment, a compound of the invention conforms to at least one of Formulas I to Vf, wherein R ^b is a 4 to 7 membered monocyclic heterocycloalkyl comprising one or more heteroatoms independently selected from O, N and S. . In certain embodiments, R ^b is oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl or thiomorpholinyl.

In one embodiment, compounds of the invention are 4 to 7 membered monocyclic compounds wherein R ^b comprises one or more heteroatoms independently selected from O, N and S, substituted with one or more independently selected halo, or oxo. according to at least one of Formulas I to Vf which is a heterocycloalkyl. In certain embodiments, R ^b is oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl or thiomorpholinyl, each of which is substituted with one or more independently selected halo or oxo. In another specific embodiment, R ^b is a 4 to 7 membered monocyclic heterocycloalkyl comprising one or more heteroatoms independently selected from O, N and S, substituted with one or more independently selected F, Cl or oxo. am. In a more specific embodiment, R ^b is oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl or thiomorpholinyl, which are each is substituted with one or more independently selected F, Cl or oxo. In the most particular embodiment, R ^b is a 4 to 7 membered monocyclic group containing one or more heteroatoms independently selected from O, N and S, substituted with 1, 2 or 3 independently selected F, Cl or oxo. Heterocycloalkyl. In another most specific embodiment, R ^b is oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl or thiomorpholinyl; , each of which is substituted with 1, 2 or 3 independently selected F, Cl or oxo.

In one embodiment, a compound of the invention is according to any of Formulas I-Vf _, wherein R ^b is -SO ₂ -C _1-4 alkyl. In certain embodiments, R ^b is -SO ₂ CH ₃ , or -SO ₂ CH ₂ CH ₃ .

In one embodiment, a compound of the invention is of formula I wherein R ^b is -C(=O)NR ^b1 R ^b2 , wherein each R ^bi or R ^b2 is independently selected from H and C _1-4 alkyl _. to Vf. In certain embodiments, each R ^bi or R ^b2 is independently selected from H, -CH ₃ and -CH ₂ CH ₃ . In a more specific embodiment, R ^b is -C(=0)NH ₂ , -C(=0)NMeH, or -C(=0)NMe ₂ .

In one embodiment, a compound of the invention is according to any of Formulas I to Vf, wherein R ^a is C _3-7 monocyclic cycloalkyl. In certain embodiments, R ^a is cyclopropyl, cyclobutyl, or cyclopentyl. In a more specific embodiment, R ^a is cyclopropyl.

In one embodiment, a compound of the invention is according to any of Formulas I-Vf, wherein R ^a is C _3-7 monocyclic cycloalkyl substituted with one or more independently selected R ^c groups. In another embodiment, R ^a is cyclopropyl, cyclobutyl, or cyclopentyl, each substituted with one or more independently selected R ^c groups. In certain embodiments, R ^a is C _3-7 monocyclic cycloalkyl substituted with 1, 2 or 3 independently selected R ^c groups. In another specific embodiment, R ^a is cyclopropyl, cyclobutyl, or cyclopentyl, each of which is substituted with 1, 2 or 3 independently selected R ^c groups. In a more specific embodiment, R ^a is C _3-7 monocyclic cycloalkyl substituted with one R ^c group. In another specific embodiment, R ^a is cyclopropyl, cyclobutyl, or cyclopentyl, each of which is substituted with one R ^c group.

In one embodiment, a compound of the invention is according to any of Formulas I to Vf, wherein R ^c is halo, CN or OH. In certain embodiments, R ^c is F, Cl, CN or OH. In a more specific embodiment, R ^c is F.

In one embodiment, a compound _of the invention is according to any one of Formulas I to Vf, wherein R ^c is C _1-4 alkyl. In certain embodiments, R ^c is -Me, -Et or -iPr.

In one embodiment, a compound of the invention conforms to any one of Formulas I to Vf, wherein R ^c is -C(=0)OH.

In one embodiment, a compound of the invention is of Formula I wherein R ^c is -C(=O)NR ^c1 R ^c2 , wherein each R ^c1 or R ^c2 is independently selected from H and C _1-4 alkyl _. to Vf. In certain embodiments, each R ^c1 or R ^c2 is independently selected from H, -CH ₃ and -CH ₂ CH ₃ . In a more specific embodiment, R ^c is -C(=0)NH ₂ , -C(=0)NMeH, or -C(=0)NMe ₂ .

In one embodiment, a compound of the invention is wherein R ^a is

according to any one of the formulas I to Vf.

In one embodiment, a compound of the invention is wherein R ^a is

according to any one of the formulas I to Vf.

In one embodiment, a compound of the invention is according to any one of Formulas I to Vf, wherein R ^a is a 4 to 7 membered monocyclic heterocycloalkyl comprising one or more heteroatoms independently selected from O, N and S. . In certain embodiments, R ^a is oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl or thiomorpholinyl.

In one embodiment, a compound of the invention is according to any one of Formulas I to Vf, wherein R ^a is a 5-6 membered monocyclic heteroaryl containing one or more heteroatoms independently selected from O, N and S. In certain embodiments, R ^a is furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, triazolyl, oxadiazolyl, tetrazole, pyridinyl, pyrazinyl or pyrimidinyl.

In one embodiment, a compound of the invention is according to Formula I, wherein said compound is selected from:

N-(6-((6-cyano-4-ethylpyridin-3-yl)(methyl)amino)-1-methyl-1H-benzo[d]imidazol-4-yl)cyclopropanecarboxamide;

N-(6-(4-cyano-2-ethyl-6-fluorophenylamino)-1-methyl-1H-benzo[d]imidazol-4-yl)cyclopropanecarboxamide;

N-(6-((4-cyano-2-ethyl-6-fluorophenyl)(methyl)amino)-1-methyl-1H-benzo[d]imidazol-4-yl)cyclopropanecarboxamide ,

methyl 6-((6-cyano-4-ethylpyridin-3-yl)(methyl)amino)-1-methyl-1H-benzo[d]imidazol-4-ylcarbamate;

methyl 6-((4-cyano-2-ethyl-6-fluorophenyl)(methyl)amino)-1-methyl-1H-benzo[d]imidazol-4-ylcarbamate;

(1R,2R)—N-[6-[(6-cyano-4-ethyl-3-pyridyl)oxy]-1-methyl-benzimidazol-4-yl]-2-fluoro-cyclopropane carboxamide,

N-(6-((4-cyano-2-ethyl-6-fluorophenyl)(methyl)amino)-1-methyl-1H-benzo[d]imidazol-4-yl)-2-fluoro Cyclopropanecarboxamide (1S,2S)/(1R,2R) racemic mixture,

(1R,2R)-N-(6-((6-cyano-4-ethylpyridin-3-yl)(methyl)amino)-1-methyl-1H-benzo[d]imidazol-4-yl) -2-fluorocyclopropanecarboxamide;

(1R,2R)-N-(6-((6-cyano-4-methylpyridin-3-yl)(methyl)amino)-1-methyl-1H-benzo[d]imidazol-4-yl) -2-fluorocyclopropanecarboxamide;

(1R,2R)—N-[6-(4-cyano-2-ethyl-phenoxy)-1-methyl-benzimidazol-4-yl]-2-fluoro-cyclopropanecarboxamide;

(1R,2R)-N-[6-(2-chloro-4-cyano-6-fluoro-N-methyl-anilino)-1-methyl-benzimidazol-4-yl]-2-fluoro rho-cyclopropanecarboxamide;

(1R,2R)-2-fluoro-N-[6-[(6-fluoro-4-methyl-3-pyridyl)-methyl-amino]-1-methyl-benzimidazol-4-yl] cyclopropanecarboxamide,

(1R,2R)-N-[6-[(2,6-difluoro-3-pyridyl)-methyl-amino]-1-methyl-benzimidazol-4-yl]-2-fluoro- cyclopropanecarboxamide,

(1R,2R)-N-[6-[(6-cyano-2-fluoro-3-pyridyl)-methyl-amino]-1-methyl-benzimidazol-4-yl]-2-fluoro rho-cyclopropanecarboxamide;

(1R,2R)—N-[6-(4-Cyano-2-fluoro-N-methyl-anilino)-1-methyl-benzimidazol-4-yl]-2-fluoro-cyclopropane carboxamide,

(1R,2R)-2-Fluoro-N-[6-(2-fluoro-N,6-dimethyl-4-methylsulfonyl-anilino)-1-methyl-benzimidazol-4-yl ]Cyclopropanecarboxamide,

(1R,2R)-2-fluoro-N-[6-(2-fluoro-N-methyl-4-methylsulfonyl-anilino)-1-methyl-benzimidazol-4-yl]cyclopropane carboxamide,

N-[6-[(4-ethyl-6-methylsulfonyl-3-pyridyl)-methyl-amino]-1-methyl-benzimidazol-4-yl]cyclopropanecarboxamide;

N-[6-(2-fluoro-N,6-dimethyl-4-methylsulfonyl-anilino)-1-methyl-benzimidazol-4-yl]cyclopropanecarboxamide;

(1R,2R)-N-[6-[(6-cyano-4-methyl-3-pyridyl)oxy]-1-methyl-benzimidazol-4-yl]-2-fluoro-cyclopropane carboxamide,

(1R,2R)-N-[6-[(6-cyano-2-fluoro-4-methyl-3-pyridyl)-methyl-amino]-1-methyl-benzimidazol-4-yl] -2-fluoro-cyclopropanecarboxamide;

(1R,2R)-N-[6-[(2-cyano-3-fluoro-5-methyl-4-pyridyl)-methyl-amino]-1-methyl-benzimidazol-4-yl] -2-fluoro-cyclopropanecarboxamide;

N-[6-[(6-cyano-4-methyl-3-pyridyl)oxy]-1-methyl-benzimidazol-4-yl]cyclopropanecarboxamide;

5-((4-(cyclopropanecarboxamido)-1-methyl-1H-benzo[d]imidazol-6-yl)(methyl)amino)-4-ethylpicolinamide;

4-ethyl-5-((4-((1R,2R)-2-fluorocyclopropanecarboxamido)-1-methyl-1H-benzo[d]imidazol-6-yl)(methyl)amino ) picolinamide,

(1R,2R)-N-[6-(N,2-Dimethyl-4-methylsulfonyl-anilino)-1-methyl-benzimidazol-4-yl]-2-fluoro-cyclopropanca box amide,

(1R,2R)-N-[6-(4-Ethylsulfonyl-N,2-dimethyl-anilino)-1-methyl-benzimidazol-4-yl]-2-fluoro-cyclopropanca box amide,

5-(7-amino-3-methyl-benzimidazol-5-yl)oxy-4-methyl-pyridine-2-carbonitrile;

N-[6-[(4-ethyl-6-methylsulfonyl-3-pyridyl)oxy]-1-methyl-benzimidazol-4-yl]cyclopropanecarboxamide;

N-[6-[4-(cyanomethyl)anilino]-1-methyl-benzimidazol-4-yl]cyclopropanecarboxamide;

N-[6-(2,3-dihydro-1,4-benzodioxin-6-ylamino)-1-methyl-benzimidazol-4-yl]cyclopropanecarboxamide, and

5-[7-[[(1R,2R)-2-fluorocyclopropanecarbonyl]amino]-3-methyl-benzimidazol-5-yl]oxy-4-methyl-pyridine-2-carboxamide .

In one embodiment, a compound of the invention is according to Formula I, wherein said compound is selected from:

1-[6-[(6-cyano-4-methyl-3-pyridyl)oxy]-1-methyl-benzimidazol-4-yl]-3-isopropyl-urea;

4-methyl-5-[3-methyl-7-(methylamino)benzimidazol-5-yl]oxy-pyridine-2-carbonitrile;

5-[7-(dimethylamino)-3-methyl-benzimidazol-5-yl]oxy-4-methyl-pyridine-2-carbonitrile;

N-[6-[(6-Cyano-4-methyl-3-pyridyl)oxy]-1-methyl-benzimidazol-4-yl]-3-hydroxy-azetidine-1-carboxamide ,

N-[6-[(6-cyano-4-methyl-3-pyridyl)oxy]-1-methyl-benzimidazol-4-yl]morpholine-4-carboxamide, and

1-[6-[(6-cyano-4-methyl-3-pyridyl)oxy]-1-methyl-benzimidazol-4-yl]-3-isopropyl-urea.

In one embodiment, a compound of the invention is according to formula I, wherein said compound is N-[6-[(6-cyano-4-methyl-3-pyridyl)oxy]-1-methyl-benz imidazol-4-yl]-2-fluoro-cyclopropanecarboxamide. In a more specific embodiment, a compound of the invention conforms to Formula I, wherein said compound is (1R,2R)-N-[6-[(6-cyano-4-methyl-3-pyridyl)oxy] -1-methyl-benzimidazol-4-yl]-2-fluoro-cyclopropanecarboxamide.

In one embodiment, a compound of the invention is according to formula I, wherein said compound is N-[6-[(6-cyano-4-methyl-3-pyridyl)oxy]-1-methyl-benz imidazol-4-yl]-2-fluoro-cyclopropanecarboxamide. In a more specific embodiment, a compound of the invention conforms to Formula I, wherein said compound is (1R,2R)-N-[6-[(6-cyano-4-methyl-3-pyridyl)oxy] -1-methyl-benzimidazol-4-yl]-2-fluoro-cyclopropanecarboxamide.

In one embodiment, a compound of the invention is according to Formula I, wherein said compound is N-(6-((6-cyano-4-methylpyridin-3-yl)(methyl)amino)-1- methyl-1H-benzo[d]imidazol-4-yl)-2-fluorocyclopropanecarboxamide. In a more specific embodiment, a compound of the invention conforms to Formula I, wherein said compound is (1R,2R)-N-(6-((6-cyano-4-methylpyridin-3-yl)(methyl )amino)-1-methyl-1H-benzo[d]imidazol-4-yl)-2-fluorocyclopropanecarboxamide.

In one embodiment, a compound of the invention is according to Formula I, wherein said compound is N-(6-((6-cyano-4-methylpyridin-3-yl)(methyl)amino)-1- Not methyl-1H-benzo[d]imidazol-4-yl)-2-fluorocyclopropanecarboxamide. In a more specific embodiment, a compound of the invention conforms to Formula I, wherein said compound is (1R,2R)-N-(6-((6-cyano-4-methylpyridin-3-yl)(methyl )amino)-1-methyl-1H-benzo[d]imidazol-4-yl)-2-fluorocyclopropanecarboxamide.

In one embodiment, the compounds of the invention are not isotopic variants.

In one aspect, a compound of the invention according to any one of the embodiments disclosed herein exists as a free base.

In one aspect, a compound of the invention according to any one of the embodiments disclosed herein is a pharmaceutically acceptable salt.

In one aspect, a compound of the invention according to any one of the embodiments disclosed herein is a solvate of said compound.

In one aspect, a compound of the invention according to any one of the embodiments disclosed herein is a solvate of a pharmaceutically acceptable salt of the compound.

Although the specified groups of each embodiment are separately and generally listed above, the compounds of the present invention are those specific members or groups each represented for each variable in multiple or individual embodiments of the formulas above as well as other formulas provided herein. is selected from one or more of these. Accordingly, the present invention is intended to include within its scope all combinations of such embodiments.

Although groups specified for each embodiment are generally listed separately above, compounds of the present invention wherein one or more variables (eg R groups) are selected from among one or more embodiments according to any one of the formula(s) listed above. it could be Accordingly, the present invention is intended to include within its scope all combinations of variables from any one of the disclosed embodiments.

Alternatively, the exclusion of one or more of the specified variables, or combinations thereof, from a group or embodiment is also contemplated by the present invention.

In some embodiments, the present invention provides prodrugs and derivatives of compounds according to the formulas above. A prodrug is a derivative of a compound of the present invention that is pharmacologically active in vivo that has a metabolically cleavable group and which yields the compound of the present invention by solvolysis or under physiological conditions. Such examples include, but are not limited to, choline ester derivatives and the like, N-alkylmorpholine esters and the like.

Other derivatives of the compounds of the present invention are active in both acid and acid derivative forms of the compounds, but often the acid-sensitive forms provide the advantages of solubility, tissue compatibility, or delayed release in mammalian organisms (Bundgard H, 1985). Prodrugs are acid derivatives well known to those skilled in the art, such as esters prepared by the reaction of the parent acid with a suitable alcohol, or amides prepared by the reaction of the parent acid compound with a substituted or unsubstituted amine; or acid anhydrides, or mixed anhydrides. Simple aliphatic or aromatic esters, amides and anhydrides derived from acidic groups pendant to the compounds of this invention are preferred prodrugs. In some cases it is desirable to prepare prodrugs of the double ester type, for example (acyloxy)alkyl esters or ((alkoxycarbonyl)oxy)alkylesters. C ₁ to C ₈ alkyl, C ₂ -C ₈ alkenyl, aryl, C ₇ -C ₁₂ substituted aryl, and C ₇ -C ₁₂ arylalkyl esters of the compounds of the present invention are particularly useful.

article

1) A compound according to Formula I, or a pharmaceutically acceptable salt or solvate, or a solvate of said pharmaceutically acceptable salt:

Formula I

In the above formula,

R ¹ is H or Me;

L ₁ is -NR ² -; -O- or -CH ₂ -;

Cy is phenyl or a 5 to 9 membered monocyclic or fused bicyclic heteroaryl containing 1 to 4 heteroatoms independently selected from N, O and S;

R ² is H or C _1-4 alkyl;

R ³ is H, halo, C _1-4 alkyl optionally substituted with one or more halo, or C _1-4 alkoxy optionally substituted with one or more halo;

R ⁴ is H or halo;

R ⁵ is -CN, halo, or -L ₂ -R ⁶ ;

-L ₂ is absent, or -C(=O)-, -C(=O)NR ⁷ -, -NR ⁷ C(=O)-, -SO ₂ -, -SO ₂ NR ⁷ -, or -NR ⁷ SO ₂ -;

R ⁶ is H or C _1-6 alkyl optionally substituted with one or more independently selected R ⁸ groups;

R ⁷ is H or C _1-4 alkyl;

R ⁸ is OH, CN, halo or C _1-4 alkoxy;

L _a is absent or -C(=0)-, -C(=0)0- or -C(=0)NH-;

R ^a is

-H,

- C _1-4 alkyl optionally substituted with one or more independently selected R ^b ,

- C _3-7 monocyclic cycloalkyl optionally substituted with one or more independently selected R ^c , or

- 4 to 7 membered monocyclic heterocycloalkyl containing one or more heteroatoms independently selected from O, N and S, or

- a 5 to 6 membered monocyclic heteroaryl containing at least one heteroatom independently selected from O, N and S;

^Rb is

- halo,

-CN,

-OH,

- C _1-4 alkoxy;

- C _3-7 cycloalkyl,

- 4 to 7 membered monocyclic heterocycloalkyl containing one or more heteroatoms independently selected from O, N and S, wherein said heterocycloalkyl is optionally substituted with one or more independently selected halo or oxo;

- -SO ₂ -C _1-4 alkyl, or

- -C(=O)NR ^b1 R ^b2 and

R ^c is

- halo,

-CN,

-OH,

- C _1-4 alkyl;

- -C(=O)OH, or

- -C(=0)NR ^c1 R ^c2 ;

Each of R ^bi , R ^b2 , R ^c1 and R ^c2 is independently selected from H and C _1-4 alkyl.

2) In the compound or pharmaceutically acceptable salt according to item 1, R ¹ is Me.

3) In the compound or pharmaceutically acceptable salt according to item 1 or item 2, Cy is phenyl.

4) In the compound according to 1 or 2 or a pharmaceutically acceptable salt thereof, Cy is a 5- to 9-membered monocyclic or condensed fused ring containing 1 to 4 heteroatoms independently selected from N, O and S It is a bicyclic heteroaryl.

5) In the compound according to item 1 or item 2 or a pharmaceutically acceptable salt thereof, the 5- to 6-membered monocyclic heteroaryl contains 1 to 4 heteroatoms independently selected from N, O and S.

6) In the compound or pharmaceutically acceptable salt according to item 1 or item 2, Cy is pyridyl.

7) A compound or pharmaceutically acceptable salt according to item 1, wherein said compound conforms to the following formulas IIa, IIb or IIc:

[Formula IIa] [Formula IIb] [Formula IIc]

In the above formulas, L ₁ , R ³ , R ⁴ , L _a , R ^a and R ⁵ are as described in item 1.

8) The compound or pharmaceutically acceptable salt according to item 1, wherein the compound conforms to the following formula IIIa, IIIb or IIIc:

[Formula IIIa] [Formula IIIb] [Formula IIIc]

In the above formulas, L ₁ , R ³ , R ⁴ , L _a , R ^a and R ⁵ are as described in item 1.

9) In the compound or pharmaceutically acceptable salt according to any one of items 1 to 8, L ₁ is -CH ₂ -.

10) In the compound or pharmaceutically acceptable salt according to any one of items 1 to 8, L ₁ is O.

11) In the compound or pharmaceutically acceptable salt according to any one of items 1 to 8, L ₁ is -NR ² -.

12) In the compound or pharmaceutically acceptable salt according to item 11, R ² is H.

13) In the compound or pharmaceutically acceptable salt according to item 11, R ² is C _1-4 alkyl.

14) In the compound or pharmaceutically acceptable salt according to item 11, R ² is Me.

15) A compound or pharmaceutically acceptable salt according to item 1, wherein said compound conforms to the following formulas IVa, IVb, IVc, IVd, IVe or IVf:

[Formula IVa] [Formula IVb] [Formula IVc]

[Formula IVd] [Formula IVe] [Formula IVf]

In the above formulas, R ³ , R ⁴ , R ⁵ , L _a and R ^a are as described in item 1.

16) In the compound or pharmaceutically acceptable salt according to any one of items 1 to 15, L _a is absent.

17) In the compound or pharmaceutically acceptable salt according to any one of items 1 to 15, L _a is -C(=O)-.

18) A compound or pharmaceutically acceptable salt according to item 1, wherein said compound conforms to the following formulas Va, Vb, Vc, Vd, Ve or Vf:

[Formula Va] [Formula Vb] [Formula Vc]

[Formula Vd] [Formula Ve] [Formula Vf]

In the above formulas, R ³ , R ⁴ , R ⁵ , L _a and R ^a are as described in item 1.

19) In the compound or pharmaceutically acceptable salt according to any one of items 1 to 18, R ⁴ is H.

20) In the compound or pharmaceutically acceptable salt according to any one of items 1 to 18, R ⁴ is F or Cl.

21) In the compound or pharmaceutically acceptable salt according to any one of items 1 to 18, R ³ is H.

₂₂ ) In the compound or pharmaceutically acceptable salt according to any one of items 1 to 18, R ³ is C _1-4 alkyl.

23) In the compound or pharmaceutically acceptable salt according to item 22, R ³ is Me or Et.

24) In the compound or pharmaceutically acceptable salt according to any one of items 1 to 23, R ⁵ is CN.

25) A compound or pharmaceutically acceptable salt according to any one of items 1 to 23, wherein R ⁵ is halo.

26) In the compound or pharmaceutically acceptable salt according to item 25, R ⁵ is F or Cl.

27) In the compound or pharmaceutically acceptable salt according to any one of items 1 to 23, R ⁵ is -L ₂ -R ⁶ , R ⁶ is as described in item 1 and L ₂ is -C( =O)NR ⁷ -, -NR ⁷ C(=O)-, -SO ₂ NR ⁷ - or -NR ⁷ SO ₂ -.

28) In the compound or pharmaceutically acceptable salt according to item 27, R ⁷ is H.

29) In the compound or pharmaceutically acceptable salt according to any one of items 1 to 23, R ⁵ is -L ₂ -R ⁶ , L ₂ is as described in item 1 and R ⁶ is H.

30) In the compound or pharmaceutically acceptable salt according to any one of items 1 to 26, R ⁵ is -L ₂ -R ⁶ , L ₂ is as described in item 1 and R ⁶ is C _{1- 6} alkyl.

31) In the compound or pharmaceutically acceptable salt according to item 30, R ⁶ is Me.

32) In the compound or pharmaceutically acceptable salt according to any one of items 1 to 23, R ⁵ is -L ₂ -R ⁶ , L ₂ is as described in item 1 and R ⁶ is one or more independent is C _1-6 alkyl substituted with _an R ⁸ group selected from .

33) The compound or pharmaceutically acceptable salt according to item 32, wherein R ⁶ is Me, Et, iPr or tBu, each of which is substituted with one or more independently selected R ⁸ groups.

34) In the compound or pharmaceutically acceptable salt according to item 33, R ⁸ is OH, CN, F, Cl, -OMe or -OEt.

35) In the compound or pharmaceutically acceptable salt according to any one of items 1 to 23, R ⁵ is -L ₂ -R ⁶ , L ₂ is -C(=O)- or -SO ₂ - and _, R ⁶ is C _1-6 alkyl.

36) In the compound or pharmaceutically acceptable salt according to item 35, R ⁵ is -C(=0)Me, -C(=0)Et, -SO ₂ Me or -SO ₂ Et.

37) In the compound or pharmaceutically acceptable salt according to any one of items 1 to 23, R ⁵ is -L ₂ -R ⁶ and L ₂ is -C(=O)NR ⁷ -, -NR ⁷ C(=0)-, -SO ₂ NR ⁷ - or -NR ⁷ SO ₂ -, R ⁷ is H, Me or Et, R ⁶ is Me, Et, iPr or tBu, each of which is independently one or more optionally substituted with selected _OH , CN, halo or C _1-4 alkoxy.

38) In the compound or pharmaceutically acceptable salt according to item 37, R ⁵ is -C(=O)NH ₂ or -SO ₂ NH ₂ .

₃₉ ) In the compound or pharmaceutically acceptable salt according to any one of items 1 to 38, R ^a is C _1-4 alkyl.

40) In the compound or pharmaceutically acceptable salt according to item 39, R ^a is Me or Et.

41) The compound or pharmaceutically acceptable salt according to any one of items 1 to 38, _wherein R ^a is C _1-4 alkyl substituted with one or more independently selected R ^b .

42) In the compound or pharmaceutically acceptable salt according to item 41, R ^a is Me, Et, iPr or tBu.

43) In the compound or pharmaceutically acceptable salt according to item 41 or item 42, R ^b is F, Cl, CN or OH.

44) In the compound or pharmaceutically acceptable salt according to item 41 or item 42, R ^b is OMe, OEt or OiPr.

45) The compound or pharmaceutically acceptable salt according to item 41 or 42, wherein R ^b is cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.

46) In the compound or pharmaceutically acceptable salt according to item 41 or item 42, R ^b is oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, azetidinyl, pyrrolidinyl, piperidinyl, pipera zinyl, morpholinyl or thiomorpholinyl.

47) In the compound or pharmaceutically acceptable salt according to item 41 or item 42, R ^b is oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, azetidinyl, pyrrolidinyl, piperidinyl, pipera zinyl, morpholinyl or thiomorpholinyl, each substituted with one or more independently selected halo or oxo.

48) In the compound or pharmaceutically acceptable salt according to item 41 or 42, R ^b is -SO ₂ CH ₃ , or -SO ₂ CH ₂ CH ₃ .

49) The compound or pharmaceutically acceptable salt according to item 41 or 42, wherein R ^b is -C(=0)NH ₂ , -C(=0)NMeH, or -C(=0)NMe ₂ .

50) In the compound or pharmaceutically acceptable salt according to any one of items 1 to 38, R ^a is C _3-7 monocyclic cycloalkyl.

51) The compound or pharmaceutically acceptable salt according to any one of items 1 to 38, wherein R ^a is C _3-7 monocyclic cycloalkyl substituted with one or more independently selected R ^c groups.

52) The compound or pharmaceutically acceptable salt according to item 50 or 51, wherein R ^a is cyclopropyl, cyclobutyl, or cyclopentyl.

53) In the compound or pharmaceutically acceptable salt according to item 51, R ^c is F, Cl, CN or OH.

54) In the compound or pharmaceutically acceptable salt according to item 51, R ^c is -Me, -Et or -iPr.

55) In the compound or pharmaceutically acceptable salt according to item 51, R ^c is -C(=0)OH.

56) The compound or pharmaceutically acceptable salt according to item 51, wherein R ^c is -C(=0)NH ₂ , -C(=0)NMeH, or -C(=0)NMe ₂ .

57) In the compound or pharmaceutically acceptable salt according to any one of items 1 to 38, R ^a is

am.

58) In the compound or pharmaceutically acceptable salt according to any one of items 1 to 38, R ^a is

am.

59) In the compound or pharmaceutically acceptable salt according to any one of items 1 to 38, R ^a is a 4 to 7 membered monocyclic heteroatom containing one or more heteroatoms independently selected from O, N and S It is cycloalkyl.

60) In the compound or pharmaceutically acceptable salt according to item 59, R ^a is oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, polynyl or thiomorpholinyl.

61) In the compound or pharmaceutically acceptable salt according to any one of items 1 to 38, R ^a is a 5 to 6 membered monocyclic heteroatom containing one or more heteroatoms independently selected from O, N and S it is aryl

62) In the compound or pharmaceutically acceptable salt according to item 61, R ^a is furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, triazolyl, oxadiazolyl, tetrazole, pyr dinyl, pyrazinyl or pyrimidinyl.

63) The compound according to item 1 or a pharmaceutically acceptable salt thereof, wherein said compound is selected from Table I.

64) The compound according to item 1 or a pharmaceutically acceptable salt thereof, wherein the compound is (1R,2R)-N-[6-[(6-cyano-4-methyl-3-pyridyl)oxy]- 1-methyl-benzimidazol-4-yl]-2-fluoro-cyclopropanecarboxamide.

65) The compound according to item 1 or a pharmaceutically acceptable salt thereof, wherein the compound is (1R,2R)-N-[6-[(6-cyano-4-methyl-3-pyridyl)oxy]- 1-methyl-benzimidazol-4-yl]-2-fluoro-cyclopropanecarboxamide.

66) In the compound according to item 1 or a pharmaceutically acceptable salt thereof,

The compound is (1R,2R)-N-(6-((6-cyano-4-methylpyridin-3-yl)(methyl)amino)-1-methyl-1H-benzo[d]imidazole-4 -yl)-2-fluorocyclopropanecarboxamide.

67) The compound according to item 1 or a pharmaceutically acceptable salt thereof, wherein the compound is (1R,2R)-N-(6-((6-cyano-4-methylpyridin-3-yl)(methyl) Amino)-1-methyl-1H-benzo[d]imidazol-4-yl)-2-fluorocyclopropanecarboxamide.

68) A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a pharmaceutically effective amount of a compound according to any one of items 1 to 67.

69) The pharmaceutical composition according to item 68 comprising an additional therapeutic agent.

70) A compound according to any one of items 1 to 67, or a pharmaceutical composition according to item 68 or 69, for use in medicine.

71) Allergic diseases, inflammatory diseases, autoimmune diseases, proliferative diseases, graft rejection, diseases involving disorders of cartilage turnover, congenital cartilage malformations, and/or diseases associated with hypersecretion of IL6 or hypersecretion of interferon A compound according to any one of claims 1 to 67, or a pharmaceutical composition according to claims 68 or 69, for use in treatment or prophylaxis.

72) A compound according to any one of items 1 to 67, or a pharmaceutical composition according to item 68 or 69, for use in the treatment or prevention of a proliferative disease.

73) In the compound according to item 72, the proliferative disease is myelofibrosis, T-cell acute lymphoblastic leukemia (T-ALL), multiple myeloma, chronic lymphoblastic leukemia (CLL), diffuse large B-cell lymphoma ( DLBCL), pancreatic cancer, liver cancer, hepatocellular carcinoma (HCC), lung cancer, breast cancer and colon cancer.

74) Allergic diseases, inflammatory diseases, autoimmune diseases, proliferative diseases, graft rejection, diseases involving disorders of cartilage turnover, congenital cartilage malformations, and/or diseases associated with hypersecretion of IL6 or hypersecretion of interferon A method for treating or preventing said disease, comprising administering a compound according to any one of claims 1 to 67, or a pharmaceutical composition according to claim 68 or 69, in an amount sufficient to effect treatment or prevention.

75) Treatment of a proliferative disease, comprising administering a compound according to any one of items 1 to 67, or a pharmaceutical composition according to item 68 or 69, in an amount sufficient to effect the treatment or prevention of said disease. or preventive methods.

76) In the method of treatment according to item 76, the proliferative disease is myelofibrosis, T-cell acute lymphoblastic leukemia (T-ALL), multiple myeloma, chronic lymphoblastic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), pancreatic cancer, liver cancer, hepatocellular carcinoma (HCC), lung cancer, breast cancer and colon cancer.

77) In the method according to any one of items 75 to 77, the compound according to any one of items 1 to 67 or the pharmaceutical composition according to items 68 or 69 is administered together with an additional therapeutic agent.

78) In the pharmaceutical composition according to item 69, or in the method according to item 78, the additional therapeutic agent is an allergic disease, an inflammatory disease, an autoimmune disease, a proliferative disease, transplant rejection, a disease involving disorders of cartilage turnover, congenital It is an agent for the treatment or prevention of a cartilage deformity, and/or a disease associated with hypersecretion of IL6 or hypersecretion of interferon.

79) The pharmaceutical composition according to item 69, or the method according to item 78, wherein the additional therapeutic agent is myelofibrosis, T-cell acute lymphoblastic leukemia (T-ALL), multiple myeloma, chronic lymphocytic leukemia (CLL), It is an agent for the treatment or prevention of diffuse large B-cell lymphoma (DLBCL), pancreatic cancer, liver cancer, hepatocellular carcinoma (HCC), lung cancer, breast cancer and colon cancer.

pharmaceutical composition

When the compounds of the present invention are used as pharmaceuticals, they are typically administered in the form of pharmaceutical compositions. Such compositions may be prepared in a manner well known in the pharmaceutical art and comprise one or more active compounds of the present invention according to formula (I). Generally, a compound of the present invention is administered in a pharmaceutically effective amount. The amount of compound actually administered will typically be determined by the physician in light of the relevant circumstances, such as the disease being treated, the route of administration selected, the compound actually administered, the age, weight and response of the individual patient, the severity of the patient's symptoms, and the like. .

The pharmaceutical composition of the present invention can be administered by various routes including oral, rectal, transdermal, subcutaneous, intra-articular, intravenous, intramuscular and intranasal. Depending on the intended route of delivery, the compounds of the present invention are preferably formulated as injectable or oral compositions or as ointments, lotions or patches for all transdermal administration.

Compositions for oral administration may take the form of bulk liquid solutions or suspensions, or bulk powders. More commonly, however, the compositions are presented in unit dosage form to facilitate precise dosing. The term "dosage unit form" refers to physically discrete units suited as unitary dosages for human patients and other mammals, each unit containing a predetermined amount of active substance calculated to produce the desired therapeutic effect. , together with a suitable pharmaceutical excipient, vehicle or carrier. Typical unit dosage forms include prefilled or premeasured ampoules or syringes of liquid compositions or, in the case of solid compositions, pills, tablets, capsules, and the like. In such compositions, the compound of the present invention according to formula (I) is usually a minor component (from about 0.1 to about 50% by weight or preferably from about 1 to about 40% by weight), with the remainder in various vehicles or carriers and the desired dosage. It is a processing aid that helps mold formation.

Liquid forms suitable for oral administration may include a suitable aqueous or non-aqueous vehicle, along with buffers, suspending and dispensing agents, coloring agents, flavoring agents and the like. A solid form may include, for example, any of the following ingredients or a compound of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch or lactose, disintegrants such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavoring.

Injectable compositions are typically based on sterile injectable saline or phosphate-buffered saline or other injectable carriers known in the art. As before, the active compound of the present invention according to formula (I) in such compositions is typically a minor component (often about 0.05 to 10% by weight), the balance being the injectable carrier and the like.

Transdermal compositions typically contain from about 0.01% to about 20%, preferably from about 0.1% to about 20%, preferably from about 0.1% to about 10%, and more preferably from about 0.1% to about 10% by weight of the active ingredient(s). It is formulated as a topical ointment or cream containing in an amount ranging from about 0.5 to about 15% by weight. When formulated as an ointment, the active ingredient will typically be combined with a paraffinic or water-miscible ointment base. Alternatively, the active ingredient may be formulated into a cream with, for example, an oil-in-water cream base. Such transdermal formulations are well known in the art and generally include additional ingredients to enhance the skin penetration stability of the active ingredient or the formulation. All such known transdermal formulations and ingredients are included within the scope of the present invention.

Compounds of the present invention may also be administered by transdermal devices. Thus, transdermal administration can be carried out using a patch of a water bath or porous membrane type, or a solid matrix lattice.

The foregoing ingredients for oral administration, injectable or topical administration compositions are exemplary only. Other materials, as well as processing techniques, are listed in Part 8 of Remington's Pharmaceutical Sciences, 17th edition, 1985, Mack Publishing Company, Easton, Pennsylvania, incorporated herein by reference.

The compounds of the present invention may also be administered in a sustained release form or from a sustained release drug delivery system. Specifications of typical slow-release materials can be found in Remington's Pharmaceutical Sciences.

The following formulation examples illustrate typical pharmaceutical compositions that can be prepared according to the present invention. However, the present invention is not limited to the following pharmaceutical compositions.

Formulation 1 - Tablets

A compound of the invention according to formula I may be mixed as a dry powder with a dry gelatine binder in an approximate 1:2 weight ratio. A small amount of magnesium stearate may be added as a lubricant. This mixture can be formed into 240-270 mg tablets (80-90 mg of active compound according to formula I per tablet) in a tablet press.

Formulation 2 - Capsule

A compound of the invention according to formula I may be mixed as a dry powder with a starch diluent in an approximate 1:1 weight ratio. This mixture can be filled into 250 mg capsules (125 mg of active compound according to formula I per capsule).

Formulation 3 - Liquid

A compound of the present invention (125 mg) was mixed with sucrose (1.75 g) and xanthan gum (4 mg) and the resulting mixture was blended and strained on a No. 10 mesh U.S. It can be passed through a sieve and then mixed with a previously prepared solution of microcrystalline cellulose and sodium carboxymethyl cellulose (11:89, 50 mg) in water. Sodium benzoate (10 mg), flavor and color may be diluted with water and added with stirring. Enough water can then be added with stirring. Sufficient water can then be added to create a total volume of 5 ml.

Formulation 4 - Tablets

A compound of the invention according to formula I may be mixed as a dry powder with a dry gelatine binder in an approximate 1:2 weight ratio. A small amount of magnesium stearate may be added as a lubricant. This mixture is formed into 450-900 mg tablets (150-300 mg of active compound of the invention according to formula I) in a tablet press.

Formulation 5 - Injectable solution

A compound of the present invention according to formula I may be dissolved or suspended in aqueous buffered sterile saline injectable aqueous medium at a concentration of approximately 5 mg/ml.

Formulation 6 - Topical

Stearyl alcohol (250 g) and white petrolatum (250 g) were melted at about 75° C. followed by a compound of the invention according to formula I (50 g) dissolved in water (about 370 g), methylparaben (0.25 g) , propylparaben (0.15 g), sodium lauryl sulfate (10 g), and propylene glycol (120 g) are added and the resulting mixture may be stirred until congealed.

treatment method

The compounds of the present invention can be used as therapeutic agents for the treatment of conditions in mammals that are causally related to or attributable to the abnormal activity of JAK. In particular, the condition is associated with aberrant activity of JAK1 and/or TYK2. Thus, the compounds and pharmaceutical compositions of the present invention can be used in mammals, including humans, for allergic diseases, inflammatory diseases, autoimmune diseases, proliferative diseases, transplant rejection, diseases involving disorders of cartilage turnover, congenital cartilage malformations, and/or or as a therapeutic agent for the prevention and/or treatment of diseases associated with hypersecretion of IL6 or hypersecretion of interferon.

In one aspect, the present invention provides a compound of the present invention, or a pharmaceutical composition comprising a compound of the present invention, for use as a medicament.

In another aspect, the present invention provides a compound of the present invention, or a pharmaceutical composition comprising a compound of the present invention, for use in the manufacture of a medicament.

In yet another aspect, the present invention provides a method for treating a mammal suffering from or at risk of suffering from a disease as described herein, said method comprising an effective condition-treating or condition-preventing amount of a condition-disclosed amount of the present invention. and administering one or more of the pharmaceutical composition and compound. In certain embodiments, the present invention relates to allergic diseases, inflammatory diseases, autoimmune diseases, proliferative diseases, graft rejection, diseases involving impaired cartilage turnover, congenital cartilage malformations, and/or hypersecretion of IL6 or secretion of interferon. A method for treating a mammal suffering from or at risk of having an overabundance-related disease is provided.

In a method of treatment aspect, the present invention provides a method for the prevention and/or treatment of a mammal susceptible to or affected by an allergic reaction, the method comprising an effective condition-treating or condition-preventing amount of the present invention. and administering one or more of the pharmaceutical compositions and compounds of the present invention as disclosed. In a particular embodiment, the allergic reaction is selected from allergic airway disease, sinusitis, eczema and urticaria, food allergy or allergy to insect poisons.

In another aspect the invention provides a compound of the invention for use in the treatment and/or prevention of an allergic reaction. In a particular embodiment, the allergic reaction is selected from allergic airway disease, sinusitis, eczema and urticaria, food allergy and allergy to insect poisons.

In yet another aspect the present invention provides a compound of the present invention or a pharmaceutical composition comprising a compound of the present invention for use in the manufacture of a medicament for the treatment or prevention of an allergic reaction. In a particular embodiment, the allergic reaction is selected from allergic airway disease, sinusitis, eczema and urticaria, food allergy and allergy to insect poisons.

In a further method of treatment aspect, the present invention provides methods for the prophylaxis and/or treatment of a mammal susceptible to or affected by an inflammatory condition. The method comprises administering an effective condition-treating or condition-preventing amount of one or more of the pharmaceutical compositions and compounds of the present invention as described herein. In a specific embodiment, the inflammatory condition is selected from rheumatoid arthritis, osteoarthritis, allergic airway disease (eg asthma) and inflammatory bowel disease.

In another aspect the invention provides a compound of the invention for use in the treatment and/or prevention of inflammatory conditions. In a specific embodiment, the inflammatory condition is selected from rheumatoid arthritis, osteoarthritis, allergic airway disease (eg asthma) and inflammatory bowel disease.

In yet another aspect, the present invention provides a compound of the present invention or a pharmaceutical composition comprising a compound of the present invention for use in the manufacture of a medicament for the treatment and/or prevention of an inflammatory condition. In a specific embodiment, the inflammatory condition is selected from rheumatoid arthritis, osteoarthritis, allergic airway disease (eg asthma) and inflammatory bowel disease (eg Crohn's disease or ulcerative colitis).

In a further method of treatment aspect, the present invention provides a method for preventing or treating a mammal susceptible to or suffering from an autoimmune disease. The method comprises administering an effective condition-treating or condition-preventing amount of one or more of the pharmaceutical compositions and compounds of the present invention as described herein. In a specific embodiment, the autoimmune disease is selected from COPD, asthma, systemic lupus erythematosus, type I diabetes, and inflammatory bowel disease.

In another aspect the invention provides a compound of the invention for use in the treatment and/or prevention of autoimmune diseases. In a specific embodiment, the autoimmune disease is selected from COPD, asthma, systemic lupus erythematosus, type I diabetes, and inflammatory bowel disease. In a more specific embodiment, the autoimmune disease is systemic lupus erythematosus.

In yet another aspect, the present invention provides a compound of the present invention, or a pharmaceutical composition comprising a compound of the present invention, for use in the manufacture of a medicament for the treatment and/or prevention of autoimmune diseases. In a specific embodiment, the autoimmune disease is selected from COPD, asthma, systemic lupus erythematosus, type I diabetes, and inflammatory bowel disease.

In a further method of treatment aspect, the present invention provides a method for the prophylaxis and/or treatment of a mammal susceptible to or suffering from a proliferative disease, said method comprising an effective condition-treating or condition-preventing amount of the present invention. and administering one or more of the pharmaceutical compositions and compounds of the present invention as described in In a specific embodiment, the proliferative disease is selected from cancer (eg solid tumors such as uterine leiomyosarcoma or prostate cancer), leukemia (eg AML, ALL or CLL), multiple myeloma and psoriasis. In a more specific embodiment, the proliferative disorder is myelofibroma, T-cell acute lymphoblastic leukemia (T-ALL), multiple myeloma, chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), It is selected from pancreatic cancer, liver cancer, hepatocellular carcinoma (HCC), lung cancer, breast cancer and colon cancer.

In another aspect the invention provides a compound of the invention for use in the treatment and/or prevention of a proliferative disease. In a specific embodiment, the proliferative disease is selected from cancer (eg solid tumors such as uterine leiomyosarcoma or prostate cancer), leukemia (eg AML, ALL or CLL), multiple myeloma and psoriasis. In a more specific embodiment, the proliferative disorder is myelofibroma, T-cell acute lymphoblastic leukemia (T-ALL), multiple myeloma, chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), It is selected from pancreatic cancer, liver cancer, hepatocellular carcinoma (HCC), lung cancer, breast cancer and colon cancer.

In yet another aspect, the present invention provides a compound of the present invention, or a pharmaceutical composition comprising a compound of the present invention, for use in the manufacture of a medicament for the treatment and/or prevention of a proliferative disease. In a specific embodiment, the proliferative disease is selected from cancer (eg solid tumors such as uterine leiomyosarcoma or prostate cancer), leukemia (eg AML, ALL or CLL), multiple myeloma and psoriasis. In a more specific embodiment, the proliferative disorder is myelofibroma, T-cell acute lymphoblastic leukemia (T-ALL), multiple myeloma, chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), It is selected from pancreatic cancer, liver cancer, hepatocellular carcinoma (HCC), lung cancer, breast cancer and colon cancer.

In a further method of treatment aspect, the present invention provides a method for the prevention and/or treatment of a mammal susceptible to or afflicted with transplant rejection, said method comprising an effective condition-treating or condition-preventing amount of the present invention. and administering one or more of the pharmaceutical compositions and compounds of the present invention as disclosed. In a specific embodiment, the transplant rejection is an organ transplant rejection.

In another aspect the invention provides a compound of the invention for use in the treatment and/or prevention of transplant rejection. In a specific embodiment, the transplant rejection is an organ transplant rejection.

In yet another aspect, the present invention provides a compound of the present invention, or a pharmaceutical composition comprising a compound of the present invention, for use in the manufacture of a medicament for the treatment and/or prevention of transplant rejection. In a specific embodiment, the transplant rejection is an organ transplant rejection.

In a method of treatment aspect, the present invention provides a method for preventing and/or treating a mammal susceptible to or suffering from a disease involving impaired cartilage turnover, the method comprising a therapeutically effective amount of a compound of the present invention; or administering one or more of the pharmaceutical compositions disclosed herein.

In another aspect, the present invention provides a compound of the present invention for use in the treatment and/or prevention of diseases involving impairment of cartilage turnover.

In yet another aspect, the present invention provides a compound of the present invention, or a pharmaceutical composition comprising a compound of the present invention, for use in the manufacture of a medicament for the treatment and/or prevention of diseases involving disorders of cartilage turnover. .

The present invention also provides a method for treating and/or preventing congenital cartilage deformities, comprising administering an effective amount of one or more of the pharmaceutical compositions and compounds of the present invention disclosed herein.

In another aspect, the present invention provides a compound of the present invention for use in the treatment and/or prevention of congenital cartilage malformations.

In yet another aspect, the present invention provides a compound of the present invention, or a pharmaceutical composition comprising a compound of the present invention, for use in the manufacture of a medicament for the treatment and/or prevention of congenital cartilage malformations.

In a further method of treatment aspect, the present invention provides a method for preventing and/or treating a mammal susceptible to or suffering from a disease associated with hypersecretion of IL6, the method comprising an effective condition-treatment or condition-prevention. and administering an amount of one or more of the pharmaceutical compositions and compounds of the present invention as described herein. In a specific embodiment, the disease associated with hypersecretion of IL6 is selected from Castleman's disease and mesangial cell proliferative glomerulonephritis.

In another aspect, the present invention provides a compound of the present invention for use in the treatment and/or prevention of diseases associated with hypersecretion of IL6. In a specific embodiment, the disease associated with hypersecretion of IL6 is selected from Castleman's disease and mesangial cell proliferative glomerulonephritis.

In another aspect, the present invention provides a compound of the present invention, or a pharmaceutical composition comprising a compound of the present invention, for use in the manufacture of a medicament for the treatment and/or prevention of diseases associated with hypersecretion of IL6. In a specific embodiment, the disease associated with hypersecretion of IL6 is selected from Castleman's disease and mesangial cell proliferative glomerulonephritis.

In a further method of treatment aspect, the present invention provides a method for preventing and/or treating a mammal susceptible to or suffering from a disease associated with hypersecretion of interferon, the method comprising an effective condition-treatment or condition-prevention and administering an amount of one or more of the pharmaceutical compositions and compounds of the present invention as described herein. In a specific embodiment, the disease associated with hypersecretion of interferons is selected from systemic and cutaneous lupus erythematosus, lupus nephritis, dermatomyositis, Sjogren's syndrome, psoriasis and rheumatoid arthritis.

In another aspect, the invention provides a compound of the invention for use in the treatment and/or prevention of diseases associated with hypersecretion of interferons. In a specific embodiment, the disease associated with hypersecretion of interferons is selected from systemic and cutaneous lupus erythematosus, lupus nephritis, dermatomyositis, Sjogren's syndrome, psoriasis and rheumatoid arthritis.

In yet another aspect, the present invention provides a compound of the present invention, or a pharmaceutical composition comprising a compound of the present invention, for use in the manufacture of a medicament for the treatment and/or prevention of diseases associated with hypersecretion of interferon. In a specific embodiment, the disease associated with hypersecretion of interferons is selected from systemic and cutaneous lupus erythematosus, lupus nephritis, dermatomyositis, Sjogren's syndrome, psoriasis and rheumatoid arthritis.

As a further aspect of the present invention there is provided a compound of the present invention for use as a medicament, in particular for the treatment and/or prophylaxis of the conditions and diseases mentioned above. Also provided herein is the use of a compound of the present invention in the manufacture of a medicament for the treatment and/or prevention of one of the conditions and diseases mentioned above.

A particular regimen of the method of the present invention provides a subject suffering from a condition involving inflammation in an effective amount of the present invention for a period of time sufficient to reduce the level of the inflammation in the subject and preferably terminate the causal process of the inflammation. and administering the compound. A particular embodiment of the method is directed to a test subject suffering from or susceptible to developing rheumatoid arthritis, respectively, to reduce or prevent inflammation in the subject's joints, preferably to terminate the causal process of the inflammation. and administering an effective amount of a compound of the present invention for a sufficient period of time.

A further particular regimen of the methods of the present invention reduces, preferably alleviates, a disease state characterized by cartilage or joint degeneration (e.g. rheumatoid arthritis and/or osteoarthritis) in a subject suffering from such degeneration. and administering an effective amount of a compound of the present invention for a period of time sufficient to terminate the spontaneously continuing causal process. A particular embodiment of the method is directed to a subject suffering from or susceptible to developing osteoarthritis, respectively, reducing or preventing cartilage degeneration in a joint of the subject, preferably terminating the spontaneously causal process of the degeneration. administering an effective amount of a compound of the present invention for a period of time sufficient to In certain embodiments, the compound may exhibit cartilage anabolic and/or catabolic-inhibiting properties.

The injection dosage levels range from about 0.1 mg/kg/h to at least 10 mg/kg/h for all about 1 to about 120 hours and especially 24 to 96 hours. A preloading bolus of about 0.1 mg/kg to about 10 mg/kg or more may also be administered to achieve suitable steady state levels. The maximum total dose is expected not to exceed about 2 g/day for a 40-80 kg human patient.

For the prevention and/or treatment of long-term conditions, eg, degenerative conditions, the treatment regimen can usually be extended over several months or years and therefore oral administration is preferred for patient convenience and tolerability. For oral administration, an oral dose of 1 to 5 times daily and especially 2 to 4 times daily and typically 3 times daily is a typical regimen. When using this dosing pattern, each dose provides from about 0.01 to about 20 mg/kg of a compound of the present invention, in particular each dose from about 0.1 to about 10 mg/kg and particularly from about 1 to about 5 mg. / kg.

Transdermal doses are generally selected to provide blood levels similar to or lower than those achieved using injection doses.

When used to prevent the development of a disease, a compound of the present invention will be administered to a patient at risk of developing the disease, typically under the advice and supervision of a physician, at the dosage levels set forth above. Patients at risk of developing a particular disease generally include those with a family history of the disease or those identified by genetic testing or screening as being particularly susceptible to developing the disease.

A compound of the present invention may be administered as the sole active agent or in combination with other therapeutic agents, for example other compounds that exhibit the same or similar therapeutic activity and are determined to be safe and efficacious for concomitant administration. In certain embodiments, simultaneous administration of two (or more) agents permits the use of significantly lower doses of each, resulting in reduced side effects.

In one embodiment, a compound of the present invention or a pharmaceutical composition comprising a compound of the present invention is administered as a medicament. In certain embodiments, the pharmaceutical composition further comprises an additional active ingredient.

In one embodiment, a compound of the invention is administered in combination with another therapeutic agent for the treatment and/or prevention of a disease with inflammation, the specific agent being an immunomodulatory agent such as but not limited to azathioprine, corticosteroids. (e.g. prednisolone or dexamethasone), cyclophosphamide, cyclosporine A, tacrolimus, mycophenolate mofetil, muromonap-CD3 (OKT3, e.g. Orthocolone®), ATG, These include aspirin, acetaminophen, ibuprofen, naproxen and piroxicam.

In one embodiment, a compound of the invention is administered in combination with another therapeutic agent for the treatment and/or prevention of arthritis (e.g. rheumatoid arthritis), the particular agent being but not limited to analgesics, non-steroidal anti-inflammatory drugs. (NSAIDS), steroids, synthetic DMARDS (such as but not limited to methotrexate, leflunomide, sulfasalazine, auranofin, sodium aurothiomalate, penicillamine, chloroquine, hydroxychloroquine, azathioprine, and cyclo sporine), and biologic DMARDS (eg, but not limited to, infliximab, etanercept, adalimumab, rituximab, and abatacept).

In one embodiment, a compound of the invention is administered in combination with another therapeutic agent for the treatment and/or prophylaxis of a proliferative disorder, the specific agent including but not limited to methotrexate, leucovorin, adriamycin, prenisone, bleomycin, Cyclophosphamide, 5-fluorouracil, paclitaxel, docetaxel, vincristine, vinblastine, vinorelbine, doxorubicin, tamoxifen, toremifene, megestrol acetate, anastrozole, goserelin, anti-HER2 monoclonal antibodies (e.g. Herceptin™), capecitabine, raloxifene hydrochloride, EGFR inhibitors (e.g. Iressa™, Tarceva™), erbitux ( Erbitux™), VEGF inhibitors (e.g. Avastin™), proteosome inhibitors (e.g. Velcade™), Glivec™ and hsp90 inhibitors (eg 17-AAG). Additionally, the compounds of the present invention may be administered in conjunction with other therapies, including but not limited to radiotherapy or surgery. In certain embodiments, the proliferative disease is selected from cancer, myeloproliferative disease and leukemia.

In one embodiment, a compound of the invention is administered in combination with another therapeutic agent for the treatment and/or prevention of an autoimmune disease, the particular agent being, but not limited to, a glucocorticoid, a cell growth inhibitor (e.g., a purine analog), Alkylating agents (e.g. nitrogen mustard (cyclophosphamide), nitrosoureas, platinum compounds, etc.), antimetabolites (e.g. methotrexate, azathioprine and mercaptopurine), cytotoxic antibiotics (e.g. dactino anthracycline mitomycin, mitomycin C, bleomycin, and mithramycin), antibodies (eg anti-CD20, anti-CD25, or anti-CD3 (OTK3) monoclonal antibodies, Atgam® and thymo) globulin (Thymoglobuline®)), cyclosporine, tacrolimus, rapamycin (sirolimus), interferons (eg INF-β), TNF binding proteins (eg infliximab (Remicade®), etanercept ( Enbrel(trademark) or adalimumab (Humira(trademark)), mycophenolate, fingolimod and myriocin.

In one embodiment, a compound of the present invention is administered in combination with another therapeutic agent for the treatment and/or prevention of transplant rejection, the particular agent including but not limited to a calcineurin inhibitor (eg cyclosporine or tacrolimus (FK506)), mTOR inhibitors (eg sirolimus, everolimus), antiproliferative agents (eg azathioprine, mycophenolic acid), corticosteroids (eg prednisolone, hydrocortisone), antibodies (eg monoclonal anti- IL-2Ra receptor antibody, basiliximab, daclizumab), polyclonal anti-T-cell antibodies (eg anti-thymocyte globulin (ATG), anti-lymphocyte globulin (ALG)).

In one embodiment, a compound of the present invention is administered in combination with an agent for the treatment and/or prevention of asthma and/or rhinitis and/or COPD, the particular agent including but not limited to a beta2-adrenergic receptor agonist (e.g. salbutamol, levalbuterol, terbutaline, and bitolterol), epinephrine (inhaled or tablet), anticholinergics (such as ipratropium bromide), glucocorticoids (oral or inhaled), long-acting β2 -Agonists (e.g. salmeterol, formoterol, bambuterol, and slow-release oral albuterol), combinations of inhaled steroids and long-acting bronchodilators (e.g. fluticasone/salmeterol, budesonide/ formoterol), leukotriene antagonists and synthesis inhibitors (eg monteleucast, zapyrleukast and zileuton), mediator release inhibitors (eg cromoglycate and ketotifen), biological modulators of the IgE response (eg eg omalizumab), antihistamines (eg ceterizine, cinnarizine, fexofenadine), and vasoconstrictors (eg oxymetazoline, xylometazoline, naphazoline and tramazoline).

In addition, the compounds of the present invention may be administered in conjunction with emergency therapy for asthma and/or COPD, such therapy may include administration of oxygen or Heliox, nebulized salbutamol or terbutaline (anticholinergics (e.g. ipratropium), systemic steroids (oral or intravenous, eg prednisone, prednisolone, methylprednisolone, dexamethasone or hydrocortisone), intravenous salbutamol, non-specific beta-agonists, Injectables or inhalants (e.g. epinephrine, isoetharine, isoproterenol, metaproterenol), anticholinergics (IV or spray form e.g. glycopyrrolate, atropine, ipratropium), methylxanthines ( theophylline, aminophylline, vamiphylline), inhalation anesthetics with bronchodilatory effects (eg isoflurane, halothane, enflurane), ketamine, and intravenous magnesium sulfate.

In one embodiment, a compound of the invention is administered in combination with another therapeutic agent for the treatment and/or prevention of inflammatory bowel disease (IBD), wherein the specific agent is non-limiting to synthesis of glucocorticoids (eg prednisone, budesonide). Disease-modifying, immunomodulatory agents (eg methotrexate, leflunomide, sulfasalazine, mesalazine, azathioprine, 6-mercaptopurine and cyclosporine) and biological disease-modifying, immunomodulatory agents (infliximab, adarimumab, rituximab) and abatacept).

In one embodiment, a compound of the invention is administered in combination with another therapeutic agent for the treatment and/or prevention of SLE, the specific agent being, but not limited to, a disease-modifying antirheumatic drug (DMARD), such as an antimalarial drug. (e.g. plaquenil, hydroxychloroquine), immunosuppressants (e.g. methotrexate and azathioprine), cyclophosphamide and mycophenolic acid, immunosuppressive drugs and analgesics such as non-steroidal anti-inflammatory drugs, opiates (eg dextropropoxyfen and co-codamol), opioids (eg hydrocodone, oxycodone, MS Contin, or methadone), and fentanyl duraze type transdermal patches.

In one embodiment, a compound of the present invention is administered in combination with another therapeutic agent for the treatment and/or prevention of psoriasis, wherein the specific agent is a topical therapeutic agent, including, but not limited to, bath solutions, moisturizers, containing coal tar. medicated creams and ointments, ditranol (Anthralin), the corticosteroid analog desoximethasone (Topicort®), fluocinonide, vitamin D3 analogues (e.g. calcipotriol), Argan oil and retinoids (etretinate, acitretin, tazarotene), systemic treatments such as methotrexate, cyclosporine, retinoids, thioguanine, hydroxyurea, sulfasalazine, mycophenolate mofetil, azathioprine, tacrolimus, puma Lric acid esters or biologics such as Amevive (trademark), Enbrel (trademark), Humira (trademark), Remicade (trademark), Raptiva (trademark) ) and ustekinumab (IL-12 and IL-23 blocker). Additionally, the compounds of the present invention may be administered in conjunction with other therapies, including but not limited to phototherapy or photochemotherapy (eg psoralen and ultraviolet A phototherapy (PUVA)).

In one embodiment, a compound of the present invention is administered in combination with another therapeutic agent for the treatment and/or prevention of an allergic reaction, the particular agent being an antihistamine, including but not limited to, cetirizine, diphenhydramine, fexofenadine. , levocetirizine), glucocorticoids (eg prednisone, betamethasone, beclomethasone, dexamethasone), epinephrine, theophylline or anti-leukotrienes (eg montelukast or zapirleukast), anti-cholinergics and congestion Contains laxatives.

Simultaneous administration includes any means of delivering two or more therapeutic agents to a patient as part of the same treatment regimen, as will be apparent to the skilled artisan. Two or more agents may be administered simultaneously in a single dosage form, although this is not required. The agents may be administered at different times in different formulations.

chemical synthesis process

general content

Compounds of the present invention can be prepared from readily available starting materials using the following general methods and procedures. It will be appreciated that where typical or preferred process conditions (ie reaction temperature, time, molar ratios of reactants, solvents, pressures, etc.) are given, other process conditions may also be used unless otherwise indicated. Optimum reaction conditions may vary depending on the particular reactants or solvents used, but such conditions can be determined by one skilled in the art by routine optimization procedures.

Additionally, as will be apparent to those skilled in the art, conventional protecting groups may be required to prevent some functional groups from undergoing undesirable reactions. The selection of suitable protecting groups for specific functional groups as well as suitable conditions for protection and deprotection are well known in the art (Greene, T W; Wuts, P G M;, 1991).

The following methods are provided herein, along with details regarding the preparation of the compounds of the invention listed above. Compounds of the present invention can be prepared by those skilled in the art of organic synthesis from known or commercially available starting materials and reagents.

All reagents were of commercial grade and used as received without further purification unless otherwise indicated. Anhydrous commercially available solvents were used for reactions conducted under an inert atmosphere. In all other cases, reagent grade solvents were used unless otherwise indicated. Column chromatography was performed on silica gel 60 (35-70 μm). Thin layer chromatography was performed using pre-coated silica gel F-254 plates (0.25 mm thick). ¹ H NMR spectra were recorded on a Brooker DPX 400 NMR spectrometer (400 MHz) or a Brooker Advance 300 NMR spectrometer (300 MHz). Chemical shifts (δ) for 1H NMR spectra are reported in parts per million (ppm) relative to tetramethylsilane (δ 0.00) as an internal standard or a suitable residual solvent peak, CHCl ₃ (δ 7.27). Multiplicities are given as singlet (s), doublet (d), triplet (t), quartet (q), quintet (quin), multiplet (m) and broad (br). Electrospray MS spectra were acquired on a Waters platform LC/MS spectrometer or with a Waters Acquity H-class UPLC coupled to a Waters Mass detector 3100 spectrometer. Columns used: Waters Acquity UPLC BEH C18 1.7 μm, 2.1 mm ID x 50 mm L, Waters Acquity UPLC BEH C18 1.7 μm, 2.1 mm ID x 30 mm L, or Waters Xterra MS 5 μm C18, 100 x 4.6 mm. The methods use a MeCN/H ₂ O gradient (H ₂ O contains 0.1% TFA or 0.1% NH ₃ ) or MeOH/H ₂ O gradient (H ₂ O contains 0.05% TFA). Microwave heating was performed with a Biotage Initiator.

Preparative HPLC purification was performed with a mass-directed auto-purification system coupled with a ZQ single quadrupole mass spectrometer. All HPLC purifications were performed with a gradient of H ₂ O (different pH)/MeCN. Preparative HPLC separations under basic conditions were usually performed using a BEH XBridge C18 (5 μm, 19 x 5 mm) precolumn and a BEH XBridge C18 (5 μm, 19 x 100 mm). Separation under acidic conditions was usually performed using a CSH Select C18 (5 μm, 19 x 5 mm) precolumn and a CSH Select C18 (5 μm, 19 x 100 mm). The focus gradient was from x% in water to x+25% acetonitrile in 5 minutes with a cycle time of 10 minutes. The column flow rate was 20 ml/min. Injection volumes ranged from 200 to 750 μl. The flow after column separation was diverted to the mass spectrometer using a capillary splitter and diluted by a make-up flow of 1 ml/min. The make-up flow is 0.1% formic acid in methanol. All samples were purified by Waters Mass Directed Fraction Collection.

[Table I]

List of abbreviations used in the experimental part

Synthetic Preparation of Compounds of the Invention

Example 1. Synthesis of Intermediate Compounds of the Invention

1.1. Synthesis of bis-(4-methoxy-benzyl)-amine (Int.1)

p-Anisaldehyde (411 mmol), 4-methoxybenzylamine (411 mmol) and toluene (500 mL) were combined in a round bottom flask equipped with a condenser and Dean-Stark trap. The reaction was refluxed for 1 hour during which time water was removed from the reaction mixture. The reaction was cooled and concentrated. The residue was dissolved in MeOH (120 mL). The mixture was cooled to 5 °C and NaBH ₄ (205 mmol) was added portionwise over 45 min. The reaction was slowly heated to reflux. After 2 hours under reflux, the reaction was cooled to room temperature and concentrated. The residue was dissolved in EtOAc. The organic layer was washed (3 x H ₂ O and brine), dried (Na ₂ SO ₄ ) and concentrated to give the desired product.

1.2. Synthesis of (6-bromo-1-methyl-1H-benzoimidazol-4-yl)-bis-(4-methoxy-benzyl)-amine (Int.2)

1.2.1. Step i: 5-Bromo-1,3-difluoro-2-nitro-benzene

A solution of 4-bromo-2,6-difluoroaniline (240 mmol) in AcOH (150 mL) was added to a suspension of NaBO ₃ 4H ₂ O (325 mmol) in AcOH (450 mL) at 70 °C for 30 min. was added to Another 2080 mmol of NaBO ₃ .4H ₂ O was added to the mixture over 5 hours. During this period the mixture was stirred at 70 °C. The mixture was poured into water and extracted with Et ₂ O. The organic layer was combined with another Et ₂ O solution from another reactant using the same conditions as described above. The mixture was concentrated. A precipitate formed and was isolated by filtration. The filtrate was concentrated to give the desired product after flash column chromatography (SiO ₂ , petroleum ether).

1.2.2. Step ii: (5-Bromo-3-fluoro-2-nitro-phenyl)-methyl-amine

2M MeNH ₂ in THF (82 mL) was added to a solution of 5-bromo-1,3-difluoro-2-nitrobenzene (164 mmol) and Cs ₂ CO ₃ (197 mmol) in THF (1 L). it was added The reaction mixture was stirred at 0 °C for 1 hour and then at room temperature for 1 hour. The solvent was removed under reduced pressure. The residue was partitioned between EtOAc and H ₂ O. The two phases were separated and the organic layer was dried (Na ₂ SO ₄ ) and concentrated to give the desired product.

1.2.3. Step iii: 5-Bromo-N,N-bis-(4-methoxy-benzyl)-N'-methyl-2-nitro-benzene-1,3-diamine

A mixture of bis-(4-methoxy-benzyl)-amine (346 mmol), 5-bromo-3-fluoro-N-methyl-2-nitroaniline (297 mmol) and Et ₃ N (891 mmol) was stirred at 120 °C for 16 hours. The mixture was then cooled. The crude material was combined with another material obtained according to the same procedure as described above. The resulting mixture was diluted in EtOAc and washed (2 x 0.2M HCl, H ₂ O and saturated NaHCO ₃ ). The organic phase was dried (Na ₂ SO ₄ ) and concentrated to give the desired product.

1.2.4. Step iv: 5-Bromo-N,N-bis-(4-methoxy-benzyl)-N"-methyl-benzene-1,2,3-triamine

5-Bromo-N,N-bis-(4-methoxy-benzyl)-N'-methyl-2-nitro-benzene-1,3-diamine ( 170 mmol), NH ₄ Cl (2038 mmol) and Zn (2034 mmol) was stirred at room temperature for 1 hour. The mixture was cooled to 0 °C and HCOOH (20 mL) was added slowly. The mixture was allowed to reach room temperature and stirred for 1 hour. The mixture was filtered and combined with another mixture obtained according to the procedure described above. The resulting mixture was concentrated. The residue was dissolved in DCM. The organic mixture was washed (saturated NH ₄ Cl), dried (Na ₂ SO ₄ ) and concentrated to give the desired product.

1.2.5. Step v: (6-Bromo-1-methyl-1H-benzoimidazol-4-yl)-bis-(4-methoxy-benzyl)-amine

5-Bromo-N,N-bis-(4-methoxy-benzyl)-N"-methyl-benzene-1,2,3-triamine (170 mmol) was reacted with HC(OEt) ₃ (100 mol) and Dissolved in the mixture of MeCN (500mL).This mixture was stirred at 85 DEG C for 5 hours and at room temperature for about 16 hours.This mixture was combined with another mixture obtained according to the procedure described above. was concentrated and the residue was purified by flash column chromatography (SiO ₂ , 15:85 to 60:40 EtOAc/petroleum ether) to give the product.

1.3. 5-{7-[bis-(4-methoxy-benzyl)-amino]-3-methyl-3H-benzoimidazol-5-yloxy}-4-methyl-pyridine-2-carbonitrile (Int. 3 ) of the synthesis.

1.3.1. Step i: Bis-(4-methoxy-benzyl)-[1-methyl-6-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)- 1H-benzoimidazol-4-yl]-amine

(6-Bromo-1-methyl-1H-benzoimidazol-4-yl)-bis-(4-methoxy-benzyl)-amine (54 mmol), bis(pinacolato) in DMF (150 mL) A mixture of diboron (81 mmol), PdCl ₂ (dppf).DCM (2.71 mmol) and KOAc (162.5 mmol) was sonicated under a stream of nitrogen for 5 min. The mixture was then stirred at 110 °C for 1 hour in a round bottom flask equipped with a condenser. The mixture was filtered through a celite pad and the filtrate was concentrated. The residue was dissolved in EtOAc and the organic layer was washed (H ₂ O), dried (Na ₂ SO ₄ ) and concentrated to give the desired product.

1.3.2. Step ii: 5-{7-[bis-(4-methoxy-benzyl)-amino]-3-methyl-3H-benzoimidazol-5-yloxy}-4-methyl-pyridine-2-carbonitrile

30 wt % H ₂ O ₂ in H ₂ O (20 mL) was dissolved in N,N-bis(4-methoxybenzyl)-1-methyl-6-(4,4,5,5- It was added to a solution of tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[d]imidazol-4-amine (58.5 mmol) and the mixture was stirred at room temperature for approximately 16 hours. The reaction was quenched with 5% aqueous Na ₂ SO ₃ . The mixture was extracted with EtOAc. The organic layer was washed (H ₂ O), dried (Na ₂ SO ₄ ) and concentrated to give the desired product.

1.4. General Method: Ortho-Directional Iodination

Wherein W can be -N=, -CH=, -C(Cl)=; Y may be -N=, -C(CN)=.

1.4.1. Method A1

A mixture of amino aromatic or heteroaromatic starting material (1 equiv.), Ag ₂ SO ₄ (1 equiv.) and I ₂ (1 equiv.) in EtOH at a temperature ranging from room temperature to 50 °C for a period varying from 1 hour to approximately 16 hours. Stir for a period of time. The mixture is filtered, concentrated and diluted in an organic solvent. The organic mixture is subjected to an aqueous work-up. The solvent is removed under reduced pressure and the residue is purified by flash column chromatography to give the desired product.

1.4.2. Illustrative Example of Method A1: Synthesis of 4-amino-3-fluoro-5-iodo-benzonitrile (Int.4).

A mixture of 4-amino-3-fluorobenzonitrile (147 mmol), I ₂ (147 mmol) and Ag ₂ SO ₄ (147 mmol) in EtOH (700 mL) was stirred at room temperature for 1.5 h. The mixture was filtered and concentrated. The residue was dissolved in EtOAc and washed (saturated Na ₂ S ₂ O ₃ x 3). The organic layer was dried (Na ₂ SO ₄ ) and concentrated. The residue was purified by flash column chromatography (SiO ₂ , 95:5 to 70:30 cyclohexane/EtOAc) to provide the desired product.

1.4.3. Method A2

A mixture of amino aromatic or heteroaromatic starting material (1 equiv.), Ag ₂ SO ₄ (3-4 equiv.) and I ₂ (3-4 equiv.) in EtOH is stirred at 70° C. for 16 h. Additional equivalents of I ₂ and Ag ₂ SO ₄ can be added to increase this conversion. The mixture is filtered and concentrated. The residue is purified by flash column chromatography to give the desired product.

1.5. Illustrative Example of Method A2: Synthesis of 5-amino-6-fluoro-4-iodo-pyridine-2-carbonitrile (Int.5).

A mixture of Int.11 (3.62 mmol), I ₂ (14.5 mmol) and Ag ₂ SO ₄ (14.5 mmol) in EtOH (200 mL) was stirred at 70 °C for 16 h. A further 5 equivalents of I ₂ and Ag ₂ SO ₄ were added and the reaction was stirred at 70 °C for an additional 72 hours. The mixture was filtered, concentrated and purified by flash column chromatography (SiO ₂ , 20:80 to 40:60 EtOAc/cyclohexane) to give the desired product.

1.6. General Method: Introduction of Nitrile Group by Negishi Reaction

wherein R _A can be aryl or heteroaryl; X can be Cl, Br or I.

1.6.1. Method B

A mixture of aryl/heteroaryl halide (1 eq.), zinc cyanide (1 to 3 eq.), and Pd(PPh ₃ ) ₄ (0.1 to 0.2 eq.) in DMF was heated at 150 °C in a microwave apparatus for a period ranging from 5 min to 0.5 h. heated at °C. The mixture is filtered and concentrated. The residue is diluted with an organic solvent. The organic mixture is worked up and the solvent is removed under reduced pressure. The residue is triturated or purified by flash column chromatography to give the desired product.

1.6.2. Illustrative Example of Method B: Synthesis of 5-fluoro-4-methyl-pyridine-2-carbonitrile (Int.7)

A microwave tube was charged with 2-chloro-5-fluoro-4-methylpyridine (5.5 mmol), Zn(CN) ₂ (16.6 mmol), Pd(PPh ₃ ) ₄ (1.1 mmol) in DMF (20 mL). . The mixture was stirred in a microwave reactor at 150 °C for 5 minutes. This mixture was combined with other crude materials obtained according to the procedure described above. The resulting mixture was filtered. The filtrate was concentrated under reduced pressure. The residue was diluted with EtOAc, washed (saturated NH ₄ Cl), dried (Na ₂ SO ₄ ) and concentrated. The residue was purified by flash column chromatography (SiO ₂ , 5:95 to 15:85 EtOAc/petroleum ether) to provide the desired product.

1.7. General Method: Suzuki with Methyl Boronic Acid

Wherein W can be -N=, -CH=, -C(Cl)=; Y can be -N=, -C(CN)=; X can be -I or -Br.

1.7.1. Method C

Aromatic/heteroaromatic halides (1 equiv.), methyl boronic acid (1.3-3 equiv.), Pd(dppf)Cl ₂ .DCM (0.11-0.2 equiv.) and Cs ₂ CO ₃ (3-5 equiv.) in 1,4-dioxane. ) is stirred at 100 °C. Dilute the mixture with an organic solvent. The organic mixture is worked up and the solvent is removed under reduced pressure. The residue is purified by flash column chromatography to give the desired product.

1.7.2. Illustrative Example of Method C: Synthesis of 5-amino-6-fluoro-4-methyl-pyridine-2-carbonitrile (Int.12)

A mixture of Int.5 (3 mmol), methyl boronic acid (9.1 mmol), Pd(dppf)Cl ₂ .DCM (0.32 mmol) and Cs ₂ CO ₃ (15.2 mmol) in 1,4-dioxane (8 mL) was Stirred at 105 °C for 5 hours. The mixture was diluted (EtOAc), washed (saturated NaHCO ₃ ), dried (Na ₂ SO ₄ ) and concentrated. The residue was purified by flash column chromatography (SiO ₂ , 10:90 to 50:50 EtOAc/petroleum ether) to provide the desired product.

1.8. Synthesis of 6-bromo-2-fluoro-pyridin-3-ylamine (Int.15)

A mixture of 2-fluoropyridin-3-amine (44.6 mmol) and KOAc (44.6 mmol) in AcOH was stirred at room temperature for 1 hour. The mixture was cooled to 0 °C and Br ₂ (44.6 mmol) was added dropwise. The mixture was stirred at 0 °C for 15 min. The mixture was concentrated and the residue was dissolved in EtOAc/MeOH. The organic solution was washed (saturated NaHCO ₃ , saturated Na ₂ S ₂ O ₃ ), dried (Na ₂ SO ₄ ) and concentrated. The residue was purified by flash column chromatography (SiO ₂ , 100:0 to 80:20 petroleum ether/EtOAc) to provide the desired product.

1.9. Synthesis of 2-bromo-6-fluoro-4-methanesulfonyl-phenylamine (Int.16)

A mixture of 2-fluoro-4-methylsulfonyl-aniline (22.76 mmol) and KOAc (22.7 mmol) in AcOH was stirred at room temperature. The mixture was cooled to 0 °C and Br ₂ (22.7 mmol) was added dropwise. The mixture was stirred at 0 °C for 30 min. The mixture was concentrated and the residue was dissolved in EtOAc. The organic mixture was washed (saturated NaHCO ₃ , saturated Na ₂ S ₂ O ₃ ), dried (Na ₂ SO ₄ ) and concentrated to give the desired product.

1.10. Synthesis of 4-amino-3-ethyl-5-fluoro-benzonitrile (Int.17)

A mixture of Cs ₂ CO ₃ (46 mmol) and Pd(dppf)Cl ₂ .DCM (0.76 mmol) was suspended in DMF (35 mL) and degassed with nitrogen. A solution of H ₂ O (400 μL), 1M Et ₃ B in THF (11.5 mL) and Int.4 (7.63 mmol) in DMF (5 mL) was added to the above mixture and the reaction was stirred at 60 °C for 30 min. The mixture was concentrated and the residue was dissolved in EtOAc, washed (saturated NaHCO ₃ , H ₂ O), dried (Na ₂ SO ₄ ) and concentrated. The residue was purified by flash column chromatography (SiO2, 100:0 to 95:5 petroleum ether/EtOAc) to provide the desired product.

1.11. General Method: Conversion of NH ₂ to Iodide

Here, R ₁₀ may be Me or Et.

1.11.1. Method D

Concentrated HCl (6 equiv.) is added dropwise to a mixture of amino aromatics/heteroaromatics (1 equiv.) in H ₂ O at 0 °C. A solution of NaNO ₂ (1.05 eq.) in H ₂ O is added dropwise. The resulting mixture is stirred at 0 °C for 15 minutes. A solution of KI (1.05 eq.) in H ₂ O is added dropwise. The mixture is stirred at 0 °C for 15 minutes and at room temperature for 1 hour. The mixture is extracted with an organic solvent. After aqueous work-up and removal of the solvent under reduced pressure, the residue can be triturated or chromatographed to give the desired product.

1.11.2. Illustrative Example of Method D: Synthesis of 5-iodo-4-methyl-pyridine-2-carbonitrile (Int.18)

Concentrated HCl (0.9 mL) was added dropwise to a mixture of Int.8 (1.88 mmol) in H ₂ O (10 mL) at 0 °C followed by a solution of NaNO ₂ (1.97 mmol) in H ₂ O (0.5 mL). did The resulting mixture was stirred at 0 °C for 15 min. A solution of KI (1.97 mmol) in H ₂ O (1 mL) was added dropwise. The resulting mixture was stirred at 0 °C for 15 minutes and at room temperature for 1 hour. The mixture was extracted with EtOAc. The organic layer was washed (H ₂ O), dried and concentrated. The residue was purified by flash column chromatography (SiO ₂ , 100:0 to 75:25 cyclohexane/EtOAc) to provide the desired product.

1.12. Synthesis of 6-bromo-4-methyl-pyridin-3-ylamine (Int.20)

A mixture of NH ₄ Cl (14.9 mmol) and iron powder (18.4 mmol) in H ₂ O (5 mL) was stirred at 90 °C. 2-Bromo-4-methyl-5-nitropyridine (2.3 mmol) was added portionwise. The mixture was stirred at 90 °C for 1 hour and 15 minutes. The reaction was stopped and extracted with EtOAc. The organic layer was dried (Na ₂ SO ₄ ) and concentrated to give the desired product.

1.13. Synthesis of 4-amino-3-chloro-5-fluoro-benzonitrile (Int.21)

A mixture of 4-amino-3-fluorobenzonitrile (184 mmol) and NCS (276 mmol) in AcOH (300 mL) was stirred at 70 °C for approximately 16 h. The mixture was concentrated. H ₂ O was added to the residue and the solid product was filtered and washed (saturated NaHCO ₃ and H ₂ O). To remove the H ₂ O, THF was added and removed under reduced pressure to give the desired product.

1.14. Synthesis of 3-ethyl-4-hydroxy-benzonitrile (Int.22)

Concentrated H ₂ SO ₄ (3.4 mL) was added dropwise to a solution of 4-amino-3-ethylbenzonitrile (6.84 mmol) in H ₂ O (12 mL) at 0 °C. A solution of NaNO ₂ (7.52 mmol) in H ₂ O (5 mL) was added dropwise to the resulting mixture at 0 °C. The reaction was stirred at 0 °C for 30 min and at 100 °C for 2 h. The mixture was cooled to room temperature and extracted with EtOAc. The organic layer was washed (H ₂ O), dried and concentrated to give the desired product.

1.15. Synthesis of 2-chloro-3-fluoro-pyridin-4-ylamine (Int.23)

1.15.1. Step i: tert-Butyl N-(2-chloro-3-fluoro-4-pyridyl)carbamate

Diphenylphosphoryl azide (DPPA) (129 mmol) was diluted with 2-chloro-3-fluoro-pyridine-4-carboxylic acid (85.7 mmol), Et ₃ N in 1:1 tert-BuOH/toluene (200 mL). (257 mmol) was added to the mixture. The mixture was heated at 110 °C for 4 hours. The mixture was diluted with H ₂ O and extracted with DCM. The organic layer was dried (Na ₂ SO ₄ ) and concentrated. The residue was purified by flash column chromatography (SiO ₂ , 100:0 to 80:20 DCM/EtOAc) to obtain the desired product tert-butyl N-(2-chloro-3-fluoro-4-pyridyl)carba A mate was provided.

1.15.2. Step ii: 2-Chloro-3-fluoro-pyridin-4-ylamine

A solution of tert-butyl N-(2-chloro-3-fluoro-4-pyridyl)carbamate (20.2 mmol) in 1:2 TFA/DCM (45 mL) was stirred at room temperature for 6 hours. The reaction mixture was concentrated and the residue was purified by flash column chromatography (SiO ₂ , 100:0 to 90:10 DCM/7N NH ₃ in MeOH) to provide the desired product.

1.16. General method: Buchwald coupling with (6-bromo-1-methyl-1H-benzoimidazol-4-yl)-bis-(4-methoxy-benzyl)-amine (Int.2).

wherein R _A can be an optionally substituted aryl or heteroaryl.

1.16.1. Method E1

A mixture of Int.2 (1 equiv.), the corresponding aniline (1.1 equiv.), Cs ₂ CO ₃ (2 equiv.) and XPhos (0.3 equiv.) in anhydrous toluene was mixed before addition of Pd(OAc) ₂ (0.1 to 0.15 equiv.). Purging with inert gas. The mixture is stirred at 110 °C for approximately 16 hours. The mixture may be partitioned between the organic solvent and the aqueous phase. The two layers are separated and the organic layer is dried and concentrated. Alternatively, the reaction mixture may be filtered and concentrated. The residue may be kept as is or purified by chromatography to give the desired product.

1.16.2. Illustrative Example of Method E1: 5-{7-[bis-(4-methoxy-benzyl)-amino]-3-methyl-3H-benzoimidazol-5-ylamino}-4-ethyl-pyridin- Synthesis of 2-carbonitrile (Int.24).

A mixture of Int.2 (0.75 mmol), Int.10 (0.79 mmol), Cs ₂ CO ₃ (1.5 mmol) and XPhos (0.225 mmol) in anhydrous toluene (6.3 mL) was dissolved in Pd(OAc) ₂ (0.075 mmol). It was purged with inert gas before addition. The mixture was stirred at 110 °C for approximately 16 hours. The mixture was diluted with H ₂ O and extracted with DCM. The organic layer was washed (brine), dried (Na ₂ SO ₄ ) and concentrated. The residue was purified by flash column chromatography (SiO ₂ , 100:0 to 50:50 cyclohexane/EtOAc) to provide the desired product.

1.16.3. Method E2

A mixture of Int.2 (1 equiv.), the corresponding aniline (1.1 equiv.), Cs ₂ CO ₃ (4-5 equiv.) and XPhos (0.4 equiv.) in anhydrous toluene was mixed with Pd(OAc) ₂ (0.2-0.3 equiv.). Purge with inert gas prior to addition. The mixture is stirred at 110 °C for a period ranging from 1 to 24 hours. The mixture may be partitioned between the organic solvent and the aqueous phase. The two layers are separated and the organic layer is dried and concentrated. Alternatively, the reaction mixture may be filtered and concentrated. The residue may be kept as is or purified by chromatography to give the desired product.

1.16.4. Illustrative Example of Method E2: 5-{7-[bis-(4-methoxy-benzyl)-amino]-3-methyl-3H-benzoimidazol-5-ylamino}-4-methyl-pyridin- Synthesis of 2-carbonitrile (Int.27).

A mixture of Int.2 (4 mmol), Int.8 (4.3 mmol), Cs ₂ CO ₃ (20 mmol) and XPhos (1.6 mmol) in anhydrous toluene (35 mL) was dissolved in Pd(OAc) ₂ (1.2 mmol). It was purged with argon before addition. The mixture was stirred at 110 °C for approximately 16 hours. The mixture was filtered through a celite pad and concentrated. The residue was purified by flash column chromatography (SiO ₂ , 98:2 to 25:75 cyclohexane/EtOAc) to provide the desired product.

1.16.5. Method E3

A mixture of Int.2 (1 equiv.), Aniline (1.1 equiv.), BINAP (0.3 equiv.), Cs ₂ CO ₃ (4 equiv.), and Pd(OAc) ₂ (0.2 equiv.) in anhydrous 1,4-dioxane was mixed with an inert gas. degas with The reaction is stirred at 100-110 °C for a period ranging from 4 hours to approximately 16 hours. The mixture may be partitioned between the organic solvent and the aqueous phase. The two layers are separated and the organic layer is dried and concentrated. Alternatively, the reaction mixture may be filtered and concentrated. The residue may be kept as is or purified by chromatography to give the desired product.

1.16.6. Illustrative Example of Method E3: N6-(2-Fluoro-4-methanesulfonyl-phenyl)-N4,N4-bis-(4-methoxy-benzyl)-1-methyl-1H-benzoimidazole- Synthesis of 4,6-diamine (Int.34)

Int.2 (1.48 mmol), 2-fluoro-4-methylsulfonyl-aniline (1.58 mmol), BINAP (0.47 mmol), Cs ₂ CO ₃ (6.32 mmol) in anhydrous 1,4-dioxane (15 mL) and Pd(OAc) ₂ (0.32 mmol) was degassed with nitrogen. The reaction was stirred at 100 °C for approximately 16 hours. The mixture was diluted with EtOAc and the organic layer was washed (H ₂ O), dried (Na ₂ SO ₄ ) and concentrated to give the desired product.

1.16.7. Method E4

A mixture of Int.2 (1 equiv.), Aniline (1.1 equiv.), Brettphos (0.1 equiv.), Cs ₂ CO ₃ (5 equiv.) and Pd(OAc) ₂ (0.05 equiv.) in anhydrous 1,4-dioxane was stirred with an inert gas. degas with The reaction is stirred at 85-95° C. for a period ranging from 2 hours to approximately 8 hours. The mixture may be partitioned between organic solvent and organic phase. The two layers are separated and the organic layer is dried and concentrated. Alternatively, the reaction mixture may be filtered and concentrated. The residue may be kept as is or purified by chromatography to give the desired product.

1.16.8. Illustrative Example of Method E4: N6-(2,3-dihydro-1,4-benzodioxin-6-yl)-N4,N4-bis[(4-methoxyphenyl)methyl]-1-methyl- Synthesis of benzimidazole-4,6-diamine (Int.78).

Int.2 (1.17 mmol), 2,3-dihydro-1,4-benzodioxin-6-amine (1.29 mmol), Brettphos (0.117 mmol), Cs ₂ CO in anhydrous 1,4-dioxane (15 mL) A mixture of ₃ (6.32 mmol) and Pd(OAc) ₂ (0.058 mmol) was degassed with nitrogen. The reaction was stirred at 90 °C for approximately 5 hours. The mixture was diluted with EtOAc and the organic layer was washed (H ₂ O), dried (Na ₂ SO ₄ ) and concentrated to give the desired product.

1.17. General Method: Methylation of Buchwald Products

wherein R _A can be an optionally substituted aryl or heteroaryl.

1.17.1. Method F

NaH (60% in mineral oil, 1.1 to 3 equiv.) is added to a solution of the intermediate obtained from Buchwald coupling (1 equiv.) in THF or DMF at 0°C. The mixture is stirred at 0 °C for 30 minutes. Mel (1.1 to 3 eq) is added and the mixture is stirred at room temperature for a period ranging from 1 hour to approximately 16 hours. The mixture is partitioned between organic solvent and aqueous phase. The two layers are separated and the organic layer is dried and concentrated. The residue is purified by silica chromatography or used as is without further purification.

1.17.2. Illustrative Example of Method F: N6-(2-Fluoro-4-methanesulfonyl-6-methyl-phenyl)-N4,N4-bis-(4-methoxy-benzyl)-1,N6-dimethyl Synthesis of -1H-benzoimidazole-4,6-diamine (Int.37)

NaH (60% in mineral oil, 6.76 mmol) was added to a solution of Int.35 (2.25 mmol) in THF (10 mL) at 0 °C. The mixture was stirred at 0 °C for 30 min. Mel (6.76 mmol) was added and the mixture was stirred at room temperature for approximately 16 hours. The mixture was diluted with EtOAc and washed with H ₂ O. The organic layer was dried (Na ₂ SO ₄ ) and concentrated. The residue was purified by flash column chromatography (SiO ₂ , 20:80 to 80:20 EtOAc/petroleum ether) to provide the desired product.

1.17.3. Illustrative Example of Method F: N4,N4-bis[(4-methoxyphenyl)methyl]-N6,1-dimethyl-N6-(2-methyl-4-methylsulfonyl-phenyl)benzimidazole- 4,6-diamine (Int.49A) and N6-(4-ethylsulfonyl-2-methyl-phenyl)-N4,N4-bis[(4-methoxyphenyl)methyl]-N6,1-dimethyl -Synthesis of benzimidazole-4,6-diamine (Int.49B).

NaH (60% in mineral oil, 4.86 mmol) was added to a solution of Int.36 (1.62 mmol) in THF (10 mL) at 0 °C. The mixture was stirred at 0 °C for 30 min. Mel (4.86 mmol) was added and the mixture was stirred at room temperature for 1 hour. The mixture was diluted with EtOAc and washed with H ₂ O. The organic layer was dried (Na ₂ SO ₄ ) and concentrated. Flash column chromatography (SiO ₂ , 20:80 to 80:20 EtOAc/petroleum ether) gave a mixture of the desired product (Int.49A, main product) with a side product (Int.49B, side product). did

1.18. Synthesis of 4-{7-[bis-(4-methoxy-benzyl)-amino]-3-methyl-3H-benzoimidazol-5-yloxy}-3-ethyl-benzonitrile (Int.50)

A mixture of Int.2 (0.4 mmol), Int.22 (0.4 mmol), CuI (0.06 mmol) and Cs ₂ CO ₃ (1 mmol) in pyridine (2 mL) was flushed with argon, sealed and heated at 200 °C. Heated in a microwave reactor for 3 hours. The mixture was diluted with H ₂ O and extracted with EtOAc. The organic layer was dried and concentrated to give the desired product.

1.19. General method: 5-{7-[bis-(4-methoxy-benzyl)-amino]-3-methyl-3H-benzoimidazol-5-yloxy}-4-methyl-pyridine-2-carbonitrile ( Ullmann coupling between Int.3) and an aromatic or heteroaromatic halide

wherein R _A can be an optionally substituted aryl or heteroaryl.

1.19.1. Method G

Int.3 (1 equiv.), aryl halide (1.2 to 1.3 equiv.), CuI (0.1 to 0.2 equiv.), N,N-dimethyl glycine (0.3 to 0.5 equiv.) and A mixture of Cs ₂ CO ₃ (3 equivalents) is flushed with inert gas and stirred at 110° C. for a period ranging from 4.5 hours to 16 hours. The mixture is filtered and partitioned between organic solvent and aqueous phase. The two layers are separated and the organic layer is dried and concentrated. The residue is purified by silica chromatography.

1.19.2. Illustrative Example of Synthetic G: 5-{7-[bis-(4-methoxy-benzyl)-amino]-3-methyl-3H-benzoimidazol-5-yloxy}-4-methyl-pyridin- Synthesis of 2-carbonitrile (Int.51).

Int.3 (0.39 mmol), Int.18 (0.47 mmol), CuI (0.08 mmol), N,N-dimethyl glycine (0.2 mmol) and Cs in 1:1 DMF/1,4-dioxane (4 mL). A mixture of ₂ CO ₃ (1.17 mmol) was flushed with argon and stirred in a sealed tube at 110 °C for approximately 16 hours. The reaction mixture was cooled to room temperature and filtered through a pad of celite. The filtrate was concentrated. Water was added and the mixture was extracted with EtOAc. The organic layer was washed with water, dried and concentrated. The residue was purified by flash column chromatography (SiO ₂ , 100:0 to 40:60 cyclohexane/EtOAc) to provide the desired product.

1.20. General method: 5-{7-[bis-(4-methoxy-benzyl)-amino]-3-methyl}-3H-benzoimidazol-5-yloxy}-4-methyl-pyridine-2-carbonitrile (Int.3) and SNAr coupling between aromatic or heteroaromatic halides

wherein R _A can be an optionally substituted aryl or heteroaryl.

1.20.1. Method H

A mixture of Int.3 (1 equiv.), aryl/heteroaryl halide (1.2 to 1.5 equiv.) and K ₂ CO ₃ (2 equiv.) in DMF is stirred at 100° C. for a period ranging from 3 hours to approximately 16 hours. The mixture is diluted with EtOAc, washed several times with H ₂ O, dried and concentrated. The residue is purified by flash column chromatography.

1.20.2. Illustrative Example of Method H: 5-{7-[bis-(4-methoxy-benzyl)-amino]-3-methyl-3H-benzoimidazol-5-yloxy}-4-methyl-pyridin- Synthesis of 2-carbonitrile (Int.51).

A mixture of Int.3 (58 mmol), Int.7 (70 mmol) and K ₂ CO ₃ (116 mmol) in DMF (160 mL) was stirred at 100 °C. After 3 hours an additional 7.35 mmol of Int.7 was added to the reaction and the mixture was stirred at 100 °C for another hour. The resulting mixture was diluted with EtOAc, washed several times with H ₂ O, dried (Na ₂ SO ₄ ) and concentrated. The residue was purified by flash column chromatography (SiO ₂ , 15:85 to 35:75 EtOAc/petroleum ether) to provide the desired product.

1.21. General Method: Bis-PMB Deprotection

wherein R _A can be an optionally substituted aryl or heteroaryl; -L- may be -NH-, -NMe- or -O-.

1.21.1. Method I1

A mixture of protected bis-PMB amine (1 equivalent) in TFA is stirred at room temperature for a period ranging from 30 minutes to approximately 16 hours. The temperature is increased to 50 or 60 °C and the mixture is further stirred for a period ranging from 0.5 to 3 hours. The mixture is worked up using a basic aqueous solution and an organic solvent. The two phases are separated and the organic layer is dried and concentrated. Alternatively, the reaction mixture is first concentrated and then the residue is subjected to the above-mentioned work-up. The residue obtained from the above work-up can be purified by silica chromatography or by ISOLUTE® SCX-3 (Biotage) ion exchange resin or stored as is.

1.21.2. Illustrative Example of Method I1: N6-(2-Fluoro-4-methanesulfonyl-6-methyl-phenyl)-1,N6-dimethyl-1H-benzoimidazole-4,6-diamine (Int .53) synthesis.

A mixture of Int.37 (0.33 mmol) in TFA (5 mL) was stirred at room temperature for 1 hour and at 50 °C for 0.5 hour. The mixture was concentrated. The residue was partitioned between saturated NaHCO ₃ and DCM. The two phases were separated and the organic layer was dried (Na ₂ SO ₄ ) and concentrated. The residue was purified by flash column chromatography (SiO ₂ , 20:80 to 100:0 EtOAc/petroleum ether) to provide the desired product.

1.21.3. Exemplary Examples of Method I1: N6,1-Dimethyl-N6-(2-methyl-4-methylsulfonyl-phenyl)benzimidazole-4,6-diamine (Int.56A) and N6-(4 Synthesis of -Ethylsulfonyl-2-methyl-phenyl)-N6,1-dimethyl-benzimidazole-4,6-diamine (Int.56B)

A mixture of Int.49A and 49B (total about 0.61 mmol) in TFA (5 mL) was stirred at room temperature for approximately 16 hours and at 50 °C for 3 hours. The mixture was concentrated. The residue was partitioned between saturated NaHCO ₃ and DCM. The two phases were separated and the organic layer was dried (Na ₂ SO ₄ ) and concentrated. The desired product (Int.56A _, A mixture of main products) was provided.

1.21.4. Method I2

A mixture of protected bis-PMB amine (1 equivalent) in DCM/TFA (3:1 to 1:1 ratio) is stirred at room temperature for a period ranging from 20 minutes to 3 hours. The mixture is worked up using an aqueous basic solution and an organic solvent. The two phases are separated and the organic layer is dried and concentrated. Alternatively, the reaction mixture is first concentrated and then the residue is subjected to the above-mentioned work-up. The residue obtained from the above work-up can be purified by silica chromatography or by Isoroot® SCX-3 (Biotage) ion exchange resin or stored as such.

1.21.5. Illustrative Example of Method I2: Synthesis of 5-(7-amino-3-methyl-benzimidazol-5-yl)oxy-4-methyl-pyridine-2-carbonitrile (Compound 28)

TFA (5 mL) was added to a solution of Int.51 (23.1 mmol) in DCM (55 mL) at 0 °C. The mixture was stirred at room temperature for 20 minutes. Toluene was added and the mixture was concentrated. The residue was partitioned between saturated NaHCO ₃ and DCM. The two phases were separated and the organic layer was dried (Na ₂ SO ₄ ) and concentrated. The residue was purified by flash column chromatography (SiO ₂ , 100:0 to 95:5 EtOAc/MeOH). The product obtained from the column was washed with THF to give the desired product (Int 79).

1.21.6. Method I3

A mixture of protected bis-PMB amine (1 equivalent) in TFA is stirred at room temperature for a period ranging from 30 minutes to approximately 16 hours. The mixture is diluted in an organic solvent and washed with a basic aqueous solution. The two phases are separated and the organic layer is dried and concentrated. Alternatively, prior to washing, the reaction mixture may first be concentrated. The organic layer is then concentrated, and the residue is purified by silica chromatography or by Isoroot® SCX-3 (Biotage) ion exchange resin, or can be used as is without any further purification.

1.21.7. Illustrative Example of Method I3: 5-[(7-amino-3-methyl-3H-benzoimidazol-5-yl)methyl-amino]-4-methyl-pyridine-2-carbonitrile (Compound 62) synthesis

A solution of Int.41 (2.4 mmol) in TFA (10 mL) was stirred at room temperature for 3 hours. The mixture was diluted (DCM) and washed with saturated NaHCO ₃ followed by 5N NaOH. The two layers were separated and the aqueous layer was further extracted with DCM. The organic layers were combined, dried (filtered through a phase separator) and concentrated. The residue was purified by flash column chromatography (SiO ₂ , 100:0 to 90:10 EtOAc/MeOH) to give the desired product.

1.22. Synthesis of 4-ethyl-6-methanesulfonyl-pyridin-3-ylamine (Int.70).

1.22.1. Step i: 4-Ethyl-2-methylsulfonyl-5-nitro-pyridine

A mixture of 2-bromo-4-ethyl-5-nitro-pyridine (4.35 mmol) and sodium methanesulfonate (4.35 mmol) in DMSO (10 mL) was stirred at room temperature for 1.5 h. The mixture was poured into ice water and stirred until the ice melted. The mixture was filtered and the solid (desired product) was collected.

1.22.2. Step ii: 4-Ethyl-6-methylsulfonyl-pyridin-3-amine

A mixture of 4-ethyl-2-methylsulfonyl-5-nitro-pyridine (4.1 mmol), NH ₄ Cl (26.65 mmol) and iron powder (33 mmol) in H ₂ O (10 mL) was heated at 90 °C to 1 Stir for an hour. The reaction was stopped, filtered and extracted with EtOAc. The organic layer was dried (Na ₂ SO ₄ ) and concentrated to give the desired product.

1.23. Synthesis of 4-ethyl-5-iodo-2-methylsulfonyl-pyridine (Int.74)

To a solution of KI (15 mmol) and NaNO ₂ (12 mmol) in H ₂ O (1.8 mL), Int.70 (6 mmol) and p-TsOH in MeCN (12 mL), maintaining the temperature at 10-15 °C. -H ₂ O (18 mmol) was added dropwise to the mixture. The mixture was stirred at 10-15 °C for 10 minutes and then at room temperature for 1 hour. The mixture was cooled to 0 °C, neutralized (saturated NaHCO ₃ ) and extracted (DCM). The organic layer was dried (Na ₂ SO ₄ ) and concentrated. The residue was purified by flash column chromatography (SiO ₂ , 70:30 petroleum ether/EtOAc) to give the desired product.

Example 2. Synthesis of compounds of the present invention

2.1. General method: acylation of amine intermediates with carbonyl chloride derivatives to give final compounds

wherein R _A can be an optionally substituted aryl or heteroaryl; L can be NH, NMe or O; R _B may be cycloalkyl, OMe.

2.1.1. Method J1

R _B COCl (1 equiv.) is added to a solution of the intermediate amine (1 equiv.) in DCM/pyridine (2:1 to 5:1 ratio) at 0°C. The mixture is stirred for a period ranging from 40 minutes to 2 hours. The mixture is partitioned between an organic solvent and an aqueous solution. The two phases are separated and the organic layer is dried and concentrated. Alternatively, the reaction mixture can be concentrated without work-up. The residue can be purified by chromatography on silica or by preparative HPLC or by precipitation using a suitable solvent mixture.

2.1.2. Illustrative Example of Method J1: N-(6-((6-cyano-4-ethylpyridin-3-yl)(methyl)amino)-1-methyl-1H-benzo[d]imidazole-4- 1) Synthesis of cyclopropanecarboxamide (Compound 1)

Cyclopropanecarbonyl chloride (0.39 mmol) was added to a solution of Int.63 (0.39 mmol) in 1:2 pyridine/DCM (2.55 mL) at 0 °C. The mixture was stirred at room temperature for 40 minutes. The reaction mixture was concentrated and the residue was purified by flash column chromatography (SiO2, 100:0 to 97:3 DCM/MeOH) to provide the desired product.

2.1.3. Method J2

R _B C(=0)Cl (1.5-3 equiv.) is added to a solution of the intermediate amine (1 equiv.) in DCM followed by pyridine (1.5-3 equiv.). The mixture is stirred for a period ranging from 2 hours to approximately 16 hours. The mixture is partitioned between an organic solvent and an aqueous solution. The two phases are separated and the organic layer is dried and concentrated. The residue can be purified by silica chromatography, preparative HPLC, or precipitation using a suitable solvent mixture.

2.1.4. Illustrative Example of Method J2: Methyl 6-((6-cyano-4-ethylpyridin-3-yl)(methyl)amino)-1-methyl-1H-benzo[d]imidazol-4-ylcarba Synthesis of Mate (Compound 4)

Methyl chloroformate (0.33 mmol) was added to a solution of Int.63 (0.22 mmol) in anhydrous DCM (2.2 mL) followed by pyridine (0.33 mmol). The mixture was stirred at room temperature for 2 hours. The mixture was partitioned between H ₂ O and DCM. The two phases were separated and the organic layer was washed (saturated NaHCO ₃ ), dried (Na ₂ SO ₄ ) and concentrated. The residue was purified by flash column chromatography (SiO ₂ , 100:0 to 95:5 DCM/MeOH) to provide the desired product.

2.2. General Method: Acylation of Amine Intermediates with Carboxylic Acid Derivatives to Obtain Final Compounds

wherein R _A can be an optionally substituted aryl or heteroaryl; L can be NH, NMe or O; R _C can be cycloalkyl, substituted cycloalkyl.

2.1.1. Method K1

(COCl) ₂ (1.4-2 equiv.) is added to a solution of the carboxylic acid (1.5-2 equiv.) in DCM at 0 °C. A catalytic amount of DMF is added and the reaction is stirred at 0° C. for a period ranging from 30 minutes to 1 hour. A solution of the intermediate amine (1 equiv.) and pyridine (2-4 equiv.) in DCM is added to the above mixture. The resulting mixture is stirred at room temperature for a period ranging from 30 minutes to 2 hours. The mixture is partitioned between an organic solvent and an aqueous solution. The two phases are separated and the organic layer is dried and concentrated. The residue can be purified by chromatography on silica, by preparative HPLC or by precipitation using a suitable solvent mixture.

2.2.2. Illustrative Example of Method K1: (1R,2R)-N-[6-[(6-cyano-4-ethyl-3-pyridyl)oxy]-1-methyl-benzimidazol-4-yl] Synthesis of -2-fluoro-cyclopropanecarboxamide (Compound 6).

(COCl) ₂ (0.186 mmol) was prepared as ((1R,2R)-(-)-2-cis-fluoro-cyclopropanecarboxylic acid (ChemCollect, Lot n. 1241399, 0.186 mmol) in DCM (1 mL). ) was then added to DMF (2 drops) The reaction mixture was stirred for 1 hour at 0° C. A solution of Int.65 (0.093 mmol) and pyridine (0.186 mmol) in DCM (1 mL) was added as above. The mixture was stirred at room temperature for 2 hours. Saturated NaHCO ₃ was added and the two phases were separated. The organic layer was concentrated. The residue was purified by flash column chromatography (SiO ₂ , 100:0 to 95:5 DCM /MeOH) gave the desired product.

2.2.3. An illustrative example of method K1: (1R,2R)-N-[6-(N,2-dimethyl-4-methylsulfonyl-anilino)-1-methyl-benzimidazol-4-yl]- 2-Fluoro-cyclopropanecarboxamide (Compound 26) and (1R,2R)-N-[6-(4-ethylsulfonyl-N,2-dimethyl-anilino)-1-methyl-benzimid Dazol-4-yl] -2-fluoro-cyclopropanecarboxamide (Compound 27)

(COCl) ₂ (0.82 mmol) was added to a solution of ((1R,2R)-(-)-cis-2-fluoro-cyclopropanecarboxylic acid (ABCR, Lot n. 1242863, 0.92 mmol) in DCM (1 mL). DMF (1 drop) was then added The reaction mixture was stirred at 0° C. for 45 minutes A solution of a mixture of Int.56A and Int56B (total approx. 0.46 mmol) and pyridine (1.85 mmol) in DCM (1 mL) was added to the mixture and the reaction was stirred at room temperature for 2 hours.The mixture was diluted with DCM, washed (H ₂ O), and the two phases were separated. The organic layer was dried (Na ₂ SO ₄ ) and concentrated. Flash column chromatography (SiO ₂ , 20:80 EtOAc/petroleum ether to 90:10 EtOAc/MeOH) provided a mixture of compound 26 and compound 27. The two components were separated by preparative HPLC.

2.2.4. Method K2

(COCl) ₂ (1.35-2.5 equiv.) is added to a solution of the carboxylic acid (1.4-3 equiv.) in DCM at 0 °C. A catalytic amount of DMF is added and the reaction is stirred at 0° C. for a period ranging from 30 minutes to 1 hour. Solutions of the intermediate amine (1 eq.) and pyridine (1.7 to 4 eq.) in NMP or NMP/DCM are prepared separately to ensure complete dissolution of the intermediate amine. To aid dissolution, heating can be applied (temperatures up to 70 °C). The latter solution is added dropwise to the solution containing the newly formed acyl chloride at 0 °C. The resulting mixture is stirred at room temperature for a period ranging from 1 to 2 hours. The mixture is partitioned between an organic solvent and an aqueous solution. The two phases are separated and the organic layer is dried and concentrated. The residue can be purified by chromatography on silica, by preparative HPLC, or by precipitation using a suitable solvent mixture.

2.2.5. Illustrative Example of Method K2: (1R,2R)-N-[6-[(6-cyano-4-methyl-3-pyridyl)oxy]-1-methyl-benzimidazol-4-yl] Synthesis of -2-fluoro-cyclopropanecarboxamide (Compound 20)

(COCl) ₂ (16.6 mmol) was added to a solution of ((1R,2R)-(-)-cis-2-fluoro-cyclopropanecarboxylic acid (ABCR, lot n. 1242863, 17.5 mmol) in DCM (70 mL). DMF (200 μl) was then added The reaction mixture was stirred at 0° C. for 45 min The solution of compound 28 (12.5 mmol) and pyridine (21.3 mmol) in NMP (15 mL) was heated at 65° C. to dissolve and allowed to cool to room temperature.The solution containing compound 28 and pyridine was added to the mixture containing the activated carboxyl derivative at 0° C. and the reaction was stirred at room temperature for 45 minutes. The mixture was mixed with EtOAc and saturated NaHCO Partitioned between _3. The two phases were separated and the organic layer was further washed (saturated NaHCO ₃ , NH ₄ Cl, H ₂ O), dried (Na ₂ SO ₄ ) and concentrated Crude was DCM/iPrOH Purified by precipitation from , and the resulting solid was washed with Et ₂ O and dried under vacuum to give the desired product.

2.3. General Method: Hydrolysis of Nitrile Groups to Obtain Final Compounds

wherein L is NH, NMe or O; R _C is cycloalkyl, substituted cycloalkyl; Z is N or CH; R ¹⁰ is Me or Et.

2.3.1. method L

A solution of the nitrile starting material (1 eq.) is dissolved in a 4:1 EtOH/DMSO mixture. The resulting organic solution was mixed with 30% H ₂ O _{2 / H 2} O and 1N NaOH in the following ratio 10:2:1 organic solution/H 2 O 2 /1N NaOH. The mixture is stirred at 50 °C for 1 hour. The mixture is partitioned between an organic solvent and an aqueous solution. The two phases are separated and the organic layer is dried and concentrated. The residue can be purified by chromatography on silica, by preparative HPLC, or by precipitation using a suitable solvent mixture.

2.3.2. Illustrative Example of Method L: 5-((4-(Cyclopropanecarboxamido)-1-methyl-1H-benzo[d]imidazol-6-yl)(methyl)amino)-4-ethylp Synthesis of Cholineamide (Compound 24).

A solution of compound 1 (0.19 mmol) was dissolved in a 4:1 EtOH/DMSO mixture (2.3 mL). The resulting organic solution was mixed with 30% H ₂ O ₂ /H ₂ O (0.4 mL) and 1N NaOH (0.23 mL). The mixture was stirred at 50 °C for 1 hour. The reaction mixture was partitioned between dichloromethane and water. The layers were separated and the organic layer was filtered through a phase separator and concentrated. The residue was purified by preparative HPLC to give the desired product.

2.3.3. Method M: general method for the synthesis of urea

Carbonylditriazole (1.5 equiv.) is added to a mixture of 28 (1.0 equiv.) and pyridine (5 equiv.) in DCM. The mixture is then stirred at 50 °C for 1 hour. Without any further treatment, the required amine R ^a NH ₂ is added to the solution at 50° C. and stirred for an additional hour. After completion of the reaction by UPLC, the reaction mixture is cooled to room temperature and diluted with DCM, then the mixture is partitioned into water. The two phases are separated and the organic layer is washed with 0.25M HCl solution, sodium bicarbonate solution, dried and concentrated. The residue can be purified by chromatography on silica, by preparative HPLC, or by precipitation using a suitable solvent mixture.

2.3.4. Illustrative Example of Method M: 1-[6-[(6-Cyano-4-methyl-3-pyridyl)oxy]-1-methyl-benzimidazol-4-yl]-3-isopropyl- Synthesis of Urea (Compound 33)

Carbonylditriazole (1.07 mmol) was added to a mixture of compound 28 (0.71 mmol) and pyridine (3.55 mmol) in DCM (3 mL) and the resulting mixture was stirred at 50 °C. A precipitate formed after 2 hours and the mixture was further stirred for 1 hour. Then methyl amine (2M solution in THF) (2.84 mmol) was added to the solution at 50 °C and stirred for an additional hour. After completion of the reaction by UPLC, the reaction mixture was cooled to room temperature and diluted with DCM, then the mixture was partitioned into water. The two phases were separated and the organic layer was washed with 0.25M HCl solution, sodium bicarbonate solution, dried and concentrated. The residue was used without any further purification.

2.4. 5-[7-[[(1R,2R)-2-fluorocyclopropanecarbonyl]amino]-3-methyl-benzimidazol-5-yl]oxy-4-methyl-pyridine-2-carboxamide Synthesis of (Compound 32)

A mixture of Compound 20 (0.27 mmol) and DMSO (0.5 mL) in H ₂ O (pH 13, 10 mL) was stirred at 50 °C for 72 h. The mixture was filtered. The solid was collected and purified by preparative HPLC to give the desired product.

2.5. 4-methyl-5-[3-methyl-7-(methylamino)benzimidazol-5-yl]oxy-pyridine-2-carbonitrile (Compound 34) and 5-[7-(dimethylamino)-3 Synthesis of -methyl-benzimidazol-5-yl]-oxy-4-methyl-pyridine-2-carbonitrile (Compound 35)

NaH was added to a mixture of compound 28 (0.36 mmol) in THF (10 mL) at 0 °C. After 30 min, Mel (0.36 mmol) was added and the reaction mixture was warmed to room temperature and stirred until complete. After monitoring completion by UPLC, the reaction was diluted in ethyl acetate and washed with water. The organic layer was dried over Na ₂ SO ₄ , filtered and evaporated. Compounds 34 and 35 were isolated by preparative HPLC.

Exemplary compounds of the present invention listed in Table III below and Comparative Examples were prepared according to the synthetic methods disclosed herein using the intermediates listed in Table II. NMR spectral data of the compounds of the present invention and some of the comparative examples are provided in Table IV.

[Table II]

Exemplary Intermediates for Compounds of the Invention

[Table III]

Exemplary Compounds of the Invention

[Table IV]

NMR data of exemplary compounds of the present invention

Example 3. Comparative compound

3.1. Compound A 2-[4-[(3-methylbenzimidazol-5-yl)amino]phenyl]acetonitrile

A mixture of Pd ₂ (dba) ₃ (0.01 mmol) and Xantphos (0.01 mmol) in 1,4-dioxane (1 mL) was sonicated and 6-bromo-1 in 1,4-dioxane (2 mL) under nitrogen. -Methyl-benzimidazole (0.45 mmol), 2-(4-aminophenyl)acetonitrile (0.58 mmol) and Cs ₂ CO ₃ (0.62 mmol) were added to the mixture. The mixture was stirred at 110 °C for 12 hours. The mixture was diluted (DCM), washed (H ₂ O), dried (phase separator) and concentrated. The residue was purified by preparative HPLC to give the desired product (Compound A).

MW: 262.3. MS Ms'd: 263.2.

NMR: 1H NMR (400 MHz, DMSO-d6): δ = 8.13 (1 H, s), 7.68 (1 H, dd), 7.60 (1 H, dd), 7.54 (1 H, d), 7.32 (1 H, d), 7.24 (1 H, t), 6.91 (1 H, dd), 3.77 (3 H, s), 3.39 (3 H, s).

3.2. compound B

A mixture of Pd ₂ (dba) ₃ (0.01 mmol) and Xantphos (0.01 mmol) in 1,4-dioxane (1 mL) was sonicated and 6-bromo-1 in 1,4-dioxane (2 mL) under nitrogen. -Methyl-benzimidazole (0.45 mmol), 2,3-dihydro-1,4-benzodioxin-6-amine (0.58 mmol) and Cs ₂ CO ₃ (0.62 mmol) were added to the mixture. The mixture was stirred at 110 °C for 12 hours. The mixture was diluted (DCM), washed (H ₂ O), dried (phase separator) and concentrated. The residue was purified by preparative HPLC to give the desired product (Compound B).

MW: 281.3. MS Ms'd: 282.1.

NMR: 1H NMR (400 MHz, DMSO-d6): δ = 7.94 (1 H, s), 7.80 (1 H, br s), 7.45 (1 H, d), 7.05 (1 H, d), 6.86 ( 1 H, dd), 6.73 (1 H, dd), 6.61–6.57 (2 H, m), 4.22–4.16 (4 H, m), 3.71 (3 H, s).

3.3. Compound C 3-fluoro-4-[methyl-(3-methylbenzimidazol-5-yl)amino]benzonitrile

3.3.1. Step i: 3-Fluoro-4-[(3-methylbenzimidazol-5-yl)amino]benzonitrile

6-Bromo-1-methyl-benzimidazole (2.38 mmol), 4-amino-3-fluoro-benzonitrile (3.57 mmol), XPhos (0.95 mmol), Cs ₂ CO in anhydrous toluene (8 mL) A mixture of ₃ (7.14 mmol) and Pd(OAc) ₂ (0.71 mmol) was stirred at 110 °C for approximately 16 h. The mixture was diluted (EtOAc), washed (H ₂ O), dried (Na ₂ SO ₄ ) and concentrated to give the desired product 3-fluoro-4-[(3-methylbenzimidazol-5-yl) amino]benzonitrile.

3.3.2. Step ii: 3-Fluoro-4-[methyl-(3-methylbenzimidazol-5-yl)amino]benzonitrile (Compound C)

NaH (7.14 mmol) was added to a solution of 3-fluoro-4-[(3-methylbenzimidazol-5-yl)amino]benzonitrile (2.38 mmol) in THF (10 mL) at 0 °C. The mixture was stirred for 30 minutes. Mel (4.76 mmol) was added and the mixture was stirred at room temperature for 3 hours. The mixture was diluted (DCM), washed (H ₂ O) and concentrated. The residue was purified by preparative HPLC to give the desired product (Compound C).

MW: 280.1. MS Ms'd: 281.0.

NMR: 1H NMR (400 MHz, DMSO-d6): δ = 8.13 (1 H, s), 7.68 (1 H, dd), 7.60 (1 H, dd), 7.54 (1 H, d), 7.32 (1 H, d), 7.24 (1 H, t), 6.91 (1 H, dd), 3.77 (3 H, s), 3.39 (3 H, s).

3.4. compound D

The synthesis of this compound is disclosed in PCT International Application (2013) WO 2013117645 (Menet et al, 2013).

biological Example

Example 4. In vitro assay

4.1. JAK1 inhibition assay

4.1.1. JAK1 assay polyGT substrate

Recombinant human JAK1 catalytic domain (amino acids 850-1154; catalog number 08-144) was purchased from Carna Biosciences. 10 ng of JAK1 was added to kinase reaction buffer (15 mM Tris-HCl pH 7.5, 1 mM DTT, 0.01% Tween-20, 10 mM MgCl ₂ , 2 μM non-radioactive ATP, 0.25 μCi ³³ P-gamma-ATP (GE Health Care, cat. no. AH9968) final concentration) in a polypropylene 96-well plate (Grayner) in a total volume of 25 μl in the presence or absence of 12.5 μg polyGT substrate (Sigma cat. no. P0275) and 5 μl of test compound or vehicle. (Greiner), V-bottom). After 45 minutes at 30° C., the reaction is stopped by adding 25 μl/well of 150 mM phosphoric acid. Transfer all terminated kinase reactions to a prewashed (75 mM phosphoric acid) 96 well filter plate (Perkin Elmer catalog number 6005177) using a cell harvester (Perkin Elmer). The plate is washed 6 times with 300 μl/well of 75 mM phosphoric acid solution and the bottom of the plate is sealed. Add 40 μl/well of Microscint-20, seal the top of the plate and read using Topcount (Perkin Elmer). Kinase activity is calculated by subtracting the counts per minute (cpm) obtained in the presence of the positive control inhibitor (10 μM staurosporine) from the cpm obtained in the presence of vehicle. The ability of a test compound to inhibit this activity is determined as follows:

Percent inhibition = ((RFU test compound - RFU control) / (RFU vehicle - RFU control)) x 100

RFU test compound = RFU measured for the sample with the test compound present

RFU control = RFU measured for sample with positive control inhibitor

RFU vehicle = RFU measured in the presence of vehicle

A series of dose dilutions are prepared for the compounds to allow for testing of dose-response effects in the JAK1 assay and calculation of the IC ₅₀ for each compound. Each compound was routinely tested at a concentration of 20 μM followed by a final concentration of 1% DMSO in a serial 1/3 dilution, 8 points (20 μM - 6.67 μM - 2.22 μM - 740 nM - 247 nM - 82 nM - 27 nM - 9 nM). do. As series compound potency increased, more dilutions were prepared and/or the highest concentration was lowered (eg 5 μM, 1 μM).

4.1.2. JAK1 Ulight-JAK1 peptide assay

Recombinant human JAK1 (catalytic domain, amino acids 866-1154; catalog number PV4774) was purchased from Invitrogen. 1 ng of JAK1 was mixed with 20 nM Ulight-JAK1 (tyr1023) peptide in kinase reaction buffer (25 mM MOPS pH 6.8, 0.01% Brij-35, 5 mM MgCl ₂ , 2 mM DTT, 7 μM ATP) in 4 μl. were incubated in white 384 Opti plates (Perkin Elmer, cat. no. 6007290) in a total volume of 20 μl in the presence or absence of test compound or vehicle (DMSO, 1% final concentration). After 60 min at room temperature, 20 μl/well of Detection Mixture (1 x Detection Buffer (Perkin Elmer, Cat. No. CR97-100C), 0.5 nM Europium-anti-Phosphotyrosine (PT66) (Perkin Elmer, Cat. No. AD0068) , 10 mM EDTA) was added to quench the reaction. The reading is performed using an Envision with excitation at 320 nm and measuring the emission at 615 nm (Perkin Elmer). Kinase activity was calculated by subtracting the relative fluorescence units (RFU) obtained in the presence of a positive control inhibitor (10 μM staurosporine) from the RFU obtained in the presence of vehicle. The ability of test compounds to inhibit this activity was determined as follows:

Percent inhibition = ((RFU measured for samples with test compound - RFU measured for samples with positive control inhibitor) / (RFU measured in the presence of vehicle - measured for samples with positive control inhibitor RFU)) x 100.

A series of dose dilutions were prepared for the compounds to allow for testing of dose-response effects in the JAK1 assay and calculation of the IC50 for the compounds. Each compound is routinely tested at a concentration of 20 μM followed by a serial 1/5 dilution, final concentration of 1% DMSO at 10 points. If the series compound potency increases, more dilutions are prepared and/or the highest concentration is lowered (eg 5 μM, 1 μM). Data are presented as mean IC ₅₀ from the assay ± standard error of the mean.

[Table V]

JAK1 IC ₅₀ values of exemplary compounds of the present invention

* > 500 nM

** > 100-500 nM

*** > 50-100 nM

****0.1-50 nM

[Table VI]

JAK1 IC ₅₀ values of comparative compounds

4.1.3. JAK1 Ki measurement assay

For the determination of Ki, different amounts of compound are mixed with an enzyme, and the enzyme reaction is tracked as a function of ATP concentration. The Ki is determined by double inverse plotting of Km versus compound concentration (Lineweber-Burk plot). 1 ng of JAK1 (Invitrogen, PV4774) is used for this assay. The substrate was 50 nM Ulight-JAK-1 (Tyr1023) peptide (Perkin Elmer, TRF0121). The reaction is performed in 25 mM MOPS pH 6.8, 0.01%, 2 mM DTT, 5 mM MgCl2 Brij-35 with various concentrations of ATP and compounds. Phosphorylated substrates are measured using Eu-labeled anti-phosphotyrosine antibody PT66 (Perkin Elmer, AD0068) as described in 1.1.2. Readings are performed on an Envision (Perkin Elmer) with excitation at 320 nm and emission at 615 nm and 665 nm.

4.2. JAK2 inhibition assay

4.2.1. JAK2 assay polyGT substrate

Recombinant human JAK2 catalytic domain (amino acids 808-1132; catalog number PV4210) was purchased from Invitrogen. 0.025 mU of JAK2 was added to kinase reaction buffer (5 mM MOPS pH 7.5, 9 mM MgAc, 0.3 mM EDTA, 0.06% Breeze, and 0.6 mM DTT, 1 μM non-radioactive ATP, 0.25 μCi ³³ P-gamma-ATP (GE Health Care, cat. # AH9968) final concentration) in the presence or absence of 2.5 μg polyGT substrate (Sigma cat. # P0275) and 5 μl test compound or vehicle (DMSO, 1% final concentration) in a total volume of 25 μl, poly Culture in propylene 96-well plates (Grainer, V-bottom). After 90 minutes at 30° C., the reaction is stopped by adding 25 μl/well of 150 mM phosphoric acid. Transfer all terminated kinase reactions to a prewashed (75 mM phosphoric acid) 96 well filter plate (Perkin Elmer catalog number 6005177) using a cell harvester (Perkin Elmer). The plate is washed 6 times with 300 μl/well of 75 mM phosphoric acid solution and the bottom of the plate is sealed. Add 40 μl/well of Microscint-20, seal the top of the plate and read using TopCount (Perkin Elmer). Kinase activity is calculated by subtracting the counts per minute (cpm) obtained in the presence of the positive control inhibitor (10 μM staurosporine) from the cpm obtained in the presence of vehicle. The ability of a test compound to inhibit this activity is determined as follows:

Percent inhibition = ((RFU test compound - RFU control) / (RFU vehicle - RFU control)) x 100

RFU test compound = RFU measured for the sample with the test compound present

RFU control = RFU measured for sample with positive control inhibitor

RFU vehicle = RFU measured in the presence of vehicle

A series of dose dilutions are prepared for the compounds to allow for testing of dose-response effects in the JAK2 assay and calculation of the IC ₅₀ for the compounds. Each compound was routinely tested at a concentration of 20 μM followed by a final concentration of 1% DMSO in a serial 1/3 dilution, 8 points (20 μM - 6.67 μM - 2.22 μM - 740 nM - 247 nM - 82 nM - 27 nM - 9 nM). do. If the series compound potency increases, more dilutions are prepared and/or the highest concentration is lowered (eg 5 μM, 1 μM).

4.2.2. JAK2 Ulight-JAK1 peptide assay

Recombinant human JAK2 (catalytic domain, amino acids 866-1154; catalog number PV4210) was purchased from Invitrogen. 0.0125 mU of JAK2 was added to 25 nM Ulight-JAK1 (tyr1023) peptide ( _Perkin Elmer catalog number TRF0121 )) and 4 μl of test compound or vehicle (DMSO, 1% final concentration) in a total volume of 20 μl, on white 384 Opti plates (Perkin Elmer, catalog number 6007290). After 60 min at room temperature, 20 μl/well of Detection Mixture (1 x Detection Buffer (Perkin Elmer, Cat. No. CR97-100C), 0.5 nM Europium-anti-Phosphotyrosine (PT66) (Perkin Elmer, Cat. No. AD0068) , 10 mM EDTA) was added to stop the reaction. The reading is performed using an Envision with excitation at 320 nm and measuring the emission at 615 nm (Perkin Elmer). Kinase activity was calculated by subtracting the relative fluorescence units (RFU) obtained in the presence of a positive control inhibitor (10 μM staurosporine) from the RFU obtained in the presence of vehicle. The ability of test compounds to inhibit this activity was determined as follows:

Percent inhibition = ((RFU measured for samples with test compound - RFU measured for samples with positive control inhibitor) / (RFU measured in the presence of vehicle - measured for samples with positive control inhibitor RFU)) x 100.

A series of dose dilutions are prepared for the compounds to allow for testing of dose-response effects in the JAK2 assay and calculation of the IC ₅₀ for the compounds. Each compound is routinely tested at a concentration of 20 μM followed by a serial 1/5 dilution, final concentration of 1% DMSO at 10 points. If the series compound potency increases, more dilutions are prepared and/or the highest concentration is lowered (eg 5 μM, 1 μM). Data are presented as mean IC ₅₀ from the assay ± standard error of the mean.

The following compounds were tested for their activity and IC ₅₀ values for JAK2 as measured using the assay disclosed herein and are provided in Table VII below.

[Table VII]

JAK2 IC ₅₀ values of exemplary compounds of the present invention

* > 500 nM

** > 100-500 nM

*** > 50-100 nM

****0.1-50 nM

[Table VIII]

JAK2 IC ₅₀ values of comparative compounds

4.2.3. JAK2 Kd measurement assay

JAK2 (Invitrogen, PV4210) is used at a final concentration of 2.5 nM. Binding experiments were performed in 50 mM Hepes pH 7.5, 0.01% Breeze-35, 10 mM using 25 nM Kinase Tracer 236 (Invitrogen, PV5592) and 2 nM Eu-anti-GST (Invitrogen, PV5594) at various compound concentrations. MgCl ₂ , 1 mM EGTA. Detection of the tracer is performed according to the manufacturer's procedure.

4.3. JAK3 inhibition assay

4.3.1. Analysis of the JAK3 Ulight-JAK1 peptide

Recombinant human JAK3 catalytic domain (amino acids 781-1124; catalog number PV3855) was purchased from Invitrogen. 0.5 ng JAK3 protein was mixed with kinase reaction buffer (25 mM Tris pH 7.5, 0.5 mM EGTA, 10 mM MgCl ₂ , 2.5 mM DTT, 0.5 mM Na ₃ VO ₄ , 5 mM b-glycerophosphate, 0.01% Triton X-100, 1 2.5 μg PolyGT Substrate (Sigma Cat. No. P0275) in μM non-radioactive ATP, 0.25 μCi ³³ P-gamma-ATP (GE Healthcare, Cat. No. AH9968) final concentration) and 5 μl of test compound or vehicle (DMSO, 1 % final concentration) in polypropylene 96-well plates (Grainer, V-bottom) in a total volume of 25 μl in the presence or absence. After 45 minutes at 30° C., the reaction was quenched by adding 25 μl/well of 150 mM phosphoric acid. All completed kinase reactions were transferred to a prewashed (75 mM phosphoric acid) 96 well filter plate (Perkin Elmer catalog number 6005177) using a cell harvester (Perkin Elmer). The plate was washed 6 times with 300 μl/well of 75 mM phosphoric acid solution and the bottom of the plate was sealed. 40 μl/well of Microscint-20 was added, the top of the plate was sealed and readings were performed using TopCount (Perkin Elmer). Kinase activity was calculated by subtracting the counts per minute (cpm) obtained in the presence of the positive control inhibitor (10 μM staurosporine) from the cpm obtained in the presence of vehicle. The ability of test compounds to inhibit this activity was determined as follows:

Percent inhibition = ((RFU test compound - RFU control) / (RFU vehicle - RFU control)) x 100

RFU test compound = RFU measured for the sample with the test compound present

RFU control = RFU measured for samples with positive control inhibitor

RFU vehicle = RFU measured in the presence of vehicle

A series of dose dilutions were prepared for each compound to allow for testing of dose-response effects in the JAK3 assay and calculation of the IC ₅₀ for that compound. Each compound was routinely tested at a concentration of 20 μM followed by a serial 1/5 dilution, final concentration of 1% DMSO at 10 points. If the series compound potency increases, more dilutions are prepared and/or the highest concentration is lowered (eg 5 μM, 1 μM).

The following compounds were tested for their activity and IC ₅₀ values for JAK3 as determined using the assay disclosed herein and are provided in Table IX below.

[Table IX]

JAK3 IC ₅₀ values of exemplary compounds of the present invention

* > 500 nM

** > 100-500 nM

*** > 50-100 nM

****0.1-50 nM

[Table X]

JAK3 IC ₅₀ values of comparative compounds

4.3.2. JAK3 Ki measurement assay

For the determination of Ki, different amounts of compound are mixed with an enzyme, and the enzyme reaction is tracked as a function of ATP concentration. The Ki is determined by double inverse plotting of Km versus compound concentration (Lineweber-Burk plot). JAK3 (Carna Biosciences, 09CBS-0625B) is used at a final concentration of 10 ng/ml. The substrate is poly(Glu,Tyr) sodium salt (4:1), MW 20 000 - 50 000 (Sigma, P0275). The reaction was performed in 25 mM Tris pH 7.5, 0.01% Triton X-100, 0.5 mM EGTA, 2.5 mM DTT, 0.5 mM Na ₃ VO ₄ , 5 mM b-glycerophosphate, 10 mM MgCl ₂ with various concentrations of ATP and compounds. and stopped by addition of 150 mM phosphoric acid. Measurement of phosphate bound to the substrate polyGT is performed by loading the samples onto a filter plate (using a harvester, Perkin Elmer) and subsequent washing. Bound ³³ P in PolyGT is measured on a TopCount scintillation counter after addition of scintillation liquid to the filter plate (Perkin Elmer).

4.4. TYK2 inhibition assay

4.4.1. TYK2 Ulight-JAK1 peptide assay

Recombinant human TYK2 catalytic domain (amino acids 871-1187; catalog number 08-147) was purchased from Karna Biosciences. 5 ng of TYK2 was added to kinase reaction buffer (25 mM Hepes pH 7.2, 50 mM NaCl, 0.5 mM EDTA, 1 mM DTT, 5 mM MnCl ₂ , 10 mM MgCl ₂ , 0.1% Breeze-35, 0.1 μM non-radioactive ATP, 0.125 μCi ³³ In the presence or absence of 12.5 μg polyGT substrate (Sigma cat # P0275) in P-gamma-ATP (GE Healthcare, cat # AH9968 final concentration) and 5 μl test compound or vehicle (DMSO, 1% final concentration) Culture in polypropylene 96-well plates (Grainer, V-bottom) in a total volume of 25 μl. After 90 minutes at 30° C., the reaction was quenched by adding 25 μl/well of 150 mM phosphoric acid. All completed kinase reactions were transferred to a prewashed (75 mM phosphoric acid) 96 well filter plate (Perkin Elmer catalog number 6005177) using a cell harvester (Perkin Elmer). The plate was washed 6 times with 300 μl/well of 75 mM phosphoric acid solution and the bottom of the plate was sealed. 40 μl/well of Microscint-20 was added, the top of the plate was sealed and readings were performed using TopCount (Perkin Elmer). Kinase activity was calculated by subtracting the counts per minute (cpm) obtained in the presence of the positive control inhibitor (10 μM staurosporine) from the cpm obtained in the presence of vehicle. The ability of test compounds to inhibit this activity was determined as follows:

Percent inhibition = ((cpm measured for samples with test compound - measured for samples with positive control inhibitor)/(cpm measured in the presence of vehicle - measured for samples with positive control inhibitor cpm)) x 100.

A series of dose dilutions were prepared for the compounds to allow for testing of dose-response effects in the TYK2 assay and calculation of the IC ₅₀ for the compounds. The compounds were routinely tested at a concentration of 20 μM followed by a final concentration of 1% DMSO in a series of 1/3 dilutions, 8 points (20 μM - 6.67 μM - 2.22 μM - 740 nM - 247 nM - 82 nM - 27 nM - 9 nM). . If the series compound potency increases, more dilutions are prepared and/or the highest concentration is lowered (eg 5 μM, 1 μM).

The following compounds were tested for their activity and IC ₅₀ values for TYK2 as determined using the assay disclosed herein and are provided in Table XI below.

[Table XI]

TYK2 IC50 values of exemplary compounds of the present invention

* > 500 nM

** > 100-500 nM

*** > 50-100 nM

****0.1-50 nM

[Table XII]

TYK2 IC ₅₀ values of comparative compounds

4.4.2. TYK2 Kd measurement assay

TYK2 (Carna Biosciences, 09CBS-0983D) is used at a final concentration of 5 nM. Binding experiments were performed in 50 mM Hepes pH 7.5, 0.01% Breeze-35, 10 mM using 50 nM Kinase Tracer 236 (Invitrogen, PV5592) and 2 nM Eu-anti-GST (Invitrogen, PV5594) at various compound concentrations. MgCl ₂ , 1 mM EGTA. Detection of the tracer is performed according to the manufacturer's procedure.

Example 5. Cell analysis:

5.1. JAK1, JAK2 and TYK2 Selective Cell Assay

5.1.1. Selective JAK1 cell assay, activation of STAT1 by IFNα in PBMCs

Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats by density gradient centrifugation using LymphoPrep™ medium (Axis-Shield) under sterile conditions, followed by Ca++Mg++ Perform 3 consecutive washing steps in PBS without water. PBMC were resuspended in plain RPMI 1640 medium containing 10% (v/v) heat-inactivated FBS, 1% Pen-Strep (100 U/mL penicillium and 100 μg/mL streptomycin) and incubated at 37°C 5 Further incubation in a humidified incubator at % CO ₂ .

PBMCs are seeded at 5.0 10 ⁰⁶ cells/well in a 200 μl volume of RPMI 1640 (Invitrogen) containing 10% (v/v) FBS and 1% Pen-Strep (Invitrogen) in 24 well plates.

PBMCs are treated with test compounds at 37° C. 5% CO ₂ for 30 min. 25 μl of 10x concentrated compound dilution is added to the medium. After 30 minutes of test compound/vehicle pre-treatment, PBMCs were treated with recombinant human IFNα (Peppro) at a final concentration of 100 ng/mL by addition of 25 μl (10x concentrated) of cytokine trigger at 37° C. 5% CO ₂ . PeproTech) for 30 minutes to obtain a final volume of 250 μl/well.

All compounds were started at 20 μM and then diluted in 0.2% DMSO in a series of 1/3 dilutions, a total of 8 points (20 μM, 6.6 μM, 2.2 μM, 0.74 μM, 0.25 μM, 0.082 μM, 0.027 μM and 0.009 μM). Test once at the final concentration.

After 30 minutes of cytokine stimulation, 250 μl of the cell suspension is transferred to a 96-well V-bottom plate and centrifuged at 1000 rpm for 5 minutes to pellet the cells, then the supernatant is removed. The cell pellet is reconstituted in 100 μl of 1x Lysis Buffer supplemented with EDTA-free protease inhibitor cocktail (Roche Applied Sciences, Cat# 11836170001) and samples are then frozen and stored at -80°C. 1x Lysis Buffer is provided in the Phospho-STAT1 ELISA kit, and the buffer contains a phosphatase inhibitor. Endogenous levels of phosphorylated STAT1 were measured using a 96-well PathScan® Phospho-STAT1 (Tyr701) sandwich ELISA kit (Cell Signaling, product #7234) according to the manufacturer's instructions. Quantitative analysis.

HRP activity (HRP is conjugated to a secondary antibody) was measured by the addition of 100 μl of freshly prepared luminol substrate (BM Chemiluminescence ELISA substrate (POD), Roche, catalog number 11582950001) at room temperature in the dark for 5 measured by minute incubation and measured on a Thermo Scientific Luminoskan ascending microplate luminometer (at integration of 200 msec).

5.1.2. Selective JAK2 Cell Assay, Activation of STAT5 by GM-CSF in PBMCs

Peripheral blood mononuclear cells (PBMC) were isolated from buffy coats by density gradient centrifugation using Lympoprep™ medium (Axis Shield) under sterile conditions followed by three consecutive washing steps in PBS without Ca++Mg++ do PBMC were resuspended in plain RPMI 1640 medium containing 10% (v/v) heat-inactivated FBS, 1% Pen-Strep (100 U/mL penicillium and 100 μg/mL streptomycin) and incubated at 37°C 5 Further incubation in a humidified incubator at % CO ₂ .

PBMCs are seeded at 5.0E06 cells/well in a 200 μl volume of RPMI 1640 (Invitrogen) containing 10% (v/v) FBS and 1% Pen-Strep (Invitrogen) in 24 well plates.

PBMCs are treated with test compounds by adding 25 μl of a 10x concentrated compound dilution to the medium and incubated at 37° C. 5% CO ₂ for 30 minutes. Subsequently, PBMCs are stimulated with recombinant human GM-CSF (Pepprotech) at a final concentration of 0.5 ng/ml by addition of 25 μl (10x concentrated) of cytokine trigger per well to obtain a final volume of 250 μl . Cells are triggered at 37° C. 5% CO ₂ for 30 min.

All compounds were started at 20 μM and then diluted in 0.2% DMSO in a series of 1/3 dilutions, a total of 8 doses (20 μM, 6.6 μM, 2.2 μM, 0.74 μM, 0.25 μM, 0.082 μM, 0.027 μM and 0.009 μM). Test once at the final concentration.

After 30 minutes of cytokine stimulation, 250 μl of the cell suspension is transferred to a 96-well V-bottom plate followed by centrifugation at 1000 rpm for 5 minutes to pellet the cells. The cell supernatant is removed and the pellet is reconstituted in 100 μl of 1x Lysis Buffer supplemented with EDTA-free protease inhibitor cocktail (Roche Applied Sciences, product number 11836170001), then samples are frozen and stored at -80°C. 1x Lysis Buffer is provided in the Phospho-STAT5 ELISA kit, and the buffer contains a phosphatase inhibitor. Endogenous levels of phosphorylated STAT5 are quantified using a 96-well PathScan® Phospho-STAT5 (Tyr694) Sandwich ELISA Kit (Cell Signaling, Product #7113) according to the manufacturer's instructions.

HRP activity (HRP is conjugated to a secondary antibody) was measured by addition of 100 μl of freshly prepared luminol substrate (BM Chemiluminescence ELISA substrate (POD), Roche, catalog # 11582950001), incubation for 5 minutes at room temperature in the dark. and measured on a Thermo Scientific Luminoscan ascending microplate photometer (at integration of 200 msec).

5.1.3. Selective TYK2 Cell Assay, Activation of STAT4 by IL-12 in NK-92 Cells

NK-92 cells (human malignant non-Hodgkin's lymphoma, an interleukin-2 (IL-2) dependent natural killer cell line, ATCC #CRL-2407).

NK-92 cells were cultured in 0.2 mM myo-inositol, 0.1 mM 2-mercapto-EtOH, 0.1 mM folic acid, 12.5% heat inactivated horse serum (Invitrogen, product number 26050-088), 12.5% heat inactivated Minimum essential medium (MEM) alpha medium w containing FBS, 1% Pen-Strep (100 U/mL Penicillium and 100 μg/mL streptomycin) and 10 ng/mL recombinant human IL-2 (R&D Systems) w /o ribonucleosides and deoxyribonucleosides, 2 mM L-glutamine, 2.2 g/L sodium bicarbonate (Invitrogen, Product No. 22561-021). IL-2 is added to the medium as each medium is freshly replenished. The cells are cultured in a humidified incubator at 37° C. 5% CO ₂ .

Subcultured fractions of NK-92 cells were first washed in plain medium without rhIL-2 and plated in 24-well plates in 0.2 mM myo-inositol, 0.1 mM 2-mercaptoethanol, 0.1 mM folic acid, 12.5% heat inactivated. In a 400 μl volume of Plain Alpha MEM medium w/o rhIL-2 containing horse serum (Invitrogen, product number 26050-088), 12.5% heat inactivated FBS, 1% Pen-Strep (Invitrogen) Seed at 0.5E06 cells/well.

NK-92 cells are treated with test compounds by adding 50 μl of 10× concentrated compound dilution for 30 minutes prior to rhIL-12 stimulation and incubated at 37° C. 5% CO ₂ . After 30 minutes of compound/vehicle pre-treatment, cells were incubated with recombinant human IL-12 (R&D Systems, cat no. 219-IL) at a final concentration of 25 ng/ml by addition of 50 μl (10x concentrated) of the cytokine trigger. ) to obtain a final volume of 500 μl/well. NK-92 cells are stimulated with rhIL-12 for 30 min at 37° C. 5% CO ₂ .

All compounds were started at 20 μM and then diluted in 0.2% DMSO in a series of 1/3 dilutions, a total of 8 doses (20 μM, 6.6 μM, 2.2 μM, 0.74 μM, 0.25 μM, 0.082 μM, 0.027 μM and 0.009 μM). Test once at the final concentration.

Levels of phospho-STAT4 in rhIL-12 stimulated NK-92 cells are quantified using flow cytometric analysis on a Gallios™ flow cytometer (Beckman Coulter). After 30 minutes of cytokine stimulation the cells are fixed by immediately adding 500 μl of forewarmed BD Cytofix fixation buffer (BD Phosflow(TM) cat # 554655) to the wells (the cells is immediately fixed to maintain the phosphorylation state rather than being spun down. It is recommended to fix the cells by adding an equal volume of forewarmed BD Cynofix buffer to the cell suspension). The cells are incubated at 37° C. for 10 minutes. The fixed cell fraction is resuspended (1 ml) and transferred to a FACS tube followed by a centrifugation step (300x g, 10 min) and supernatant removal. Permeability was achieved by mixing (vortexing) the cell pellet and adding 1 ml of BD Phosflow Perm Buffer III (BD Phosflow™, product number 558050) followed by incubation on ice for 30 minutes. make it After the permeabilization step, the cells are washed twice with BD Pharmingen ® staining buffer (BD Pharmingen, product number 554656) with intermediate centrifugation at 300x g for 10 minutes and removal of the supernatant. . The pellet (0.5E06 cells) was resuspended in 100 μl of BD Pharmingen (trademark) staining buffer and stained by mixing 20 μl of PE mouse anti-STAT4 (pY693) to the cells (BD Phosflow (trademark) , PE mouse anti-STAT4 (pY693), catalog number 558249), followed by incubation for 30 minutes at room temperature in the dark. The stained cells were washed once with 2 ml of BD Pharmingen (trademark) staining buffer, resuspended in 500 μl of BD Pharmingen (trademark) staining buffer and analyzed on a Galios (trademark) flow cytometer (Beckman Coulter) did

For all analyses, dead cells and cell debris are excluded by forward scatter (FSC) and side scatter (SSC). Changes in phosphorylation of the STAT4 protein following cytokine stimulation were evaluated by X-median or X-mean fluorescence intensity (MFI) per cell based on 100% of the gated fraction for all cytokine-stimulated test compounds and unstimulated samples. Calculate and get an approximate value.

5.1.4. JAK1, JAK2 and TYK2 assay results:

An unstimulated sample (no trigger/vehicle (0.2% DMSO)) is used as a positive control (100% inhibition). As a negative control (0% inhibition), a stimulated sample (trigger/vehicle (0.2% DMSO)) is used. Calculate 'Z' and 'Percent Inhibition (PIN)' values using the above positive and negative controls.

Percent inhibition is calculated from:

Percent Inhibition = ((RCLU (trigger/vehicle) - RCLU (test compound))/((RCLU (trigger/vehicle) - RCLU (no trigger/vehicle)) x 100

From here

RCLU (trigger/vehicle): Relative chemiluminescent signal measured in the presence of vehicle and triggering agent

RCLU (test compound): Relative chemiluminescent signal measured in the presence of test compound

RCLU (no trigger/vehicle): Relative chemiluminescent signal measured in the presence of vehicle without trigger

When readout signals are presented as X-means (flow cytometric analysis of pSTAT4 levels in cytokine stimulated NK-92 cells), the RCLU is replaced by X-means.

PIN values are plotted for compounds tested as dose-response and EC ₅₀ values are derived using GraphPad Prism software applying non-linear regression (sigmoidal) curve fitting.

5.2. JAK1 mutations in lung cancer and hepatocellular carcinoma cell line analysis

5.2.1. JAK1 mutations induced constitutive signaling.

Cancer cell lines with and without JAK1 mutation (Table I - lung cancer cell lines) were incubated without or with serum for 4 to 6 hours and incubated with cytokine cocktails (IFNγ, IL2, IL4 and IL6 for 5, 10, 30 and 45 minutes). ) stimulated or not stimulated. Phosphorylation of JAK1, STAT1, STAT3 and STAT5 is evaluated by immunoblot (cell signaling antibody).

5.2.2. Targeting JAK1 mutants with JAK inhibitors

5.2.2.1. JAK-STAT pathway phosphorylation:

Cancer cell lines with and without JAK1 mutations are cultured in the presence or absence of different concentrations of JAK inhibitors. Cells are analyzed at 24 and 48 hours for effective JAK-STAT pathway inhibition by immunoblot.

[Table XIII]

Table I: Exemplary lung cancer cell lines

5.2.2.2. cell viability

2D-Analysis:

Cancer cell lines with and without JAK1 mutations are cultured in the presence or absence of increasing concentrations of JAK inhibitors. After 48-72 hours, cell viability is measured using the Cell Titer-Glo Luminescent Cell Viability Assay (Promega) or MTT Assay. Alternatively, cancer cell lines are assayed for cell viability using a Cell Titer-Glo Luminescent Cell Viability Assay (Promega) or MTT assay with a fixed concentration of a JAK inhibitor at different culture time points.

3D-Analysis:

Cancer cell lines with and without the JAK1 mutation are seeded on semi-solid agar medium. The formation of multi-cell colonies is measured by measuring cell viability using a fluorescent dye at different culture time points. Addition of potential inhibitors after cell seeding allows analysis of tumorigenicity inhibitory effects.

5.2.3. Investigation of human JAK1 mutations in murine Ba/F3 cells

(as exemplified by Kan et al., 2013; Staerk et al., 2005; Zenatti et al., 2011)

Construction of JAK1 expression vectors: Wild-type and mutant human JAK1 sequences are cloned into retroviral vectors and clones are identified by sequencing.

Retroviral infection of Ba/F3 cells: Ba/F3 cells are infected with the retroviral supernatant produced in 293T cells.

Ba/F3 cells expressing human WT or mutated JAK1 are incubated with or without IL-3 for 4 hours and phosphorylation of the JAK-STAT pathway is assessed by immunoblot.

The transforming potential of JAK1 mutants is evaluated by measuring the ability of each mutant to induce autonomous growth when expressed in cytokine-dependent Ba/F3 cells. Cell growth is assessed in the absence of the cytokine IL-3.

The sensitivity of the mutant JAK1 transduced Ba/F3 cell line to the JAK inhibitor is assessed by culturing it in the presence or absence of increasing concentrations of the JAK inhibitor. After 48-72 hours, cell viability is measured using the Cell Titer-Glo Luminescent Cell Viability Assay (Promega) or MTT Assay. Alternatively, cancer cell lines are assayed for cell viability using a Cell Titer-Glo Luminescent Cell Viability Assay (Promega) or MTT assay with a fixed concentration of a JAK inhibitor at different culture time points.

5.2.4. In vivo tumorigenic potential of JAK1 mutations

5.2.4.1. Xenograft models:

Mutant JAK1 expressing cells are injected subcutaneously in CD1 nu/nu mice or Rag1−/− mice and evaluated for tumor progression. A subcutaneous tumor volume growth curve is established. Implantability of primary tumors into secondary recipient animals is determined.

5.2.4.2. PDX model

Patient-derived xenograft (PDX) is based on the transfer of a primary tumor (containing a JAK1 mutation) directly from the patient into an immunodeficient mouse. To do this, the patient's tumor must be freshly obtained from surgery, at which point the tumor is either mechanically or chemically excised, at which time a small portion is isolated as a primary stock and established in NOD-SCID mice. The PDX model is maintained by subculturing the cells directly from mouse to mouse once the tumor size becomes too large. Tumors can be implanted either heterotopically (the tumor is implanted subcutaneously into the flank of the mouse) or orthotopically (implanted directly into the mouse's organ of choice).

Phosphorylation of JAK1, STAT1, STAT3 and STAT5 in primary and secondary tumors is evaluated by immunoblot.

5.3. PBL proliferation assay

Human peripheral blood lymphocytes (PBL) are stimulated with IL-2 and proliferation is measured using a BrdU binding assay. The PBLs were first stimulated with PHA for 72 hours to induce IL-2 receptor induction, followed by fasting for 24 hours to arrest cell proliferation, followed by IL-2 stimulation for an additional 72 hours (including 24 hours BrdU labeling). Cells are pre-incubated with test compounds 1 hour prior to IL-2 addition. Cells are cultured in RPMI 1640 containing 10% (v/v) FBS.

5.4. Human Whole Blood Analysis (hWBA)

5.4.1. protocol 1

5.4.1.1. IL-6 stimulation protocol

Flow cytometric analysis is performed to establish JAK1 for JAK2 compound selectivity ex vivo in human whole blood. Therefore, blood is drawn from human volunteers who have given informed consent. The blood is then equilibrated at 37° C. for 30 minutes with gentle shaking, and then aliquoted into Eppendorf tubes. Compounds were added at different concentrations and incubated at 37 °C for 30 min with gentle shaking, followed by interleukin 6 (IL-6) for JAK1-dependent pathway stimulation or GM-CSF for JAK2-dependent pathway stimulation with gentle shaking. Stimulate at 37 °C for 20 min. Phospho-STAT1 and phospho-STAT5 are then evaluated using FACS analysis.

5.4.1.2. Phospho-STAT1 assay

5.4.1.2.1. Preparation of reagents

5X Lysis/Fixation Buffer (BD PhosFlow, catalog number 558049) is diluted 5-fold with distilled water and forewarmed at 37°C. Discard the remaining diluted Lysis/Fixation Buffer.

10 μg rhIL-6 (R&D Systems, catalog number 206-IL) was dissolved in 1 ml of PBS 0.1% BSA to obtain a 10 μg/ml stock solution. The mother liquor is aliquoted and stored at -80 °C.

Three-fold serial dilutions of the compounds are prepared in DMSO (10 mM stock solution). Control-treated samples receive DMSO instead of compound. All samples are incubated with 1% final DMSO concentration.

5.4.1.2.2. Incubation with blood and compounds and stimulation with IL-6

Human blood is collected in heparinized tubes. The blood is divided into aliquots of 148.5 μl. Then, 1.5 μl of the test compound dilution is added to each blood aliquot and the blood sample is incubated for 30 minutes at 37° C. with gentle shaking. One and a half microliters of 10-fold diluted IL-6 stock solution is added to the blood sample (final concentration 10 ng/ml) and the sample is incubated at 37° C. for 20 minutes with gentle shaking.

5.4.1.2.3. leukocyte preparation

At the end of the stimulation period, 3 ml of 1× forewarm lysis/fixation buffer is added directly to the blood sample to lyse red blood cells and fix white blood cells, briefly vortexed and incubated at 37° C. for 15 minutes in a water bath.

Centrifuge the tube at 400xg for 5 minutes at 4°C. The cell pellet is washed with 3 ml of cold 1X PBS, and after centrifugation, the cell pellet is resuspended in 100 μl of ice-cold 1X PBS and 900 μl of ice-cold 100% MeOH is added. Cells are then incubated at 4 °C for 30 min for infiltration.

The infiltrated cells are then washed with 1x PBS containing 3% BSA and finally resuspended in 80 μl of 1X PBX containing 3% BSA.

5.4.1.2.4. Cell labeling with anti-phospho-STAT1 and anti-CD4 antibodies

20 μl of PE mouse anti-STAT1 (pY701) or PE mouse IgG2aκ isotype control antibody (BD Biosciences, catalog numbers 612564 and 559319, respectively) and FITC-conjugated anti-CD4 antibody or control FITC-conjugated isotype Type antibody is added and mixed, then incubated for 30 minutes at 4°C in the dark.

Cells are then washed once with 1X PBS and analyzed on a FACSCanto II flow cytometer (BD Biosciences).

5.4.1.2.5. Fluorescence analysis on FACSCanto II

A total of 50,000 events are counted and phospho-STAT1 positive cells are measured after gating on CD4 ⁺ cells in a lymphocyte gate. Data are analyzed using FACSDiva software and the percentage inhibition of IL-6 stimulation is calculated from the percentage of cells positive for phospho-STAT1 on CD4+ cells.

5.4.1.3. Phospho-STAT5 assay

Preparation of reagents

5X Lysis/Fixation Buffer (BD Phosflow, catalog number 558049) is diluted 5-fold with distilled water and forewarmed at 37°C. Discard the remaining diluted Lysis/Fixation Buffer.

10 μg rhGM-CSF (AbCys S.A., catalog number P300-03) was dissolved in 100 μl PBS 0.1% BSA to obtain a 100 μg/ml stock solution. The mother liquor is aliquoted and stored at -80 °C.

Three-fold serial dilutions of the compounds are prepared in DMSO (10 mM stock solution). Control-treated samples receive DMSO without test compound. All samples are incubated with 1% final DMSO concentration.

5.4.1.3.1. Incubation with blood and compounds and stimulation with GM-CSF

Human blood is collected in heparinized tubes. The blood is divided into aliquots of 148.5 μl. Then, 1.5 μl of compound dilution is added to each aliquot and the blood sample is incubated for 30 minutes at 37° C. with gentle shaking. 5,000-fold diluted GM-CSF stock solution (1.5 μl) was added to the blood sample (final concentration 20 ng/ml) and the sample was incubated at 37° C. for 20 minutes with gentle shaking.

5.4.1.3.2. leukocyte preparation

At the end of the stimulation period, 3 ml of 1× forewarm lysis/fixation buffer is added directly to the blood sample to lyse red blood cells and fix white blood cells, briefly vortexed and incubated at 37° C. for 15 minutes in a water bath.

Centrifuge the tube at 400xg for 5 minutes at 4°C. The cell pellet is washed with 3 ml of cold 1X PBS, and after centrifugation, the cell pellet is resuspended in 100 μl of ice-cold 1X PBS and 900 μl of ice-cold 100% MeOH is added. Cells are then incubated at 4 °C for 30 min for infiltration.

5.4.1.3.3. Cell labeling with anti-phospho-STAT5 and anti-CD33 antibodies

20 μl of PE mouse anti-STAT5 (pY694) or PE mouse IgG1κ isotype control antibody (BD Biosciences, catalog numbers 612567 and 554680, respectively) and APC mouse anti-CD33 antibody (BD Biosciences #345800) or control APC mouse IgG1 isotype antibody (BD Biosciences #345818) is added and mixed, then incubated in the dark at 4°C for 30 minutes.

Cells are then washed once with 1X PBS and analyzed on a FACSCanto II flow cytometer (BD Biosciences).

5.4.1.3.4. Fluorescence analysis on FACSCanto II

A total of 50,000 events are counted and phospho-STAT5 positive cells are determined after gate control on lymphoid CD33 ⁺ cells. Data are analyzed using FACSDiva software and correspond to the percentage of inhibition of GM-CSF stimulation calculated from the percentage of cells positive for phospho-STAT5 on CD33 ⁺ cells.

5.5. protocol 2

5.5.1. stimulation protocol

Flow cytometric analysis is performed to establish JAK1 for JAK2 compound selectivity ex vivo in human whole blood. Therefore, blood is drawn from informed human volunteers. The blood is then equilibrated at 37° C. for 30 minutes with gentle shaking, and then aliquoted into Eppendorf tubes. Compounds were added at different concentrations and incubated at 37 °C for 30 min with gentle shaking, followed by interleukin 6 (IL-6) for JAK1-dependent pathway stimulation, interferon alpha (IFNα) for JAK1/TYK2 pathway stimulation, and JAK1/TYK2 pathway stimulation. Stimulate with interleukin 2 (IL-2) for JAK3 pathway stimulation or GM-CSF for JAK2-dependent pathway stimulation at 37°C for 20 minutes with gentle shaking. Phospho-STAT1 (for IL-6- and IFNa-stimulated cells) and phospho-STAT5 (for IL-2- and GM-CSF-stimulated cells) are then evaluated using FACS analysis.

5.5.2. Phospho-STAT assay

5.5.2.1. Preparation of reagents

5X Lysis/Fixation Buffer (BD Phosflow, catalog number 558049) is diluted 5-fold with distilled water and forewarmed at 37°C. Discard the remaining diluted Lysis/Fixation Buffer.

10 μg rhIL-6 (R&D Systems, catalog number 206-IL) was dissolved in 1 ml of PBS + 0.1% BSA to obtain a 10 μg/ml stock solution. The mother liquor is aliquoted and stored at -80 °C.

10 μg rhIL-2 (R&D Systems, catalog number 202-IL) was dissolved in 1 ml of PBS + 0.1% BSA to obtain a 10 μg/ml stock solution. The mother liquor is aliquoted and stored at -80 °C.

5 μg rhGM-CSF (AbCys S.A., catalog number P300-03) was dissolved in 12.5 ml of PBS + 0.1% BSA to obtain 400 ng/ml stock solution. The mother liquor is aliquoted and stored at -80 °C.

Three-fold serial dilutions of the compounds are prepared in DMSO (10 mM stock solution). Control-treated samples receive DMSO instead of compound. All samples are incubated with 1% final DMSO concentration.

5.5.2.2. Incubation with blood and compounds and stimulation with triggers

Human blood is collected in heparinized tubes. The blood is divided into aliquots of 148.5 μl. Then, 1.5 μl of the test compound dilution is added to each blood aliquot and the blood sample is incubated for 30 minutes at 37° C. with gentle shaking. 1 and 1/2 microliters of 10-fold diluted IL-6 stock, 1.5 μl of uIFNα (PBL Biomedical, catalog number 11200-1) stock, 1.5 μl of 25-fold diluted IL-2 stock, or 1.5 μl of 25-fold diluted IL-2 stock μl of 200-fold diluted GM-CSF stock solution is added to the blood samples and the samples are incubated at 37° C. for 20 minutes with gentle shaking.

5.5.2.3. leukocyte preparation

At the end of the stimulation period, 3 ml of 1× forewarm lysis/fixation buffer is added directly to the blood sample to lyse red blood cells and fix white blood cells, briefly vortexed and incubated at 37° C. for 15 minutes in a water bath.

Centrifuge the tube at 400xg for 5 minutes at 4°C. The cell pellet is washed with 3 ml of cold 1X PBS, and after centrifugation, the cell pellet is resuspended in 100 μl of ice-cold 1X PBS and 900 μl of ice-cold 100% MeOH is added. Cells are then incubated at 4 °C for 30 min for infiltration.

The infiltrated cells are then washed with 1x PBS containing 3% BSA and finally resuspended in 80 μl of 1X PBX containing 3% BSA.

5.5.2.4. cell labeling

20 μl of PE mouse anti-STAT1 (pY701) or PE mouse IgG2aκ isotype control antibody (BD Biosciences, catalog numbers 612564 and 559319, respectively) and APC-conjugated anti-CD4 antibody or control APC-conjugated isotype Type antibodies (BD Biosciences, catalog numbers 555349 and 555751, respectively) are added to the IL-6 and IFNα-stimulated tubes, mixed, and then incubated in the dark at 4° C. for 20 minutes.

20 μl of PE mouse anti-STAT5 (pY694) or PE mouse IgG1κ isotype control antibody (BD Biosciences, catalog numbers 612567 and 554680, respectively) and APC-conjugated anti-CD4 antibody or control APC-conjugated isotype Type antibody (BD Biosciences, catalog numbers 555349 and 555751, respectively) is added to the IL-2-stimulated tube, mixed, and then incubated in the dark at 4° C. for 20 minutes.

20 μl of PE mouse anti-STAT5 (pY694) or PE mouse IgG1κ isotype control antibody (BD Biosciences, catalog numbers 612567 and 554680, respectively) and APC mouse anti-CD33 antibody (BD Biosciences #345800) or control APC mouse IgG1 isotype antibody (BD Biosciences, catalog number 345818) is added to the GM-CSF-stimulated tube and mixed, then incubated in the dark at 4°C for 20 minutes.

Cells are then washed once with 1X PBS and analyzed on a FACSCanto II flow cytometer (BD Biosciences).

5.5.2.5. Fluorescence analysis on FACSCanto II

A total of 50,000 events are counted and phospho-STAT1 positive cells are counted after gating on CD4+ cells in a lymphocyte gate for IL-6- and IFNα-stimulated cells. Phospho-STAT5 positive cells are measured after gate control on CD4+ cells in the lymphocyte gate for IL-2-stimulated cells. Phospho-STAT5 positive cells are measured after gate control on CD33+ cells. Data are analyzed using FACSDiva software and the percentage inhibition of IL-6 or IFNα stimulation is calculated from the percentage of cells positive for phospho-STAT1 on CD4+ cells. For IL-2 stimulated cells, data are analyzed using FACSDiva software and the percentage inhibition of IL-2 stimulation is calculated from the percentage of cells positive for phospho-STAT1 on CD4+ cells. For GM-CSF stimulated cells, the percentage inhibition of GM-CSF stimulation is calculated from the percentage of cells positive for phospho-STAT5 on CD33+ cells.

Example 6. In vivo model

6.1. CIA model

6.1.1. matter

Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) were purchased from Difco. bovine collagen type II (CII), lipopolysaccharide (LPS), and Enbrel, respectively, from Chondrex (Isle d'Abeau, France); Sigma (P4252, L'Isle d'Abeau, France), Whyett (25 mg injectable syringe, France) Acros Organics (Palo Alto, CA). All other reagents used were of reagent grade and all solvents were of analytical grade.

6.1.2. animal

Black agouti rats (male, 7-8 weeks old) were obtained from Harlan Laboratories (Maisons-Alfort, France). Rats were maintained on a 12 hour light/dark cycle (0700-1900). The temperature was maintained at 22° C., and feed and water were provided ad libitum.

6.1.3. Collagen Induced Arthritis (CIA)

One day prior to the experiment, a CII solution (2 mg/ml) was prepared in 0.05 M acetic acid and stored at 4°C. Immediately prior to immunization, equal parts of the adjuvant (IFA) and CII were mixed by a homogenizer in a glass vial pre-chilled in an ice-water bath. An excess of adjuvant and prolonged homogenization may be necessary if an emulsion is not formed. 0.2 ml of this emulsion was injected intradermally into the tail base of each rat on day 1, and a second additional intradermal injection (CFA solution of 2 mg/ml CII in 0.1 ml saline) was performed on day 9. The immunization method was modified from published methods (Jou et al., 2005; Sims et al, 2004).

6.1.4. study design

The therapeutic effects of the compounds were tested in the rat CIA model. Rats were randomly divided into equal groups, each group containing 10 rats. All rats were immunized on day 1 and boosted on day 9. Treatment dosing continued from day 16 to day 30. Negative controls were treated with vehicle (MC 0.5%) and positive controls were treated with Enbrel (10 mg/kg, 3x weeks, s.c.). A compound of interest is typically tested in 3 doses, eg 3, 10, 30 mg/kg, p.o.

6.1.5. Clinical Assessment of Arthritis

Arthritis is described in [Khachigian 2006], [Lin et al 2007] and [Nishida et al. 2004)]. Swelling of each of the four paws is ranked by the arthritis score as follows: 0 - no symptoms; 1 - mild but distinct redness and swelling of one type of joint, such as the ankle or wrist, or apparent redness and swelling confined to an individual digit, regardless of the number of digits affected; 2 - Moderate redness and swelling of 2 or more types of joints; 3 - Severe redness and swelling of the entire foot, including the toes; 4 - maximally inflamed limb with multiple joints (maximum cumulative clinical joint score is 16 points per animal) (Nishida et al., 2004).

To allow meta-analysis of multiple studies, the clinical score values were standardized as follows:

AUC of clinical score (AUC score): The area under the curve (AUC) from day 1 to day 14 was calculated for each individual rat. Each animal's AUC was divided by the average AUC obtained for vehicle in the study for which data for that animal was obtained and multiplied by 100 (i.e., AUC was expressed as a percentage of average vehicle AUC per study).

Clinical scores increase from day 1 to day 14 (endpoint score): the clinical score difference for each animal is divided by the average clinical score difference obtained for vehicle in the study for which data for that animal was obtained and 100 is calculated. multiplied (i.e. the difference was expressed as a percentage of the mean clinical score difference for the vehicle per study).

6.1.6. Weight change after onset of arthritis (%)

Clinically, weight loss is associated with arthritis (Rall and Roubenoff, 2004; Shelton et al., 2005; Walsmith et al., 2004). Thus, body weight change after onset of arthritis can be used as a non-specific endpoint for evaluation of treatment effect in this rat model. Weight change (%) after onset of the arthritis was calculated as follows:

Mice: (body weight _{(week 6)} - body weight _{(week 5)} )/body weight _{(week 5)} x 100%

Rats: (body weight _{(week 4)} - body weight _{(week 3)} )/body weight _{(week 3)} x 100%

6.1.7. radiology

X-ray pictures were taken of the hind paws of each individual animal. A random blinded identification number was assigned to each photograph, and the severity of bone erosion was ranked by two independent scores with a radiographic Larsen scoring system as follows: 0-complete bone contour and normal joint space. normal with; 1-slight abnormality of any one or two of the external metatarsals with mild bone erosion; 2-clear early abnormalities of 3 to 5 of the external metatarsals showing bone erosion; 3-moderate destructive anomaly of all of the external metatarsals as well as any one or two of the internal metatarsals with clear bone erosion; 4 - severe destructive abnormality in which all metatarsals show clear bone erosion and at least one of the inner metatarsals is completely eroded, partially preserving some bone joint contours; 5 - Abnormal cut without bone contour. This scoring system is a modification of: Bush et al., 2002; Jou et al., 2005; Salvemini et al., 2001; Sims et al., 2004.

6.1.8. histology

After radiographic analysis, hind paws of mice were fixed in 10% phosphate-buffered formalin (pH 7.4), decalcified with rapid bone demineralization (Laboratories Eurobio) and embedded in paraffin for precise histology. For reliable extensive evaluation of arthritic joints, at least 4 series of segments (5 μm thick) were cut with 100 μm between each series of segments. The sections were stained with hematoxylin and eosin (H&E). Biopsies for synovial inflammation and bone and cartilage damage were performed using a double-blind protocol. In each paw, 4 parameters were evaluated on a 4-point scale. The parameters were cell invasion, pannus severity, cartilage erosion and bone erosion. Correspondingly scored as follows: 1-normal, 2-mild, 3-moderate, 4-severe. The four scores are summed together and presented as an additional score, 'RA total score'.

6.1.9. Microcomputed tomography (μCT) analysis of the calcaneus (heel bone)

Bone damage observed in RA occurs particularly in the bone cortex and can be revealed by μCT analysis (Oste et al., 2007; Sims et al., 2004). After scanning and 3D volume reconstruction of the calcaneus, bone damage is measured as the number of identified objects present per slide, isolated in silico perpendicular to the longitudinal axis of the bone. The more the bone is damaged, the more identification objects are measured. 1000 slides evenly distributed along the calcaneus (spaced approximately 10.8 μm) are analyzed.

6.1.10. steady state PK

On day 7 or 11, blood samples were taken from the posterior-orbital cavity using lithium heparin as anticoagulant at the following time points: predose, 1, 3 and 6 hours. Whole blood samples were centrifuged and the resulting plasma samples were stored at -20 °C until analysis. The plasma concentration of each test compound was determined by the LC-MS/MS method, in which the mass spectrometer was operated in positive electrospray mode. Pharmacokinetic parameters were calculated using Winnonlin® (Pharsight®, USA) and the pre-dose plasma levels were assumed to equal 24 hour plasma levels.

6.2. oncology model

In vivo models for confirming the efficacy of small molecules against JAK2-driven myeloproliferative diseases are described [Geron et al., 2008]; See Wernig et al. 2008].

6.3. Mouse IBD model

In vitro and in vivo models for determining the efficacy of small molecules against IBD are disclosed in Wirtz and Neurath, 2007.

6.4. mouse asthma model

In vitro and in vivo models for determining the efficacy of small molecules for asthma are described in Ip et al. 2006]; [Kudlacz et al., 2008]; [Nials and Uddin, 2008]; [Pernis and Rothman, 2002].

6.5. A murine model of psoriasis-type epithelial hyperplasia induced by intradermal injection of IL22 or IL23

6.5.1. matter

Carrier-free, mouse recombinant IL22 (582-ML-CF) is provided by R&D Systems. Carrier-free, mouse recombinant IL23 (14-8231, CF) is provided by e-Bioscience.

6.5.2. animal

Balb/c mice (female, 18-20 g body weight) are obtained from CERJ (France). Mice are maintained on a 12 hour light/dark cycle (07:00 - 19:00). The temperature is maintained at 22 °C, and food and water are provided ad libitum.

6.5.3. study design

The design of this study is adapted from the literature [Rizzo et al, 2011].

On day 1 (D1), the mice are shaved around both ears.

For 4 consecutive days (D1 to D4), the mice were injected with mouse recombinant IL22 or IL23 (1 μg/20 μl in PBS/0.1% BSA) in the right auricle and left auricle under anesthesia induced by inhalation of isoflurane. A daily intradermal dose of 20 μl of PBS/0.1% BSA was given.

During D1-D5, mice are dosed with test-compound (10, 30 or 100 mg/kg in MC 0.5%, po, qd) 1 hour prior to IL23/IL22 injection or vehicle.

6.5.4. evaluation of disease

The thickness of both ears is measured daily with an automatic caliper. Body weight is assessed initially and at death. On day 5, the mice are sacrificed 2 hours after the final administration. The pinna of the ear is resected except for the cartilage. The pinna is weighed and then placed in a vial or formaldehyde containing 1 ml of RNAlater solution.

On D4, blood samples are also collected from the posterior-orbital cavity for PK profiles immediately prior to dosing (T0) and at 1, 3, and 6 hours post-dose.

There are 8 mice per group. Results are presented as mean±sem and statistical analysis is performed using one-way Anova followed by Dunnett's post hoc test for IL22 or IL23 vehicle groups.

6.5.5. histology

After killing, ears are collected and fixed in 3.7% paraformaldehyde and then embedded in paraffin. Cut to 2 μm thickness and stain with hematoxylin and eosin. Auricular epidermal thickness is measured by image analysis (Sis'Ncom software) by capturing 6 images per ear at ×20 magnification. Data are presented as mean±sem and statistical analysis is performed using one-way Anova followed by Dunnett's post hoc test for IL22 or IL23 vehicle groups.

6.5.6. RNA extraction, RT-PCR and real-time PCR

IL-17a, IL-22, IL-1β, LCN2 and S100A9 transcript levels in ear tissues are measured using real-time quantitative PCR.

Example 7. Pharmacokinetics, ADME and toxicity analysis

7.1. thermodynamic solubility

The test compound is added to 0.2 M phosphate buffer pH 7.4 or 0.1 M citrate buffer pH 3.0 at a concentration of 1 mg/ml in a glass vial.

Samples are spun on a Rotator drive STR 4 (Stuart Scientific, Bibby) at a speed of 3.0 at room temperature for 24 hours.

After 24 hours, 800 μl of the sample is transferred to an Eppendorf tube and centrifuged at 14000 rpm for 5 minutes. 200 μl of the supernatant of the sample was then transferred to a MultiscreenR solubility plate (Millipore, MSSLBPC50) and the supernatant was transferred to a clean Grainer polypropylene V-bottom 96 well plate (Cat. No. 651201) (10-12" Hg). 5 μl of the filtrate is diluted in 95 μl (F20) of the same buffer used for incubation in the plate containing the standard curve (Grayner, catalog number 651201). .

Standard curves for these compounds are freshly prepared in DMSO starting from a 10 mM DMSO stock solution diluted by factor 2 in DMSO (5000 μM) and then further diluted to 19.5 μM in DMSO. Then 3 μl of serial dilution from 5000 μM was transferred to 97 μl acetonitrile-buffer mixture (50/50). The final concentration range is 2.5 to 150 μM.

The plate was sealed with a sealing mat (MA96RD-04S, www.kinesis.co.uk) and samples were measured on LC-MS (ZQ 1525, Waters) under optimized conditions using Quanoptimize at room temperature. Determine the appropriate mass of the molecule.

The sample is analyzed on LC-MS at a flow rate of 1 ml/min. Solvent A is 15 mM ammonia and solvent B is acetonitrile. The sample is run under positive ion spray on an Xbridge C ₁₈ 3.5 μM (2.1 x 30 mm) column (Waters). The solvent gradient ranged from 5% B to 95% B with a total run time of 2 minutes.

The peak area is analyzed with the help of Masslynx software and the peak area of the sample is plotted against a standard curve to obtain the solubility of the compound.

Solubility values are reported as μM or μg/mL.

7.2. aqueous solubility

Starting from a 10 mM stock solution in DMSO, a series of dilutions of these compounds are prepared in DMSO. The serial dilutions are transferred to a 96 NUNC MaxiSolve Plate F-Bottom (catalog number 442404) and 0.2 M Phosphate Buffer pH 7.4 or 0.1 M Citrate Buffer pH 3.0 is added at room temperature.

Final concentrations range from 200 μM to 2.5 μM in 5 equal dilution steps. The final DMSO concentration does not exceed 2%. 200 μM pyrene is applied to the corner points of each 96 well plate and serves as a reference point for Z-axis calibration on the microscope.

The assay plate is sealed and incubated for 1 hour at 37° C. with shaking at 230 rpm. The plate is then scanned under a white light microscope, providing an individual picture of the precipitate per concentration. The precipitate is analyzed and converted to numbers and plotted on a graph. The first concentration at which the compound appears to be completely dissolved is the concentration reported below, but the true concentration will lie somewhere between that concentration and one higher dilution step.

Solubility values measured according to the above protocol are reported as μg/ml.

7.3. Plasma protein binding (equilibrium dialysis)

A 10 mM stock solution of the compound in DMSO is diluted by a factor of 5 in DMSO. The solution is further diluted to a final concentration of 5 μM and a final DMSO concentration of 0.5% in freshly thawed human, rat, mouse or dog plasma (BioReclamation INC) (PP-MasterBlock 96 wells (grey) You, 5.5 μl of 1094.5 μl plasma from catalog number 780285).

Prepare a Pierce Red Device (Thermo Scientific, catalog number 89809) with an insert and fill the buffer chamber with 750 μl PBS and the plasma chamber with 500 μl of spiked plasma. The plate is incubated for 4 hours at 37° C. with shaking at 230 rpm. After incubation, 120 μl of both chambers is transferred to 360 μl acetonitrile in a 96-well round-bottom, PP deep well plate (Nunc, catalog number 278743) and sealed with an aluminum foil lid. The sample is mixed and placed on ice for 30 minutes. The plate is then centrifuged 30 minutes at 4° C. at 1200 rcf and the supernatant is transferred to a 96 v-bottom PP plate (Grainer, 651201) for analysis on LC-MS.

The plate was sealed with a sealing mat (MA96RD-04S) from www.kinesis.co.kr and the samples were measured on LC-MS (ZQ 1525, Waters) under optimized conditions using quant optimize at room temperature to determine the molecular Measure the appropriate mass.

The sample is analyzed on LC-MS at a flow rate of 1 ml/min. Solvent A is 15 mM ammonia and solvent B is acetonitrile. The sample is run under positive ion spray on an Xbridge C ₁₈ 3.5 μM (2.1 x 30 mm) column (Waters). The solvent gradient ranged from 5% B to 95% B with a total run time of 2 minutes.

Peak areas from compounds in the buffer chamber and plasma chamber are considered to be 100% compound. The percentage bound to plasma is derived from these results and is reported as the percentage bound to plasma.

The solubility of the compound at the final test concentration in PBS is examined microscopically to indicate whether a precipitate is observed.

7.4. Aldehyde oxidase stability

A 10 mM stock solution of the test compound in DMSO is first diluted with water (5-fold) to obtain a 50 μM working solution. A selective inhibitor of aldehyde oxidase (hydrarazine) is prepared in water as a 5 mM solution.

An incubation mixture is prepared by adding 10 μl of liver S9 suspension (human and rat, BD Biosciences Gentest, 20 mg/ml) to 86 μl of 50 mM potassium phosphate buffer, pH 7.4 at 37°C. 2 μl of 5 mM hydrarazine is added (in the case of incubation with the addition of a selective inhibitor) or 2 μl of water (in the case of incubation without the addition of the inhibitor).

After 5 minutes of forewarming, the reaction is initiated by adding 2 μl of 50 μM test compound to the incubation mixture. After 0, 3, 6, 12, 18 and 30 minutes of incubation, the reaction (100 μl) was treated with 300 μl of MeCN:MeOH ( 2:1).

Samples are mixed, centrifuged and the supernatant analyzed by LC-MS.

A test compound is considered a substrate of aldehyde oxidase if clearance by S9 is inhibited by hydralazine. Removal of the species specificity of the test compound may also suggest metabolism by aldehyde oxidase.

Phthalazine is included as a positive control.

The instrument response (peak area ratio of test compound to internal standard) is referenced to the zero time point sample (considered as 100%) to determine the percentage of compound remaining. Using the plot of the percentage of test compound remaining, half-life (T _½ ) and specific clearance in the S9 culture are determined with GraphPad Prism software.

To calculate the intrinsic clearance in vitro (CL _int (μl/min/mg)), the following formula is used:

CLint = (0.693/T½) x (culture volume/protein amount) x 1000

7.5. Liver microsomal stability

10 mM stock solutions of compounds in DMSO are diluted to 6 μM in 105 mM phosphate buffer pH 7.4 in a 96 deep well plate (Grainer, catalog number 780285) and forewarmed at 37°C.

Glucose-6-phosphate-dehydrogenase (G6PDH, Roche, 10127671001) working stock solution at 700 U/mL is diluted by a factor of 1:700 in 105 mM phosphate buffer pH 7.4. A cofactor mixture containing 0.528 M MgCl ₂ .6H ₂ O (Sigma, M2670), 0.528 M glucose-6-phosphate (Sigma, G-7879) and 0.208 M NADP+ (Sigma, N-0505) was mixed with 105 mM phosphate buffer. , diluted by a factor of 1:8 in pH 7.4.

Liver microsomes (Genotech) of the species of interest (human, mouse, rat, dog...) at 1 mg/ml, 0.8 U/ml G6PDH and cofactor mixture (6.6 mM MgCl2, 6.6 mM glucose-6-phosphate, A working solution containing 2.6 mM NADP+) is prepared. Pre-incubate the mixture at room temperature for 15 minutes, but never more than 20 minutes.

After pre-incubation, equal amounts of the compound dilution and the mixture containing the microsomes are added together and incubated at 300 rpm for 30 minutes. For the 0 min time point, 2 volumes of MeOH are added to the compound dilution followed by the microsomal mixture. Final concentrations in culture are 3 μM compound or control compound, 0.5 mg/mL microsomes, 0.4 U/mL G6PDH, 3.3 mM MgCl ₂ , 3.3 mM glucose-6-phosphate and 1.3 mM NADP+.

After 30 minutes of incubation, the reaction is stopped with 2 volumes of MeOH.

At both of these time points, samples are mixed, centrifuged, and the supernatant is harvested for analysis on LC-MS/MS. The instrument response (i.e. peak height) is referenced to the zero time point sample (as 100%) to determine the percentage of compound remaining. The standard compounds propanol and verapamil are included in the assay design.

Data for microsomal stability are presented as a percentage of the total amount of compound remaining after 30 minutes.

7.6. Hepatocellular stability

A model for evaluating metabolic clearance in hepatocytes is disclosed in McGinnity et al., Drug Metabolism and Disposition 2008, 32, 11, 1247.

7.7. Caco2 permeability

A bidirectional Caco-2 assay is performed as described below. Caco-2 cells are obtained from the European Collection of Cell Cultures (ECACC, cat 86010202) and used after 21 days of cell culture in 24-well transwell plates (Fisher TKT-545-020B).

2 x 10 ⁵ cells/well are seeded in a plating medium consisting of DMEM + GlutaMAXI + 1% NEAA + 10% FBS (FetalClone II) + 1% Pen/Strep. The medium is exchanged every 2 to 3 days.

Test and comparative compounds (propranolol and rhodamine 123 or vinblastine, both purchased from Sigma) were prepared in Hank's balanced salt solution containing 25 mM HEPES, pH 7.4, to a final DMSO concentration of 0.25%. , at a concentration of 10 μM to the top (125 μL) or bottom (600 μL) chamber of the transwell plate assembly.

Integrity of the cell layer is assessed by adding 50 μM Lucifer Yellow (Sigma) to the donor buffer in all wells to monitor Lucifer Yellow penetration. As lucifer yellow (LY) cannot freely penetrate the lipophilic barrier, a high degree of LY transport indicates poor integrity of the cell layer.

After 1 hour incubation at 37° C. with shaking on an orbital shaker at 150 rpm, an aliquot of 70 μl was taken from the normal (A) and basal (B) chambers and assay internal standard (0.5 μM carbamazepine) in a 96 well plate. 100 μl of a 50:50 acetonitrile:water solution containing

Lucifer yellow is measured with a Spectramax Gemini XS (Ex 426 nm and Em 538 nm) in a clear 96 well plate containing 150 μl of liquid from the basolateral and apex sides.

Concentrations of compounds in the samples are determined by high-performance liquid-chromatography/mass spectroscopy (LC-MS/MS).

The apparent permeability (Papp) value is calculated from the relationship:

Papp = [compound] _{acceptor final} x V _acceptor /([compound] _{donor initial} x V _donor )/T _inc x V _donor /surface area x 60 x 10 ^-6 cm/s

V = chamber volume

T _inc = incubation time.

surface area = 0.33 cm 3

As an indicator of active efflux from the normal cell surface, the efflux ratio is calculated using the ratio P _app B>A/P _app A>B.

The following analytical acceptance criteria are used:

Propranolol: P _app (A>B) value ≥ 20 (x10 ^-6 cm/s)

Rhodamine 123 or vinblastine: P _app (A>B) value < 5 (x10 ^-6 cm/s), efflux ratio ≥ 5.

Lucifer Yellow Permeability: ≤ 100 nm/s

7.8. MDCKII-MDR1 permeability

MDCKII-MDR1 cells are Madin-Dalby canine kidney epithelial cells overexpressing the human multidrug resistance (MDR1) gene, encoding P-glycoprotein (P-gp). Cells are obtained from the Netherlands Cancer Institute and used after 3-4 days cell culture in 24-well Millicell cell culture insert plates (Millipore, PSRP010R5). A bidirectional MDCKII-MDR1 permeability assay is performed as described below.

3 x 10 ⁵ cells/ml (1.2 x 10 ⁵ cells/well) consisting of DMEM + 1% Glutamax-100 + 1% antibiotics/antimycotics + 10% FBS (Biowest, S1810) Seed on smear medium. The cells are placed in a CO ₂ incubator for 3-4 days. The medium is changed 24 hours after seeding and on the day of the experiment.

Test and reference compounds (amprenavir and propranolol) were prepared in Dulbecco's phosphate buffered saline (D-PBS, pH 7.4) and top (400 μl) or bottom (800 μl) of the minicell cell culture insert plate assembly. Add to the chamber at a final concentration of 10 µM (0.5 µM for Amprenavir) with a final DMSO concentration of 1%.

100 μM Lucifer Yellow (Sigma) is added to all donor buffer solutions to assess the integrity of the cell monolayer by monitoring Lucifer Yellow penetration. Lucifer Yellow is a fluorescent marker for intercellular pathways and is used as an internal control on all monolayers to confirm tight junction integrity during the assay.

After 1 hour incubation at 37 °C with shaking at 150 rpm on an orbital shaker, aliquots of 75 μl were taken from both the normal (A) and basal (B) chambers and placed in 96 well plates with analytical internal standard (10 ng/ml warfarin). ) in 225 μl acetonitrile:water solution (2:1). An aliquot is also run at the beginning of the experiment from the donor solution to obtain the initial (Co) concentration.

The concentration of the compound in the sample is determined by high-performance liquid-chromatography/mass spectroscopy (LC-MS/MS).

Lucifer Yellow is measured with a Fluoroscan Ascent FL Thermo Scientific (Ex 485 nm and Em 530 nm) in a 96 well plate containing 150 μl of liquid from all recipient wells (basal or top) do.

7.9. Pharmacokinetic studies in rodents

7.9.1. animal

Sprague-Dawley rats (male, 5-6 weeks old) are obtained from Janvier (France). Rats are acclimatized for at least 7 days prior to treatment and maintained on a 12 hour light/dark cycle (0700 - 1900). The temperature is maintained at approximately 22 °C, and water and food are provided ad libitum. Two days before the administration of the test compound, rats under isoflurane anesthesia underwent an operation to implant a catheter in the jugular vein. After the surgery, rats are individually housed. At least 16 hours before and 6 hours after oral administration, animals are deprived of food. Water is provided freely.

7.9.2. Pharmacokinetic studies

Compounds are formulated in PEG200/normal saline (60/40) for the intravenous route and 0.5% methylcellulose and 10% hydroxypropyl-β-cyclodextrin pH 3 for the oral route. The test compound is administered orally at 5 mg/kg (dosage volume of 5 ml/kg) as a single esophageal feed and administered intravenously at 1 mg/kg (dosage volume of 5 ml/kg) as a bolus via the tail vein. Each group consisted of 3 rats. Blood samples are drawn via the jugular vein, using lithium-heparin as an anticoagulant, at the following time points: 0.05, 0.25, 0.5, 1, 3, 5 and 8 hours (intravenous route) and 0.25, 0.5, 1, 3 , 5, 8 and 24 hours (oral route). Alternatively, blood samples are taken in the postero-orbital cavity using lithium-heparin as an anticoagulant at the following time points: 0.25, 1, 3 and 6 hours (oral route). Whole blood samples are centrifuged at 5000 rpm for 10 minutes and the resulting plasma samples are stored at -20°C until analysis.

7.9.3. Quantitative analysis of compound levels in plasma

The plasma concentration of each test compound is determined by the LC-MS/MS method, in which the mass spectrometer operates in a positive electrospray mode.

7.9.4. Measurement of Pharmacokinetic Parameters

Pharmacokinetic parameters are calculated using Winnolin® (Farsight®, USA).

7.10. 7-Day Rat Toxicity Study

A 7-day oral toxicity study for the test compound was conducted in Sprague-Dawley male rats by gavage at a constant dose-volume of 5 ml/kg/day, daily at 100, 300 and 500 mg/kg/day. Doses are performed to evaluate their toxic potential and toxic pharmacokinetics.

Test compounds are formulated in 30% (v/v) HPpCD in purified water. Each group included 5 primary male rats as well as 3 satellite animals for toxico-pharmacokinetics. A fourth group is given only 30% (v/v) HPpCD in water at the same frequency, dose volume, by the same route of administration, and this group serves as the vehicle control group.

The purpose of this study is to determine the lowest dose that does not produce side effects (no observable side effect level - NOAEL).

7.11. Responsibilities for QT prolongation

The potential for QT prolongation is assessed in hERG patch clamp assays.

Whole-cell patch-clamp recordings are performed using an EPC10 amplifier controlled by Pulse v8.77 software (HEKA). Series resistance is typically less than 10 MΩ and is compensated up to greater than 60%, and records are not missing deductions. Electrodes are made from GC150TF pipette glass (Harvard).

The external bath solution contains 135 mM NaCl, 5 mM KCl, 1.8 mM CaCl ₂ , 5 mM glucose, 10 mM HEPES, pH 7.4.

The internal patch pipette solution contains 100 mM K-gluconate, 20 mM KCl, 1 mM CaCl ₂ , 1 mM MgCl ₂ , 5 mM Na ₂ ATP, 2 mM glutathione, 11 mM EGTA, 10 mM HEPES, pH 7.2.

Drugs are perfused using a Biologic MEV-9/EVH-9 high-speed perfusion system.

All recordings are performed on HEK293 cells stably expressing the hERG channel. Cells are cultured on 12 mm round coverslips (German glass, Bellco) fixed in a recording chamber using two platinum rods (Goodfellow). The hERG current is generated at +40 mV for 1000 m using an activation pulse and then at -50 mV for 2000 m with a tail current pulse, with a holding potential of -80 mV. Pulses are applied every 20 seconds and all experiments are performed at room temperature.

final opinion

Those skilled in the art will appreciate that the above description is illustrative and explanatory in nature, and is intended to illustrate the present invention and its preferred embodiments. Through routine experimentation, the skilled artisan will recognize obvious modifications and variations that can be made without departing from the spirit of the invention. It is intended to embrace all such modifications as fall within the scope of the appended claims. Accordingly, it is intended that the present invention be limited not by the foregoing description, but by the following claims and equivalents thereof.

All publications, including but not limited to patents and patent applications, cited herein are incorporated herein by reference as if each individual publication were specifically and individually indicated to be incorporated by reference as if fully disclosed.

Of course, factors such as the ability of different compounds to penetrate cells differently may contribute to discrepancies between the activity of compounds in in vitro biochemistry and cellular assays.

At least some of the chemical names of the compounds of the invention as provided and shown herein can be generated on an automated basis by use of commercially available chemical naming software programs and have not been independently verified. Typical programs that perform these functions are the Lexichem naming tool sold by Open Eye Software, Inc. and the Autonom software tool sold by MDL, Inc. includes In case the chemical name shown above and the structure shown are different, the structure shown above will be suppressed.

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Claims (16)

A compound according to Formula I, or a pharmaceutically acceptable salt or solvate, or a solvate of said pharmaceutically acceptable salt:
Formula I

In the above formula,
R ¹ is H or Me;
L ₁ is -NR ² -; -O- or -CH ₂ -;
Cy is phenyl, pyridine or dihydrobenzodioxin;
R ² is H or C _1-4 alkyl;
R ³ is H, halo, C _1-4 alkyl optionally substituted with one or more halo, or C _1-4 alkoxy optionally substituted with one or more halo;
R ⁴ is H or halo;
R ⁵ is -CN, halo, or -L ₂ -R ⁶ ;
-L ₂ is absent, or -C(=O)-, -C(=O)NR ⁷ -, -NR ⁷ C(=O)-, -SO ₂ -, -SO ₂ NR ⁷ -, or -NR ⁷ SO ₂ -;
R ⁶ is H or C _1-6 alkyl optionally substituted with one or more independently selected R ⁸ groups;
R ⁷ is H or C _1-4 alkyl;
R ⁸ is OH, CN, halo or C _1-4 alkoxy;
L _a is absent or -C(=0)-, -C(=0)0- or -C(=0)NH-;
R ^a is
-H,
- C _1-4 alkyl optionally substituted with one or more independently selected R ^b ,
- C _3-7 monocyclic cycloalkyl optionally substituted with one or more independently selected R ^c , or
- 4 to 7 membered monocyclic heterocycloalkyl containing one or more heteroatoms independently selected from O, N and S, or
- a 5 to 6 membered monocyclic heteroaryl containing at least one heteroatom independently selected from O, N and S;
^Rb is
- halo,
-CN,
-OH,
- C _1-4 alkoxy;
- C _3-7 cycloalkyl,
- 4 to 7 membered monocyclic heterocycloalkyl containing one or more heteroatoms independently selected from O, N and S, wherein said heterocycloalkyl is optionally substituted with one or more independently selected halo or oxo;
- -SO ₂ -C _1-4 alkyl, or
- -C(=O)NR ^b1 R ^b2 and
R ^c is
- halo,
-CN,
-OH,
- C _1-4 alkyl;
- -C(=O)OH, or
- -C(=0)NR ^c1 R ^c2 ;
Each of R ^bi , R ^b2 , R ^c1 and R ^c2 is independently selected from H and C _1-4 alkyl. According to claim 1,
A compound or pharmaceutically acceptable salt wherein R ¹ is Me. According to claim 1,
A compound according to Formula IIa, IIb or IIc or a pharmaceutically acceptable salt thereof:

In the above formulas, L ₁ , R ³ , R ⁴ , L _a , R ^a and R ⁵ are as described in claim 1. According to claim 1,
A compound or pharmaceutically acceptable salt according to formula IVa, IVb, IVc, IVd, IVe or IVf:

In the above formulas, R ³ , R ⁴ , R ⁵ , L _a and R ^a are as described in claim 1. According to claim 1,
A compound or pharmaceutically acceptable salt according to formula Va, Vb, Vc, Vd, Ve or Vf:

In the above formulas, R ³ , R ⁴ , R ⁵ and R ^a are as described in claim 1 . According to claim 1,
A compound or pharmaceutically acceptable salt wherein R ⁴ is H, F or Cl. According to claim 1,
A compound or pharmaceutically acceptable salt wherein R ³ is H, Me or Et. According to claim 1,
A compound or pharmaceutically acceptable salt wherein R ⁵ is CN, F, Cl, -SO ₂ Me or -SO ₂ Et. According to claim 1,
R ^a is

A phosphorus compound or pharmaceutically acceptable salt. According to claim 1,
(1R,2R)-N-[6-[(6-cyano-4-methyl-3-pyridyl)oxy]-1-methyl-benzimidazol-4-yl]-2-fluoro-cyclopropane A compound that is a carboxamide or a pharmaceutically acceptable salt thereof. Allergic diseases, rheumatoid arthritis, osteoarthritis, comprising a pharmaceutically acceptable carrier and a pharmaceutically effective amount of the compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof; Children's idiopathic arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, allergic airway disease, chronic obstructive pulmonary disease, ulcerative colitis, Crohn's disease, asthma, systemic lupus erythematosus, type I diabetes, transplant rejection, disorders of cartilage turnover A pharmaceutical composition for use in the treatment or prevention of concomitant diseases or congenital cartilage malformations. Castleman's disease, multiple myeloma, psoriasis, Kaposi's sarcoma, comprising a pharmaceutically acceptable carrier and a pharmaceutically effective amount of the compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof. or a pharmaceutical composition for use in the treatment or prevention of mesangial cell proliferative glomerulonephritis. Systemic and cutaneous lupus erythematosus, lupus nephritis, comprising a pharmaceutically acceptable carrier and a pharmaceutically effective amount of the compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof; A pharmaceutical composition for use in the treatment or prevention of dermatomyositis, Sjogren's syndrome, psoriasis or rheumatoid arthritis. delete delete delete