WO2013117649A1 - Imidazo [4, 5 -c] pyridine derivatives useful for the treatment of degenerative and inflammatory diseases - Google Patents

Imidazo [4, 5 -c] pyridine derivatives useful for the treatment of degenerative and inflammatory diseases Download PDF

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WO2013117649A1
WO2013117649A1 PCT/EP2013/052441 EP2013052441W WO2013117649A1 WO 2013117649 A1 WO2013117649 A1 WO 2013117649A1 EP 2013052441 W EP2013052441 W EP 2013052441W WO 2013117649 A1 WO2013117649 A1 WO 2013117649A1
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compound
alkyl
independently selected
optionally substituted
arh
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French (fr)
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Christel MENET
Benoît SCHMITT
Raphaël GENEY
Kevin Doyle
Joanne Peach
Nicholas Palmer
Graham Jones
David Hardy
James Duffy
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Galapagos Nv
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D407/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00
    • C07D407/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings
    • C07D407/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D411/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen and sulfur atoms as the only ring hetero atoms
    • C07D411/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen and sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D411/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen and sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/06—Peri-condensed systems

Definitions

  • the present invention relates to compounds that are inhibitors of JAK, a family of tyrosine kinases that are involved in allergic or inflammatory conditions, autoimmune diseases, proliferative diseases, transplantation rejection, diseases involving impairment of cartilage turnover, congenital cartilage malformations, and/or diseases associated with hypersecretion of IL6 or interferons.
  • the compounds of the invention inhibit JAK1 and/or JAK2.
  • the present invention also provides methods for the production of the compounds of the invention, pharmaceutical compositions comprising the compounds of the invention, methods for the prevention and/or treatment of diseases involving allergic or inflammatory conditions, autoimmune diseases, proliferative diseases, transplantation rejection, diseases involving impairment of cartilage turnover, congenital cartilage malformations, and/or diseases associated with hypersecretion of IL6 or interferons by administering a compound of the invention.
  • JAK3 is validated by mouse and human genetics as an immune- suppression target (O'Shea J. et al. (2004)). JAK3 inhibitors were successfully taken into clinical development, initially for organ transplant rejection but later also in other immuno-inflammatory indications such as rheumathoid arthritis (RA), psoriasis and Crohn's disease (http://clinicaltrials.gov/).
  • RA rheumathoid arthritis
  • psoriasis http://clinicaltrials.gov/.
  • TYK2 is a potential target for immuno-inflammatory diseases, being validated by human genetics and mouse knock-out studies (Levy D. and Loomis C. (2007)).
  • JAK1 and JAK2 are implicated in intracellular signal transduction for many cytokines and hormones. Pathologies associated with any of these cytokines and hormones can be ameliorated by JAK1 and JAK2 inhibitors.
  • JAK1 and JAK2 inhibitors might benefit from treatment with compounds described in this invention including rheumatoid arthritis, systemic lupus erythematosis, juvenile idiopathic arthritis, osteoarthritis, asthma, chronic obstructive pulmonary disease COPD, tissue fibrosis, eosinophilic inflammation, eosophagitis, inflammatory bowel diseases (e.g. Crohn's, ulcerative colitis), transplantation, graft-versus-host disease, psoriasis, myositis, multiple sclerosis (Kopf et al., 2010).
  • Osteoarthritis is difficult to treat. At present, no cure is available and treatment focuses on relieving pain and preventing the affected joint from becoming deformed. Common treatments include the use of non-steroidal anti-inflammatory drugs (NSAIDs). Although dietary supplements such as chondroitin and glucosamine sulphate have been advocated as safe and effective options for the treatment of osteoarthritis, a recent clinical trial revealed that both treatments did not reduce pain associated to osteoarthritis. (Clegg et al., 2006).
  • NSAIDs non-steroidal anti-inflammatory drugs
  • Stimulation of the anabolic processes, blocking catabolic processes, or a combination of these two, may result in stabilization of the cartilage, and perhaps even reversion of the damage, and therefore prevent further progression of the disease.
  • Therapeutic methods for the correction of the articular cartilage lesions that appear during the osteoarthritic disease have been developed, but so far none of them have been able to mediate the regeneration of articular cartilage in situ and in vivo. Taken together, no disease modifying osteoarthritic drugs are available.
  • JAK family members have been implicated in additional conditions including myeloproliferative disorders (O'Sullivan et al, 2007, Mol Immunol. 44(10):2497-506), where mutations in JAK2 have been identified. This indicates that inhibitors of JAK in particular JAK2 may also be of use in the treatment of myeloproliferative disorders. Additionally, the JAK family, in particular JAKl, JAK2 and JAK3, has been linked to cancers, in particular leukaemias e.g. acute myeloid leukaemia (O'Sullivan et al, 2007, Mol Immunol.
  • the current therapies are not satisfactory and therefore there remains a need to identify further compounds that may be of use in the treatment of allergic or inflammatory conditions, autoimmune diseases, proliferative diseases, transplantation rejection, diseases involving impairment of cartilage turnover, congenital cartilage malformations, and/or diseases associated with hypersecretion of IL6 or interferons.
  • the present invention therefore provides compounds, methods for their manufacture and pharmaceutical compositions comprising the compounds of the invention together with a suitable pharmaceutical carrier.
  • the present invention also provides for the use of a compound of the invention in the preparation of a medicament for the treatment of allergic or inflammatory conditions, autoimmune diseases, proliferative diseases, transplantation rejection, diseases involving impairment of cartilage turnover, congenital cartilage malformations, and/or diseases associated with hypersecretion of IL6 or interferons.
  • the present invention is based on the identification that the compounds of the invention are able to act as inhibitors of JAK and that they are useful for the treatment of allergic or inflammatory conditions, autoimmune diseases, proliferative diseases, transplantation rejection, diseases involving impairment of cartilage turnover, congenital cartilage malformations, and/or diseases associated with hypersecretion of IL6 or interferons.
  • the compounds of the invention are inhibitors of JAK1 and/or JAK2.
  • the present invention also provides methods for the production of these compounds, pharmaceutical compositions comprising these compounds and methods for treating allergic or inflammatory conditions, autoimmune diseases, proliferative diseases, transplantation rejection, diseases involving impairment of cartilage turnover, congenital cartilage malformations, and/or diseases associated with hypersecretion of IL6 or interferons by administering the compounds of the invention.
  • R is C3.7 cycloalkyl, or C 1 .4 alkyl optionally substituted with one or more halo;
  • each of w, x, y, and z is independantly N, CH or is absent;
  • the ring A is a fused 5-6-membered saturated or 5-membered unsaturated ring, each of which optionally comprises 1 or 2 heteroatoms independently selected from N, O, and S; to afford a 8- 10 membered bicyclic group;
  • each R 2b is independently selected from:
  • 5- 10 membered heteroaryl optionally substituted with one or more independently groups selected from CN, halo, OH, C M alkyl, C M alkoxy, and NR 3a R 3 ⁇ 4
  • heterocycloalkyl is optionally substituted with one or more CM alkyl (optionally substituted with one or more CN));
  • the compounds of the invention are inhibitors of JAK1 and/or JAK2.
  • the present invention provides a compound of the invention, or a pharmaceutical composition comprising a compound of the invention, for use as a medicament.
  • this invention provides methods for synthesizing the compounds of the invention, with representative synthetic protocols and pathways disclosed later on herein. [0027] Accordingly, it is a principal object of this invention to provide novel compounds, which can modify the activity of JAK and thus prevent or treat any conditions that may be causally related thereto. In a specific aspect the compounds of the invention modulate the activity of JAKl and/or JAK2.
  • analogue means one analogue or more than one analogue.
  • JAK Janus kinases
  • JAKs Janus kinases
  • JAKl cytoplasmic tyrosine kinases that transduce cytokine signaling from membrane receptors to STAT transcription factors.
  • JAKl cytoplasmic tyrosine kinases that transduce cytokine signaling from membrane receptors to STAT transcription factors.
  • JAKl cytoplasmic tyrosine kinases that transduce cytokine signaling from membrane receptors to STAT transcription factors.
  • JAKl cytoplasmic tyrosine kinases
  • JAK3 cytoplasmic tyrosine kinases that transduce cytokine signaling from membrane receptors to STAT transcription factors.
  • JAK may refer to all the JAK family members collectively or one or more of the JAK family members as the context indicates.
  • alkoxy' refers to the group -OR 26 where R 26 is alkyl with the number of carbon atoms specified.
  • Particular alkoxy groups are methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, and 1 ,2-dimethylbutoxy.
  • Particular alkoxy groups are lower alkoxy, i.e. with between 1 and 6 carbon atoms. Further particular alkoxy groups have between 1 and 4 carbon atoms.
  • 'Alkylene' refers to divalent alkene radical groups having the number of carbon atoms specified, in particular having 1 to 6 carbon atoms and more particularly 1 to 4 carbon atoms which can be straight-chained or branched. This term is exemplified by groups such as methylene (-CH2-), ethylene (-CH2-CH2-), or -CH(CH3)- and the like.
  • alkyl means straight or branched aliphatic hydrocarbon with the number of carbon atoms specified. Particular alkyl groups have 1 to 8 carbon atoms. More particular is lower alkyl which has 1 to 6 carbon atoms. A further particular group has 1 to 4 carbon atoms. Exemplary straight chained 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 is attached to a linear alkyl chain, exemplary branched chain groups include isopropyl, iso-butyl, t-butyl and isoamyl.
  • alkenyl' refers to monovalent olefinically (unsaturated) hydrocarbon groups with the number of carbon atoms specified.
  • Particular alkenyl has 2 to 8 carbon atoms, and more particularly, from 2 to 6 carbon atoms, which can be straight-chained or branched and having at least 1 and particularly from 1 to 2 sites of olefinic unsaturation.
  • 'Amino' refers to the radical -NH 2 .
  • 'Cycloalkyl refers to a non-aromatic hydrocarbyl ring structure, monocyclic or polycyclic, with the number of ring atoms specified.
  • a cycloalkyl may have from 3 to 10 carbon atoms, and in particular from 3 to 7 carbon atoms.
  • Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.
  • 'Halo' or 'halogen' refers to fluoro (F), chloro (CI), bromo (Br) and iodo (I). Particular halo groups are either fluoro or chloro.
  • 'Hetero' when used to describe a compound or a 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 may be applied to any of the hydrocarbyl groups described above such as alkyl, e.g. heteroalkyl, cycloalkyl, e.g. heterocycloalkyl, aryl, e.g. heteroaryl, and the like having from 1 to 4, and particularly from 1 to 3 heteroatoms, more typically 1 or 2 heteroatoms, for example a single heteroatom.
  • Heteroaryl means an aromatic ring structure, monocyclic or polycyclic, that includes one or more heteroatoms independently selected from O, N and S and the number of ring atoms specified.
  • the aromatic ring structure may have from 5 to 10 ring members.
  • the heteroaryl group can be, for example, a five membered or six membered monocyclic ring or a bicyclic structure formed from fused five and six membered rings or two fused six membered rings or, by way of a further example, two fused five membered rings.
  • Each ring may contain up to four heteroatoms typically selected from nitrogen, sulphur and oxygen.
  • the heteroaryl ring will contain up to 4 heteroatoms, more typically up to 3 heteroatoms, more usually up to 2, for example a single heteroatom.
  • the heteroaryl ring contains at least one ring nitrogen atom.
  • the nitrogen atoms in the heteroaryl rings can 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. In general the number of basic nitrogen atoms present in the heteroaryl group, including any amino group substituents of the ring, will be less than five.
  • heteroaryls examples include the following:
  • each W is selected from CH 2 , NH, O and S; and each Y is selected from NH, O, CO, S0 2 , and S.
  • 'heterocycloalkenyl' means a 'heterocycloalkyl, wherein one bond of the ring is reduced, thus the ring comprises a double bond.
  • heterocycloalkenyl groups are shown in the following illustrative examples:
  • 'Thiol' refers to the group -SH.
  • 'Thioalkoxy' refers to the group -SR 26 where R 26 has the number of carbon atoms specified and particularly Ci_g alkyl.
  • Particular thioalkoxy groups are thiomethoxy, thioethoxy, n-thiopropoxy, isothiopropoxy, n-thiobutoxy, tert-thiobutoxy, sec-thiobutoxy, n-thiopentoxy, n-thiohexoxy, and 1,2- dimethylthiobutoxy.
  • Particular thioalkoxy groups are lower thioalkoxy, i.e. with between 1 and 6 carbon atoms. Further particular alkoxy groups have between 1 and 4 carbon atoms.
  • heterocyclic ring may have one to four heteroatoms so long as the heteroaromatic ring is chemically feasible and stable.
  • 'Pharmaceutically acceptable means approved or approvable by a regulatory agency of the Federal or a state government or the corresponding agency in countries other than the United States, or that is listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly, in humans.
  • 'Pharmaceutically acceptable salt' refers to a salt of a compound of the invention that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound.
  • such salts are non-toxic may be inorganic or organic acid addition salts and base addition salts.
  • Prodrugs' refers to compounds, including derivatives of the compounds of the invention, which have cleavable groups and become by solvolysis or under physiological conditions the compounds of the invention which are pharmaceutically active in vivo.
  • Such examples include, but are not limited to, choline ester derivatives and the like, N-alkylmorpholine esters and the like.
  • 'Solvate' encompasses both solution-phase and isolable solvates.
  • Representative solvates include hydrates, ethanolates and methanolates.
  • 'Subject' includes humans. The terms 'human', 'patient' and 'subject' are used interchangeably herein.
  • 'Preventing' or 'prevention' refers to a reduction in risk of acquiring or developing a disease or disorder (i.e. causing at least one of the clinical symptoms of the disease not to develop in a subject that may be exposed to a disease-causing agent, or predisposed to the disease in advance of disease onset.
  • autoimmune disease(s)' refers to the group of diseases including obstructive airways disease, including conditions such as COPD, asthma (e.g intrinsic asthma, extrinsic asthma, dust asthma, infantily asthma) particularly chronic or inveterate asthma (for example late asthma and airway hyperreponsiveness), bronchitis, including bronchial asthma, systemic lupus erythematosus (SLE), cutaneous lupus erythrematosis, lupus nephritis, dermatomyositis, Sjogren's syndrome, multiple sclerosis, psoriasis, dry eye disease, type I diabetes mellitus and complications associated therewith, atopic eczema (atopic dermatitis), contact dermatitis and further eczematous dermatitis, inflammatory bowel disease (e.g.
  • COPD chronic or inveterate asthma
  • bronchitis including bronchial asthma, systemic lup
  • the term 'diseases involving impairment of cartilage turnover' includes conditions such as osteoarthritis, psoriatic arthritis, juvenile rheumatoid arthritis, gouty arthritis, septic or infectious arthritis, reactive arthritis, reflex sympathetic dystrophy, algodystrophy, Tietze syndrome or costal chondritis, fibromyalgia, osteochondritis, neurogenic or neuropathic arthritis, arthropathy, endemic forms of arthritis like osteoarthritis deformans endemica, Mseleni disease and Handigodu disease; degeneration resulting from fibromyalgia, systemic lupus erythematosus, scleroderma and ankylosing spondylitis.
  • R 1 is C3.7 cycloalkyl, or C1.4 alkyl optionally substituted with one or more halo;
  • each of w, x, y, and z is independantly N, CH or is absent;
  • 5- 10 membered heteroaryl optionally substituted with one or more groups independently selected from CN, halo, OH, C M alkyl, C M alkoxy, and NR 3a R 3 ⁇ 4 C3. 7 cycloalkyl (optioinally substituted with one or more -CN),
  • heterocycloalkyl is optionally substituted with one or more CM alkyl (optionally substituted with one or more CN));
  • CM alkyl optionally substituted with one or more independently selected halo, or
  • CM alkoxy optionally substituted with one or more independently selected halo
  • each R 3a , R 3b , R 4a , and R 4b is independently selected from H, and CM alkyl;
  • R 2a is not CI, Me or OMe
  • R is C3. 7 cycloalkyl, or C alkyl optionally substituted with one or more halo;
  • each of w, x, y, and z is independantly N, CH or is absent;
  • the ring A is a fused 5-6-membered saturated or 5-membered unsaturated ring, each of which optionally comprises 1 or 2 heteroatoms independently selected from N, O, and S; to afford a 8-10 membered bicyclic group;
  • each R 2b is independently selected from:
  • CM alkyl optionally substituted with one or more groups independently selected from halo, - CN, -OH, C6-io aryl (optionally substituted with one or more groups independently selected from CM alkoxy), and amino (optionally substituted with one or more C alkyl),
  • Ci_ 6 alkoxy substituted with one or more groups independently selected from OH, CN, halo, CM alkoxy, C6-10 aryl (optionally substituted with one or more groups independently selected from CM alkoxy), C 3 . 7 cycloalkyl, alkoxy, -C( 0)-CM alkyl, and 5-10 membered heteroaryl (optionally substituted with one or more CM alkyl),
  • phenyl optionally substituted with one or more groups independently selected from CN, halo, OH, C M alkyl, C M alkoxy, and NR 3a R 3 ⁇ 4
  • heterocycloalkenyl comprising 1 double bond, and comprising 1 or 2 heteroatoms independently selected from N, O, and S, optionally substituted with one or more groups independently selected from:
  • o alkyl optionally substituted with one or more independently selected CN, OH, and halo, o alkyl, and
  • heteroaryl optionally substituted with one or more groups independently selected from CN, halo, OH, C M alkyl, C M alkoxy, and NR 3a R 3 ⁇ 4
  • alkyl (optionally substituted with CN);
  • each R 2c is independently selected from oxo, CM alkyl and halo;
  • CM alkyl optionally substituted with one or more independently selected halo, or
  • CM alkoxy optionally substituted with one or more independently selected halo
  • each R 3a , R 3b , R 4a , and R 4b is independently selected from H, and CM alkyl;
  • R 2a is not CI, Me or OMe
  • the compound of the invention is according to Formula I, wherein R 1 is C alkyl substituted with one or more independently selected halo.
  • R 1 is CM alkyl substituted with one or more independently selected F, or CI.
  • R 1 is Me, Et, n-Pr, or n-Bu, each of which is substituted with one or more independently selected F or CI.
  • R 1 is -CF 3 .
  • the compound of the invention is according to Formula I, wherein R 1 is C alkyl.
  • R 1 is Me, Et, n-Pr, z ' -Pr, or ⁇ -Bu.
  • R 1 is Me.
  • the compound of the invention is according to Formula I, wherein R 1 is C3. 7 cycloalkyl. In a particular embodiment, R 1 is cyclopropyl.
  • the compound of the invention is according to Formula I, wherein Cy is Cyi, and wherein the subscript m is 0, 1, 2, 3, or 4. In a particular embodiment, m is 0, 1 or 2. In a more particular embodiment, m is 0. In another more particular embodiment, m is 1.
  • the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 0, and wherein R 2a is F, Br, I, cyclopropyl, C 2 - 4 alkyl, CM alkyl substituted with one or more independently selected halo, C 2 - 4 alkoxy or CM alkoxy substituted with one or more independently selected halo.
  • R a is F, Br, I, C 2 _ 4 alkyl, C M alkyl substituted with one or more independently selected halo, C 2 _ 4 alkoxy or C M alkoxy substituted with one or more independently selected halo.
  • R 2a is F, Et, z ' -Pr, -CF 3 , -OEt, -OCHF 2 , or -OCF 3 .
  • R 2a is Et.
  • the compound of the invention is according to Formula I, wherein Cy is Cyi, R 2b is as described above, the subscript m is 1 or 2, and R 2a is halo, cyclopropyl, C M alkyl optionally substituted with one or more independently selected halo, or C M alkoxy optionally substituted with one or more independently selected halo.
  • R 2a is halo, C alkyl optionally substituted with one or more independently selected halo, or C M alkoxy optionally substituted with one or more independently selected halo.
  • R 2a is CI, Me, Et, z ' -Pr, -CF 3 , -OMe, -OEt, -OCHF 2 , or -OCF 3 .
  • R 2a is Et.
  • the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R 2b is CN, halo, or OH. In a particular embodiment, R 2b is CN, F, CI, or OH.
  • the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R 2b is C M alkyl.
  • R 2b is Me, Et, n-Pr, z ' -Pr or t- Bu.
  • R 2b is Me, Et, or i-Bu.
  • the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R 2b is C M alkyl substituted with one or more groups independently selected from halo, CN, OH, and Ce-io aryl (optionally substituted with one or more independently selected C M alkoxy).
  • R 2b is C M alkyl substituted with one or more groups independently selected from F, CN, OH, phenyl and 4-OMe-phenyl.
  • R 2b is Me, Et, n-Pr, or z ' -Pr, each of which is substituted with one or more groups independently selected from halo, CN, OH, and Ce-io aryl (optionally substituted with one or more independently selected C M alkoxy).
  • R 2b is -CH 2 -CN, -CH 2 -OH, -CF 3 , -CH 2 -Ph, or -CH 2 -(4- OMe-Ph).
  • the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R 2b is C 2 _ 4 alkenyl comprising 1 double bond (optionally substituted with one CN group).
  • the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R 2b is C 2 - 6 alkoxy. In a particular embodiment, R 2b is C 2 - 6 alkoxy. In a more particular embodiment, R 2b is OEt, On-Pr, or Oz ' -Pr.
  • the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R 2b is Ci_ 6 alkoxy substituted with one or more groups independently selected from OH, CN, halo, CM alkoxy, C6- 10 aryl (optionally substituted with one or more independently selected CM alkoxy), C3. 7 cycloalkyl, alkyl, and 5-10 membered heteroaryl (optionally substituted with one or more independently selected C 1 . 4 alkyl).
  • R 2b is OMe, OEt, On-Pr, or Oz ' -Pr, each of which is substituted with one or more groups independently selected from OH, CN, halo, C 1 . 4 alkoxy, C6- 10 aryl (optionally substituted with one or more independently selected Ci_ 4 alkoxy), C3. 7 cycloalkyl, alkyl, and 5-10 membered heteroaryl (optionally substituted with one or more C 1 . 4 alkyl).
  • R 2b is -OCH 2 - CN, -OCH 2 -CH 2 -OH, -OCH 2 -CH 2 -OMe, -OCH 2 -CH 2 -OEt, -OCHF 2 , -OCF 3 , -OCH 2 -Ph, or -OCH 2 -(4- OMe-Ph).
  • the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R 2b is alkoxy.
  • the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R 2b is -O-C6- 10 aryl. In a particular embodiment, R 2b is -OPh.
  • the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R 2b is 4-10 membered heterocycloalkyl. In a particular embodiment, R 2b is azetidine, pyrrolidine, piperidinyl piperazinyl, morpholinyl, or thiomorpholinyl.
  • the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R 2b is 4-10 membered heterocycloalkyl substituted with one to three oxo, halo, CM alkyl (optionally substituted with one or more -CN), alkyl, or -SO2-C 1 .4 alkyl.
  • R 2b is azetidine, pyrrolidine, piperidinyl piperazinyl, morpholinyl, or thiomorpholinyl, each of which is substituted with one to three oxo, halo, C 1 .4 alkyl (optionally substituted with one or more -CN), alkyl, or -SO2-C 1 .4 alkyl.
  • the subscript m is 1
  • the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R 2b is 5-10 membered heteroaryl.
  • R 2b is furanyl, thienyl, pyrrazolyl, imidazolyl, triazolyl, thiazolyl, pyridinyl, pyrimidinyl, or pyrazinyl.
  • the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R 2b is 5-10 membered heteroaryl substituted with one or more independently groups groups independently selected from CN, halo, OH, CM alkyl, and CM alkoxy.
  • R 2b is 5-10 membered heteroaryl substituted with one group selected from CN, halo, OH, CM alkyl, and C 1 .4 alkoxy.
  • R 2b is furanyl, thienyl, pyrrazolyl, imidazolyl, triazolyl, thiazolyl, pyridinyl, pyrimidinyl, or pyrazinyl, each of which is substituted with one or more groups independently selected from CN, halo, OH, CM alkyl, and C alkoxy.
  • R 2b is furanyl, thienyl, pyrrazolyl, imidazolyl, triazolyl, thiazolyl, pyridinyl, pyrimidinyl, or pyrazinyl, each of which is substituted with one group selected from CN, halo, OH, C alkyl, and C alkoxy.
  • R 2b is furanyl, thienyl, pyrrazolyl, imidazolyl, triazolyl, thiazolyl, pyridinyl, pyrimidinyl, or pyrazinyl, each of which is substituted with one or more groups independently selected from CN, F, CI, OH, Me, Et, z ' -Pr, -OMe, - OEt, and -Oz ' -Pr.
  • R 2b is furanyl, thienyl, pyrrazolyl, imidazolyl, triazolyl, thiazolyl, pyridinyl, pyrimidinyl, or pyrazinyl, each of which is substituted with one group selected from CN, F, CI, OH, Me, Et, z ' -Pr, -OMe, -OEt, and -Oz ' -Pr.
  • R 2b is thienyl, pyrrazolyl, imidazolyl, pyridinyl, or pyrimidinyl, each of which is substituted with one group selected from CN, Me, Et, z ' -Pr, -OMe, -OEt, and -Oz ' -Pr.
  • the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R 2b is 5-10 membered heteroaryl substituted with one or more NR 3a R 3b groups, wherein each R 3a or R 3b is independently selected from H and CM alkyl.
  • R 2b is 5-10 membered heteroaryl substituted with one NR 3a R 3b group.
  • R 2b is furanyl, thienyl, pyrrazolyl, imidazolyl, triazolyl, thiazolyl, pyridinyl, pyrimidinyl, or pyrazinyl, each of which is substituted with one substituted with one NR 3a R 3b group.
  • R 2b is furanyl, thienyl, pyrrazolyl, imidazolyl, triazolyl, thiazolyl, pyridinyl, pyrimidinyl, or pyrazinyl, each of which is substituted with one NR 3a R 3b group, wherein R 3a and R 3b are both H.
  • R 2b is furanyl, thienyl, pyrrazolyl, imidazolyl, triazolyl, thiazolyl, pyridinyl, pyrimidinyl, or pyrazinyl, each of which is substituted with one NR 3a R 3b group, wherein R 3a is H and R 3b is C alkyl.
  • R 2b is furanyl, thienyl, pyrrazolyl, imidazolyl, triazolyl, thiazolyl, pyridinyl, pyrimidinyl, or pyrazinyl, each of which is substituted with one NR 3a R 3b group, wherein R 3a is H and R 3b is Me, or Et.
  • the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R 2b is phenyl substituted with one or more groups independently selected from CN, halo, OH, CM alkyl, and C alkoxy.
  • R 2b is phenyl substituted with one group selected from CN, halo, OH, CM alkyl, and CM alkoxy.
  • R 2b is phenyl, substituted with one or more groups independently selected from CN, F, CI, OH, Me, Et, z ' -Pr, -OMe, -OEt, and -Oz ' -Pr.
  • R 2b is phenyl substituted with one group selected from CN, F, CI, OH, Me, Et, z ' -Pr, -OMe, -OEt, and -Oz ' -Pr.
  • R 2b is phenyl substituted with one group selected from CN, Me, Et, z ' -Pr, -OMe, -OEt, and -Oz ' -Pr.
  • the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R 2b is phenyl substituted with one or more NR 3a R 3b groups, wherein each R 3a or R 3b is independently selected from H and CM alkyl.
  • R 2b is phenyl substituted with one NR 3a R 3b group.
  • R 2b is phenyl substituted with one NR 3a R 3b group, wherein R 3a and R 3b are both H.
  • R 2b is phenyl substituted with one NR 3a R 3b group, wherein R 3a is H and R 3b is CM alkyl.
  • R 2b is phenyl substituted with one NR 3a R 3b group, wherein R 3a is H and R 3b is Me, or Et.
  • the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R 2b is 4-7 membered heterocycloalkenyl comprising 1 double bond, and comprising 1 or 2 heteroatoms independently selected from N, O, and S.
  • R 2b is dihydropyran or tetrahydropyridinyl.
  • the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R 2b is 4-7 membered heterocycloalkenyl comprising 1 double bond, and comprising 1 or 2 heteroatoms independently selected from N, O, and S, substituted with one or more groups independently selected from C alkyl, CM alkoxy, and C 3 .7 cycloalkyl.
  • R 2b is 4-7 membered heterocycloalkenyl, substituted with one group independently selected from CM alkyl, C 1 .4 alkoxy, and C 3 .7 cycloalkyl.
  • R 2b is dihydropyran or tetrahydropyridinyl, each of which is substituted with one or more groups independently selected from C alkyl, C 1 .4 alkoxy, and C 3 .7 cycloalkyl.
  • R 2b is dihydropyran or tetrahydropyridinyl, each of which is substituted with one or more groups independently selected from Me, Et, z ' Pr, iBu, -OMe, -OEt, -Oz ' Pr, -OiBu, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.
  • R 2b is dihydropyran or tetrahydropyridinyl, each of which is substituted with one group selected from Me, Et, z ' Pr, iBu, -OMe, -OEt, -Oz ' Pr, -OiBu, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.
  • R 2b is dihydropyran or tetrahydropyridinyl, each of which is substituted with one or more groups independently selected from - alkyl optionally substituted with one or more independently selected CN, OH, and halo.
  • the compound of the invention is according to Formula I, wherein Cy is Cyi, R 2a , R 2b , and the subscript m are as described above, x is N, and each of w, y and z are CH.
  • R 2a and R 2b are as described above, the subscript m is 1, x is N, and each of w, y and z are CH.
  • the compound of the invention is according to Formula Ila, lib, lie or lid, wherein R 2b is CM alkyl substituted with one or more groups independently selected from halo, CN, OH, and Ce-io aryl (optionally substituted with one or more independently selected C alkoxy).
  • R 2b is CM alkyl substituted with one or more groups independently selected from F, CN, OH, phenyl and 4-OMe-phenyl.
  • the compound of the invention is according to Formula Ila, lib, lie or lid, wherein R 2b is 4-10 membered heterocycloalkyl.
  • R 2b is azetidine, pyrrolidine, piperidinyl piperazinyl, morpholinyl, or thiomorpholinyl.
  • R 2b is furanyl, thienyl, pyrrazolyl, imidazolyl, triazolyl, thiazolyl, pyridinyl, pyrimidinyl, or pyrazinyl, each of which is substituted with one NR 3a R 3b group, wherein R 3a is H and R 3b is CM alkyl.
  • the compound of the invention is according to Formula Ila, lib, lie or lid, wherein R 2B is phenyl substituted with one or more groups independently selected from CN, halo, OH, CM alkyl, and CM alkoxy.
  • R 2B is phenyl substituted with one group selected from CN, halo, OH, C alkyl, and C alkoxy.
  • R 2B is phenyl, substituted with one or more groups independently selected from CN, F, CI, OH, Me, Et, z ' -Pr, -OMe, - OEt, and -Oz ' -Pr.
  • the compound of the invention is according to Formula Ila, lib, lie or lid, wherein R 2B is 4-7 membered heterocycloalkenyl comprising 1 double bond, and comprising 1 or 2 heteroatoms independently selected from N, O, and S.
  • R 2B is dihydropyran or tetrahydropyridinyl.
  • the compound of the invention is according to Formula Ila, lib, lie or lid, wherein R 2B is 4-7 membered heterocycloalkenyl comprising 1 double bond, and comprising 1 or 2 heteroatoms independently selected from N, O, and S, substituted with one or more groups independently selected from C alkyl, CM alkoxy,C3_ 7 cycloalkyl.
  • R 2B is 4-7 membered heterocycloalkenyl, substituted with one group independently selected from CM alkyl, CM alkoxy, and C3. 7 cycloalkyl.
  • R 2b is dihydropyran or tetrahydropyridinyl, each of which is substituted with one or more groups independently selected from alkyl optionally substituted with one or more independently selected CN, OH, and halo.
  • the compound of the invention is according to Formula Ila, lib, lie or lid, wherein R 2b is 4-7 membered heterocycloalkenyl comprising 1 double bond, and comprising 1 or 2 heteroatoms independently selected from N, O, and S, substituted with one or more independently selected alkyl.
  • R 2b is 4-7 membered heterocycloalkenyl, substituted with one alkyl.
  • R 2b is dihydropyran or tetrahydropyridinyl, each of which is substituted with one or more independently selected
  • R 5 is selected from C1.4 alkyl, CM alkoxy, C3.7 cycloalkyl, l (optionally substituted with one or more independently selected CN, OH, and halo); a alkyl.
  • the compound of the invention is according to Formula III, wherein R 5 is alkyl optionally substituted with one or more groups independently selected from CN, OH, and halo.
  • the compound of the invention according to Formula I is compound to Formula IV:
  • R 6 is Ci_ 4 alkyl (optionally substituted with one or more -CN), alkyl, or -S0 2 -Ci_ 4 alkyl.
  • R 6 is-S0 2 -Me, -S0 2 -Et, -S0 2 -zPr, or -S0 2 -iBu.
  • the compound of the invention is according to Formula I, wherein Cy is selected from Cy 2 , and Cy 3 ; and the ring A is a fused 5-6 membered saturated ring optionally comprising 1 or 2 heteroatoms independently selected from N, O, and S; to afford a 8-10 membered bicyclic group.
  • the ring A is a fused 5-6 membered saturated ring optionally comprising 1 or 2 heteroatoms independently selected from N, O, and S; to afford a 9-10 membered bicyclic group.
  • the compound of the invention is according to Formula I, wherein Cy is Cy 2 , wherein the ring A is a fused 5-6 membered saturated ring optionally comprising 1 or 2 heteroatoms independently selected from N, O, and S; to afford a 9-10 membered bicyclic group, wherein the subscript p is 1, R 2d is as described above, and R 2c is oxo.
  • the compound of the invention is according to Formula I, wherein Cy is Cy 2 , wherein the ring A is a fused 5-6 membered saturated ring optionally comprising 1 or 2 heteroatoms independently selected from N, O, and S; to afford a 9-10 membered bicyclic group, wherein the subscript p, and R 2c are as described above, and R 2d is H, halo, C alkyl optionally substituted with one or more halo, or CM alkoxy optionally substituted with one or more halo. In a particular embodiment, R 2d is H.
  • R 2d is F, CI, Me, Et, z ' -Pr, -CF 3 , - OMe, -OEt, -OCHF 2 , or -OCF 3 .
  • R 2d is CI, Me or Et.
  • R is H, halo, CM alkyl optionally substituted with one or more independently selected halo, or C alkoxy optionally substituted with one or more independently selected halo.
  • R is H.
  • R is F, CI, Me, Et, z ' -Pr, -CF 3 , -OMe, -OEt, - OCHF 2 , or -OCF 3 .
  • R 2d is CI, Me or Et.
  • the compound of the invention is according to Formula I, wherein Cy is selected from Cy 2 , and Cy 3 ; and the ring A is a fused 5-membered unsaturated ring optionally comprising 1 or 2 heteroatoms independently selected from N, O, and S; to afford a 8-10 membered bicyclic group.
  • the ring A is a fused 5-membered unsaturated ring optionally comprising 1 or 2 heteroatoms independently selected from N, O, and S; to afford a 9-10 membered bicyclic group.
  • the compound of the invention is according to Formula I, wherein Cy is Cy 2 , wherein the ring A is a fused 5-membered unsaturated ring optionally comprising 1 or 2 heteroatoms independently selected from N, O, and S; to afford a 9-10 membered bicyclic group, wherein the subscript p is 1 or 2, R 2d is as described above, and each R 2c is independently selected from C alkyl or halo. In a particular embodiment, R 2c is Me, Et, or F.
  • the compound of the invention is according to Formula I, wherein Cy is Cy 2 , wherein the ring A is a fused 5-membered unsaturated ring optionally comprising 1 or 2 heteroatoms independently selected from N, O, and S; to afford a 9-10 membered bicyclic group, wherein the subscript p, and R 2c are as described above, and R 2d is H, halo, C 1 . 4 alkyl optionally substituted with one or more independently selected halo, or CM alkoxy optionally substituted with one or more independently selected halo. In a particular embodiment, R 2d is H.
  • R 2d is F, CI, Me, Et, z-Pr, -CF 3 , -OMe, -OEt, -OCHF 2 , or -OCF 3 .
  • R 2d is CI, Me or Et.
  • the compound of the invention is according to Formula I, wherein Cy is
  • R is H, CM alkyl, or halo; and R e is selected from H, and CM alkyl.
  • R 2e is as previously defined, and R 2d is H, Me, Et, or CI.
  • R 2d is as previously defined, and R 2e is H, Me or Et.
  • R 2d is selected from H, Me, Et, or CI, and R 2e is H, Me or Et.
  • the compound of the invention according to Formula I is selected from:
  • the compound of the invention is not an isotopic variant.
  • a compound of the invention according to any one of the embodiments herein described is a pharmaceutically acceptable salt.
  • a compound of the invention according to any one of the embodiments herein described is a solvate of the compound.
  • a compound of the invention according to any one of the embodiments herein described is a solvate of a pharmaceutically acceptable salt of a compound.
  • a compound of the invention may be one for which one or more variables (for example, R groups) is selected from one or more embodiments according to any of the Formula(e) listed above. Therefore, the present invention is intended to include all combinations of variables from any of the disclosed embodiments within its scope.
  • the present invention provides prodrugs and derivatives of the compounds according to the formulae above.
  • Prodrugs are derivatives of the compounds of the invention, which have metabolically cleavable groups and become by solvolysis or under physiological conditions the compounds of the invention, which are pharmaceutically active, in vivo.
  • Such examples include, but are not limited to, choline ester derivatives and the like, N-alkylmorpholine esters and the like.
  • Prodrugs include acid derivatives well know to practitioners of the art, such as, for example, esters prepared by reaction of the parent acid with a suitable alcohol, or amides prepared by 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 on the compounds of this invention are preferred prodrugs.
  • double ester type prodrugs such as (acyloxy)alkyl esters or ((alkoxycarbonyl)oxy)alkylesters.
  • Particularly useful are the Ci_g alkyl, C2-8 alkenyl, aryl, C7.12 substituted aryl, and C7.12 arylalkyl esters of the compounds of the invention.
  • the compounds of the invention are novel inhibitors of JAK.
  • the compounds are potent inhibitors of JAKl and/or JAK2; however they may inhibit TYK2 and JAK3 with a lower potency.
  • a compound of the invention When employed as a pharmaceutical, a compound of the invention is typically administered in the form of a pharmaceutical composition. Such compositions can be prepared in a manner well known in the pharmaceutical art and comprise at least one active compound. Generally, a compound of this invention is administered in a pharmaceutically effective amount. The amount of the compound actually administered will typically be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.
  • compositions of the invention can be administered by a variety of routes including oral, rectal, transdermal, subcutaneous, intra-articular, intravenous, intramuscular, and intranasal.
  • routes including oral, rectal, transdermal, subcutaneous, intra-articular, intravenous, intramuscular, and intranasal.
  • a compound of this invention is preferably formulated as either injectable or oral compositions or as salves, as lotions or as patches all for transdermal administration.
  • compositions for oral administration can take the form of bulk liquid solutions or suspensions, or bulk powders. More commonly, however, the compositions are presented in unit dosage forms to facilitate accurate dosing.
  • unit dosage forms refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient, vehicle or carrier.
  • Typical unit dosage forms include prefilled, premeasured ampules or syringes of the liquid compositions or pills, tablets, capsules or the like in the case of solid compositions.
  • the compound of the invention 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 being various vehicles or carriers and processing aids helpful for forming the desired dosing form.
  • a minor component from about 0.1 to about 50% by weight or preferably from about 1 to about 40%
  • Liquid forms suitable for oral administration may include a suitable aqueous or nonaqueous vehicle with buffers, suspending and dispensing agents, colorants, flavors and the like.
  • Solid forms may include, for example, any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate
  • a glidant such as colloidal silicon dioxide
  • a sweetening agent such as sucrose or saccharin
  • Injectable compositions are typically based upon injectable sterile saline or phosphate- buffered saline or other injectable carriers known in the art.
  • the active compound in such compositions is typically a minor component, often being from about 0.05 to 10% by weight with the remainder being the injectable carrier and the like.
  • Transdermal compositions are typically formulated as a topical ointment or cream containing the active ingredient(s), generally in an amount ranging from about 0.01 to about 20% by weight, preferably from about 0.1 to about 20% by weight, preferably from about 0.1 to about 10% by weight, and more preferably from about 0.5 to about 15% by weight.
  • the active ingredients When formulated as a ointment, the active ingredients will typically be combined with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredients may be formulated in 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 dermal penetration of stability of the active ingredients or the formulation. All such known transdermal formulations and ingredients are included within the scope of this invention.
  • a compound of the invention can also be administered by a transdermal device. Accordingly, transdermal administration can be accomplished using a patch either of the reservoir or porous membrane type, or of a solid matrix variety.
  • a compound of the invention can also be administered in sustained release forms or from sustained release drug delivery systems.
  • sustained release materials can be found in Remington's Pharmaceutical Sciences.
  • a compound of the invention may be admixed as a dry powder with a dry gelatin binder in an approximate 1 :2 weight ratio.
  • a minor amount of magnesium stearate may be added as a lubricant.
  • the mixture may be formed into 240-270 mg tablets (80-90 mg of active amide compound per tablet) in a tablet press.
  • a compound of the invention may be admixed as a dry powder with a starch diluent in an approximate 1 : 1 weight ratio.
  • the mixture may be filled into 250 mg capsules (125 mg of active amide compound per capsule).
  • a compound of the invention (125 mg), may be admixed with sucrose (1.75 g) and xanthan gum (4 mg) and the resultant mixture may be blended, passed through a No. 10 mesh U.S. sieve, and then mixed with a previously made 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. Sufficient water may then be added with stirring. Further sufficient water may be then added to produce a total volume of 5 mL.
  • a compound of the invention may be admixed as a dry powder with a dry gelatin binder in an approximate 1 :2 weight ratio.
  • a minor amount of magnesium stearate may be added as a lubricant.
  • the mixture may be formed into 450-900 mg tablets (150-300 mg of active amide compound) in a tablet press.
  • a compound of the invention may be dissolved or suspended in a buffered sterile saline injectable aqueous medium to a concentration of approximately 5 mg/mL.
  • Stearyl alcohol (250 g) and a white petrolatum (250 g) may be melted at about 75°C and then a mixture of the compound of the invention (50 g) methylparaben (0.25 g), propylparaben (0.15 g), sodium lauryl sulfate (10 g), and propylene glycol (120 g) dissolved in water (about 370 g) may be added and the resulting mixture may be stirred until it congeals.
  • a compound of the invention may be used as a therapeutic agent for the treatment of conditions in mammals that are causally related or attributable to aberrant activity of JAK.
  • the compounds and pharmaceutical compositions of the invention find use as therapeutics for preventing and/or treating allergic or inflammatory conditions, autoimmune diseases, proliferative diseases, transplantation rejection, diseases involving impairment of cartilage turnover, congenital cartilage malformations, and/or diseases associated with hypersecretion of IL6 or interferons in mammals including humans.
  • the present invention provides a compound of the invention, or a pharmaceutical composition comprising a compound of the invention for use as a medicament.
  • the present invention provides a compound of the invention, or a pharmaceutical composition comprising a compound of the invention for use in the manufacture of a medicament.
  • the present invention provides a method of treating a mammal having, or at risk of having a disease disclosed herein, said method comprising administering an effective condition-treating or condition-preventing amount of one or more of the pharmaceutical compositions or compound of the invention herein described.
  • the present invention provides a method of treating a mammal having, or at risk of having allergic or inflammatory conditions, autoimmune diseases, proliferative diseases, transplantation rejection, diseases involving impairment of cartilage turnover, congenital cartilage malformations, and/or diseases associated with hypersecretion of IL6 or interferons.
  • this invention provides methods of treatment and/or prophylaxis of a mammal susceptible to or afflicted with an allergic reaction, said method comprising administering an effective condition-treating or condition-preventing amount of one or more of the pharmaceutical compositions or compound of the invention herein described.
  • the allergic reaction is selected from allergic airway disease, sinusitis, eczema and hives, food allergies and allergies to insect venom.
  • the present invention provides a compound of the invention for use in the treatment, and/or prophylaxis of an allergic reaction.
  • the allergic reaction is selected from allergic airway disease, sinusitis, eczema and hives, food allergies and allergies to insect venom.
  • the present invention provides the compound of the invention, or a pharmaceutical composition comprising the compound of the invention for use in the manufacture of a medicament for the treatment, or prophylaxis of an allergic reaction.
  • the allergic reaction is selected from allergic airway disease, sinusitis, eczema and hives, food allergies and allergies to insect venom.
  • this invention provides methods of treatment and/or prophylaxis of a mammal susceptible to or afflicted with an inflammatory condition, said methods comprise administering an effective condition-treating or condition-preventing amount of one or more of the pharmaceutical compositions or compound of the invention herein described.
  • the inflammatory condition is selected from rheumatoid arthritis, osteoarthritis, allergic airway disease (e.g. asthma) and inflammatory bowel diseases.
  • the present invention provides a compound of the invention for use in the treatment, and/or prophylaxis of an inflammatory condition.
  • the inflammatory condition is selected from rheumatoid arthritis, osteoarthritis, allergic airway disease (e.g. asthma) and inflammatory bowel diseases.
  • the present invention provides the compound of the invention, or a pharmaceutical composition comprising the compound of the invention for use in the manufacture of a medicament for the treatment, and/or prophylaxis of an inflammatory condition.
  • the inflammatory condition is selected from rheumatoid arthritis, osteoarthritis, allergic airway disease (e.g. asthma) and inflammatory bowel diseases.
  • this invention provides methods of treatment and/or prophylaxis of a mammal susceptible to or afflicted with an autoimmune disease, saidmethods comprise administering an effective condition-treating or condition-preventing amount of one or more of the pharmaceutical compositions or compounds of the invention herein described.
  • the autoimmune disease is selected from COPD, asthma, systemic lupus erythematosis, type I diabetes mellitus and inflammatory bowel disease.
  • the present invention provides a compound of the invention for use in the treatment, and/or prophylaxis of an autoimmune disease.
  • the autoimmune disease is selected from COPD, asthma, systemic lupus erythematosis, type I diabetes mellitus and inflammatory bowel disease.
  • the autoimmune disease is systemic lupus erythematosis.
  • the present invention provides the compound of the invention, or a pharmaceutical composition comprising the compound of the invention for use in the manufacture of a medicament for the treatment, and/or prophylaxis of an autoimmune disease.
  • the autoimmune disease is selected from COPD, asthma, systemic lupus erythematosis, type I diabetes mellitus and inflammatory bowel disease.
  • this invention provides methods of treatment and/or prophylaxis of a mammal susceptible to or afflicted with a proliferative disease, said methods comprising administering an effective condition-treating or condition-preventing amount of one or more of the pharmaceutical compositions or compound of the invention herein described.
  • the proliferative disease is selected from cancer (e.g. solid tumors such as uterine leiomyosarcoma or prostate cancer), leukemia (e.g. AML, ALL or CLL), multiple myeloma and psoriasis.
  • the present invention provides a compound of the invention for use in the treatment, and/or prophylaxis of a proliferative disease.
  • the proliferative disease is selected from cancer (e.g. solid tumors such as uterine leiomyosarcoma or prostate cancer), leukemia (e.g. AML, ALL or CLL), multiple myeloma and psoriasis.
  • the present invention provides the compound of the invention, or a pharmaceutical composition comprising the compound of the invention for use in the manufacture of a medicament for the treatment, and/or prophylaxis of a proliferative disease.
  • the proliferative disease is selected from cancer (e.g. solid tumors such as uterine leiomyosarcoma or prostate cancer), leukemia (e.g. AML, ALL or CLL), multiple myeloma and psoriasis.
  • this invention provides methods of treatment and/or prophylaxis of a mammal susceptible to or afflicted with transplantation rejection, said methods comprising administering an effective condition-treating or condition-preventing amount of one or more of the pharmaceutical compositions or compound of the invention herein described.
  • the transplantation rejection is organ transplant rejection.
  • the present invention provides a compound of the invention for use in the treatment, and/or prophylaxis of transplantation rejection.
  • the transplantation rejection is organ transplant rejection.
  • the present invention provides the compound of the invention, or a pharmaceutical composition comprising the compound of the invention for use in the manufacture of a medicament for the treatment and/or prophylaxis of of transplantation rejection.
  • the transplantation rejection is organ transplant rejection.
  • this invention provides a method of treatment, and/or prophylaxis in a mammal susceptible to or afflicted with diseases involving impairment of cartilage turnover, which method comprises administering a therapeutically effective amount of a compound of the invention, or one or more of the pharmaceutical compositions herein described.
  • the present invention provides a compound of the invention for use in the treatment, and/or prophylaxis of diseases involving impairment of cartilage turnover.
  • the present invention provides the compound of the invention, or a pharmaceutical composition comprising the compound of the invention for use in the manufacture of a medicament for the treatment, and/or prophylaxis of diseases involving impairment of cartilage turnover.
  • the present invention also provides a method of treatment and/or prophylaxis of congenital cartilage malformations, which method comprises administering an effective amount of one or more of the pharmaceutical compositions or compounds of the invention herein described.
  • the present invention provides a compound of the invention for use in the treatment, and/or prophylaxis of congenital cartilage malformations.
  • the present invention provides the compound of the invention, or a pharmaceutical composition comprising the compound of the invention for use in the manufacture of a medicament for the treatment, and/or prophylaxis of congenital cartilage malformations.
  • this invention provides methods of treatment and/or prophylaxis of a mammal susceptible to or afflicted with diseases associated with hypersecretion of IL6, said methods comprising administering an effective condition-treating or condition-preventing amount of one or more of the pharmaceutical compositions or compound of the invention herein described.
  • the disease associated with hypersecretion of IL6 is selected from Castleman's disease and mesangial proliferative glomerulonephritis.
  • the present invention provides a compound of the invention for use in the treatment, and/or prophylaxis of diseases associated with hypersecretion of IL6.
  • the disease associated with hypersecretion of IL6 is selected from Castleman's disease and mesangial proliferative glomerulonephritis.
  • the present invention provides the compound of the invention, or a pharmaceutical composition comprising the compound of the invention for use in the manufacture of a medicament for the treatment, and/or prophylaxis of diseases associated with hypersecretion of IL6.
  • the disease associated with hypersecretion of IL6 is selected from Castleman's disease and mesangial proliferative glomerulonephritis.
  • this invention provides methods of treatment and/or prophylaxis of a mammal susceptible to or afflicted with diseases associated with hypersecretion of interferons, said methods comprising administering an effective condition-treating or condition- preventing amount of one or more of the pharmaceutical compositions or compound of the invention herein described.
  • the disease associated with hypersecretion of interferons is selected from systemic and cutaneous lupus erythematosis, lupus nephritis, dermatomyositis, Sjogren's syndrome, psoriasis, and rheumatoid arthritis.
  • the present invention provides a compound of the invention for use in the treatment, and/or prophylaxis of diseases associated with hypersecretion of interferons.
  • the disease associated with hypersecretion of interferons is selected from systemic and cutaneous lupus erythematosis, lupus nephritis, dermatomyositis, Sjogren's syndrome, psoriasis, and rheumatoid arthritis.
  • the present invention provides the compound of the invention, or a pharmaceutical composition comprising the compound of the invention for use in the manufacture of a medicament for the treatment, and/or prophylaxis of diseases associated with hypersecretion of interferons.
  • the disease associated with hypersecretion of interferons is selected from systemic and cutaneous lupus erythematosis, lupus nephritis, dermatomyositis, Sjogren's syndrome, psoriasis, and rheumatoid arthritis.
  • a compound of the invention for use as a pharmaceutical especially in the treatment and/or prophylaxis of the aforementioned conditions and diseases. Also provided herein is the use of the present compounds in the manufacture of a medicament for the treatment and/or prophylaxis of one of the aforementioned conditions and diseases.
  • a particular regimen of the present method comprises the administration to a subject suffering from a disease involving inflammation, of an effective amount of a compound of the invention for a period of time sufficient to reduce the level of inflammation in the subject, and preferably terminate the processes responsible for said inflammation.
  • a special embodiment of the method comprises administering of an effective amount of a compound of the invention to a subject patient suffering from or susceptible to the development of rheumatoid arthritis, for a period of time sufficient to reduce or prevent, respectively, inflammation in the joints of said patient, and preferably terminate, the processes responsible for said inflammation.
  • a further particular regimen of the present method comprises the administration to a subject suffering from a disease condition characterized by cartilage or joint degradation (e.g. rheumatoid arthritis and/or osteoarthritis) of an effective amount of a compound of the invention for a period of time sufficient to reduce and preferably terminate the self-perpetuating processes responsible for said degradation.
  • a particular embodiment of the method comprises administering of an effective amount of a compound of the invention to a subject patient suffering from or susceptible to the development of osteoarthritis, for a period of time sufficient to reduce or prevent, respectively, cartilage degradation in the joints of said patient, and preferably terminate, the self-perpetuating processes responsible for said degradation.
  • said compound may exhibit cartilage anabolic and/or anti- catabolic properties.
  • Injection dose levels range from about 0.1 mg/kg/h to at least 10 mg/kg/h, all for from about 1 to about 120 h and especially 24 to 96 h.
  • a preloading bolus of from about 0.1 mg/kg to about 10 mg/kg or more may also be administered to achieve adequate steady state levels.
  • the maximum total dose is not expected to exceed about 2 g/day for a 40 to 80 kg human patient.
  • each dose provides from about 0.01 to about 20 mg/kg of a compound of the invention, with particular doses each providing from about 0.1 to about 10 mg/kg and especially about 1 to about 5 mg/kg.
  • Transdermal doses are generally selected to provide similar or lower blood levels than are achieved using injection doses.
  • a compound of the invention When used to prevent the onset of a condition, a compound of the invention will be administered to a patient at risk for developing the condition, typically on the advice and under the supervision of a physician, at the dosage levels described above.
  • Patients at risk for developing a particular condition generally include those that have a family history of the condition, or those who have been identified by genetic testing or screening to be particularly susceptible to developing the condition.
  • a compound of the invention can be administered as the sole active agent or it can be administered in combination with other therapeutic agents, including other compounds that demonstrate the same or a similar therapeutic activity and that are determined to safe and efficacious for such combined administration.
  • co-administration of two (or more) agents allows for significantly lower doses of each to be used, thereby reducing the side effects seen.
  • a compound of the invention or a pharmaceutical composition comprising a compound of the invention is administered as a medicament.
  • said pharmaceutical composition additionally comprises a further active ingredient.
  • a compound of the invention is co-administered with another therapeutic agent for the treatment and/or prophylaxis of a disease involving inflammation;
  • agents include, but are not limited to, immunoregulatory agents e.g. azathioprine, corticosteroids (e.g. prednisolone or dexamethasone), cyclophosphamide, cyclosporin A, tacrolimus, Mycophenolate Mofetil, muromonab-CD3 (OKT3, e.g. Orthocolone®), ATG, aspirin, acetaminophen, ibuprofen, naproxen, and piroxicam.
  • immunoregulatory agents e.g. azathioprine, corticosteroids (e.g. prednisolone or dexamethasone), cyclophosphamide, cyclosporin A, tacrolimus, Mycophenolate Mofetil, muromonab-CD3 (OK
  • a compound of the invention is co-administered with another therapeutic agent for the treatment and/or prophylaxis of arthritis (e.g. rheumatoid arthritis); particular agents include but are not limited to analgesics, non-steroidal anti-inflammatory drugs (NSAIDS), steroids, synthetic DMARDS (for example but without limitation methotrexate, leflunomide, sulfasalazine, auranofin, sodium aurothiomalate, penicillamine, chloroquine, hydroxychloroquine, azathioprine, and ciclosporin), and biological DMARDS (for example but without limitation Infliximab, Etanercept, Adalimumab, Rituximab, and Abatacept).
  • NSAIDS non-steroidal anti-inflammatory drugs
  • DMARDS for example but without limitation methotrexate, leflunomide, sulfasalazine, auranofin, sodium aurothiomalate, penici
  • a compound of the invention is co-administered with another therapeutic agent for the treatment and/or prophylaxis of proliferative disorders; particular agents include but are not limited to: methotrexate, leukovorin, adriamycin, prenisone, bleomycin, cyclophosphamide, 5- fluorouracil, paclitaxel, docetaxel, vincristine, vinblastine, vinorelbine, doxorubicin, tamoxifen, toremifene, megestrol acetate, anastrozole, goserelin, anti-HER2 monoclonal antibody (e.g.
  • a compound of the invention may be administered in combination with other therapies including, but not limited to, radiotherapy or surgery.
  • the proliferative disorder is selected from cancer, myeloproliferative disease or leukaemia.
  • a compound of the invention is co-administered with another therapeutic agent for the treatment and/or prophylaxis of autoimmune diseases
  • agents include but are not limited to: glucocorticoids, cytostatic agents (e.g. purine analogs), alkylating agents, (e.g nitrogen mustards (cyclophosphamide), nitrosoureas, platinum compounds, and others), antimetabolites (e.g. methotrexate, azathioprine and mercaptopurine), cytotoxic antibiotics (e.g. dactinomycin anthracyclines, mitomycin C, bleomycin, and mithramycin), antibodies (e.g.
  • anti-CD20, anti-CD25 or anti-CD3 (OTK3) monoclonal antibodies Atgam® and Thymoglobuline®
  • cyclosporin tacrolimus, rapamycin (sirolimus), interferons (e.g. IFN- ⁇ ), TNF binding proteins (e.g. infliximab (RemicadeTM), etanercept (EnbrelTM), or adalimumab (HumiraTM)), mycophenolate, Fingolimod and Myriocin.
  • IFN- ⁇ interferons
  • TNF binding proteins e.g. infliximab (RemicadeTM), etanercept (EnbrelTM), or adalimumab (HumiraTM)
  • mycophenolate Fingolimod and Myriocin.
  • a compound of the invention is co-administered with another therapeutic agent for the treatment and/or prophylaxis of transplantation rejection
  • therapeutic agents include but are not limited to: calcineurin inhibitors (e.g. cyclosporin or tacrolimus (FK506)), mTOR inhibitors (e.g. sirolimus, everolimus), anti-proliferatives (e.g. azathioprine, mycophenolic acid), corticosteroids (e.g. prednisolone, hydrocortisone), Antibodies (e.g.
  • monoclonal anti-IL-2Ra receptor antibodies basiliximab, daclizumab
  • polyclonal anti-T-cell antibodies e.g. anti-thymocyte globulin (ATG), anti- lymphocyte globulin (ALG)).
  • a compound of the invention is co-administered with another therapeutic agent for the treatment and/or prophylaxis of asthma and/or rhinitis and/or COPD
  • particular agents include but are not limited to: beta2-adrenoceptor agonists (e.g. salbutamol, levalbuterol, terbutaline and bitolterol), epinephrine (inhaled or tablets), anticholinergics (e.g. ipratropium bromide), glucocorticoids (oral or inhaled) Long-acting p2-agonists (e.g.
  • bronchodilators e.g. fluticasone/salmeterol, budesonide/formoterol
  • leukotriene antagonists and synthesis inhibitors e.g. montelukast, zafirlukast and zileuton
  • inhibitors of mediator release e.g. cromoglycate and ketotifen
  • biological regulators of IgE response e.g. omalizumab
  • antihistamines e.g. ceterizine, cinnarizine, fexofenadine
  • vasoconstrictors e.g. oxymethazoline, xylomethazoline, nafazoline and tramazoline.
  • a compound of the invention may be administered in combination with emergency therapies for asthma and/or COPD, such therapies include oxygen or heliox administration, nebulized salbutamol or terbutaline (optionally combined with an anticholinergic (e.g. ipratropium), systemic steroids (oral or intravenous, e.g. prednisone, prednisolone, methylprednisolone, dexamethasone, or hydrocortisone), intravenous salbutamol, non-specific beta-agonists, injected or inhaled (e.g.
  • oxygen or heliox administration ebulized salbutamol or terbutaline
  • an anticholinergic e.g. ipratropium
  • systemic steroids oral or intravenous, e.g. prednisone, prednisolone, methylprednisolone, dexamethasone, or hydrocortisone
  • intravenous salbutamol e.g. pred
  • epinephrine isoetharine, isoproterenol, metaproterenol
  • anticholinergics IV or nebulized, e.g. glycopyrrolate, atropine, ipratropium
  • methylxanthines theophylline, aminophylline, bamiphylline
  • inhalation anesthetics that have a bronchodilatory effect (e.g. isoflurane, halothane, enflurane), ketamine and intravenous magnesium sulfate.
  • a compound of the invention is co-administered with another therapeutic agent for the treatment and/or prophylaxis of inflammatory bowel disease (IBD), particular agents include but are not limited to: glucocorticoids (e.g. prednisone, budesonide) synthetic disease modifying, immunomodulatory agents (e.g. methotrexate, leflunomide, sulfasalazine, mesalazine, azathioprine, 6- mercaptopurine and ciclosporin) and biological disease modifying, immunomodulatory agents (infliximab, adalimumab, rituximab, and abatacept).
  • glucocorticoids e.g. prednisone, budesonide
  • immunomodulatory agents e.g. methotrexate, leflunomide, sulfasalazine, mesalazine, azathioprine, 6- mercaptopurine and
  • a compound of the invention is co-administered with another therapeutic agent for the treatment and/or prophylaxis of SLE
  • particular agents include but are not limited to: Disease-modifying antirheumatic drugs (DMA Ds) such as antimalarials (e.g. plaquenil, hydroxychloroquine), immunosuppressants (e.g. methotrexate and azathioprine), cyclophosphamide and mycophenohc acid; immunosuppressive drugs and analgesics, such as nonsteroidal anti-inflammatory drugs, opiates (e.g. dextropropoxyphene and co-codamol), opioids (e.g. hydrocodone, oxycodone, MS Contin, or methadone) and the fentanyl duragesic transdermal patch.
  • DMA Ds Disease-modifying antirheumatic drugs
  • antimalarials e.g. plaquenil, hydroxychloroquine
  • immunosuppressants
  • a compound of the invention is co-administered with another therapeutic agent for the treatment and/or prophylaxis of psoriasis
  • particular agents include but are not limited to: topical treatments such as bath solutions, moisturizers, medicated creams and ointments containing coal tar, dithranol (anthralin), corticosteroids like desoximetasone (TopicortTM), fluocinonide, vitamin D3 analogues (for example, calcipotriol), Argan oiland retinoids (etretinate, acitretin, tazarotene), systemic treatments such as methotrexate, cyclosporine, retinoids, tioguanine, hydroxyurea, sulfasalazine, mycophenolate mofetil, azathioprine, tacrolimus, fumaric acid esters or biologies such as AmeviveTM, EnbrelTM, Humir
  • a compound of the invention is co-administered with another therapeutic agent for the treatment and/or prophylaxis of allergic reaction
  • therapeutic agents include but are not limited to: antihistamines (e.g. cetirizine, diphenhydramine, fexofenadine, levocetirizine), glucocorticoids (e.g. prednisone, betamethasone, beclomethasone, dexamethasone), epinephrine, theophylline or anti-leukotrienes (e.g. montelukast or zafirlukast), anti-cholinergics and decongestants.
  • antihistamines e.g. cetirizine, diphenhydramine, fexofenadine, levocetirizine
  • glucocorticoids e.g. prednisone, betamethasone, beclomethasone, dexamethasone
  • epinephrine e
  • any means of delivering two or more therapeutic agents to the patient as part of the same treatment regime is included any means of delivering two or more therapeutic agents to the patient as part of the same treatment regime, as will be apparent to the skilled person. Whilst the two or more agents may be administered simultaneously in a single formulation this is not essential. The agents may be administered in different formulations and at different times.
  • a compound of the 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 (i.e. reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given; other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.
  • a compound of the invention may be prepared from known or commercially available starting materials and reagents by one skilled in the art of organic synthesis.
  • LC-MS were recorded on a Waters Micromass ZQ coupled to a HPLC Waters 2795, equipped with a UV detector Waters 2996. LC were also run on a HPLC Agilent 1100 coupled to a UV detector Agilent G1315A.
  • a solution of the product obtained in Step i) (1 eq.) in MeOH (0.02 M) is passed through an H-Cube (60°C, 60 Bar, flow rate: 1 mL.min "1 ) on 5 wt.% ruthenium on carbon.
  • the solution is collected and passed through the H-Cube (under identical conditions) a further two times.
  • the resultant solution is concentrated in vacuo.
  • the reaction is cooled to room temperature and filtered through Celite, washed through with DCM and the organics are washed with water, the layers are separated and the aqueous layer further extracted with DCM. The organics are combined, dried (hydrophobic filter) and concentrated in vacuo.
  • the resulting residue is dissolved in DCM and allowed to load under gravity onto a 10 g SCX column, washed with DCM and MeOH and eluted with 7 N NH 3 in MeOH : MeOH (1 :5).
  • the eluent is concentrated in vacuo and the resulting residue is purified using column chromatography on silica gel. The fractions containing product are combined and concentrated in vacuo to give 4-Aryl-2-Ethylaniline.
  • the aqueous phase was extracted with DCM and the combined organics were dried (MgSO i), filtered and concentrated in vacuo.
  • the crude product was purified by column chromatography using silica gel and eluting with 0 - 20% EtOAc in isohexanes to give the desired compound.
  • reaction mixture was filtered through celite, washed through with EtOAc and partitioned between EtOAc and water. The layers were separated and the aqueous layer extracted with EtOAc, the organics were combined and dried (MgSO i), filtered and concentrated in vacuo to give the desired compound.
  • a 100 mg sample was purified by preparative HPLC to give the desired compound.
  • Tris(dibenzylideneacetone)dipalladium(0) (0.68 g, 0.00075 mol) was added and the mixture was stirred at 100°C for 4.5 h. The mixture was concentrated in vacuo and the residue was treated with water (50 mL) and extracted into EtOAc (2 x 300 mL). The combined extracts were washed with brine, dried (MgSO i), filtered and concentrated in vacuo to give a solid which was recrystalised from isopropanol to give the desired compound.
  • reaction mixture was cooled to room temperature, filtered through a PL-Thiol MP SPE+ column (pre-conditioned with 1 mL MeOH) using MeOH (3 x 2 mL) to wash the column.
  • MeOH 3 x 2 mL
  • Iron powder (5.46 g, 0.095 mol) was added to a stirred suspension of ammonium chloride (1.52 g, 0.028 mol) plus 4-(4-methoxybenzyl)-7-nitro-2H-benzo[ ⁇ ][l,4]oxazin-3(4H)-one (6.15 g, 0.019 mol) in a mixture of THF (22 mL), ethanol (22 mL) and water (10 mL). The resulting suspension was stirred at 90 °C for 2.5 h and cooled to room temperature.
  • Tris(dibenzylideneacetone)dipalladium(0) (0.23 g, 0.25 mmol) was added and the reaction was stirred at 100 °C for 5 h. The mixture was concentrated in vacuo and the residue was treated with water (50 mL) and extracted into EtOAc (2 x 200 mL). The combined extracts were washed with brine, dried (MgSO i), filtered and concentrated in vacuo to give the desired compound. For analytical purpose, a sample of this material (100 mg) was purified by preparative HPLC to give the desired compound.
  • the reaction mixture was heated to 100°C for 1.5 h, cooled to room temperature, filtered through Celite and washed through with DCM. The filtrate was washed with water, dried (MgSO i), filtered and concentrated in vacuo and the resulting residue was purified by column chromatography using silica gel and eluting with 0-3%> MeOH in DCM. The fractions containing product were combined and concentrated in vacuo to give the desired compound.
  • Step 2 N-(4-Ethyl-6-(thiophen-2-yl)pyridin-3-yl)-l-methyl-lH-imidazo[ 4, 5-c]pyridin-6-amine
  • Step i) N-[ 1 -(4-Nitro-phenyl)-meth-(E)-ylidene] -methanesulfonamide
  • Step ii) N-[2-Cyano-l-(4-nitro-phenyl) -ethyl] -methanesulfonamide
  • Iron powder (3 eq, 124 mg) was added to a stirred suspension of ammonium chloride (3 eq, 119 mg) and the nitrobenzene (1 eq, 200 mg) in a mixture of acetone (5 mL) and water (1 mL) and the resulting mixture was stirred at 65°C for 4 h.
  • the mixture was filtered through Celite and washed with EtOAc (5 x 2 mL).
  • the filtrate was washed with aq. sat. NaHC0 3 (15 mL) and the organic layer was dried and concentrated.
  • the residue was added on a previously conditioned SCX Column (1 g), washed with MeOH (3x5 ml) and then with 7N NH 3 solution in MeOH/MeOH (10 mL) to retrieve the desired compound.
  • Step Hi (2-Difluoromethoxy-4-methyl-phenyl)-(l -methyl- 1 H-imidazo[ 4, 5-cj pyridin-6-yl) -amine
  • Step ii) 2-[ 3-Ethyl-4-(l -methyl- lH-imidazo[ 4, 5-cj pyridin-6-ylamino) -phenyl) '-3-oxo-propionitrile
  • IC1 (1M in DCM, 1.4 eq, 15 mL) was added to a solution of the ester (1 eq, 2.0 g) in DCM (200 mL). After 4 h, aq. sat. Na 2 S203 solution was added and the solution was extracted with DCM (3x). The combined organics were washed with water, brine, dried and concentrated. The residue was purified by silica chromatography (EtOAc/cyclohexane; 3:97 to 30:70) to afford the desired product.
  • TFAA (4.8 eq, 34 mL) was added to a solution of H 2 0 2 (4 eq, 20 niL) in DCM (100 niL) at 0°C. After 5 min, a solution 4-bromo-2-ethyl aniline (1 eq, 7.1 mL) in DCM (200 mL) was added dropwise over 30 min and the reaction was heated to reflux for 2 h. Aq. sat NaHC0 3 solution was added, organic layer separated, dried and concentrated. The residue was purified by silica chromatography (EtOAc/cyclohexane) to afford the intermediate.
  • Step (Hi) [6-(3,3-Difluoro-azetidin-l-yl)-4-ethyl-pyridin-3-yl]-(l-methyl-lH-imidazo[4,5-c]pyridm ⁇ yl)-amine
  • reaction mixture from the first flask was added dropwised via a cannule to the second flask.
  • the resulting reaction mixture was stirred at 85°C for 18 h.
  • the reaction was then quenched with water and extracted with EtOAc (3x).
  • the combined organics was dried and concentrated under reduced pressure.
  • the residue was purified by column chromatography (MeOH/DCM; 0:100 to 10:90) to give the expected aniline.
  • Step (ii) ( 6-Cyclopentyl-4-ethyl-pyridin-3-yl)-(l -methyl- 1 H-imidazo[ 4, 5-cj pyridin-6-yl) -amine
  • Step ( i) 5-Amino-4-ethyl-3 ',4 ',5 ' 6' -tetrahydro-2 'H-f 2, 4 'Jbipyridinyl-1 '-carboxylic acid tert-butyl ester
  • reaction mixture from the first flask was added dropwised via a cannule to the second flask.
  • the resulting reaction mixture was stirred at 85°C for 2 h.
  • the reaction was then quenched with water and extracted with EtOAc (3x).
  • the combined organics was dried and concentrated under reduced pressure.
  • the residue was purified by column chromatography (MeOH/DCM; 0:100 to 10:90) to give the expected aniline.
  • Step ii) ( ( 6-Cyclohexyl-4-ethyl-pyridin-3-yl)-(l -methyl- 1 H-imidazo[ 4, 5-cj pyridin-6-yl) -amine
  • Fe powder (2.86 eq, 317 mg) was added to a solution of 4-bromo-5-nitro-phthalonitrile (1 eq, 500mg) in MeOH (4 mL) and HCl (1.4 mL) at 60°C and the reaction was heated at reflux for 2 h. The reaction mixture was hot filtered through Celite and the filtrate was concentrated. The residue was dissolved in Hot MeOH, precipitate and collected by filtration to afford the desired product.
  • Benzyltrimethylammonium tribromide (1.1 eq, 4.5 g) was added portionwised to a stirred solution of 5-aminopyrimidine (1 eq, 1 g) in DCM (50 mL) and MeOH (10 mL) at 0°C. The mixture was warmed up to room temperature and stirred for 90 min. Aq. sat. NaHCC>3 solution was added (pH ⁇ 8), the organic was separated and the aqueous extracted with EtOAc (3x). The combined organics were dried and concentrated. Purification by silica chromatography (EtOAc/cyclohexane; 0:100 to 50:50) gave the desired compound.

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Abstract

Novel imidazolopyridines according to Formula I, able to inhibit JAK are disclosed, wherein R1 and Cy are as disclosed herein. These compounds may be prepared as a pharmaceutical composition, and may be used for the prevention and treatment of a variety of conditions in mammals including humans, including by way of non-limiting example, allergic or inflammatory conditions, autoimmune diseases, proliferative diseases, transplantation rejection, diseases involving impairment of cartilage turnover, congenital cartilage malformations, and/or diseases associated with hypersecretion of IL6 or interferons.

Description

IMIDAZO [4 , 5 -C] PYRIDINE DERIVATIVES USEFUL FOR THE TREATMENT OF
DEGENERATIVE AND INFLAMMATORY DISEASES
FIELD OF THE INVENTION
[0001] The present invention relates to compounds that are inhibitors of JAK, a family of tyrosine kinases that are involved in allergic or inflammatory conditions, autoimmune diseases, proliferative diseases, transplantation rejection, diseases involving impairment of cartilage turnover, congenital cartilage malformations, and/or diseases associated with hypersecretion of IL6 or interferons. In particular, the compounds of the invention inhibit JAK1 and/or JAK2. The present invention also provides methods for the production of the compounds of the invention, pharmaceutical compositions comprising the compounds of the invention, methods for the prevention and/or treatment of diseases involving allergic or inflammatory conditions, autoimmune diseases, proliferative diseases, transplantation rejection, diseases involving impairment of cartilage turnover, congenital cartilage malformations, and/or diseases associated with hypersecretion of IL6 or interferons by administering a compound of the invention.
[0002] Janus kinases (JAKs) are cytoplasmic tyrosine kinases that transduce cytokine signaling from membrane receptors to STAT transcription factors. Four JAK family members are described, JAK1 , JAK2, JAK3 and TYK2. Upon binding of the cytokine to its receptor, JAK family members auto- and/or transphosphorylate each other, followed by phosphorylation of STATs that then migrate to the nucleus to modulate transcription. JAK-STAT intracellular signal transduction serves the interferons, most interleukins, as well as a variety of cytokines and endocrine factors such as EPO, TPO, GH, OSM, LIF, CNTF, GM-CSF and PRL (Vainchenker W. et al. (2008)).
[0003] The combination of genetic models and small molecule JAK inhibitor research revealed the therapeutic potential of several JAKs. JAK3 is validated by mouse and human genetics as an immune- suppression target (O'Shea J. et al. (2004)). JAK3 inhibitors were successfully taken into clinical development, initially for organ transplant rejection but later also in other immuno-inflammatory indications such as rheumathoid arthritis (RA), psoriasis and Crohn's disease (http://clinicaltrials.gov/).
[0004] TYK2 is a potential target for immuno-inflammatory diseases, being validated by human genetics and mouse knock-out studies (Levy D. and Loomis C. (2007)).
[0005] JAKl is a target in the immuno-inflammatory disease area. JAK1 heterodimerizes with the other JAKs to transduce cytokine- driven pro-inflammatory signaling. Therefore, inhibition of JAKl is of interest for immuno-inflammatory diseases with pathology-associated cytokines that use JAKl signaling, such as IL-6, IL-4, IL-5, IL-12, IL-13, IL-23, or IFNgamma, as well as for other diseases driven by JAK-mediated signal transduction. BACKGROUND OF THE INVENTION
[0006] The degeneration of cartilage is the hallmark of various diseases, among which rheumatoid arthritis and osteoarthritis are the most prominent. Rheumatoid arthritis (RA) is a chronic joint degenerative disease, characterized by inflammation and destruction of the joint structures. When the disease is unchecked, it leads to substantial disability and pain due to loss of joint functionality and even premature death. The aim of an RA therapy, therefore, is not only to slow down the disease but to attain remission in order to stop the joint destruction. Besides the severity of the disease outcome, the high prevalence of RA (~ 0.8% of adults are affected worldwide) means a high socio-economic impact. (For reviews on RA, we refer to Smolen and Steiner (2003); Lee and Weinblatt (2001); Choy and Panayi (2001); O'Dell (2004) and Firestein (2003)).
[0007] JAK1 and JAK2 are implicated in intracellular signal transduction for many cytokines and hormones. Pathologies associated with any of these cytokines and hormones can be ameliorated by JAK1 and JAK2 inhibitors. Hence, several allergy, inflammation and autoimmune disorders might benefit from treatment with compounds described in this invention including rheumatoid arthritis, systemic lupus erythematosis, juvenile idiopathic arthritis, osteoarthritis, asthma, chronic obstructive pulmonary disease COPD, tissue fibrosis, eosinophilic inflammation, eosophagitis, inflammatory bowel diseases (e.g. Crohn's, ulcerative colitis), transplantation, graft-versus-host disease, psoriasis, myositis, multiple sclerosis (Kopf et al., 2010).
[0008] Osteoarthritis (also referred to as OA, or wear-and-tear arthritis) is the most common form of arthritis and is characterized by loss of articular cartilage, often associated with hypertrophy of the bone and pain. For an extensive review on osteoarthritis, we refer to Wieland et al. (2005).
[0009] Osteoarthritis is difficult to treat. At present, no cure is available and treatment focuses on relieving pain and preventing the affected joint from becoming deformed. Common treatments include the use of non-steroidal anti-inflammatory drugs (NSAIDs). Although dietary supplements such as chondroitin and glucosamine sulphate have been advocated as safe and effective options for the treatment of osteoarthritis, a recent clinical trial revealed that both treatments did not reduce pain associated to osteoarthritis. (Clegg et al., 2006).
[0010] Stimulation of the anabolic processes, blocking catabolic processes, or a combination of these two, may result in stabilization of the cartilage, and perhaps even reversion of the damage, and therefore prevent further progression of the disease. Therapeutic methods for the correction of the articular cartilage lesions that appear during the osteoarthritic disease have been developed, but so far none of them have been able to mediate the regeneration of articular cartilage in situ and in vivo. Taken together, no disease modifying osteoarthritic drugs are available.
[0011] Vandeghinste et al. (WO 2005/124342) discovered JAK1 as a target whose inhibition might have therapeutic relevance for several diseases including OA. Knockout of the JAK1 gene in mice demonstrated that JAK1 plays essential and non-redundant roles during development: JAK1-/- mice died within 24h after birth and lymphocyte development was severely impaired. Moreover, JAK1 -/- cells were not, or less, reactive to cytokines that use class II cytokine receptors, cytokine receptors that use the gamma-c subunit for signaling and the family of cytokine receptors that use the gpl30 subunit for signaling (Rodig et al, 1998).
[0012] Various groups have implicated JAK-STAT signaling in chondrocyte biology. Li et al. (2001) showed that Oncostatin M induces MMP and TIMP3 gene expression in primary chondrocytes by activation of JAK/STAT and MAPK signaling pathways. Osaki et al. (2003) showed that interferon- gamma mediated inhibition of collagen II in chondrocytes involves JAK-STAT signaling. ILl-beta induces cartilage catabolism by reducing the expression of matrix components, and by inducing the expression of collagenases and inducible nitric oxide synthase (NOS2), which mediates the production of nitric oxide (NO). Otero et al., (2005) showed that leptin and ILl-beta synergistically induced NO production or expression of NOS2 mRNA in chondrocytes, and that that was blocked by a JAK inhibitor. Legendre et al. (2003) showed that IL6/IL6Receptor induced downregulation of cartilage- specific matrix genes collagen II, aggrecan core and link protein in bovine articular chondrocytes, and that this was mediated by JAK/STAT signaling. Therefore, these observations suggest a role for JAK kinase activity in cartilage homeostasis and therapeutic opportunities for JAK kinase inhibitors.
[0013] JAK family members have been implicated in additional conditions including myeloproliferative disorders (O'Sullivan et al, 2007, Mol Immunol. 44(10):2497-506), where mutations in JAK2 have been identified. This indicates that inhibitors of JAK in particular JAK2 may also be of use in the treatment of myeloproliferative disorders. Additionally, the JAK family, in particular JAKl, JAK2 and JAK3, has been linked to cancers, in particular leukaemias e.g. acute myeloid leukaemia (O'Sullivan et al, 2007, Mol Immunol. 44(10):2497-506; Xiang et al, 2008, "Identification of somatic JAKl mutations in patients with acute myeloid leukemia" Blood First Edition Paper, prepublished online December 26, 2007; DOI 10.1182/blood-2007-05-090308) and acute lymphoblastic leukaemia (Mullighan et al, 2009) or solid tumours e.g. uterine leiomyosarcoma (Constantinescu et al, 2007, Trends in Biochemical Sciences 33(3): 122-131), prostate cancer (Tarn et al, 2007, British Journal of Cancer, 97, 378 - 383). These results indicate that inhibitors of JAK, in particular of JAKl and/or JAK2, may also have utility in the treatment of cancers (leukaemias and solid tumours e.g. uterine leiomyosarcoma, prostate cancer).
[0014] In addition, Castleman's disease, multiple myeloma, mesangial proliferative glomerulonephritis, psoriasis, and Kaposi's sarcoma are likely due to hypersecretion of the cytokine IL- 6, whose biological effects are mediated by intracellular JAK-STAT signaling (Tetsuji Naka, Norihiro Nishimoto and Tadamitsu Kishimoto, Arthritis Res 2002, 4 (suppl 3):S233-S242). This result shows that inhibitors of JAK, may also find utility in the treatment of said diseases.
[0015] The current therapies are not satisfactory and therefore there remains a need to identify further compounds that may be of use in the treatment of allergic or inflammatory conditions, autoimmune diseases, proliferative diseases, transplantation rejection, diseases involving impairment of cartilage turnover, congenital cartilage malformations, and/or diseases associated with hypersecretion of IL6 or interferons. The present invention therefore provides compounds, methods for their manufacture and pharmaceutical compositions comprising the compounds of the invention together with a suitable pharmaceutical carrier. The present invention also provides for the use of a compound of the invention in the preparation of a medicament for the treatment of allergic or inflammatory conditions, autoimmune diseases, proliferative diseases, transplantation rejection, diseases involving impairment of cartilage turnover, congenital cartilage malformations, and/or diseases associated with hypersecretion of IL6 or interferons.
SUMMARY OF THE INVENTION
[0016] The present invention is based on the identification that the compounds of the invention are able to act as inhibitors of JAK and that they are useful for the treatment of allergic or inflammatory conditions, autoimmune diseases, proliferative diseases, transplantation rejection, diseases involving impairment of cartilage turnover, congenital cartilage malformations, and/or diseases associated with hypersecretion of IL6 or interferons. In a specific aspect the compounds of the invention are inhibitors of JAK1 and/or JAK2. The present invention also provides methods for the production of these compounds, pharmaceutical compositions comprising these compounds and methods for treating allergic or inflammatory conditions, autoimmune diseases, proliferative diseases, transplantation rejection, diseases involving impairment of cartilage turnover, congenital cartilage malformations, and/or diseases associated with hypersecretion of IL6 or interferons by administering the compounds of the invention.
[0017] Accordingly, in a first aspect of the invention, compounds of the invention are provided having a Formula (I):
Figure imgf000006_0001
wherein
R is C3.7 cycloalkyl, or C1.4 alkyl optionally substituted with one or more halo;
Cy is
Figure imgf000007_0001
wherein
each of w, x, y, and z, is independantly N, CH or is absent;
the ring A is a fused 5-6-membered saturated or 5-membered unsaturated ring, each of which optionally comprises 1 or 2 heteroatoms independently selected from N, O, and S; to afford a 8- 10 membered bicyclic group;
R2a is
halo,
cyclopropyl,
C alkyl optionally substituted with one or more halo, or
C alkoxy optionally substituted with one or more halo;
each R2b is independently selected from:
- CN,
halo,
- OH,
amino optionally substituted with one or two groups independently selected from CM alkyl, - alkyl, and -S(0)2Ci_4 alkyl,
-C(=0)NH2 optionally substituted with one or two independently selected CM alkyl,
-S(0)2NH2 optionally substituted with one or two independently selected CM alkyl,
- alkoxy,
CM alkyl optionally substituted with one or more groups independently selected from halo, - CN, -C(=0)H,
Figure imgf000007_0002
alkyl, -OH, Ce-io aryl (optionally substituted with one or more groups independently selected from CM alkoxy), -C(=0)-(4-6 membered heterocycloalkyl comprising 1 to 3 heterotaoms independently selected from N, S, or O) (which heterocycloalkyl is optionally substituted with one or more oxo, or CM alkyl) and amino (optionally substituted with one or more CM alkyl),
C2-4 alkenyl comprising 1 double bond (optionally substituted with one CN group),
C2-6 alkoxy,
Ci_6 alkoxy substituted with one or more groups independently selected from OH, CN, halo, C alkoxy, Ce-io aryl (optionally substituted with one or more groups independently selected from CM alkoxy), C3.7 cycloalkyl, alkoxy, -C(=0)-CM alkyl, and 5-10 membered heteroaryl (optionally substituted with one or more CM alkyl),
- -O-Ce-io aryl,
4-10 membered heterocycloalkyl (optionally substituted with oxo, halo, CM alkyl (optionally substituted with one or more -CN), -C(=0)0-CM alkyl, or -SO2-C1.4 alkyl),
phenyl optionally substituted with one or more groups independently selected from CN, halo, OH, Ci_4 alkyl, CM alkoxy, and NR3aR¾
4- 7 membered heterocycloalkenyl comprising 1 double bond, and comprising 1 or 2 heteroatoms independently selected from N, O, and S, optionally substituted with one or more groups independently selected from:
o CM alkyl,
o CM alkoxy,
o C3.7 cycloalkyl,
o alkyl optionally substituted with one or more independently selected CN,
OH, or halo,
o
Figure imgf000008_0001
alkyl, and
o -C(=0)NR4aR4b,
5- 10 membered heteroaryl optionally substituted with one or more independently groups selected from CN, halo, OH, CM alkyl, CM alkoxy, and NR3aR¾
C3.7 cycloalkyl (optionally substituted with one or more -CN),
C thioalkyl,
alkyl (optionally substituted with CN),
-C(=0)-(4-6 membered heterocycloalkyl), which heterocycloalkyl is optionally substituted with one or more CM alkyl (optionally substituted with one or more CN));
each R2c is independently selected from oxo, CM alkyl and halo;
R2d is
- H,
halo,
C alkyl optionally substituted with one or more independently selected halo, or
CM alkoxy optionally substituted with one or more independently selected halo;
each R3a, R3b, R4a, and R4b is independently selected from H, and CM alkyl;
the subscript m is 0, 1, 2, 3, or 4; and
the subscript p is 0, 1, or 2;
provided that
when the subscript m is 0, R2a is not CI, Me or OMe
no more than two of w, x, y, or z are simultaneously N; and
only one of w, x, y, or z may be absent. [0018] In a particular embodiment the compounds of the invention are inhibitors of JAK1 and/or JAK2.
[0019] In a further aspect, the present invention provides pharmaceutical compositions comprising the compounds of the invention, and a pharmaceutical carrier, excipient or diluent. Moreover, the compounds of the invention, useful in the pharmaceutical compositions and treatment methods disclosed herein, are pharmaceutically acceptable as prepared and used. In this aspect of the invention, the pharmaceutical composition may additionally comprise further active ingredients suitable for use in combination with the compounds of the invention.
[0020] In a further aspect, the invention provides a compound of the invention or a pharmaceutical composition comprising a compound of the invention for use as a medicament. In a specific embodiment, said pharmaceutical composition additionally comprises a further active ingredient.
[0021] In a further aspect of the invention, this invention provides a method of treating a mammal susceptible to or afflicted with a condition from among those listed herein, and particularly, such condition as may be associated with aberrant JAK activity, e.g. allergic or inflammatory conditions, autoimmune diseases, proliferative diseases, transplantation rejection, diseases involving impairment of cartilage turnover, congenital cartilage malformations, and/or diseases associated with hypersecretion of IL6 or interferons, which method comprises administering an effective amount of the pharmaceutical composition or compound of the invention as described herein. In a specific embodiment the condition is associated with aberrant JAK1 and/or JAK2 activity.
[0022] In a further aspect, the present invention provides the compounds of the invention for use in the treatment or prophylaxis of a condition selected from those listed herein, particularly such conditions as may be associated with aberrant JAK activity, e.g. allergic or inflammatory conditions, autoimmune diseases, proliferative diseases, transplantation rejection, diseases involving impairment of cartilage turnover, congenital cartilage malformations, and/or diseases associated with hypersecretion of IL6 or interferons.
[0023] In yet another method of treatment aspect, this invention provides a method for treating a mammal susceptible to or afflicted with a condition that is causally related to abnormal JAK activity as described herein, and comprises administering an effective condition-treating or condition-preventing amount of the pharmaceutical composition or a compound of the invention described herein. In a specific aspect the condition is causally related to abnormal JAKl and/or JAK2 activity.
[0024] In a further aspect, the present invention provides a compound of the invention, or a pharmaceutical composition comprising a compound of the invention, for use as a medicament.
[0025] In a further aspect, the present invention provides the compounds of the invention for use in the treatment or prophylaxis of a condition that is causally related to abnormal JAK activity.
[0026] In additional aspects, this invention provides methods for synthesizing the compounds of the invention, with representative synthetic protocols and pathways disclosed later on herein. [0027] Accordingly, it is a principal object of this invention to provide novel compounds, which can modify the activity of JAK and thus prevent or treat any conditions that may be causally related thereto. In a specific aspect the compounds of the invention modulate the activity of JAKl and/or JAK2. It is a further object of this invention to provide compounds that can treat or alleviate conditions or symptoms of same, such as allergic or inflammatory conditions, autoimmune diseases, proliferative diseases, transplantation rejection, diseases involving impairment of cartilage turnover, congenital cartilage malformations, and/or diseases associated with hypersecretion of IL6 or interferons, that may be causally related to the activity of JAK, in particular JAKl and/or JAK2.
[0028] A still further object of this invention is to provide a pharmaceutical composition that may be used in the treatment or prophylaxis of a variety of conditions, including the diseases associated with JAK activity such as allergic or inflammatory conditions, autoimmune diseases, proliferative diseases, transplantation rejection, diseases involving impairment of cartilage turnover, congenital cartilage malformations, and/or diseases associated with hypersecretion of IL6 or interferons. In a specific embodiment the disease is associated with JAKl and/or JAK2 activity.
[0029] Other objects and advantages will become apparent to those skilled in the art from a consideration of the ensuing detailed description.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0030] The following terms are intended to have the meanings presented therewith below and are useful in understanding the description and intended scope of the present invention.
[0031] When describing the invention, which may include compounds, pharmaceutical compositions containing such compounds and methods of using such compounds and compositions, the following terms, if present, have the following meanings unless otherwise indicated. It should also be understood that when described herein any of the moieties defined forth below may be substituted with a variety of substituents, and that the respective definitions are intended to include such substituted moieties within their scope as set out below. Unless otherwise stated, the term "substituted" is to be defined as set out below. It should be further understood that the terms "groups" and "radicals" can be considered interchangeable when used herein.
[0032] The articles "a" and "an" may be used herein to refer to one or to more than one (i.e. at least one) of the grammatical objects of the article. By way of example "an analogue" means one analogue or more than one analogue.
[0033] As used herein the term 'JAK' relates to the family of Janus kinases (JAKs) which are cytoplasmic tyrosine kinases that transduce cytokine signaling from membrane receptors to STAT transcription factors. Four JAK family members are described, JAKl, JAK2, JAK3 and TYK2 and the term JAK may refer to all the JAK family members collectively or one or more of the JAK family members as the context indicates.
[0034] 'Alkoxy' refers to the group -OR26 where R26 is alkyl with the number of carbon atoms specified. Particular alkoxy groups are methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, and 1 ,2-dimethylbutoxy. Particular alkoxy groups are lower alkoxy, i.e. with between 1 and 6 carbon atoms. Further particular alkoxy groups have between 1 and 4 carbon atoms.
[0035] 'Alkylene' refers to divalent alkene radical groups having the number of carbon atoms specified, in particular having 1 to 6 carbon atoms and more particularly 1 to 4 carbon atoms which can be straight-chained or branched. This term is exemplified by groups such as methylene (-CH2-), ethylene (-CH2-CH2-), or -CH(CH3)- and the like.
[0036] 'Alkyl' means straight or branched aliphatic hydrocarbon with the number of carbon atoms specified. Particular alkyl groups have 1 to 8 carbon atoms. More particular is lower alkyl which has 1 to 6 carbon atoms. A further particular group has 1 to 4 carbon atoms. Exemplary straight chained 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 is attached to a linear alkyl chain, exemplary branched chain groups include isopropyl, iso-butyl, t-butyl and isoamyl.
[0037] 'Alkenyl' refers to monovalent olefinically (unsaturated) hydrocarbon groups with the number of carbon atoms specified. Particular alkenyl has 2 to 8 carbon atoms, and more particularly, from 2 to 6 carbon atoms, which can be straight-chained or branched and having at least 1 and particularly from 1 to 2 sites of olefinic unsaturation. Particular alkenyl groups include ethenyl (- CH=CH2), n-propenyl (-CH2CH=CH2), isopropenyl (-C(CH3)=CH2) and the like.
[0038] 'Amino' refers to the radical -NH2.
[0039] '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. In particular aryl refers to an aromatic ring structure, monocyclic or polyyclic, with the number of ring atoms specified. Specifically, the term includes groups that include from 6 to 10 ring members. Where the aryl group is a monocyclic ring system it preferentially contains 6 carbon atoms. Particularly aryl groups include phenyl, naphthyl, indenyl, and tetrahydronaphthyl.
[0040] 'Cycloalkyl'refers to a non-aromatic hydrocarbyl ring structure, monocyclic or polycyclic, with the number of ring atoms specified. A cycloalkyl may have from 3 to 10 carbon atoms, and in particular from 3 to 7 carbon atoms. Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.
[0041] 'Cyano' refers to the radical -CN.
[0042] 'Halo' or 'halogen' refers to fluoro (F), chloro (CI), bromo (Br) and iodo (I). Particular halo groups are either fluoro or chloro. [0043] 'Hetero' when used to describe a compound or a 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 may be applied to any of the hydrocarbyl groups described above such as alkyl, e.g. heteroalkyl, cycloalkyl, e.g. heterocycloalkyl, aryl, e.g. heteroaryl, and the like having from 1 to 4, and particularly from 1 to 3 heteroatoms, more typically 1 or 2 heteroatoms, for example a single heteroatom.
[0044] 'Heteroaryl' means an aromatic ring structure, monocyclic or polycyclic, that includes 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 from 5 to 10 ring members. The heteroaryl group can be, for example, a five membered or six membered monocyclic ring or a bicyclic structure formed from fused five and six membered rings or two fused six membered rings or, by way of a further example, two fused five membered rings. Each ring may contain up to four heteroatoms typically selected from nitrogen, sulphur and oxygen. Typically the heteroaryl ring will contain up to 4 heteroatoms, more typically up to 3 heteroatoms, more usually up to 2, for example a single heteroatom. In one embodiment, the heteroaryl ring contains at least one ring nitrogen atom. The nitrogen atoms in the heteroaryl rings can 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. In general the number of basic nitrogen atoms present in the heteroaryl group, including any amino group substituents of the ring, will be less than five. Examples of five membered monocyclic heteroaryl groups include but are not limited to pyrrole, furan, thiophene, imidazole, furazan, oxazole, oxadiazole, oxatriazole, isoxazole, thiazole, isothiazole, pyrazole, triazole and tetrazole groups. Examples of six membered monocyclic heteroaryl groups include but are not limited to pyridine, pyrazine, pyridazine, pyrimidine and triazine. Particular examples of bicyclic heteroaryl groups containing a five membered ring fused to another five membered ring include but are not limited to imidazothiazole and imidazoimidazole. Particular examples of bicyclic heteroaryl groups containing a six membered ring fused to a five membered ring include but are not limited to benzfuran, benzthiophene, benzimidazole, benzoxazole, isobenzoxazole, benzisoxazole, benzthiazole, benzisothiazole, isobenzofuran, indole, isoindole, isoindolone, indolizine, indoline, isoindoline, purine (e.g., adenine, guanine), indazole, pyrazolopyrimidine, triazolopyrimidine, benzodioxole and pyrazolopyridine groups. Particular examples of bicyclic heteroaryl groups containing two fused six membered rings include but are not limited to quinoline, isoquinoline, chroman, thiochroman, chromene, isochromene, chroman, isochroman, benzodioxan, quinolizine, benzoxazine, benzodiazine, pyridopyridine, quinoxaline, quinazoline, cinnoline, phthalazine, naphthyridine and pteridine groups. Particular heteroaryl groups are those derived from thiophene, pyrrole, benzothiophene, benzofuran, indole, pyridine, quinoline, imidazole, oxazole and pyrazine.
Examples of representative heteroaryls include the following:
Figure imgf000013_0001
wherein each Y is selected from >C=0, NH, O and S.
[0045] As used herein, the term 'heterocycloalkyl' means a stable non-aromatic ring structure, mono-cyclic or polycyclic, that includes one or more heteroatoms independently selected from O, N and S and the number of ring atoms specified. The non-aromatic ring structure may have from 4 to 10 ring members, and in particular from 4 to 7 ring members. A fused heterocyclic ring system may include carbocyclic rings and need only to include one heterocyclic ring. Examples of heterocyclic rings include, but are not limited to, morpholine, piperidine (e.g. 1 -piperidinyl, 2-piperidinyl, 3-piperidinyl and 4-piperidinyl), pyrrolidine (e.g. 1 -pyrrolidinyl, 2-pyrrolidinyl and 3-pyrrolidinyl), pyrrolidone, pyran, , tetrahydrofuran, tetrahydrothiophene, dioxane, tetrahydropyran (e.g. 4-tetrahydro pyranyl), imidazoline, imidazolidinone, oxazoline, thiazoline, 2-pyrazoline, pyrazolidine, piperazine, and N-alkyl piperazines such as N-methyl piperazine. Further examples include thiomorpholine and its S-oxide and S,S-dioxide (particularly thiomorpholine). Still further examples include azetidine, piperidone, piperazone, and N-alkyl piperidines such as N-methyl piperidine. Particular examples of heterocycloa
Figure imgf000013_0002
wherein each W is selected from CH2, NH, O and S; and each Y is selected from NH, O, CO, S02, and S. [0046] As used herein, the term 'heterocycloalkenyl' means a 'heterocycloalkyl, wherein one bond of the ring is reduced, thus the ring comprises a double bond. Particular examples of heterocycloalkenyl groups are shown in the following illustrative examples:
Figure imgf000014_0001
wherein each W is selected from CH2, NH, O and S; and each Y is selected from NH, O, CO, SO2, and S.
[0047] 'Hydroxyl' refers to the radical -OH.
[0048] 'Substituted' refers to a group in which one or more hydrogen atoms are each independently replaced with the same or different substituent(s).
[0049] 'Sulfo' or 'sulfonic acid' refers to a radical such as -SO3H.
[0050] 'Thiol' refers to the group -SH.
[0051] As used herein, term 'substituted with one or more' refers to one to four substituents. In one embodiment it refers to one to three substituents. In further embodiments it refers to one or two substituents. In a yet further embodiment it refers to one substituent.
[0052] 'Thioalkoxy' refers to the group -SR26 where R26 has the number of carbon atoms specified and particularly Ci_g alkyl. Particular thioalkoxy groups are thiomethoxy, thioethoxy, n-thiopropoxy, isothiopropoxy, n-thiobutoxy, tert-thiobutoxy, sec-thiobutoxy, n-thiopentoxy, n-thiohexoxy, and 1,2- dimethylthiobutoxy. Particular thioalkoxy groups are lower thioalkoxy, i.e. with between 1 and 6 carbon atoms. Further particular alkoxy groups have between 1 and 4 carbon atoms.
[0053] One having ordinary skill in the art of organic synthesis will recognize that the maximum number of heteroatoms in a stable, chemically feasible heterocyclic ring, whether it is aromatic or non aromatic, is determined by the size of the ring, the degree of unsaturation and the valence of the heteroatoms. In general, a heterocyclic ring may have one to four heteroatoms so long as the heteroaromatic ring is chemically feasible and stable.
[0054] 'Pharmaceutically acceptable' means approved or approvable by a regulatory agency of the Federal or a state government or the corresponding agency in countries other than the United States, or that is listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly, in humans. [0055] 'Pharmaceutically acceptable salt' refers to a salt of a compound of the invention that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. In particular, such salts are non-toxic 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 formed with organic acids 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-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-l-carboxylic acid, glucoheptonic acid, 3- phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g. an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine and the like. Salts further include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the compound contains a basic functionality, salts of non toxic organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like. 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.
[0056] 'Pharmaceutically acceptable vehicle' refers to a diluent, adjuvant, excipient or carrier with which a compound of the invention is administered.
[0057] 'Prodrugs' refers to compounds, including derivatives of the compounds of the invention, which have cleavable groups and become by solvolysis or under physiological conditions the compounds of the invention which are pharmaceutically active in vivo. Such examples include, but are not limited to, choline ester derivatives and the like, N-alkylmorpholine esters and the like.
[0058] 'Solvate' refers to forms of the compound that are associated with a solvent, usually by a solvolysis reaction. This physical association includes hydrogen bonding. Conventional solvents include water, ethanol, acetic acid and the like. The compounds of the invention may be prepared e.g. in crystalline form and may be solvated or hydrated. Suitable solvates include pharmaceutically acceptable solvates, such as hydrates, and further include both stoichiometric solvates and non-stoichiometric solvates. In certain instances the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. 'Solvate' encompasses both solution-phase and isolable solvates. Representative solvates include hydrates, ethanolates and methanolates. [0059] 'Subject' includes humans. The terms 'human', 'patient' and 'subject' are used interchangeably herein.
[0060] 'Therapeutically effective amount' means the amount of a compound that, when administered to a subject for treating a disease, is sufficient to effect such treatment for the disease. The 'therapeutically effective amount' can vary depending on the compound, the disease and its severity, and the age, weight, etc., of the subject to be treated.
[0061] 'Preventing' or 'prevention' refers to a reduction in risk of acquiring or developing a disease or disorder (i.e. causing at least one of the clinical symptoms of the disease not to develop in a subject that may be exposed to a disease-causing agent, or predisposed to the disease in advance of disease onset.
[0062] The term 'prophylaxis' is related to 'prevention', and refers to a measure or procedure the purpose of which is to prevent, rather than to treat or cure a disease. Non-limiting examples of prophylactic measures may include the administration of vaccines; the administration of low molecular weight heparin to hospital patients at risk for thrombosis due, for example, to immobilization; and the administration of an anti-malarial agent such as chloroquine, in advance of a visit to a geographical region where malaria is endemic or the risk of contracting malaria is high.
[0063] 'Treating' or 'treatment' of any disease or disorder refers, in one embodiment, to ameliorating the disease or disorder (i.e. arresting the disease or reducing the manifestation, extent or severity of at least one of the clinical symptoms thereof). In another embodiment 'treating' or 'treatment' refers to ameliorating at least one physical parameter, which may not be discernible by the subject. In yet another embodiment, 'treating' or 'treatment' refers to modulating the disease or disorder, either physically, (e.g. stabilization of a discernible symptom), physiologically, (e.g. stabilization of a physical parameter), or both. In a further embodiment, "treating" or "treatment" relates to slowing the progression of the disease.
[0064] As used herein the term 'allergy' refers to the group of conditions characterized by a hypersensitivity disorder of the immune system including, allergic airway disease (e.g. asthma, rhinitis), sinusitis, eczema and hives, as well as food allergies or allergies to insect venom.
[0065] As used herein the term 'inflammatory condition(s)' refers to the group of conditions including, rheumatoid arthritis, osteoarthritis, juvenile idiopathic arthritis, psoriasis, psoriatic arthritis, allergic airway disease (e.g. asthma, rhinitis), inflammatory bowel diseases (e.g. Crohn's disease, ulcerative colitis), endotoxin-driven disease states (e.g. complications after bypass surgery or chronic endotoxin states contributing to e.g. chronic cardiac failure), and related diseases involving cartilage, such as that of the joints. Partcicularly the term refers to rheumatoid arthritis, osteoarthritis, allergic airway disease (e.g. asthma) and inflammatory bowel diseases.
[0066] As used herein the term 'autoimmune disease(s)' refers to the group of diseases including obstructive airways disease, including conditions such as COPD, asthma (e.g intrinsic asthma, extrinsic asthma, dust asthma, infantily asthma) particularly chronic or inveterate asthma (for example late asthma and airway hyperreponsiveness), bronchitis, including bronchial asthma, systemic lupus erythematosus (SLE), cutaneous lupus erythrematosis, lupus nephritis, dermatomyositis, Sjogren's syndrome, multiple sclerosis, psoriasis, dry eye disease, type I diabetes mellitus and complications associated therewith, atopic eczema (atopic dermatitis), contact dermatitis and further eczematous dermatitis, inflammatory bowel disease (e.g. Crohn's disease and ulcerative colitis), atherosclerosis and amyotrophic lateral sclerosis. Particularly the term refers to COPD, asthma, systemic lupus erythematosis, type I diabetes mellitus and inflammatory bowel disease.
[0067] As used herein the term 'proliferative disease(s)' refers to conditions such as cancer (e.g. uterine leiomyosarcoma or prostate cancer), myeloproliferative disorders (e.g. polycythemia vera, essential thrombocytosis and myelofibrosis), leukemia (e.g. acute myeloid leukaemia, acute and chronic lymphoblastic leukemia), multiple myeloma, psoriasis, restenosis, scleroderma or fibrosis. In particular the term refers to cancer, leukemia, multiple myeloma and psoriasis.
[0068] As used herein, the term 'cancer' refers to a malignant or benign growth of cells in skin or in body organs, for example but without limitation, breast, prostate, lung, kidney, pancreas, stomach or bowel. A cancer tends to infiltrate into adjacent tissue and spread (metastasise) to distant organs, for example to bone, liver, lung or the brain. As used herein the term cancer includes both metastatic rumour cell types, such as but not limited to, melanoma, lymphoma, leukaemia, fibrosarcoma, rhabdomyosarcoma, and mastocytoma and types of tissue carcinoma, such as but not limited to, colorectal cancer, prostate cancer, small cell lung cancer and non-small cell lung cancer, breast cancer, pancreatic cancer, bladder cancer, renal cancer, gastric cancer, glioblastoma, primary liver cancer, ovarian cancer, prostate cancer and uterine leiomyosarcoma.
[0069] As used herein the term 'leukemia' refers to neoplastic diseases of the blood and blood forming organs. Such diseases can cause bone marrow and immune system dysfunction, which renders the host highly susceptible to infection and bleeding. In particular the term leukemia refers to acute myeloid leukaemia (AML), and acute lymphoblastic leukemia (ALL) and chronic lymphoblastic leukaemia (CLL).
[0070] As used herein the term 'transplantation rejection' refers to the acute or chronic rejection of cells, tissue or solid organ alio- or xenografts of e.g. pancreatic islets, stem cells, bone marrow, skin, muscle, corneal tissue, neuronal tissue, heart, lung, combined heart-lung, kidney, liver, bowel, pancreas, trachea or oesophagus, or graft-versus-host diseases.
[0071] As used herein the term 'diseases involving impairment of cartilage turnover' includes conditions such as osteoarthritis, psoriatic arthritis, juvenile rheumatoid arthritis, gouty arthritis, septic or infectious arthritis, reactive arthritis, reflex sympathetic dystrophy, algodystrophy, Tietze syndrome or costal chondritis, fibromyalgia, osteochondritis, neurogenic or neuropathic arthritis, arthropathy, endemic forms of arthritis like osteoarthritis deformans endemica, Mseleni disease and Handigodu disease; degeneration resulting from fibromyalgia, systemic lupus erythematosus, scleroderma and ankylosing spondylitis.
[0072] As used herein the term 'congenital cartilage malformation(s)' includes conditions such as hereditary chondrolysis, chondrodysplasias and pseudochondrodysplasias, in particular, but without limitation, microtia, anotia, metaphyseal chondrodysplasia, and related disorders.
[0073] 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 mesangial proliferative glomerulonephritis.
[0074] As used herein the term 'disease(s) associated with hypersecretion of interferons includes conditions such as systemic and cutaneous lupus erythematosis, lupus nephritis, dermatomyositis, Sjogren's syndrome, psoriasis, and/or rheumatoid arthritis.
[0075] 'Compound(s) of the invention', and equivalent expressions, are meant to embrace compounds of the Formula(e) as herein described, which expression includes the pharmaceutically acceptable salts, and the solvates, e.g. hydrates, and the solvates of the pharmaceutically acceptable salts where the context so permits. Similarly, reference to intermediates, whether or not they themselves are claimed, is meant to embrace their salts, and solvates, where the context so permits.
[0076] When ranges are referred to herein, for example but without limitation, Ci_g alkyl, the citation of a range should be considered a representation of each member of said range.
[0077] Other derivatives of the compounds of this invention have activity in both their acid and acid derivative forms, but in the acid sensitive form often offers advantages of solubility, tissue compatibility, or delayed release in the mammalian organism (see, Bundgard, H., Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam 1985). Prodrugs include acid derivatives well know to practitioners of the art, such as, for example, esters prepared by reaction of the parent acid with a suitable alcohol, or amides prepared by 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 on the compounds of this invention are particularly useful prodrugs. In some cases it is desirable to prepare double ester type prodrugs such as (acyloxy)alkyl esters or ((alkoxycarbonyl)oxy)alkylesters. Particular such prodrugs are the Ci_g alkyl, C2-8 alkenyl, C6-10 optionally substituted aryl, and (C6-10 aryl)-(Ci_4 alkyl) esters of the compounds of the invention.
[0078] As used herein, the term 'isotopic variant' refers to a compound that contains unnatural proportions of isotopes at one or more of the atoms that constitute such compound. For example, an 'isotopic variant' of a compound can contain one or more non-radioactive isotopes, such as for example, deuterium (2H or D), carbon- 13 (13C), nitrogen- 15 (15N), or the like. It will be understood that, in a compound where such isotopic substitution is made, the following atoms, where present, may vary, so that for example, any hydrogen may be 2H/D, any carbon may be 13C, or any nitrogen may be 15N, and that the presence and placement of such atoms may be determined within the skill of the art. Likewise, the invention may include the preparation of isotopic variants with radioisotopes, in the instance for example, where the resulting compounds may be used for drug and/or substrate tissue distribution studies. The radioactive isotopes tritium, i.e. 3H, and carbon- 14, i.e. 14C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection. Further, compounds may be prepared that are substituted with positron emitting isotopes, such as UC, 18F, 150 and 13N, and would be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy.
[0079] All isotopic variants of the compounds provided herein, radioactive or not, are intended to be encompassed within the scope of the invention.
[0080] It is also to be understood that compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space are termed 'isomers'. Isomers that differ in the arrangement of their atoms in space are termed 'stereoisomers'.
[0081] Stereoisomers that are not mirror images of one another are termed 'diastereomers' and those that are non-superimposable mirror images of each other are termed 'enantiomers'. When a compound has an asymmetric center, for example, it is bonded to four different groups, a pair of enantiomers is possible. An enantiomer can be characterized by the absolute configuration of its asymmetric center and is described by the R- and S-sequencing rules of Cahn and Prelog, or by the manner in which the molecule rotates the plane of polarized light and designated as dextrorotatory or levorotatory (i.e. as (+) or (-)-isomers respectively). A chiral compound can exist as either individual enantiomer or as a mixture thereof. A mixture containing equal proportions of the enantiomers is called a 'racemic mixture'.
[0082] 'Tautomers' refer to compounds that are interchangeable forms of a particular compound structure, and that vary in the displacement of hydrogen atoms and electrons. Thus, two structures may be in equilibrium through the movement of π electrons and an atom (usually H). For example, enols and ketones are tautomers because they are rapidly interconverted by treatment with either acid or base. Another example of tautomerism is the aci- and nitro- forms of phenylnitromethane, that are likewise formed by treatment with acid or base.
[0083] Tautomeric forms may be relevant to the attainment of the optimal chemical reactivity and biological activity of a compound of interest.
[0084] The compounds of the invention may possess one or more asymmetric centers; such compounds can therefore be produced as individual (R)- or (S)- stereoisomers or as mixtures thereof.
[0085] Unless indicated otherwise, the description or naming of a particular compound in the specification and claims is intended to include both individual enantiomers and mixtures, racemic or otherwise, thereof. The methods for the determination of stereochemistry and the separation of stereoisomers are well-known in the art.
[0086] It will be appreciated that compounds of the invention may be metabolized to yield biologically active metabolites. THE COMPOUNDS
[0087] The present invention is based on the identification that the compounds of the invention are inhibitors of JAK and that they are useful for the treatment of allergic or inflammatory conditions, autoimmune diseases, proliferative diseases, transplantation rejection, diseases involving impairment of cartilage turnover, congenital cartilage malformations, and/or diseases associated with hypersecretion of IL6 or interferons. The present invention also provides methods for the production of the compounds of the invention, pharmaceutical compositions comprising a compound of the invention and methods for treating allergic or inflammatory conditions, autoimmune diseases, proliferative diseases, transplantation rejection, diseases involving impairment of cartilage turnover, congenital cartilage malformations, and/or diseases associated with hypersecretion of IL6 or interferons by administering a compound of the invention. In a specific embodiment the compounds of the invention are inhibitors of JAKl and JAK2.
[0088] Accordingly, in a first aspect of the invention, compounds of the invention are provided having a Formula (I):
Figure imgf000020_0001
wherein
R1 is C3.7 cycloalkyl, or C1.4 alkyl optionally substituted with one or more halo;
Cy is
Figure imgf000020_0002
OU
Cyi Cy2 Cy3
wherein
each of w, x, y, and z, is independantly N, CH or is absent;
the ring A is a fused 5-6-membered saturated or 5-membered unsaturated ring, each of which optionally comprises 1 or 2 heteroatoms independently selected from N, O, and S; to afford a 8-10 membered bicyclic group;
R2a is
halo,
cyclopropyl,
C alkyl optionally substituted with one or more halo, or
C alkoxy optionally substituted with one or more halo; each R is independently selected from:
- CN,
halo,
- OH,
amino optionally substituted with one or two groups independently selected from CM alkyl, - alkyl, and -S(0)2Ci_4 alkyl,
-C(=0)NH2 optionally substituted with one or two independently selected CM alkyl,
-S(0)2NH2 optionally substituted with one or two independently selected CM alkyl,
- alkoxy,
CM alkyl optionally substituted with one or more groups independently selected from halo, - CN, -C(=0)H,
Figure imgf000021_0001
alkyl, -OH, C6-10 aryl (optionally substituted with one or more groups independently selected from C1.4 alkoxy), -C(=0)-(4-6 membered heterocycloalkyl) (which heterocycloalkyl is optionally substituted with one or more CM alkyl) and amino (optionally substituted with one or more CM alkyl),
C2-4 alkenyl comprising 1 double bond (optionally substituted with one CN group),
C2-6 alkoxy,
Ci_6 alkoxy substituted with one or more groups independently selected from OH, CN, halo, CM alkoxy, C6-10 aryl (optionally substituted with one or more groups independently selected from C alkoxy), C3.7 cycloalkyl,
Figure imgf000021_0002
alkoxy, -C(=0)-CM alkyl, and 5-10 membered heteroaryl (optionally substituted with one or more CM alkyl),
- -O-Ce-io aryl,
4-10 membered heterocycloalkyl (optionally substituted with oxo, halo, CM alkyl (optionally substituted with one or more -CN), -C(=0)0-CM alkyl, or -SO2-C1.4 alkyl),
phenyl optionally substituted with one or more groups independently selected from CN, halo, OH, CM alkyl, CM alkoxy, and NR3aR¾
4- 7 membered heterocycloalkenyl comprising 1 double bond, and comprising 1 or 2 heteroatoms independently selected from N, O, and S, optionally substituted with one or more groups independently selected from:
o CM alkyl,
o CM alkoxy,
o C3.7 cycloalkyl,
o alkyl optionally substituted with one or more independently selected CN,
OH, or halo,
o
Figure imgf000021_0003
alkyl, and
o -C(=0)NR4aR4b,
5- 10 membered heteroaryl optionally substituted with one or more groups independently selected from CN, halo, OH, CM alkyl, CM alkoxy, and NR3aR¾ C3.7 cycloalkyl (optioinally substituted with one or more -CN),
C thioalkyl,
alkyl (optionally substituted with CN), and
-C(=0)-(4-6 membered heterocycloalkyl), which heterocycloalkyl is optionally substituted with one or more CM alkyl (optionally substituted with one or more CN));
each R2c is independently selected from oxo, CM alkyl and halo;
R2d is
- H,
halo,
CM alkyl optionally substituted with one or more independently selected halo, or
CM alkoxy optionally substituted with one or more independently selected halo;
each R3a, R3b, R4a, and R4b is independently selected from H, and CM alkyl;
the subscript m is 0, 1, 2, 3, or 4; and
the subscript p is 0, 1, or 2;
provided that
when the subscript m is 0, R2a is not CI, Me or OMe
no more than two of w, x, y, or z are simultaneously N; and
only one of w, x, y, or z may be absent.
[0089] Accordingly, in a second aspect of the invention, the compounds of the invention are provided having a Formula (I):
Figure imgf000022_0001
wherein
R is C3.7 cycloalkyl, or C alkyl optionally substituted with one or more halo;
Cy is
Figure imgf000022_0002
Cyi Cy2 Cy3
wherein
each of w, x, y, and z, is independantly N, CH or is absent; the ring A is a fused 5-6-membered saturated or 5-membered unsaturated ring, each of which optionally comprises 1 or 2 heteroatoms independently selected from N, O, and S; to afford a 8-10 membered bicyclic group;
R2a is
halo,
cyclopropyl,
C alkyl optionally substituted with one or more halo, or
C alkoxy optionally substituted with one or more halo;
each R2b is independently selected from:
- CN,
halo,
- OH,
amino optionally substituted with one or two groups independently selected from CM alkyl, - alkyl, and -S(0)2Ci_4 alkyl,
-C(=0)NH2 optionally substituted with one or two independently selected CM alkyl,
-S(0)2NH2 optionally substituted with one or two independently selected CM alkyl,
- alkoxy,
CM alkyl optionally substituted with one or more groups independently selected from halo, - CN, -OH, C6-io aryl (optionally substituted with one or more groups independently selected from CM alkoxy), and amino (optionally substituted with one or more C alkyl),
C2-6 alkoxy
Ci_6 alkoxy substituted with one or more groups independently selected from OH, CN, halo, CM alkoxy, C6-10 aryl (optionally substituted with one or more groups independently selected from CM alkoxy), C3.7 cycloalkyl,
Figure imgf000023_0001
alkoxy, -C(=0)-CM alkyl, and 5-10 membered heteroaryl (optionally substituted with one or more CM alkyl),
- -O-Ce-io aryl,
4-10 membered heterocycloalkyl (optionally substituted with oxo),
phenyl optionally substituted with one or more groups independently selected from CN, halo, OH, CM alkyl, CM alkoxy, and NR3aR¾
4-7 membered heterocycloalkenyl comprising 1 double bond, and comprising 1 or 2 heteroatoms independently selected from N, O, and S, optionally substituted with one or more groups independently selected from:
o CM alkyl,
o CM alkoxy,
o C3.7 cycloalkyl,
o alkyl optionally substituted with one or more independently selected CN, OH, and halo, o
Figure imgf000024_0001
alkyl, and
o -C(=0)NR4aR4b,
5-10 membered heteroaryl optionally substituted with one or more groups independently selected from CN, halo, OH, CM alkyl, CM alkoxy, and NR3aR¾
C3.7 cycloalkyl,
C1.4 thioalkyl, and
alkyl (optionally substituted with CN);
each R2c is independently selected from oxo, CM alkyl and halo;
R2d is
- H,
halo,
CM alkyl optionally substituted with one or more independently selected halo, or
CM alkoxy optionally substituted with one or more independently selected halo;
each R3a, R3b, R4a, and R4b is independently selected from H, and CM alkyl;
the subscript m is 0, 1, 2, 3, or 4; and
the subscript p is 0, 1, or 2;
provided that
when the subscript m is 0, R2a is not CI, Me or OMe
no more than two of w, x, y, or z are simultaneously N; and
only one of w, x, y, or z may be absent.
[0090] In one embodiment, the compound of the invention is according to Formula I, wherein R1 is C alkyl substituted with one or more independently selected halo. In a particular embodiment, R1 is CM alkyl substituted with one or more independently selected F, or CI. In a more particular embodiment, R1 is Me, Et, n-Pr, or n-Bu, each of which is substituted with one or more independently selected F or CI. In a most particular embodiment, R1 is -CF3.
[0091] In one embodiment, the compound of the invention is according to Formula I, wherein R1 is C alkyl. In a particular embodiment, R1 is Me, Et, n-Pr, z'-Pr, or ί-Bu. In a more particular embodiment, R1 is Me.
[0092] In one embodiment, the compound of the invention is according to Formula I, wherein R1 is C3.7 cycloalkyl. In a particular embodiment, R1 is cyclopropyl.
[0093] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cyi, and wherein the subscript m is 0, 1, 2, 3, or 4. In a particular embodiment, m is 0, 1 or 2. In a more particular embodiment, m is 0. In another more particular embodiment, m is 1.
[0094] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 0, and wherein R2a is F, Br, I, cyclopropyl, C2-4 alkyl, CM alkyl substituted with one or more independently selected halo, C2-4 alkoxy or CM alkoxy substituted with one or more independently selected halo. In a particular embodiment, R a is F, Br, I, C2_4 alkyl, CM alkyl substituted with one or more independently selected halo, C2_4 alkoxy or CM alkoxy substituted with one or more independently selected halo. In a more particular embodiment, R2a is F, Et, z'-Pr, -CF3, -OEt, -OCHF2, or -OCF3. In a most particular embodiment, R2a is Et.
[0095] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cyi, R2b is as described above, the subscript m is 1 or 2, and R2a is halo, cyclopropyl, CM alkyl optionally substituted with one or more independently selected halo, or CM alkoxy optionally substituted with one or more independently selected halo. In a particular embodiment, R2a is halo, C alkyl optionally substituted with one or more independently selected halo, or CM alkoxy optionally substituted with one or more independently selected halo. In a more particular embodiment, R2a is CI, Me, Et, z'-Pr, -CF3, -OMe, -OEt, -OCHF2, or -OCF3. In a most particular embodiment, R2a is Et.
[0096] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R2b is CN, halo, or OH. In a particular embodiment, R2b is CN, F, CI, or OH.
[0097] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R2b is CM alkyl. In a particular embodiment, R2b is Me, Et, n-Pr, z'-Pr or t- Bu. In a more particular embodiment, R2b is Me, Et, or i-Bu.
[0098] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R2b is CM alkyl substituted with one or more groups independently selected from halo, CN, OH, and Ce-io aryl (optionally substituted with one or more independently selected CM alkoxy). In a particular embodiment, R2b is CM alkyl substituted with one or more groups independently selected from F, CN, OH, phenyl and 4-OMe-phenyl. In another particular embodiment, R2b is Me, Et, n-Pr, or z'-Pr, each of which is substituted with one or more groups independently selected from halo, CN, OH, and Ce-io aryl (optionally substituted with one or more independently selected CM alkoxy). In a more particular embodiment, R2b is -CH2-CN, -CH2-OH, -CF3, -CH2-Ph, or -CH2-(4- OMe-Ph).
[0099] In another embodiment, the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R2b is CM alkyl substituted with one or more groups independently selected from -C(=0)H,
Figure imgf000025_0001
alkyl, or -C(=0)-(4-6 membered heterocycloalkyl) (which heterocycloalkyl is optionally substituted with one or more oxo,or CM alkyl). In a particular embodiment, R2b is C alkyl substituted with one or more groups independently selected from - C(=0)H, -C(=0)OCH3, -C(=0)OCH2CH3, -C(=0)azetidinyl, -C(=0)pyrrolidinyl, -C(=0)piperidinyl, - C(=0)piperazinyl, and -C(=0)morpholinyl, which azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl or morpholinyl are optionally substituted with one or two independently selected -CH3, -CH2CH3, and oxo. In another particular embodiment, R2b is Me, Et, n-Pr, or z'-Pr, each of which is substituted with one or more groups independently selected from -C(=0)H, -C(=0)OCH3, -C(=0)OCH2CH3, -C(=0)azetidinyl, -C(=0)pyrrolidinyl, -C(=0)piperidinyl, -C(=0)piperazinyl, or -C(=0)morpholinyl„ which azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl and morpholinyl are optionally substituted with one or two independently selected -CH3, -CH2CH3, and oxo.
[00100] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R2b is C2_4 alkenyl comprising 1 double bond (optionally substituted with one CN group). In a particular embodiment, R2b is -CH=CH-CH3, -CH=CH-CN, or -CH2-CH=CH2.
[00101] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R2b is C2-6 alkoxy. In a particular embodiment, R2b is C2-6 alkoxy. In a more particular embodiment, R2b is OEt, On-Pr, or Oz'-Pr.
[00102] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R2b is Ci_6 alkoxy substituted with one or more groups independently selected from OH, CN, halo, CM alkoxy, C6-10 aryl (optionally substituted with one or more independently selected CM alkoxy), C3.7 cycloalkyl,
Figure imgf000026_0001
alkyl, and 5-10 membered heteroaryl (optionally substituted with one or more independently selected C1.4 alkyl). In a particular embodiment, R2b is Ci_6 alkoxy substituted with one or more groups independently selected from OH, CN, F, OMe, OEt, phenyl, 4-OMe-phenyl, cyclopropyl, -C(=0)OMe, -C(=0)OEt, -C(=0)Oi- Bu, -C(=0)Me, -C(=0)Et, -C(=0)z-Pr, and -C(=0)i-Bu. In another particular embodiment, R2b is OMe, OEt, On-Pr, or Oz'-Pr, each of which is substituted with one or more groups independently selected from OH, CN, halo, C1.4 alkoxy, C6-10 aryl (optionally substituted with one or more independently selected Ci_ 4 alkoxy), C3.7 cycloalkyl,
Figure imgf000026_0002
alkyl, and 5-10 membered heteroaryl (optionally substituted with one or more C1.4 alkyl). In a more particular embodiment, R2b is -OCH2- CN, -OCH2-CH2-OH, -OCH2-CH2-OMe, -OCH2-CH2-OEt, -OCHF2, -OCF3, -OCH2-Ph, or -OCH2-(4- OMe-Ph).
[00103] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R2b is -C(=0)NH2 optionally substituted with one or two independently selected C1.4 alkyl. In a particular embodiment, R2b is -C(=0)NH2. In another particular embodiment, R2b is-C(=0)NH2 substituted with one or two CM alkyl. In a more particular embodiment, R2b is- C(=0)NH2 substituted with one or two groups independently selected from Me, Et, n-Pr, z'-Pr, and i-Bu. In a most particular embodiment, R2b is -C(=0)NHMe, -C(=0)NMe2, -C(=0)NHEt, -C(=0)NHz-Pr or - C(=0)NHi-Bu.
[00104] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R2b is
Figure imgf000026_0003
alkoxy. In a particular embodiment, R2b is - C(=0)OMe, -C(=0)OEt, or -C(=0)Oi-Bu.
[00105] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R2b is -O-C6-10 aryl. In a particular embodiment, R2b is -OPh. [00106] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R2b is 4-10 membered heterocycloalkyl. In a particular embodiment, R2b is azetidine, pyrrolidine, piperidinyl piperazinyl, morpholinyl, or thiomorpholinyl.
[00107] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R2b is 4-10 membered heterocycloalkyl substituted with one to three oxo, halo, CM alkyl (optionally substituted with one or more -CN),
Figure imgf000027_0001
alkyl, or -SO2-C1.4 alkyl. In a particular embodiment, R2b is azetidine, pyrrolidine, piperidinyl piperazinyl, morpholinyl, or thiomorpholinyl, each of which is substituted with one to three oxo, halo, C1.4 alkyl (optionally substituted with one or more -CN),
Figure imgf000027_0002
alkyl, or -SO2-C1.4 alkyl. In a more particular embodiment, the subscript m is 1, and R2b is 4-10 membered heterocycloalkyl substituted with one to three oxo, F, CI, Me, Et, iPr, tBu, -CH2CN, -CH2-CH2-CN, -C(=0)OMe, -C(=0)OEt, -C(=0)Oz'Pr, - C(=0)OiBu, -S02-Me, -S02-Et, -S02-z'Pr, or -S02-iBu. In a most particular embodiment, the subscript m is 1 , and R2b is azetidine, pyrrolidine, piperidinyl piperazinyl, morpholinyl, or thiomorpholinyl, each of which is substituted with one to three oxo, F, CI, Me, Et, iPr, tBu, -CH2CN, -CH2-CH2-CN, - C(=0)OMe, -C(=0)OEt, -C(=0)Oz'Pr, -C(=0)OiBu, -S02-Me, -S02-Et, -S02-z'Pr, or -S02-iBu.
[00108] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R2b is 5-10 membered heteroaryl. In a particular embodiment, R2b is furanyl, thienyl, pyrrazolyl, imidazolyl, triazolyl, thiazolyl, pyridinyl, pyrimidinyl, or pyrazinyl.
[00109] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R2b is 5-10 membered heteroaryl substituted with one or more independently groups groups independently selected from CN, halo, OH, CM alkyl, and CM alkoxy. In a particular embodiment, R2b is 5-10 membered heteroaryl substituted with one group selected from CN, halo, OH, CM alkyl, and C1.4 alkoxy. In another particular embodiment, R2b is furanyl, thienyl, pyrrazolyl, imidazolyl, triazolyl, thiazolyl, pyridinyl, pyrimidinyl, or pyrazinyl, each of which is substituted with one or more groups independently selected from CN, halo, OH, CM alkyl, and C alkoxy. In a more particular embodiment, R2b is furanyl, thienyl, pyrrazolyl, imidazolyl, triazolyl, thiazolyl, pyridinyl, pyrimidinyl, or pyrazinyl, each of which is substituted with one group selected from CN, halo, OH, C alkyl, and C alkoxy. In a most particular embodiment, R2b is furanyl, thienyl, pyrrazolyl, imidazolyl, triazolyl, thiazolyl, pyridinyl, pyrimidinyl, or pyrazinyl, each of which is substituted with one or more groups independently selected from CN, F, CI, OH, Me, Et, z'-Pr, -OMe, - OEt, and -Oz'-Pr. In another most particular embodiment, R2b is furanyl, thienyl, pyrrazolyl, imidazolyl, triazolyl, thiazolyl, pyridinyl, pyrimidinyl, or pyrazinyl, each of which is substituted with one group selected from CN, F, CI, OH, Me, Et, z'-Pr, -OMe, -OEt, and -Oz'-Pr. In a further most paricular embodiment, R2b is thienyl, pyrrazolyl, imidazolyl, pyridinyl, or pyrimidinyl, each of which is substituted with one group selected from CN, Me, Et, z'-Pr, -OMe, -OEt, and -Oz'-Pr. [00110] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R2b is 5-10 membered heteroaryl substituted with one or more NR3aR3b groups, wherein each R3a or R3b is independently selected from H and CM alkyl. In a particular embodiment, R2b is 5-10 membered heteroaryl substituted with one NR3aR3b group. In another particular embodiment, R2b is furanyl, thienyl, pyrrazolyl, imidazolyl, triazolyl, thiazolyl, pyridinyl, pyrimidinyl, or pyrazinyl, each of which is substituted with one substituted with one NR3aR3b group. In a more particular embodiment, R2b is furanyl, thienyl, pyrrazolyl, imidazolyl, triazolyl, thiazolyl, pyridinyl, pyrimidinyl, or pyrazinyl, each of which is substituted with one NR3aR3b group, wherein R3a and R3b are both H. In another more particular embodiment, R2b is furanyl, thienyl, pyrrazolyl, imidazolyl, triazolyl, thiazolyl, pyridinyl, pyrimidinyl, or pyrazinyl, each of which is substituted with one NR3aR3b group, wherein R3a is H and R3b is C alkyl. In yet another more particular embodiment, R2b is furanyl, thienyl, pyrrazolyl, imidazolyl, triazolyl, thiazolyl, pyridinyl, pyrimidinyl, or pyrazinyl, each of which is substituted with one NR3aR3b group, wherein R3a is H and R3b is Me, or Et.
[00111] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R2b is phenyl substituted with one or more groups independently selected from CN, halo, OH, CM alkyl, and C alkoxy. In a particular embodiment, R2b is phenyl substituted with one group selected from CN, halo, OH, CM alkyl, and CM alkoxy. In a most particular embodiment, R2b is phenyl, substituted with one or more groups independently selected from CN, F, CI, OH, Me, Et, z'-Pr, -OMe, -OEt, and -Oz'-Pr. In another most particular embodiment, R2b is phenyl substituted with one group selected from CN, F, CI, OH, Me, Et, z'-Pr, -OMe, -OEt, and -Oz'-Pr. In a further most paricular embodiment, R2b is phenyl substituted with one group selected from CN, Me, Et, z'-Pr, -OMe, -OEt, and -Oz'-Pr.
[00112] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R2b is phenyl substituted with one or more NR3aR3b groups, wherein each R3a or R3b is independently selected from H and CM alkyl. In a particular embodiment, R2b is phenyl substituted with one NR3aR3b group. In a more particular embodiment, R2b is phenyl substituted with one NR3aR3b group, wherein R3a and R3b are both H. In another more particular embodiment, R2b is phenyl substituted with one NR3aR3b group, wherein R3a is H and R3b is CM alkyl. In yet another more particular embodiment, R2b is phenyl substituted with one NR3aR3b group, wherein R3a is H and R3b is Me, or Et.
[00113] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R2b is 4-7 membered heterocycloalkenyl comprising 1 double bond, and comprising 1 or 2 heteroatoms independently selected from N, O, and S. In a particular embodiment, R2b is dihydropyran or tetrahydropyridinyl.
[00114] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R2b is 4-7 membered heterocycloalkenyl comprising 1 double bond, and comprising 1 or 2 heteroatoms independently selected from N, O, and S, substituted with one or more groups independently selected from C alkyl, CM alkoxy, and C3.7 cycloalkyl. In another embodiment, R2b is 4-7 membered heterocycloalkenyl, substituted with one group independently selected from CM alkyl, C1.4 alkoxy, and C3.7 cycloalkyl. In a particular embodiment, R2b is dihydropyran or tetrahydropyridinyl, each of which is substituted with one or more groups independently selected from C alkyl, C1.4 alkoxy, and C3.7 cycloalkyl. In a more particular embodiment, R2b is dihydropyran or tetrahydropyridinyl, each of which is substituted with one or more groups independently selected from Me, Et, z'Pr, iBu, -OMe, -OEt, -Oz'Pr, -OiBu, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. In another more particular embodiment, R2b is dihydropyran or tetrahydropyridinyl, each of which is substituted with one group selected from Me, Et, z'Pr, iBu, -OMe, -OEt, -Oz'Pr, -OiBu, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.
[00115] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cyl , the subscript m is 1 , and R2b is 4-7 membered heterocycloalkenyl comprising 1 double bond, and comprising 1 or 2 heteroatoms independently selected from N, O, and S, substituted with one or more groups independently selected from -C(=0)-CM alkyl optionally substituted with one or more independently selected CN, OH, or halo. In another embodiment, R2b is 4-7 membered heterocycloalkenyl, substituted with -C(=0)-C M alkyl optionally substituted with one or more independently selected CN, OH, or halo. In a particular embodiment, R2b is dihydropyran or tetrahydropyridinyl, each of which is substituted with one or more groups independently selected from - alkyl optionally substituted with one or more independently selected CN, OH, and halo. In a more particular embodiment, R2b is dihydropyran or tetrahydropyridinyl, each of which is substituted with -C(=0)CH3, -C(=0)Et, -C(=0)z'Pr, -C(=0)iBu, -C(=0)CH2-CN, -C(=0)CH2-CH2-CN, or - C(=0)CH2-OH. In another more particular embodiment, R2b is dihydropyran or tetrahydropyridinyl, each of which is substituted with -C(=0)CH3, -C(=0)iBu, -C(=0)CH2-CN, or -C(=0)CH2-OH. In a most particular embodiment, R2b is tetrahydropyridinyl substituted with -C(=0)CH3, -C(=0)iBu, - C(=0)CH2-CN, or -C(=0)CH2-OH.
[00116] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cyl , the subscript m is 1 , and R2b is 4-7 membered heterocycloalkenyl comprising 1 double bond, and comprising 1 or 2 heteroatoms independently selected from N, O, and S, substituted with one or more independently selected -C(=0)-OCM alkyl. In another embodiment, R2b is 4-7 membered heterocycloalkenyl, substituted with one -C(=0)-OC M alkyl. In a particular embodiment, R2b is dihydropyran or tetrahydropyridinyl, each of which is substituted with one or more independently selected -C(=0)-OCM alkyl. In a more particular embodiment, R2b is dihydropyran or tetrahydropyridinyl, each of which is substituted with one group selected from -C(=0)OCH3, - C(=0)OEt, -C(=0)Oz'Pr, and -C(=0)OiBu . In another more particular embodiment, R2b is dihydropyran or tetrahydropyridinyl, each of which is substituted with one -C(=0)OiBu. In a most particular embodiment, R2b is tetrahydropyridinyl substituted with one -C(=0)OiBu. [00117] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cyl , the subscript m is 1 , and R2b is 4-7 membered heterocycloalkenyl comprising 1 double bond, and comprising 1 or 2 heteroatoms independently selected from N, O, and S, substituted with one or more independently selected -C(=0)NR4aR4b, wherein each R4a and R4b is independently selected from H and C alkyl. In another embodiment, R2b is 4-7 membered heterocycloalkenyl, substituted with one - C(=0)NR4aR4b, wherein each R4a and R4b is independently selected from H and CM alkyl. In a particular embodiment, R2b is dihydropyran or tetrahydropyridinyl, each of which is substituted with one or more independently selected -C(=0)NR4aR4b, wherein each R4a and R4b is independently selected from H and CM alkyl. In a more particular embodiment, R2b is dihydropyran or tetrahydropyridinyl, each of which is substituted with one -C(=0)NR4aR4b, wherein each R4a and R4b is independently selected from H and CM alkyl. In a more particular embodiment, R2b is dihydropyran or tetrahydropyridinyl, each of which is substituted with one -C(=0)NR4aR4b, wherein each R4a and R4b is independently selected from H, Me, Et, z'Pr. In another more particular embodiment, R2b is dihydropyran or tetrahydropyridinyl, each of which is substituted with one -C(=0)NH2, -C(=0)NHMe, or -C(=0)NMe2.
[00118] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R2b is C3.7 cycloalkyl. In a particular embodiment, R2b is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. In a more particular embodiment, R2b is cyclopropyl.
[00119] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R2b is C3.7 cycloalkyl substituted with one CN. In a particular embodiment, R2b is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, each of which may be substituted with one CN.
[00120] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R2b is CM thioalkoxy. In a particular embodiment, R2b is -SMe, -SEt, -Sn- Pr, or Si-Bu. In a more particular embodiment, R2b is -SMe.
[00121] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R2b is
Figure imgf000030_0001
alkyl (optionally substituted with CN). In a particular embodiment, R2b is -C(=0)Me, -C(=0)Et, -C(=0)Pr, or -C(=0)CH2CN.
[00122] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R2b is -C(=0)-(4-6 membered heterocycloalkyl). In a particular embodiment, R2b is -C(=0)-azetidinyl, -C(=0)-pyrrolidinyl, -C(=0)-piperidinyl, -C(=0)-piperazinyl, - C(=0)-morpholinyl.
[00123] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cyi, the subscript m is 1, and R2b is -C(=0)-(4-6 membered heterocycloalkyl), which heterocycloalkyl is substituted with one or more CM alkyl (optionally substituted with one or more CN)). In a particular embodiment, R2b is -C(=0)-azetidinyl, -C(=0)-pyrrolidinyl, -C(=0)-piperidinyl, -C(=0)-piperazinyl, - C(=0)-morpholinyl, each of which azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, or morpholinyl is substituted with one or more CM alkyl (optionally substituted with one or more CN)). In another more particular embodiment, R2b is -C(=0)-(4-6 membered heterocycloalkyl), which heterocycloalkyl is substituted with one or more -CH3, -CH2-CH3, CH(CH3)2, -CH2-CN, or -CH2-CH2-CN. In another more particular embodiment, R2b is -C(=0)-azetidinyl, -C(=0)-pyrrolidinyl, -C(=0)-piperidinyl, - C(=0)-piperazinyl, -C(=0)-morpholinyl, each of which azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, or morpholinyl is substituted with one or more -CH3, -CH2-CH3, CH(CH3)2, -CH2-CN, or -CH2-CH2- CN.
[00124] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cyi, R2a, R2b, and the subscript m are as described above, w is N, and each of x, y and z are CH. In a particular embodiment, R2a and R2b, are as described above, the subscript m is 1, w is N, and each of x, y and z are CH.
[00125] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cyi, R2a, R2b, and the subscript m are as described above, x is N, and each of w, y and z are CH. In a particular embodiment, R2a and R2b, are as described above, the subscript m is 1, x is N, and each of w, y and z are CH.
[00126] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cyi, R2a, R2b, and the subscript m are as described above, y is N, and each of w, x and z are CH. In a particular embodiment, R2a and R2b, are as described above, the subscript m is 1, y is N, and each of w, x and z are CH.
[00127] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cyi, R2a, R2b, and the subscript m are as described above, z is N, and each of w, x and y are CH. In a particular embodiment, R2a and R2b, are as described above, the subscript m is 1, z is N, and each of w, x and y are CH.
[00128] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cyi, R2a, R2b, and the subscript m are as described above, and each of w, x, y and z are CH. In a particular embodiment, R2a and R2b, are as described above, the subscript m is 1, and each of w, x, y and z are CH.
[00129] In one embodiment, the compound of the invention according to Formula I is according to Formula Ila or lib:
Figure imgf000031_0001
I la Mb
wherein R a and R , are as described in any one of the embodiments above. [00130] In one embodiment, the compound of the invention according to Formula I is according to Formula lie or lid:
Figure imgf000032_0001
l ie lid
wherein R2a and R2b, are as described in any one of the embodiments above.
[00131] In one embodiment, the compound of the invention is according to Formula Ila, lib, lie or lid, wherein R2a is halo, cyclopropyl, CM alkyl optionally substituted with one or more halo, or CM alkoxy optionally substituted with one or more halo. In a particular embodiment, R2a is halo, CM alkyl optionally substituted with one or more independently selected halo, or CM alkoxy optionally substituted with one or more halo. In a more particular embodiment, R2a is CI, Me, Et, z'-Pr, -CF3, -OMe, -OEt, -OCHF2, or -OCF3. In a most particular embodiment, R2a is F, CI, Me, Et, or -OCHF2. In a further most particular embodiment, R2a is Et.
[00132] In one embodiment, the compound of the invention is according to Formula Ila, lib, lie or lid, wherein R2b is as described in any one of the embodiments above, the subscript m is 1 or 2, and R2a is halo, cyclopropyl, CM alkyl optionally substituted with one or more independently selected halo, or CM alkoxy optionally substituted with one or more independently selected halo. In a particular embodiment, R2a is halo, CM alkyl optionally substituted with one or more independently selected halo, or CM alkoxy optionally substituted with one or more independently selected halo. In a more particular embodiment, R2a is CI, Me, Et, z'-Pr, -CF3, -OMe, -OEt, -OCHF2, or -OCF3. In a most particular embodiment, R2a is Et.
[00133] In one embodiment, the compound of the invention is according to Formula Ila, lib, lie or lid, wherein R2b is CN, halo, or OH. In a particular embodiment, R2b is CN, F, CI, or OH.
[00134] In one embodiment, the compound of the invention is according to Formula Ila, lib, lie or lid, wherein R2b is CM alkyl. In a particular embodiment, R2b is Me, Et, n-Pr, z'-Pr or ί-Bu. In a more particular embodiment, R2b is Me, Et, or i-Bu.
[00135] In one embodiment, the compound of the invention is according to Formula Ila, lib, lie or lid, wherein R2b is CM alkyl substituted with one or more groups independently selected from halo, CN, OH, and Ce-io aryl (optionally substituted with one or more independently selected C alkoxy). In a particular embodiment, R2b is CM alkyl substituted with one or more groups independently selected from F, CN, OH, phenyl and 4-OMe-phenyl. In another particular embodiment, R2b is Me, Et, n-Pr, or z'- Pr, each of which is substituted with one or more groups independently selected from halo, CN, OH, and Ce-io aryl (optionally substituted with one or more independently selected CM alkoxy). In a more particular embodiment, R2b is -CH2-CN, -CH2-OH, -CF3, -CH2-Ph, or -CH2-(4-OMe-Ph). [00136] In one embodiment, the compound of the invention is according to Formula Ila, lib, lie or lid, wherein R2b is C2_6 alkoxy. In a particular embodiment, R2b is C2_6 alkoxy. In a more particular embodiment, R2b is OEt, On-Pr, or Oz'-Pr.
[00137] In one embodiment, the compound of the invention is according to Formula Ila, lib, lie or lid, wherein R2b is Ci_6 alkoxy substituted with one or more groups independently selected from OH, CN, halo, C alkoxy, C6-10 aryl (optionally substituted with one or more independently selected CM alkoxy), C3.7 cycloalkyl, -C(=0)-Ci_4 alkoxy,
Figure imgf000033_0001
alkyl, and 5-10 membered heteroaryl (optionally substituted with one or more independently selected CM alkyl). In a particular embodiment, R2b is Ci_6 alkoxy substituted with one or more groups independently selected from OH, CN, F, OMe, OEt, phenyl, 4-OMe-phenyl, cyclopropyl, -C(=0)OMe, -C(=0)OEt, -C(=0)Oi-Bu, -C(=0)Me, - C(=0)Et, -C(=0)z-Pr, and -C(=0)i-Bu. In another particular embodiment, R2b is OMe, OEt, On-Pr, or Oz'-Pr, each of which is substituted with one or more groups independently selected from OH, CN, halo, CM alkoxy, Ce-io aryl (optionally substituted with one or more groups independently selected from CM alkoxy), C3.7 cycloalkyl, -C(=0)-CM alkoxy, -C(=0)-CM alkyl, and 5-10 membered heteroaryl (optionally substituted with one or more C alkyl). In a more particular embodiment, R2b is -OCH2- CN, -OCH2-CH2-OH, -OCH2-CH2-OMe, -OCH2-CH2-OEt, -OCHF2, -OCF3, -OCH2-Ph, or -OCH2-(4- OMe-Ph).
[00138] In one embodiment, the compound of the invention is according to Formula Ila, lib, lie or lid, wherein R2b is -C(=0)NH2 optionally substituted with one or two independently selected CM alkyl. In a particular embodiment, R2b is -C(=0)NH2. In another particular embodiment, R2b is-C(=0)NH2 substituted with one or two CM alkyl. In a more particular embodiment, R2b is-C(=0)NH2 substituted with one or two groups independently selected from Me, Et, n-Pr, z'-Pr, and ί-Bu. In a most particular embodiment, R2b is -C(=0)NHMe, -C(=0)NMe2, -C(=0)NHEt, -C(=0)NHz-Pr or -C(=0)NHi-Bu.
[00139] In one embodiment, the compound of the invention is according to Formula Ila, lib, lie or lid, wherein R2b is -C(=0)-CM alkoxy. In a particular embodiment, R2b is -C(=0)OMe, -C(=0)OEt, or -C(=0)Oi-Bu.
[00140] In one embodiment, the compound of the invention is according to Formula Ila, lib, lie or Ildwherein R2b is -O-Ce-io aryl. In a particular embodiment, R2b is -OPh.
[00141] In one embodiment, the compound of the invention is according to Formula Ila, lib, lie or lid, wherein R2b is 4-10 membered heterocycloalkyl. In a particular embodiment, R2b is azetidine, pyrrolidine, piperidinyl piperazinyl, morpholinyl, or thiomorpholinyl.
[00142] In one embodiment, the compound of the invention is according to Formula Ila, lib, lie or lid, wherein R2b is 4-10 membered heterocycloalkyl substituted with one oxo, halo, CM alkyl (optionally substituted with one or more -CN), -C(=0)0-CM alkyl, or -S02-CM alkyl. In a particular embodiment, R2b is azetidine, pyrrolidine, piperidinyl piperazinyl, morpholinyl, or thiomorpholinyl, each of which is substituted with one oxo, halo, CM alkyl (optionally substituted with one or more -CN), -C(=0)0-CM alkyl, or -SO2-C1.4 alkyl. In a more particular embodiment, the subscript m is 1, and R is 4-10 membered heterocycloalkyl substituted with one oxo, F, CI, Me, Et, iPr, tBu, -CH2CN, -CH2-CH2-CN, - C(=0)OMe, -C(=0)OEt, -C(=0)Oz'Pr, -C(=0)OiBu, -S02-Me, -S02-Et, -S02-z'Pr, or -S02-iBu. In a most particular embodiment, the subscript m is 1 , and R2b is azetidine, pyrrolidine, piperidinyl piperazinyl, morpholinyl, or thiomorpholinyl, each of which is substituted with one oxo, F, CI, Me, Et, iPr, tBu, -CH2CN, -CH2-CH2-CN, -C(=0)OMe, -C(=0)OEt, -C(=0)Oz'Pr, -C(=0)OiBu, -S02-Me, - S02-Et, -S02-z'Pr, or -S02-iBu.
[00143] In one embodiment, the compound of the invention is according to Formula Ila, lib, lie or lid, wherein R2b is 5-10 membered heteroaryl. In a particular embodiment, R2b is furanyl, thienyl, pyrrazolyl, imidazolyl, triazolyl, thiazolyl, pyridinyl, pyrimidinyl, or pyrazinyl.
[00144] In one embodiment, the compound of the invention is according to Formula Ila, lib, lie or lid, wherein R2b is 5-10 membered heteroaryl substituted with one or more groups independently selected from CN, halo, OH, C1.4 alkyl, and C1.4 alkoxy. In a particular embodiment, R2b is 5-10 membered heteroaryl substituted with one group selected from CN, halo, OH, C1.4 alkyl, and CM alkoxy. In another particular embodiment, R2b is furanyl, thienyl, pyrrazolyl, imidazolyl, triazolyl, thiazolyl, pyridinyl, pyrimidinyl, or pyrazinyl, each of which is substituted with one or more groups independently selected from CN, halo, OH, C1.4 alkyl, and CM alkoxy. In a more particular embodiment, R2b is furanyl, thienyl, pyrrazolyl, imidazolyl, triazolyl, thiazolyl, pyridinyl, pyrimidinyl, or pyrazinyl, each of which is substituted with one group selected from CN, halo, OH, CM alkyl, and CM alkoxy. In a most particular embodiment, R2b is furanyl, thienyl, pyrrazolyl, imidazolyl, triazolyl, thiazolyl, pyridinyl, pyrimidinyl, or pyrazinyl, each of which is substituted with one or more groups independently selected from CN, F, CI, OH, Me, Et, z'-Pr, -OMe, -OEt, and -Oz'-Pr. In another most particular embodiment, R2b is furanyl, thienyl, pyrrazolyl, imidazolyl, triazolyl, thiazolyl, pyridinyl, pyrimidinyl, or pyrazinyl, each of which is substituted with one group selected from CN, F, CI, OH, Me, Et, z'-Pr, -OMe, -OEt, and -Oz'-Pr. In a further most paricular embodiment, R2b is thienyl, pyrrazolyl, imidazolyl, pyridinyl, or pyrimidinyl, each of which is substituted with one group selected from CN, Me, Et, z'-Pr, -OMe, -OEt, and -Oz'-Pr.
[00145] In one embodiment, the compound of the invention is according to Formula Ila, lib, lie or lid, wherein R2b is 5-10 membered heteroaryl substituted with one or more NR3aR3b groups, wherein each R3a or R3b is independently selected from H and CM alkyl. In a particular embodiment, R2b is 5-10 membered heteroaryl substituted with one NR3aR3b group. In another particular embodiment, R2b is furanyl, thienyl, pyrrazolyl, imidazolyl, triazolyl, thiazolyl, pyridinyl, pyrimidinyl, or pyrazinyl, each of which is substituted with one NR3aR3b group. In a more particular embodiment, R2b is furanyl, thienyl, pyrrazolyl, imidazolyl, triazolyl, thiazolyl, pyridinyl, pyrimidinyl, or pyrazinyl, each of which is substituted with one NR3aR3b group, wherein R3a and R3b are both H. In another more particular embodiment, R2b is furanyl, thienyl, pyrrazolyl, imidazolyl, triazolyl, thiazolyl, pyridinyl, pyrimidinyl, or pyrazinyl, each of which is substituted with one NR3aR3b group, wherein R3a is H and R3b is CM alkyl. In yet another more particular embodiment, R is furanyl, thienyl, pyrrazolyl, imidazolyl, triazolyl, thiazolyl, pyridinyl, pyrimidinyl, or pyrazinyl, each of which is substituted with one NR3AR3B group, wherein R3A is H and R3B is Me, or Et.
[00146] In one embodiment, the compound of the invention is according to Formula Ila, lib, lie or lid, wherein R2B is phenyl substituted with one or more groups independently selected from CN, halo, OH, CM alkyl, and CM alkoxy. In a particular embodiment, R2B is phenyl substituted with one group selected from CN, halo, OH, C alkyl, and C alkoxy. In a most particular embodiment, R2B is phenyl, substituted with one or more groups independently selected from CN, F, CI, OH, Me, Et, z'-Pr, -OMe, - OEt, and -Oz'-Pr. In another most particular embodiment, R2B is phenyl substituted with one group selected from CN, F, CI, OH, Me, Et, z'-Pr, -OMe, -OEt, and -Oz'-Pr. In a further most paricular embodiment, R2B is phenyl substituted with one group selected from CN, Me, Et, z'-Pr, -OMe, -OEt, and - Oz'-Pr.
[00147] In one embodiment, the compound of the invention is according to Formula Ila, lib, lie or lid, wherein R2B is phenyl substituted with one or more NR3AR3B groups, wherein each R3A or R3B is independently selected from H or CM alkyl. In a particular embodiment, R2B is phenyl substituted with one NR3AR3B group. In a more particular embodiment, R2B is phenyl substituted with one NR3AR3B group, wherein R3A and R3B are both H. In another more particular embodiment, R2B is phenyl substituted with one NR3AR3B group, wherein R3A is H and R3B is C alkyl. In yet another more particular embodiment, R2B is phenyl substituted with one NR3AR3B group, wherein R3A is H and R3B is Me, or Et.
[00148] In one embodiment, the compound of the invention is according to Formula Ila, lib, lie or lid, wherein R2B is 4-7 membered heterocycloalkenyl comprising 1 double bond, and comprising 1 or 2 heteroatoms independently selected from N, O, and S. In a particular embodiment, R2B is dihydropyran or tetrahydropyridinyl.
[00149] In one embodiment, the compound of the invention is according to Formula Ila, lib, lie or lid, wherein R2B is 4-7 membered heterocycloalkenyl comprising 1 double bond, and comprising 1 or 2 heteroatoms independently selected from N, O, and S, substituted with one or more groups independently selected from C alkyl, CM alkoxy,C3_7 cycloalkyl. In another embodiment, R2B is 4-7 membered heterocycloalkenyl, substituted with one group independently selected from CM alkyl, CM alkoxy, and C3.7 cycloalkyl. In a particular embodiment, R2B is dihydropyran or tetrahydropyridinyl, each of which is substituted with one or more groups independently selected from CM alkyl, CM alkoxy, and C3.7 cycloalkyl. In a more particular embodiment, R2B is dihydropyran or tetrahydropyridinyl, each of which is substituted with one or more groups independently selected from Me, Et, z'Pr, iBu, -OMe, -OEt, -Oz'Pr, -OiBu, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. In another more particular embodiment, R2B is dihydropyran or tetrahydropyridinyl, each of which is substituted with one group independently selected from Me, Et, z'Pr, iBu, -OMe, -OEt, -Oz'Pr, -OiBu, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. [00150] In one embodiment, the compound of the invention is according to Formula Ila, lib, lie or lid, wherein R2b is 4-7 membered heterocycloalkenyl comprising 1 double bond, and comprising 1 or 2 heteroatoms independently selected from N, O, and S, substituted with one or more groups independently selected from
Figure imgf000036_0001
alkyl optionally substituted with one or more independently selected CN, OH, or halo. In another embodiment, R2b is 4-7 membered heterocycloalkenyl, substituted with -C(=0)-Ci_4 alkyl optionally substituted with one or more independently selected CN, OH, or halo. In a particular embodiment, R2b is dihydropyran or tetrahydropyridinyl, each of which is substituted with one or more groups independently selected from
Figure imgf000036_0002
alkyl optionally substituted with one or more independently selected CN, OH, and halo. In a more particular embodiment, R2b is dihydropyran or tetrahydropyridinyl, each of which is substituted with -C(=0)CH3, -C(=0)Et, -C(=0)z'Pr, - C(=0)iBu, -C(=0)CH2-CN, -C(=0)CH2-CH2-CN, or -C(=0)CH2-OH. In another more particular embodiment, R2b is dihydropyran or tetrahydropyridinyl, each of which is substituted with -C(=0)CH3, -C(=0)iBu, -C(=0)CH2-CN, or -C(=0)CH2-OH . In a mo st particular emb odiment, R2b is tetrahydropyridinyl substituted with -C(=0)CH3, -C(=0)iBu, -C(=0)CH2-CN, or -C(=0)CH2-OH.
[00151] In one embodiment, the compound of the invention is according to Formula Ila, lib, lie or lid, wherein R2b is 4-7 membered heterocycloalkenyl comprising 1 double bond, and comprising 1 or 2 heteroatoms independently selected from N, O, and S, substituted with one or more independently selected
Figure imgf000036_0003
alkyl. In another embodiment, R2b is 4-7 membered heterocycloalkenyl, substituted with one
Figure imgf000036_0004
alkyl. In a particular embodiment, R2b is dihydropyran or tetrahydropyridinyl, each of which is substituted with one or more independently selected
Figure imgf000036_0005
alkyl. In a more particular embodiment, R2b is dihydropyran or tetrahydropyridinyl, each of which is substituted with one group selected from -C(=0)OCH3, -C(=0)OEt, -C(=0)OzPr, and -C(=0)OiBu. In another more particular embodiment, R2b is dihydropyran or tetrahydropyridinyl, each of which is substituted with one -C(=0)OiBu. In a most particular embodiment, R2b is tetrahydropyridinyl substituted with one -C(=0)OiBu.
[00152] In one embodiment, the compound of the invention is according to Formula Ila, lib, lie or lid, wherein R2b is 4-7 membered heterocycloalkenyl comprising 1 double bond, and comprising 1 or 2 heteroatoms independently selected from N, O, and S, substituted with one or more independently selected -C(=0)NR4aR4b, wherein each R4a and R4b is independently selected from H and CM alkyl. In another embodiment, R2b is 4-7 membered heterocycloalkenyl, substituted with one -C(=0)NR4aR4b, wherein each R4a and R4b is independently selected from H and CM alkyl. In a particular embodiment, R2b is dihydropyran or tetrahydropyridinyl, each of which is substituted with one or more independently selected -C(=0)NR4aR4b, wherein each R4a and R4b is independently selected from H and CM alkyl. In a more particular embodiment, R2b is dihydropyran or tetrahydropyridinyl, each of which is substituted with one -C(=0)NR4aR4b, wherein each R4a and R4b is independently selected from H and CM alkyl. In a more particular embodiment, R2b is dihydropyran or tetrahydropyridinyl, each of which is substituted with one -C(=0)NR4aR4b, wherein each R4a and R4b is independently selected from H, Me, Et, and zPr. In another more particular embodiment, R is dihydropyran or tetrahydropyridinyl, each of which is substituted with one group selected from -C(=0)NH2, -C(=0)NHMe, and -C(=0)NMe2.
[00153] In one embodiment, the compound of the invention is according to Formula Ila, lib, lie or lid, wherein R2b is C3.7 cycloalkyl. In a particular embodiment, R2b is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. In a more particular embodiment, R2b is cyclopropyl.
[00154] In one embodiment, the compound of the invention is according to Formula Ila Ila, lib, lie or lid, wherein R2b is C thioalkoxy. In a particular embodiment, R2b is -SMe, -SEt, -Sn-Pr, or Si-Bu. In a more particular embodiment, R2b is -SMe.
[00155] In one embodiment, the compound of the invention is according to Formula Ila, lib, lie or lid, wherein R2b is -C(=0)-Ci_4 alkyl (optionally substituted with CN). In a particular embodiment, R2b is -C(=0)Me, -C(=0)Et, -C(=0)Pr, or -C(=0)CH2CN.
[00156] In one embodiment, the compound of the invention according to Formula I is compound to Formula III:
Figure imgf000037_0001
wherein R5 is selected from C1.4 alkyl, CM alkoxy, C3.7 cycloalkyl, l (optionally substituted with one or more independently selected CN, OH, and halo); a
Figure imgf000037_0002
alkyl.
[00157] In one embodiment, the compound of the invention is according to Formula III, wherein R5 is
Figure imgf000037_0003
or - C(=0)OiBu.
[00158] In one embodiment, the compound of the invention is according to Formula III, wherein R5 is alkyl optionally substituted with one or more groups independently selected from CN, OH, and halo. In a particular embodiment, R5 is -C(=0)CH3, -C(=0)Et, -C(=0)zPr, -C(=0)iBu, - C(=0)CH2-CN, -C(=0)CH2-CH2-CN, or -C(=0)CH2-OH.
[00159] In one embodiment, the compound of the invention according to Formula I is compound to Formula IV:
Figure imgf000037_0004
wherein R6 is Ci_4 alkyl (optionally substituted with one or more -CN),
Figure imgf000037_0005
alkyl, or -S02-Ci_4 alkyl. [00160] In one embodiment, the compound of the invention is according to Formula IV, wherein R6 is Me, Et, iPr, tBu, -CH2CN, -CH2-CH2-CN, -C(=0)OMe, -C(=0)OEt, -C(=0)OzPr, -C(=0)OiBu, - S02-Me, -S02-Et, -S02-zPr, or -S02-iBu. In a more particular embodiment, R6 is -C(=0)OMe, - C(=0)OEt, -C(=0)OzPr, -C(=0)OiBu, -S02-Me, -S02-Et, -S02-zPr, or -S02-iBu. In a most particular embodiment, R6 is-S02-Me, -S02-Et, -S02-zPr, or -S02-iBu.
[00161] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is selected from Cy2, and Cy3; and the ring A is a fused 5-6 membered saturated ring optionally comprising 1 or 2 heteroatoms independently selected from N, O, and S; to afford a 8-10 membered bicyclic group. In a particular embodiment, the ring A is a fused 5-6 membered saturated ring optionally comprising 1 or 2 heteroatoms independently selected from N, O, and S; to afford a 9-10 membered bicyclic group.
[00162] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cy2, wherein the ring A is a fused 5-6 membered saturated ring optionally comprising 1 or 2 heteroatoms independently selected from N, O, and S; to afford a 9-10 membered bicyclic group, wherein the subscript p is 2, R2d is as described above, and R2c is CM alkyl or halo. In a particular embodiment, R2c is Me, Et, or F.
[00163] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cy2, wherein the ring A is a fused 5-6 membered saturated ring optionally comprising 1 or 2 heteroatoms independently selected from N, O, and S; to afford a 9-10 membered bicyclic group, wherein the subscript p is 1, R2d is as described above, and R2c is oxo.
[00164] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cy2, wherein the ring A is a fused 5-6 membered saturated ring optionally comprising 1 or 2 heteroatoms independently selected from N, O, and S; to afford a 9-10 membered bicyclic group, wherein the subscript p, and R2c are as described above, and R2d is H, halo, C alkyl optionally substituted with one or more halo, or CM alkoxy optionally substituted with one or more halo. In a particular embodiment, R2d is H. In another particular embodiment, R2d is F, CI, Me, Et, z'-Pr, -CF3, - OMe, -OEt, -OCHF2, or -OCF3. In more particular embodiment, R2d is CI, Me or Et.
[00165] In one la I, wherein Cy is:
Figure imgf000038_0001
wherein R is H, halo, CM alkyl optionally substituted with one or more independently selected halo, or C alkoxy optionally substituted with one or more independently selected halo. In a particular embodiment, R is H. In another particular embodiment, R is F, CI, Me, Et, z'-Pr, -CF3, -OMe, -OEt, - OCHF2, or -OCF3. In more particular embodiment, R2d is CI, Me or Et.
[00166] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is selected from Cy2, and Cy3; and the ring A is a fused 5-membered unsaturated ring optionally comprising 1 or 2 heteroatoms independently selected from N, O, and S; to afford a 8-10 membered bicyclic group. In a particular embodiment, the ring A is a fused 5-membered unsaturated ring optionally comprising 1 or 2 heteroatoms independently selected from N, O, and S; to afford a 9-10 membered bicyclic group.
[00167] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cy2, wherein the ring A is a fused 5-membered unsaturated ring optionally comprising 1 or 2 heteroatoms independently selected from N, O, and S; to afford a 9-10 membered bicyclic group, wherein the subscript p is 1 or 2, R2d is as described above, and each R2c is independently selected from C alkyl or halo. In a particular embodiment, R2c is Me, Et, or F.
[00168] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cy2, wherein the ring A is a fused 5-membered unsaturated ring optionally comprising 1 or 2 heteroatoms independently selected from N, O, and S; to afford a 9-10 membered bicyclic group, wherein the subscript p is 0.
[00169] In one embodiment, the compound of the invention is according to Formula I, wherein Cy is Cy2, wherein the ring A is a fused 5-membered unsaturated ring optionally comprising 1 or 2 heteroatoms independently selected from N, O, and S; to afford a 9-10 membered bicyclic group, wherein the subscript p, and R2c are as described above, and R2d is H, halo, C1.4 alkyl optionally substituted with one or more independently selected halo, or CM alkoxy optionally substituted with one or more independently selected halo. In a particular embodiment, R2d is H. In another particular embodiment, R2d is F, CI, Me, Et, z-Pr, -CF3, -OMe, -OEt, -OCHF2, or -OCF3. In more particular embodiment, R2d is CI, Me or Et.
[00170] In one embodiment the compound of the invention is according to Formula I, wherein Cy is
Figure imgf000039_0001
wherein R is H, CM alkyl, or halo; and R e is selected from H, and CM alkyl. In a particular embodiment, R2e is as previously defined, and R2d is H, Me, Et, or CI. In another particular embodiment, R2d is as previously defined, and R2e is H, Me or Et. In a more particular embodiment, R2d is selected from H, Me, Et, or CI, and R2e is H, Me or Et.
[00171] In one embodiment, the comp n is according to Formula I, wherein Cy is:
Figure imgf000039_0002
wherein R is H, C alkyl, or halo; and R is selected from H, and C alkyl. In a particular embodiment, R2f is as previously defined, and R2d is H, Me, Et, or CI. In another particular embodiment, R2d is as previously defined, and R2f is H, Me or Et. In a more particular embodiment, R2d is selected from H, Me, Et, or CI, and R2f is H, Me or Et.
[00172] In one embodiment, the compound of the invention according to Formula I is selected from:
(2-Ethyl-phenyl)-(l -methyl-lH-imidazo[4,5-c]pyridin-6-yl)-amine,
(2-Isopropyl-phenyl)-(l-methyl-lH-imidazo[4,5-c]pyridin-6-yl)-amine,
N-(2,3-dihydrobenzo[b][l,4]dioxin-6-yl)-l-methyl-lH-imidazo[4,5-c]pyridin-6-amine,
N-(4-methoxy-2-(trifluoromethyl)phenyl)- 1 -methyl- 1 H-imidazo[4,5-c]pyridin-6-amine,
l-methyl-N-(2,4,6-trimethoxyphenyl)-lH-imidazo[4,5-c]pyridin-6-amine,
N-(benzo[d] [l,3]dioxol-5-yl)-l-methyl-lH-imidazo[4,5-c]pyridin-6-amine,
N-(3-cyclopropyl- 1 -methyl- 1 H-pyrazol-5-yl)- 1 -methyl- 1 H-imidazo[4,5-c]pyridin-6-amine,
N-(l -ethyl-3-methyl- 1 H-pyrazol-5-yl)- 1 -methyl- 1 H-imidazo[4,5-c]pyridin-6-amine,
N-(3-tert-butyl- 1 -methyl- 1 H-pyrazol-5-yl)- 1 -methyl- 1 H-imidazo[4,5-c]pyridin-6-amine,
N-(l -ethyl- 1 H-pyrazol-4-yl)- 1 -methyl- 1 H-imidazo[4,5-c]pyridin-6-amine,
N-(l -benzyl- 1 H-pyrazol-4-yl)- 1 -methyl- 1 H-imidazo[4,5-c]pyridin-6-amine,
1- methyl-N-(l-methyl-lH-pyrazol-3-yl)-lH-imidazo[4,5-c]pyridin-6-amine,
N-(l -(methoxymethyl)- 1 H-pyrazol-4-yl)- 1 -methyl- 1 H-imidazo[4,5-c]pyridin-6-amine,
N-(2,2-dimethylbenzo[d][l,3]dioxol-5-yl)-l -methyl-lH-imidazo[4,5-c]pyridin-6-amine,
N-(2,2-difluorobenzo[d][l,3]dioxol-5-yl)-l-methyl-lH-imidazo[4,5-c]pyridin-6-amine,
6-(l -methyl- 1 H-imidazo[4,5-c]pyridin-6-ylamino)-2,3-dihydro- 1 H-inden- 1 -one,
N-(2,3-dihydro- 1 H-inden-5-yl)- 1 -methyl- 1 H-imidazo[4,5-c]pyridin-6-amine,
2- methyl-N-(l-methyl-lH-imidazo[4,5-c]pyridin-6-yl)benzo[d]oxazol-6-amine,
6-(l -methyl- 1 H-imidazo[4,5-c]pyridin-6-ylamino)isobenzofuran- 1 (3H)-one,
N-(l-methyl-lH-imidazo[4,5-c]pyridin-6-yl)benzo[d]oxazol-5-amine,
2-methyl-N-(l-methyl-lH-imidazo[4,5-c]pyridin-6-yl)benzo[d]oxazol-5-amine,
1- methyl-N-(2-methylbenzofuran-5-yl)-lH-imidazo[4,5-c]pyridin-6-amine,
N-(benzofuran-5-yl)- 1 -methyl- 1 H-imidazo[4,5-c]pyridin-6-amine,
1 -methyl-N-(l -methyl- 1 H-indazol-5-yl)- 1 H-imidazo[4,5-c]pyridin-6-amine,
2- methyl-N-(l-methyl-lH-imidazo[4,5-c]pyridin-6-yl)benzo[d]thiazol-5-amine,
3- ethyl-4-(l -methyl-lH-imidazo[4,5-c]pyridin-6-ylamino)benzonitrile,
3,3-dimethyl-6-(l-methyl-lH-imidazo[4,5-c]pyridin-6-ylamino)-2,3-dihydro-lH-inden-l -one,
N-(l-methyl-lH-imidazo[4,5-c]pyridin-6-yl)benzo[c][l ,2,5]thiadiazol-5-amine,
(7-Methyl-2,3-dihydro-benzo[l,4]dioxin-6-yl)-(l-methyl-lH-imidazo[4,5-c]pyridin-6-yl)-amine,
(2,3-Dihydro-benzo[l,4]dioxin-5-yl)-(l-methyl-lH-imidazo[4,5-c]pyridin-6-yl)-amine,
(3-Ethyl-pyridin-2-yl)-(l-methyl-lH-imidazo[4,5-c]pyridin-6-yl)-amine,
(2,3-Dihydro-benzo[l,4]dioxin-6-yl)-(l-ethyl-lH-imidazo[4,5-c]pyridin-6-yl)-amine, (7-Ethyl-2,3-dihydro-benzo[l,4]dioxin-6-yl)-(l-meth^
(2-Ethyl-6-methyl-phenyl)-(l-methyl-lH-imidazo[4,5-c]pyridin-6-yl)-amine,
(l-Cyclopropyl-lH-imidazo[4,5-c]pyridin-6-yl)-(2,3-dihydro-benzo[l,4]dioxin-6-yl)-amine,
6- (l-Methyl-lH-imidazo[4,5-c]pyridin-6-ylamino)-4H-benzo[l,4]oxazin-3-one,
7- (l-Methyl-lH-imidazo[4,5-c]pyridin-6-ylamino)-4H-benzo[l,4]oxazin-3-one,
(2-Ethyl-4-fluoro-phenyl)-(l-methyl-lH-imidazo[4,5-c]pyridin-6-yl)-amine,
(l-Methyl-lH-imidazo[4,5-c]pyridin-6-yl)-(2-trifluoromethyl-phenyl)-amine,
Indan-4-yl-(l-methyl-lH-imidazo[4,5-c]pyridin-6-yl)-amine,
[4-(4-Methoxy-benzyloxy)-2-methyl-phenyl]-(l-methyl-lH-imidazo[4,5-c]pyridin-6-yl)-amin^
Benzothiazol-6-yl-(l-methyl-lH-imidazo[4,5-c]pyridin-6-yl)-amine,
Benzothiazol-5-yl-(l-methyl-lH-imidazo[4,5-c]pyridin-6-yl)-amine,
(2-Difluoromethoxy-phenyl)-(l-methyl-lH-imidazo[4,5-c]pyridin-6-yl)-amine,
(l-Methyl-lH-imidazo[4,5-c]pyridin-6-yl)-(2-trifluoromethoxy-phenyl)-amine,
(7-Chloro-2,3-dihydro-benzo[l,4]dioxin-6-yl)-(l-methy
[5-(4-Methoxy-benzyloxy)-2-methyl-phenyl]-(l-methyl-lH-i^
[3-(4-Methoxy-benzyloxy)-2-methyl-phenyl]-(l-methyl-lH-i^
6-Chloro-7-(l-methyl-lH-imidazo[4,5-c]pyridin-6-ylamino)-4H-benzo[l,4]oxazin-3-one,
6-(l -Methyl- 1 H-imidazo[4,5-c]pyridin-6-ylamino)-3,4-dihydro- 1 H-quinolin-2-one,
4-ethyl-N,N-dimethyl-3-(l-methyl-lH-imidazo[4,5-c]pyridin-6-ylamino)benzamide,
N-(2-ethyl-4-(pyrimidin-5-yl)phenyl)- 1 -methyl- 1 H-imidazo[4,5-c]pyridin-6-amine,
N-(2-ethyl-4-(l -methyl- 1 H-pyrazol-4-yl)phenyl)- 1 -methyl- 1 H-imidazo[4,5-c]pyridin-6-amine,
N-(2-ethyl-4-(pyridin-3-yl)phenyl)-l-methyl-lH-imidazo[4,5-c]pyridin-6-amine,
(4-ethyl-3-(l-methyl-lH-imidazo[4,5-c]pyridin-6-ylamino)phenyl)methanol,
methyl 4-ethyl-3-(l-methyl-lH-imidazo[4,5-c]pyridin-6-ylamino)benzoate,
N-(2-ethyl-4-(2-methoxypyrimidin-5-yl)phenyl)- 1 -methyl- 1 H-imidazo[4,5-c]pyridin-6-amine,
N-(2-ethyl-4-(thiophen-3-yl)phenyl)-l-methyl-lH-imidazo[4,5-c]pyridin-6-amine,
N-(4-(2-(dimethylamino)pyrimidin-5-yl)-2-ethylphenyl)- 1 -methyl- 1 H-imidazo[4,5-c]pyridin-6-amine,
N-(2-ethyl-4-(thiophen-2-yl)phenyl)- 1 -methyl- 1 H-imidazo[4,5-c]pyridin-6-amine,
N-(2-ethyl-4-(l ,3,5-trimethyl- 1 H-pyrazol-4-yl)phenyl)- 1 -methyl- 1 H-imidazo[4,5-c]pyridin-6-amine,
N-(4-(l -benzyl- 1 H-pyrazol-4-yl)-2-ethylphenyl)- 1 -methyl- 1 H-imidazo[4,5-c]pyridin-6-amine,
N-(2-ethyl-4-(2-isopropylpyrimidin-5-yl)phenyl)-l-methyl-lH-imidazo[4,5-c]pyridin-6-amine, tert-butyl 4-(3-ethyl-4-(l-methyl-lH-imidazo[4,5-c]pyridin-6-ylamino)phenyl)-5,6-dihydropyridine-
1 (2H)-carboxylate,
3'-ethyl-4'-(l-methyl-lH-imidazo[4,5-c]pyridin-6-ylamino)biphenyl-4-carbonitrile, and
3'-ethyl-4'-(l-methyl-lH-imidazo[4,5-c]pyridin-6-ylamino)biphenyl-3-carbonitrile.
[00173] In one embodiment, the compound of the invention according to Formula I is selected from:
(4-Methoxy-6-thiophen-2-yl-pyridin-3-yl)-(l-methyl-lH-imidazo[4,5-c]pyridin-6-yl)-amine, [4-Methoxy-6-(l,3,5 rimethyl-lH-pyrazol-4-yl)-pyridin-3-yl]-(l-methyl-lH-imidazo[4,5-c]p yl)-amine,
[3-Ethyl-4-(l-methyl-lH-imidazo[4,5-c]pyridin-6-ylamino)-phenyl]-acetonitrile,
[4-Methoxy-5-(l-methyl-lH-imidazo[4,5-c]pyridin-6-ylamino)-pyridin-2-yl]-acetonitril^ and
(E)-3-[4-(l-Methyl-lH-imidazo[4,5-c]pyridin-6-ylamino)-phenyl]-acrylonitrile.
[00174] In one embodiment the compound of the invention is not an isotopic variant.
[00175] In one aspect a compound of the invention according to any one of the embodiments herein described is present as the free base.
[00176] In one aspect a compound of the invention according to any one of the embodiments herein described is a pharmaceutically acceptable salt.
[00177] In one aspect a compound of the invention according to any one of the embodiments herein described is a solvate of the compound.
[00178] In one aspect a compound of the invention according to any one of the embodiments herein described is a solvate of a pharmaceutically acceptable salt of a compound.
[00179] While specified groups for each embodiment have generally been listed above separately, a compound of the invention includes one in which several or each embodiment in the above Formula, as well as other formulae presented herein, is selected from one or more of particular members or groups designated respectively, for each variable. Therefore, this invention is intended to include all combinations of such embodiments within its scope.
[00180] While specified groups for each embodiment have generally been listed above separately, a compound of the invention may be one for which one or more variables (for example, R groups) is selected from one or more embodiments according to any of the Formula(e) listed above. Therefore, the present invention is intended to include all combinations of variables from any of the disclosed embodiments within its scope.
[00181] Alternatively, the exclusion of one or more of the specified variables from a group or an embodiment, or combinations thereof is also contemplated by the present invention.
[00182] In certain aspects, the present invention provides prodrugs and derivatives of the compounds according to the formulae above. Prodrugs are derivatives of the compounds of the invention, which have metabolically cleavable groups and become by solvolysis or under physiological conditions the compounds of the invention, which are pharmaceutically active, in vivo. Such examples include, but are not limited to, choline ester derivatives and the like, N-alkylmorpholine esters and the like.
[00183] Other derivatives of the compounds of this invention have activity in both their acid and acid derivative forms, but the acid sensitive form often offers advantages of solubility, tissue compatibility, or delayed release in the mammalian organism (see, Bundgard, H. Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam 1985). Prodrugs include acid derivatives well know to practitioners of the art, such as, for example, esters prepared by reaction of the parent acid with a suitable alcohol, or amides prepared by 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 on the compounds of this invention are preferred prodrugs. In some cases it is desirable to prepare double ester type prodrugs such as (acyloxy)alkyl esters or ((alkoxycarbonyl)oxy)alkylesters. Particularly useful are the Ci_g alkyl, C2-8 alkenyl, aryl, C7.12 substituted aryl, and C7.12 arylalkyl esters of the compounds of the invention.
[00184] The compounds of the invention are novel inhibitors of JAK. In particular, the compounds are potent inhibitors of JAKl and/or JAK2; however they may inhibit TYK2 and JAK3 with a lower potency.
PHARMACEUTICAL COMPOSITIONS
[00185] When employed as a pharmaceutical, a compound of the invention is typically administered in the form of a pharmaceutical composition. Such compositions can be prepared in a manner well known in the pharmaceutical art and comprise at least one active compound. Generally, a compound of this invention is administered in a pharmaceutically effective amount. The amount of the compound actually administered will typically be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.
[00186] The pharmaceutical compositions of the invention can be administered by a variety of routes including oral, rectal, transdermal, subcutaneous, intra-articular, intravenous, intramuscular, and intranasal. Depending on the intended route of delivery, a compound of this invention is preferably formulated as either injectable or oral compositions or as salves, as lotions or as patches all for transdermal administration.
[00187] The compositions for oral administration can take the form of bulk liquid solutions or suspensions, or bulk powders. More commonly, however, the compositions are presented in unit dosage forms to facilitate accurate dosing. The term 'unit dosage forms' refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient, vehicle or carrier. Typical unit dosage forms include prefilled, premeasured ampules or syringes of the liquid compositions or pills, tablets, capsules or the like in the case of solid compositions. In such compositions, the compound of the invention 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 being various vehicles or carriers and processing aids helpful for forming the desired dosing form. [00188] Liquid forms suitable for oral administration may include a suitable aqueous or nonaqueous vehicle with buffers, suspending and dispensing agents, colorants, flavors and the like. Solid forms may include, for example, any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
[00189] Injectable compositions are typically based upon injectable sterile saline or phosphate- buffered saline or other injectable carriers known in the art. As before, the active compound in such compositions is typically a minor component, often being from about 0.05 to 10% by weight with the remainder being the injectable carrier and the like.
[00190] Transdermal compositions are typically formulated as a topical ointment or cream containing the active ingredient(s), generally in an amount ranging from about 0.01 to about 20% by weight, preferably from about 0.1 to about 20% by weight, preferably from about 0.1 to about 10% by weight, and more preferably from about 0.5 to about 15% by weight. When formulated as a ointment, the active ingredients will typically be combined with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredients may be formulated in 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 dermal penetration of stability of the active ingredients or the formulation. All such known transdermal formulations and ingredients are included within the scope of this invention.
[00191] A compound of the invention can also be administered by a transdermal device. Accordingly, transdermal administration can be accomplished using a patch either of the reservoir or porous membrane type, or of a solid matrix variety.
[00192] The above-described components for orally administrable, injectable or topically administrable compositions are merely representative. Other materials as well as processing techniques and the like are set forth in Part 8 of Remington's Pharmaceutical Sciences, 17th edition, 1985, Mack Publishing Company, Easton, Pennsylvania, which is incorporated herein by reference.
[00193] A compound of the invention can also be administered in sustained release forms or from sustained release drug delivery systems. A description of representative sustained release materials can be found in Remington's Pharmaceutical Sciences.
[00194] The following formulation examples illustrate representative pharmaceutical compositions that may be prepared in accordance with this invention. The present invention, however, is not limited to the following pharmaceutical compositions.
Formulation 1 - Tablets [00195] A compound of the invention may be admixed as a dry powder with a dry gelatin binder in an approximate 1 :2 weight ratio. A minor amount of magnesium stearate may be added as a lubricant. The mixture may be formed into 240-270 mg tablets (80-90 mg of active amide compound per tablet) in a tablet press.
Formulation 2 - Capsules
[00196] A compound of the invention may be admixed as a dry powder with a starch diluent in an approximate 1 : 1 weight ratio. The mixture may be filled into 250 mg capsules (125 mg of active amide compound per capsule).
Formulation 3 - Liquid
[00197] A compound of the invention (125 mg), may be admixed with sucrose (1.75 g) and xanthan gum (4 mg) and the resultant mixture may be blended, passed through a No. 10 mesh U.S. sieve, and then mixed with a previously made 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. Sufficient water may then be added with stirring. Further sufficient water may be then added to produce a total volume of 5 mL.
Formulation 4 - Tablets
[00198] A compound of the invention may be admixed as a dry powder with a dry gelatin binder in an approximate 1 :2 weight ratio. A minor amount of magnesium stearate may be added as a lubricant. The mixture may be formed into 450-900 mg tablets (150-300 mg of active amide compound) in a tablet press.
Formulation 5 - Injection
[00199] A compound of the invention may be dissolved or suspended in a buffered sterile saline injectable aqueous medium to a concentration of approximately 5 mg/mL.
Formulation 6 - Topical
[00200] Stearyl alcohol (250 g) and a white petrolatum (250 g) may be melted at about 75°C and then a mixture of the compound of the invention (50 g) methylparaben (0.25 g), propylparaben (0.15 g), sodium lauryl sulfate (10 g), and propylene glycol (120 g) dissolved in water (about 370 g) may be added and the resulting mixture may be stirred until it congeals.
METHODS OF TREATMENT [00201] A compound of the invention may be used as a therapeutic agent for the treatment of conditions in mammals that are causally related or attributable to aberrant activity of JAK. In particular, conditions related to aberrant activity of JAK1 and/or JAK2. Accordingly, the compounds and pharmaceutical compositions of the invention find use as therapeutics for preventing and/or treating allergic or inflammatory conditions, autoimmune diseases, proliferative diseases, transplantation rejection, diseases involving impairment of cartilage turnover, congenital cartilage malformations, and/or diseases associated with hypersecretion of IL6 or interferons in mammals including humans.
[00202] In one aspect, the present invention provides a compound of the invention, or a pharmaceutical composition comprising a compound of the invention for use as a medicament.
[00203] In another aspect, the present invention provides a compound of the invention, or a pharmaceutical composition comprising a compound of the invention for use in the manufacture of a medicament.
[00204] In yet another aspect, the present invention provides a method of treating a mammal having, or at risk of having a disease disclosed herein, said method comprising administering an effective condition-treating or condition-preventing amount of one or more of the pharmaceutical compositions or compound of the invention herein described. In a particular aspect, the present invention provides a method of treating a mammal having, or at risk of having allergic or inflammatory conditions, autoimmune diseases, proliferative diseases, transplantation rejection, diseases involving impairment of cartilage turnover, congenital cartilage malformations, and/or diseases associated with hypersecretion of IL6 or interferons.
[00205] In a method of treatment aspect, this invention provides methods of treatment and/or prophylaxis of a mammal susceptible to or afflicted with an allergic reaction, said method comprising administering an effective condition-treating or condition-preventing amount of one or more of the pharmaceutical compositions or compound of the invention herein described. In a specific embodiment, the allergic reaction is selected from allergic airway disease, sinusitis, eczema and hives, food allergies and allergies to insect venom.
[00206] In another aspect the present invention provides a compound of the invention for use in the treatment, and/or prophylaxis of an allergic reaction. In a specific embodiment, the allergic reaction is selected from allergic airway disease, sinusitis, eczema and hives, food allergies and allergies to insect venom.
[00207] In yet another aspect, the present invention provides the compound of the invention, or a pharmaceutical composition comprising the compound of the invention for use in the manufacture of a medicament for the treatment, or prophylaxis of an allergic reaction. In a specific embodiment, the allergic reaction is selected from allergic airway disease, sinusitis, eczema and hives, food allergies and allergies to insect venom. [00208] In additional method of treatment aspects, this invention provides methods of treatment and/or prophylaxis of a mammal susceptible to or afflicted with an inflammatory condition, said methods comprise administering an effective condition-treating or condition-preventing amount of one or more of the pharmaceutical compositions or compound of the invention herein described. In a specific embodiment, the inflammatory condition is selected from rheumatoid arthritis, osteoarthritis, allergic airway disease (e.g. asthma) and inflammatory bowel diseases.
[00209] In another aspect the present invention provides a compound of the invention for use in the treatment, and/or prophylaxis of an inflammatory condition. In a specific embodiment, the inflammatory condition is selected from rheumatoid arthritis, osteoarthritis, allergic airway disease (e.g. asthma) and inflammatory bowel diseases.
[00210] In yet another aspect, the present invention provides the compound of the invention, or a pharmaceutical composition comprising the compound of the invention for use in the manufacture of a medicament for the treatment, and/or prophylaxis of an inflammatory condition. In a specific embodiment, the inflammatory condition is selected from rheumatoid arthritis, osteoarthritis, allergic airway disease (e.g. asthma) and inflammatory bowel diseases.
[00211] In additional method of treatment aspects, this invention provides methods of treatment and/or prophylaxis of a mammal susceptible to or afflicted with an autoimmune disease, saidmethods comprise administering an effective condition-treating or condition-preventing amount of one or more of the pharmaceutical compositions or compounds of the invention herein described. In a specific embodiment, the autoimmune disease is selected from COPD, asthma, systemic lupus erythematosis, type I diabetes mellitus and inflammatory bowel disease.
[00212] In another aspect the present invention provides a compound of the invention for use in the treatment, and/or prophylaxis of an autoimmune disease. In a specific embodiment, the autoimmune disease is selected from COPD, asthma, systemic lupus erythematosis, type I diabetes mellitus and inflammatory bowel disease. In a more specific embodiment, the autoimmune disease is systemic lupus erythematosis.
[00213] In yet another aspect, the present invention provides the compound of the invention, or a pharmaceutical composition comprising the compound of the invention for use in the manufacture of a medicament for the treatment, and/or prophylaxis of an autoimmune disease. In a specific embodiment, the autoimmune disease is selected from COPD, asthma, systemic lupus erythematosis, type I diabetes mellitus and inflammatory bowel disease.
[00214] In further method of treatment aspects, this invention provides methods of treatment and/or prophylaxis of a mammal susceptible to or afflicted with a proliferative disease, said methods comprising administering an effective condition-treating or condition-preventing amount of one or more of the pharmaceutical compositions or compound of the invention herein described. In a specific embodiment, the proliferative disease is selected from cancer (e.g. solid tumors such as uterine leiomyosarcoma or prostate cancer), leukemia (e.g. AML, ALL or CLL), multiple myeloma and psoriasis.
[00215] In another aspect the present invention provides a compound of the invention for use in the treatment, and/or prophylaxis of a proliferative disease. In a specific embodiment, the proliferative disease is selected from cancer (e.g. solid tumors such as uterine leiomyosarcoma or prostate cancer), leukemia (e.g. AML, ALL or CLL), multiple myeloma and psoriasis.
[00216] In yet another aspect, the present invention provides the compound of the invention, or a pharmaceutical composition comprising the compound of the invention for use in the manufacture of a medicament for the treatment, and/or prophylaxis of a proliferative disease. In a specific embodiment, the proliferative disease is selected from cancer (e.g. solid tumors such as uterine leiomyosarcoma or prostate cancer), leukemia (e.g. AML, ALL or CLL), multiple myeloma and psoriasis.
[00217] In further method of treatment aspects, this invention provides methods of treatment and/or prophylaxis of a mammal susceptible to or afflicted with transplantation rejection, said methods comprising administering an effective condition-treating or condition-preventing amount of one or more of the pharmaceutical compositions or compound of the invention herein described. In a specific embodiment, the transplantation rejection is organ transplant rejection.
[00218] In another aspect the present invention provides a compound of the invention for use in the treatment, and/or prophylaxis of transplantation rejection. In a specific embodiment, the transplantation rejection is organ transplant rejection.
[00219] In yet another aspect, the present invention provides the compound of the invention, or a pharmaceutical composition comprising the compound of the invention for use in the manufacture of a medicament for the treatment and/or prophylaxis of of transplantation rejection. In a specific embodiment, the transplantation rejection is organ transplant rejection.
[00220] In a method of treatment aspect, this invention provides a method of treatment, and/or prophylaxis in a mammal susceptible to or afflicted with diseases involving impairment of cartilage turnover, which method comprises administering a therapeutically effective amount of a compound of the invention, or one or more of the pharmaceutical compositions herein described.
[00221] In another aspect the present invention provides a compound of the invention for use in the treatment, and/or prophylaxis of diseases involving impairment of cartilage turnover.
[00222] In yet another aspect, the present invention provides the compound of the invention, or a pharmaceutical composition comprising the compound of the invention for use in the manufacture of a medicament for the treatment, and/or prophylaxis of diseases involving impairment of cartilage turnover.
[00223] The present invention also provides a method of treatment and/or prophylaxis of congenital cartilage malformations, which method comprises administering an effective amount of one or more of the pharmaceutical compositions or compounds of the invention herein described. [00224] In another aspect the present invention provides a compound of the invention for use in the treatment, and/or prophylaxis of congenital cartilage malformations.
[00225] In yet another aspect, the present invention provides the compound of the invention, or a pharmaceutical composition comprising the compound of the invention for use in the manufacture of a medicament for the treatment, and/or prophylaxis of congenital cartilage malformations.
[00226] In further method of treatment aspects, this invention provides methods of treatment and/or prophylaxis of a mammal susceptible to or afflicted with diseases associated with hypersecretion of IL6, said methods comprising administering an effective condition-treating or condition-preventing amount of one or more of the pharmaceutical compositions or compound of the invention herein described. In a specific embodiment, the disease associated with hypersecretion of IL6 is selected from Castleman's disease and mesangial proliferative glomerulonephritis.
[00227] In another aspect the present invention provides a compound of the invention for use in the treatment, and/or prophylaxis 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 proliferative glomerulonephritis.
[00228] In yet another aspect, the present invention provides the compound of the invention, or a pharmaceutical composition comprising the compound of the invention for use in the manufacture of a medicament for the treatment, and/or prophylaxis 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 proliferative glomerulonephritis.
[00229] In further method of treatment aspects, this invention provides methods of treatment and/or prophylaxis of a mammal susceptible to or afflicted with diseases associated with hypersecretion of interferons, said methods comprising administering an effective condition-treating or condition- preventing amount of one or more of the pharmaceutical compositions or compound of the invention herein described. In a specific embodiment, the disease associated with hypersecretion of interferons is selected from systemic and cutaneous lupus erythematosis, lupus nephritis, dermatomyositis, Sjogren's syndrome, psoriasis, and rheumatoid arthritis.
[00230] In another aspect the present invention provides a compound of the invention for use in the treatment, and/or prophylaxis 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 erythematosis, lupus nephritis, dermatomyositis, Sjogren's syndrome, psoriasis, and rheumatoid arthritis.
[00231] In yet another aspect, the present invention provides the compound of the invention, or a pharmaceutical composition comprising the compound of the invention for use in the manufacture of a medicament for the treatment, and/or prophylaxis 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 erythematosis, lupus nephritis, dermatomyositis, Sjogren's syndrome, psoriasis, and rheumatoid arthritis.
[00232] As a further aspect of the invention there is provided a compound of the invention for use as a pharmaceutical especially in the treatment and/or prophylaxis of the aforementioned conditions and diseases. Also provided herein is the use of the present compounds in the manufacture of a medicament for the treatment and/or prophylaxis of one of the aforementioned conditions and diseases.
[00233] A particular regimen of the present method comprises the administration to a subject suffering from a disease involving inflammation, of an effective amount of a compound of the invention for a period of time sufficient to reduce the level of inflammation in the subject, and preferably terminate the processes responsible for said inflammation. A special embodiment of the method comprises administering of an effective amount of a compound of the invention to a subject patient suffering from or susceptible to the development of rheumatoid arthritis, for a period of time sufficient to reduce or prevent, respectively, inflammation in the joints of said patient, and preferably terminate, the processes responsible for said inflammation.
[00234] A further particular regimen of the present method comprises the administration to a subject suffering from a disease condition characterized by cartilage or joint degradation (e.g. rheumatoid arthritis and/or osteoarthritis) of an effective amount of a compound of the invention for a period of time sufficient to reduce and preferably terminate the self-perpetuating processes responsible for said degradation. A particular embodiment of the method comprises administering of an effective amount of a compound of the invention to a subject patient suffering from or susceptible to the development of osteoarthritis, for a period of time sufficient to reduce or prevent, respectively, cartilage degradation in the joints of said patient, and preferably terminate, the self-perpetuating processes responsible for said degradation. In a particular embodiment said compound may exhibit cartilage anabolic and/or anti- catabolic properties.
[00235] Injection dose levels range from about 0.1 mg/kg/h to at least 10 mg/kg/h, all for from about 1 to about 120 h and especially 24 to 96 h. A preloading bolus of from about 0.1 mg/kg to about 10 mg/kg or more may also be administered to achieve adequate steady state levels. The maximum total dose is not expected to exceed about 2 g/day for a 40 to 80 kg human patient.
[00236] For the prophylaxis and/or treatment of long-term conditions, such as degenerative conditions, the regimen for treatment usually stretches over many months or years so oral dosing is preferred for patient convenience and tolerance. With oral dosing, one to five and especially two to four and typically three oral doses per day are representative regimens. Using these dosing patterns, each dose provides from about 0.01 to about 20 mg/kg of a compound of the invention, with particular doses each providing from about 0.1 to about 10 mg/kg and especially about 1 to about 5 mg/kg.
[00237] Transdermal doses are generally selected to provide similar or lower blood levels than are achieved using injection doses. [00238] When used to prevent the onset of a condition, a compound of the invention will be administered to a patient at risk for developing the condition, typically on the advice and under the supervision of a physician, at the dosage levels described above. Patients at risk for developing a particular condition generally include those that have a family history of the condition, or those who have been identified by genetic testing or screening to be particularly susceptible to developing the condition.
[00239] A compound of the invention can be administered as the sole active agent or it can be administered in combination with other therapeutic agents, including other compounds that demonstrate the same or a similar therapeutic activity and that are determined to safe and efficacious for such combined administration. In a specific embodiment, co-administration of two (or more) agents allows for significantly lower doses of each to be used, thereby reducing the side effects seen.
[00240] In one embodiment, a compound of the invention or a pharmaceutical composition comprising a compound of the invention is administered as a medicament. In a specific embodiment, said pharmaceutical composition additionally comprises a further active ingredient.
[00241] In one embodiment, a compound of the invention is co-administered with another therapeutic agent for the treatment and/or prophylaxis of a disease involving inflammation; particular agents include, but are not limited to, immunoregulatory agents e.g. azathioprine, corticosteroids (e.g. prednisolone or dexamethasone), cyclophosphamide, cyclosporin A, tacrolimus, Mycophenolate Mofetil, muromonab-CD3 (OKT3, e.g. Orthocolone®), ATG, aspirin, acetaminophen, ibuprofen, naproxen, and piroxicam.
[00242] In one embodiment, a compound of the invention is co-administered with another therapeutic agent for the treatment and/or prophylaxis of arthritis (e.g. rheumatoid arthritis); particular agents include but are not limited to analgesics, non-steroidal anti-inflammatory drugs (NSAIDS), steroids, synthetic DMARDS (for example but without limitation methotrexate, leflunomide, sulfasalazine, auranofin, sodium aurothiomalate, penicillamine, chloroquine, hydroxychloroquine, azathioprine, and ciclosporin), and biological DMARDS (for example but without limitation Infliximab, Etanercept, Adalimumab, Rituximab, and Abatacept).
[00243] In one embodiment, a compound of the invention is co-administered with another therapeutic agent for the treatment and/or prophylaxis of proliferative disorders; particular agents include but are not limited to: methotrexate, leukovorin, adriamycin, prenisone, bleomycin, cyclophosphamide, 5- fluorouracil, paclitaxel, docetaxel, vincristine, vinblastine, vinorelbine, doxorubicin, tamoxifen, toremifene, megestrol acetate, anastrozole, goserelin, anti-HER2 monoclonal antibody (e.g. HerceptinTM), capecitabine, raloxifene hydrochloride, EGFR inhibitors (e.g. Iressa®, Tarceva™, Erbitux™), VEGF inhibitors (e.g. Avastin™), proteasome inhibitors (e.g. Velcade™), Glivec® and hsp90 inhibitors (e.g. 17-AAG). Additionally, a compound of the invention may be administered in combination with other therapies including, but not limited to, radiotherapy or surgery. In a specific embodiment the proliferative disorder is selected from cancer, myeloproliferative disease or leukaemia.
[00244] In one embodiment, a compound of the invention is co-administered with another therapeutic agent for the treatment and/or prophylaxis of autoimmune diseases, particular agents include but are not limited to: glucocorticoids, cytostatic agents (e.g. purine analogs), alkylating agents, (e.g nitrogen mustards (cyclophosphamide), nitrosoureas, platinum compounds, and others), antimetabolites (e.g. methotrexate, azathioprine and mercaptopurine), cytotoxic antibiotics (e.g. dactinomycin anthracyclines, mitomycin C, bleomycin, and mithramycin), antibodies (e.g. anti-CD20, anti-CD25 or anti-CD3 (OTK3) monoclonal antibodies, Atgam® and Thymoglobuline®), cyclosporin, tacrolimus, rapamycin (sirolimus), interferons (e.g. IFN-β), TNF binding proteins (e.g. infliximab (Remicade™), etanercept (Enbrel™), or adalimumab (Humira™)), mycophenolate, Fingolimod and Myriocin.
[00245] In one embodiment, a compound of the invention is co-administered with another therapeutic agent for the treatment and/or prophylaxis of transplantation rejection, particular agents include but are not limited to: calcineurin inhibitors (e.g. cyclosporin or tacrolimus (FK506)), mTOR inhibitors (e.g. sirolimus, everolimus), anti-proliferatives (e.g. azathioprine, mycophenolic acid), corticosteroids (e.g. prednisolone, hydrocortisone), Antibodies (e.g. monoclonal anti-IL-2Ra receptor antibodies, basiliximab, daclizumab), polyclonal anti-T-cell antibodies (e.g. anti-thymocyte globulin (ATG), anti- lymphocyte globulin (ALG)).
[00246] In one embodiment, a compound of the invention is co-administered with another therapeutic agent for the treatment and/or prophylaxis of asthma and/or rhinitis and/or COPD, particular agents include but are not limited to: beta2-adrenoceptor agonists (e.g. salbutamol, levalbuterol, terbutaline and bitolterol), epinephrine (inhaled or tablets), anticholinergics (e.g. ipratropium bromide), glucocorticoids (oral or inhaled) Long-acting p2-agonists (e.g. salmeterol, formoterol, bambuterol, and sustained-release oral albutero l) , combinations o f inhaled steroids and long-acting bronchodilators (e.g. fluticasone/salmeterol, budesonide/formoterol), leukotriene antagonists and synthesis inhibitors (e.g. montelukast, zafirlukast and zileuton), inhibitors of mediator release (e.g. cromoglycate and ketotifen), biological regulators of IgE response (e.g. omalizumab), antihistamines (e.g. ceterizine, cinnarizine, fexofenadine) and vasoconstrictors (e.g. oxymethazoline, xylomethazoline, nafazoline and tramazoline).
[00247] Additionally, a compound of the invention may be administered in combination with emergency therapies for asthma and/or COPD, such therapies include oxygen or heliox administration, nebulized salbutamol or terbutaline (optionally combined with an anticholinergic (e.g. ipratropium), systemic steroids (oral or intravenous, e.g. prednisone, prednisolone, methylprednisolone, dexamethasone, or hydrocortisone), intravenous salbutamol, non-specific beta-agonists, injected or inhaled (e.g. epinephrine, isoetharine, isoproterenol, metaproterenol), anticholinergics (IV or nebulized, e.g. glycopyrrolate, atropine, ipratropium), methylxanthines (theophylline, aminophylline, bamiphylline), inhalation anesthetics that have a bronchodilatory effect (e.g. isoflurane, halothane, enflurane), ketamine and intravenous magnesium sulfate. [00248] In one embodiment, a compound of the invention is co-administered with another therapeutic agent for the treatment and/or prophylaxis of inflammatory bowel disease (IBD), particular agents include but are not limited to: glucocorticoids (e.g. prednisone, budesonide) synthetic disease modifying, immunomodulatory agents (e.g. methotrexate, leflunomide, sulfasalazine, mesalazine, azathioprine, 6- mercaptopurine and ciclosporin) and biological disease modifying, immunomodulatory agents (infliximab, adalimumab, rituximab, and abatacept).
[00249] In one embodiment, a compound of the invention is co-administered with another therapeutic agent for the treatment and/or prophylaxis of SLE, particular agents include but are not limited to: Disease-modifying antirheumatic drugs (DMA Ds) such as antimalarials (e.g. plaquenil, hydroxychloroquine), immunosuppressants (e.g. methotrexate and azathioprine), cyclophosphamide and mycophenohc acid; immunosuppressive drugs and analgesics, such as nonsteroidal anti-inflammatory drugs, opiates (e.g. dextropropoxyphene and co-codamol), opioids (e.g. hydrocodone, oxycodone, MS Contin, or methadone) and the fentanyl duragesic transdermal patch.
[00250] In one embodiment, a compound of the invention is co-administered with another therapeutic agent for the treatment and/or prophylaxis of psoriasis, particular agents include but are not limited to: topical treatments such as bath solutions, moisturizers, medicated creams and ointments containing coal tar, dithranol (anthralin), corticosteroids like desoximetasone (Topicort™), fluocinonide, vitamin D3 analogues (for example, calcipotriol), Argan oiland retinoids (etretinate, acitretin, tazarotene), systemic treatments such as methotrexate, cyclosporine, retinoids, tioguanine, hydroxyurea, sulfasalazine, mycophenolate mofetil, azathioprine, tacrolimus, fumaric acid esters or biologies such as Amevive™, Enbrel™, Humira™, Remicade™, Raptiva™ and ustekinumab (a IL-12 and IL-23 blocker). Additionally, a compound of the invention may be administered in combination with other therapies including, but not limited to phototherapy, or photochemotherapy (e.g. psoralen and ultraviolet A phototherapy (PUVA)).
[00251] In one embodiment, a compound of the invention is co-administered with another therapeutic agent for the treatment and/or prophylaxis of allergic reaction, particular agents include but are not limited to: antihistamines (e.g. cetirizine, diphenhydramine, fexofenadine, levocetirizine), glucocorticoids (e.g. prednisone, betamethasone, beclomethasone, dexamethasone), epinephrine, theophylline or anti-leukotrienes (e.g. montelukast or zafirlukast), anti-cholinergics and decongestants.
[00252] By co-administration is included any means of delivering two or more therapeutic agents to the patient as part of the same treatment regime, as will be apparent to the skilled person. Whilst the two or more agents may be administered simultaneously in a single formulation this is not essential. The agents may be administered in different formulations and at different times.
GENERAL SYNTHETIC PROCEDURES
General [00253] A compound of the 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 (i.e. reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given; other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.
[00254] Additionally, as will be apparent to those skilled in the art, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions. The choice of a suitable protecting group for a particular functional group as well as suitable conditions for protection and deprotection are well known in the art. For example, numerous protecting groups, and their introduction and removal, are described in T. W. Greene and P. G. M. Wuts, Protecting Groups in Organic Synthesis, Second Edition, Wiley, New York, 1991, and references cited therein.
[00255] The following methods are presented with details as to the preparation of a compound of the invention as defined hereinabove and the comparative examples. A compound of the invention may be prepared from known or commercially available starting materials and reagents by one skilled in the art of organic synthesis.
[00256] All reagents were of commercial grade and were used as received without further purification, unless otherwise stated. Commercially available anhydrous solvents were used for reactions conducted under inert atmosphere. Reagent grade solvents were used in all other cases, unless otherwise specified. Column chromatography was performed on silica gel 60 (35-70 μιη). Thin layer chromatography was carried out using pre-coated silica gel F-254 plates (thickness 0.25 mm). lH NMR spectra were recorded on a Bruker DPX 400 NMR spectrometer (400 MHz). Chemical shifts (δ) for 1H NMR spectra are reported in parts per million (ppm) relative to tetramethylsilane (δ 0.00) or the appropriate residual solvent peak, i.e. CHCI3 (δ 7.27), as internal reference. Multiplicities are given as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m) and broad (br). Coupling constants (J) are given in Hz. Electrospray MS spectra were obtained on a Micromass platform LC/MS spectrometer. Columns Used for LCMS analysis: Hichrom, Kromasil Eternity, 2.5μιη C18, 150 x 4.6mm, Waters Xbridge 5μιη C18 (2), 250 x 4.6mm (ref 86003117), Waters Xterra MS 5μιη C18, 100 x 4.6mm (Plus guard cartridge) (ref 186000486), Gemini-NX 3 μιη C 1 8 1 00 x 3.0 mm (ref 00D-4453-Y0), Phenomenex Luna 5μιη C18 (2), 100 x 4.6mm. (Plus guard cartridge) (ref 00D-4252-E0), Kinetix fused core 2.7μιη C18 100 x 4.6 mm (ref 00D-4462-E0), Supelco, Ascentis® Express C18 (ref 53829-U), or Hichrom Halo C18, 2.7μιη C18, 150 x 4.6mm (ref 92814-702). LC-MS were recorded on a Waters Micromass ZQ coupled to a HPLC Waters 2795, equipped with a UV detector Waters 2996. LC were also run on a HPLC Agilent 1100 coupled to a UV detector Agilent G1315A. Preparative HPLC: Waters XBridge Prep C18 5μιη ODB 19mm ID x 100mm L (Part No.186002978). All the methods are using MeCN/H20 gradients. H20 contains either 0.1% TFA or 0.1% NH3. [00257] List of abbreviations used in the experimental section:
Figure imgf000055_0002
Figure imgf000055_0001
Glyceraldehyde phosphate High pressure liquid
GAPDH HPLC
dehydrogenase chromatography
h hour NMP N-Methylpyrrolidone
d day SNAr Nucleophilic aromatic substitution mmol millimoles Strong cation exchange silica-
SCX
bound tosic acid
[00258]
Synthetic Preparation of the Compound of the Invention.
Synthesis of intermediates
Intermediate 1 / Intermediate 2
Figure imgf000056_0001
Intermediate 1 Intermediate 2
Step (i): (2-Chloro-5-nitro-pyridin-4-yl)-methyl-amine (Intermediate 1)
[00259] To a solution of 2-chloro-4-methoxy-5-nitro-pyridine (26 mmol) in dry THF (50 mL) at room temperature was added methyl amine (25 mL) (2M in THF). The mixture was allowed to stir for a further 2 h at room temperature. After completion of reaction as seen by TLC and LCMS, solvent was evaporated under reduced pressure to give 5 g of desired Intermediate 1.
[00260] 'H-NMR (400 MHz, DMSO-i¾: δ 2.95 (d, 3H), 7.01 (s, 1H), 8.57 (bs, 1H), 8.86, 1H).
[00261] Mass (M+l): m/z 188.
Step (ii): 6-Chloro-N-methyl-pyridine-3, 4-diamine
[00262] To a stirred solution of intermediate 1 (26 mmol) in acetic acid (100 mL) was added iron powder (9 g, 0.16 mL) at 50°C. The reaction mixture was then heated at 80°C for about 1 h when TLC showed the completion of reaction; it was cooled, filtered and washed with ethyl acetate (3x100 mL). Evaporation of organic layer gave residual mass, which was then neutralized with aq. NaHC03 solution and extracted with ethyl acetate (3x100 mL). Combined organic layers were washed with water (2x100 mL) dried over anhydrous sodium sulphate and concentrated under reduced pressure to give the desired product.
[00263] 'H-NMR (400 MHz, DMSO-i¾: δ 2.74 (d, 3H), 4.66 (s, 2H), 6.25 (s, 1H), 7.36 (s, 1H).
[00264] Mass (M+l): m/z 158. Step (Hi) 6-Chloro-l-methyl-lH-imidazo[4,5-c]pyridine: (intermediate 2)
[00265] To a stirred solution of 6-Chloro-N-methyl-pyridine-3, 4-diamine (22 mmol) in trimethyl orthoformate (25 mL) was added formic acid (1 mL) and was heated ay 100°C for nearly 4 h when TLC showed the completion of reaction. The reaction was allowed to cool to room temperature and water (50 mL) was added and the mixture was extracted with ethyl acetate (4x50 mL), the combined organic layers were washed with aq. NaHC03 solution, dried over anhydrous sodium sulphate and concentration under reduced pressure gave the desired product Intermediate 2.
[00266] 'H-NMR (400 MHz, DMSO-i¼): δ 3.84 (s, 3H), 7.83 (s, 1H), 8.39 (s, 1H), 8.74 (s, 1H).
[00267] Mass (M+l): w/z 168.
Interm
Figure imgf000057_0001
Step i): Pd-mediated coupling
[00268] The (hetero)aryl chloride (6.31 mmol), potassium vinyltrifluoroborate (7.57 mmol), cesium carbonate (18.93 mmol) and bis(diphenylphosphino)ferrocene]dichloropalladium(II) (0.631 mmol) are suspended in THF (50 mL) and water (5 mL) and the stirred reaction mixture is heated to 75°C for 1.5 h. The reaction mixture is filtered through celite and partitioned between diethyl ether and water. The layers are separated and the aqueous layer is extracted with diethyl ether, the organics are combined, dried (MgSO i) and concentrated in vacuo.
Step ii): reduction
[00269] To a stirred solution of the nitro compound (6.31 mmol) in MeOH (30 mL) is added ammonium formate (63.1 mmol) and the reaction mixture is degassed (N2) for 5 min. Pd/C (10% wt, 50% water, 200 mg) is added and the reaction mixture is heated to 75°C under an atmosphere of N2 for 4 h. The reaction mixture is filtered through celite and washed through with DCM and the filtrate washed with water. The aqueous layer is extracted with DCM and the organics are combined, filtered through a hydrophobic filter and concentrated in vacuo. The resulting residue is dissolved in DCM and loaded onto a 20 g SCX column. DCM and MeOH are passed through the column and the target material eluted with 7 N NH3 in MeOH:MeOH (1 :5). The eluent is concentrated in vacuo.
Intermediate 4: 6-Chloro-7-nitro-2H-benzo[b][l,4]oxazin-3(4H)-one.
Figure imgf000058_0001
[00270] To a stirred suspension of potassium fluoride (150 mmol) and ethyl bromoacetate (60 mmol) in DMF (240 mL) was added 2-amino-4-chloro-5-nitrophenol (60 mmol) portionwise over 15 min. After this time, the resulting mixture was heated to 60°C. After 2.5 h, the reaction mixture was cooled to room temperature and poured into ice-water (200 mL). The resulting precipitate was collected and dried in vacuo to give the desired compound.
[00271] Ή NMR δ (ppm)(DMSO-d6): 1 1.31 (1 H, s, NH), 7.79 (1 H, s, ArH), 7.1 1 (1 H, s, ArH), 4.77 (2 H, s, CH2).
General Synthetic Methods for Preparation of the Compounds of Invention
Method A
Figure imgf000058_0002
[00272] The corresponding amine (0.58 mmol) is added to a solution of 6-chloro-l -methyl- 1H- imidazo[4,5-c]pyridine (intermediate 2) (0.45 mmol) and cesium carbonate (0.62 mmol) in dioxane (3 mL). Degassing is done for 5 min, followed by addition of a solution previously sonicated for 10 min of Xantphos (0.06 eq), Pd2(dba)3 (0.03eq) in dioxane (1 mL) under nitrogen. The reaction heated at 110°C for 16 h. After completion, water and DCM are added and this mixture is filtered through a phase separator. The organic layers are concentrated under reduced pressure. The crude product is purified by preparative HPLC to afford the expected compound.
Method A'
Figure imgf000058_0003
[00273] To a solution of 6-chloro-l -methyl- lH-imidazo[4,5-c]pyridine (intermediate 2) (14.9 mmol) in dioxane (100 mL) is added the corresponding aniline (22.4 mmol), cesium carbonate (22.4 mmol), BINAP (0.9mmol) and tris(dibenzylideneacetone)dipalladium (0.45 mmol). The reaction mixture is refluxed for 15 h, filtrated on celite, evaporated to dryness and purified on silica gel to give the expected product.
Method A"
Figure imgf000059_0001
[00274] A mixture of 6-chloro-l-methyl-lH-imidazo[4,5-c]pyridine (intermediate 2) (84 mg, 0.5 mmol), 7-methyl-2,3-dihydrobenzo[6][l,4]dioxin-6-amine ( 1 .5 eq. ) , dib enzylidene ac etone bis(triphenylphiosphine) palladium (0) (5mol.%), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (10 mol.%), sodium tert-butoxide (1.5 eq.) and DMF (0.33 M) is stirred at 120 °C for 16 h. The reaction mixture is allowed to cool to room temperature, filtered and the solvent removed in vacuo. The crude product is purified by preparative HPLC to afford the desired product.
Method B: Synthesis of A^l-substituted lH-imidazo 4,5-c]pyridin-6-amines
Figure imgf000059_0002
Step i)
[00275] A 2 M solution of RaNH2 in THF (3 eq.) is added to a stirred solution of 2-chloro-4-methoxy- 5-nitropyridine (Intermediate 1) (1 eq.) and THF (0.5 M) at 0 °C. The reaction mixture is allowed to warm to room temperature. The reaction mixture is stirred at room temperature for 16 h. The solvent was removed in vacuo. The crude product is purified by column chromatography using silica gel and eluting with 0 - 100% DCM in isohexanes to afford the desired compound.
Step ii)
[00276] A solution of the product obtained in Step i) (1 eq.) in MeOH (0.02 M) is passed through an H-Cube (60°C, 60 Bar, flow rate: 1 mL.min"1) on 5 wt.% ruthenium on carbon. The solution is collected and passed through the H-Cube (under identical conditions) a further two times. The resultant solution is concentrated in vacuo.
Step Hi)
[00277] A mixture of the product obtained in Step ii) (1 eq.), triethylortho formate (20 eq.) and formic acid (1.5 eq.) is stirred at 105 °C for 0.5 h. The reaction mixture is allowed to cool to room temperature and concentrated in vacuo. The crude product is purified by column chromatography using silica gel and eluting with 0 - 100% EtOAc in isohexanes to to afford the desired compound. Step iv)
[00278] A mixture of the product obtained in Step iii) (1 eq.), RbNH2 (1.4 eq.), dibenzylideneacetone bis(triphenylphiosphine) palladium(O) (3 mol.%), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (6 mol.%), cesium carbonate (1.5 eq.) and dioxane (0.2 M) is stirred at 90 °C for 16 h. The reaction mixture is allowed to cool to room temperature. Water is added and the mixture was extracted with EtOAc. The combined organic extracts are washed with sat. NaCl (aq.), dried (MgSO i), filtered and the solvent removed in vacuo. The crude product is purified by preparative HPLC to give the desired compound.
Method C
Figure imgf000060_0001
Step i)
[00279] The aryl bromide (7.4 mmol), ethylboronic acid (0.6 g, 8.14 mmol), potassium carbonate (3.1 g, 22.2 mmol) and [l,l '-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (302 mg, 0.37 mmol) are suspended in dioxane (25 mL) and water (5 mL). The stirred reaction mixture is degassed (N2 (g)) and heated to 100 °C for 1 d. The reaction mixture is filtered through celite and partitioned between EtOAc and water. The layers are separated and the aqueous layer is extracted with EtOAc, the organics are combined, dried (MgSO i) and concentrated in vacuo. The resulting residue is purified by column chromatography using silica gel.
Step ii)
[00280] The nitro-benzene (2.7 mmol), NH4C1 (217 mg, 4.05 mmol) and iron powder (753 mg, 13.5 mmol) are suspended in THF (3 mL), ethanol (3 mL) and water (1 mL). The stirred reaction mixture is heated to 90 °C for 1 h. The reaction mixture is filtered through celite, washed through with DCM and concentrated in vacuo. The resulting residue is partitioned between DCM and sat. aq. NaHCOs solution. The layers are separated and the aqueous layer extracted with DCM, the organics are combined and dried (hydrophobic filter) and concentrated in vacuo. The resulting residue is purified using column chromatography on silica gel.
Method D
Figure imgf000061_0001
Step i): 4-Aryl-2-Ethylaniline
[00281] 4-Bromo-2-ethylaniline (0.5 g, 2.5 mmol), boronic acid or boronate pinacol ester (1.2 equiv., 3 mmol) and cesium carbonate (2.4 g, 7.5 mmol) are suspended in 1,4-dioxane (10 mL) and water (2 rriL) and the reaction mixture is then degassed (N2) fo r 5 min . A ft er wh ic h [ 1 , Γ- bis(diphenylphosphino)ferrocene]dichloropalladium(II) (0.102 g, 0.125 mmol) is added and the reaction mixture is heated to 95°C for 1 d. The reaction is cooled to room temperature and filtered through Celite, washed through with DCM and the organics are washed with water, the layers are separated and the aqueous layer further extracted with DCM. The organics are combined, dried (hydrophobic filter) and concentrated in vacuo. The resulting residue is dissolved in DCM and allowed to load under gravity onto a 10 g SCX column, washed with DCM and MeOH and eluted with 7 N NH3 in MeOH : MeOH (1 :5). The eluent is concentrated in vacuo and the resulting residue is purified using column chromatography on silica gel. The fractions containing product are combined and concentrated in vacuo to give 4-Aryl-2-Ethylaniline.
Step ii): (2-Ethyl-4-aryl-phenyl)-(3-methyl-3H-benzoimidazol-5-yl)-amine
[00282] To stirred degassed (N2) 1,4-dioxane (10 mL) is added 4-Aryl-2-Ethylaniline obtained in step i) (2.00 mmol), 6-chloro- 1 -methyl- lH-imidazo[4,5-c] p yri d i n e ( 3 03 m g , 1 . 8 mm o l ) , tris(dibenzylideneacetone)dipalladium(0) (82 mg, 0.09 mmol), 2-dicyclohexylphosphino-2',4',6'- triisopropylbiphenyl (86 mg, 0.18 mmol) and sodium teri-butoxide (260 mg, 2.7 mmol). The reaction mixture is heated to 100°C for 1 d, cooled to room temperature, filtered through Celite and washed through with DCM. The reaction mixture is washed with water and the layers are separated and the aqueous layer further extracted with DCM. The organics are combined, dried (hydrophobic filter) and concentrated in vacuo and the resulting residue is purified by column chromatography using silica gel. The fractions containing product are combined and concentrated in vacuo to give the final product (2- Ethyl-4-aryl-phenyl)-(3-methyl-3H-benzoimidazol-5-yl)-amine.
Representative Examples for the Preparation of the Compounds of Invention
Compound 29: Synthesis of l-Methyl-N-(7-methyl-2,3-dihydrobenzo[b][l,4]dioxin-i imidazo[4,5-c]pyridin-6-amine.
Figure imgf000062_0001
[00283] A mixture of 6-chloro-l -methyl- lH-imidazo[4,5-c]pyridine (84 mg, 0.5 mmol), 7-methyl- 2,3-dihydrobenzo[6][l,4]dioxin-6-amin e ( 1 24 mg , 0 . 7 5 mm o l ) , d ib e nzy l i d en e ac e t o n e bis(triphenylphiosphine) palladium (0) (23 mg, 0.025 mmol), 4,5-bis(diphenylphosphino)-9,9- dimethylxanthene (29 mg, 0.05 mmol), sodium teri-butoxide (72 mg, 0.75 mmol) and DMF (1.5 mL) was stirred at 120 °C for 16 h. The reaction mixture was allowed to cool to room temperature, filtered and the solvent removed in vacuo. The crude product was purified by preparative HPLC to give the desired compound.
[00284] ¾ NMR δ (ppm)(CHCl3-d): 8.64 (1 H, s, ArH), 7.68 (1 H, s, ArH), 6.93 (1 H, s, ArH), 6.79 (1 H, m, ArH), 6.39 (1 H, s, NH), 6.34 (1 H, s, ArH), 4.26-4.29 (4 H, m, CH2), 3.67 (3 H, s, CH3), 2.17 (3 H, s, CH3).
[00285] LCMS (1 Ocm ESI Bicarb MeCN) tR 2.48 (min) m/z 297 (MH+).
Compound 32: Synthesis of N-(2,3-dihydrobenzo[b][l,4]dioxin-6-yl)-l-ethyl-lH-imidazo[4,5- cJpyridin-6-amin
Figure imgf000062_0002
Step i): 2-Chloro-N-ethyl-5-nitropyridin-4-amine
[00286] A 2 M solution of ethylamine in THF (8 mL, 16 mmol) was added to a stirred solution of 2- chloro-4-methoxy-5-nitropyridine (1.0 g, 5.4 mmol) and THF (10 mL) at 0 °C. The reaction mixture was allowed to warm to room temperature. The reaction mixture was stirred at room temperature for 16 h. The solvent was removed in vacuo. The crude product was purified by column chromatography using silica gel and eluting with 0 - 100% DCM in isohexanes to give the desired compound.
Step ii): 6-Chloro-N*-ethylpyridine-3,4-diamine
[00287] A solution of 2-chloro-N-ethyl-5-nitropyridin-4-amine (310 mg, 1.5 mmol) in MeOH (15 mL) was passed through an H-Cube (60°C, 60 Bar, flow rate: 1 mL.min 1) fitted with a 5 wt.% ruthenium on carbon CatCart® (ThalesNano Nanotechnology Inc, Graphisoft Park, Zahony u. 7., H- 1031 Budapest, Catalogue n# THS 021 12). The solution was collected and passed through the H-Cube (under identical conditions) a further two times. The resultant solution was concentrated in vacuo to give the desired compound.
Step Hi): 6-Chloro-l-ethyl-lH-imidazo[4,5-c] pyridine
[00288] A mixture of 6-chloro-iV4-ethylpyridine-3,4-diamine (352 mg, 2. 1 mmol) , triethyl orthoformate (6.4 mL, 38.4 mmol) and formic acid (113 iL, 2.96 mmol) was stirred at 105 °C for 0.5 h. The reaction mixture was allowed to cool to room temperature and concentrated in vacuo. The crude product was purified by column chromatography using silica gel and eluting with 0 - 100% EtOAc in isohexanes to give the desired compound as a yellow solid.
Step iv): N-(2, 3-Dihydrobenzo[b] [ 1, 4]dioxin-6-yl)-l -ethyl- lH-imidazo[ 4, 5-c]pyridin-6-amine (Compound 32)
[00289] A mixture of 6-chloro-l -ethyl- lH-imidazo[4,5-c]pyridine (86 mg, 0.47 mmol), 2,3- dihydrobenzo[6] [l,4]dioxin-6-am i n e ( 1 0 1 m g , 0 . 6 8 m m o l ) , d i b e n z y l i d e n e a c e t o n e bis(triphenylphiosphine) palladium (0) (13 mg, 0.014 mmol), 4,5-bis(diphenylphosphino)-9,9- dimethylxanthene (16 mg, 0.028 mmol), cesium carbonate (230 mg, 0.71 mmol) and dioxane (2.5 mL) was stirred at 90 °C for 16 h. The reaction mixture was allowed to cool to room temperature. Water (6 mL) was added and the mixture was extracted with EtOAc (4 x 2 mL). The combined organic extracts were washed with sat. NaCl (aq.) (2 mL), dried (MgSO i), filtered and the solvent removed in vacuo. The crude product was purified by preparative HPLC to give the desired compound.
[00290] Ή NMR δ (ppm)(CHCl3-d): 8.68 (1 H, s, ArH), 7.75 (1 H, s, ArH), 6.84-6.90 (2 H, m, ArH), 6.75-6.79 (1 H, m, ArH), 6.71 (1 H, s, ArH), 6.43 (1 H, s, NH), 4.25-4.31 (4 H, m, CH2), 4.08 (2 H, q, CH2), 1.47 (3 H, t, CH3).
[00291] LCMS (1 Ocm ESI Bicarb MeCN) tR 2.54 (min) m/z 297 (MH+).
Compound 33: N-(7-Ethyl-2,3-dihydrobenzo[b][l,4]dioxin-6-yl)-l-methyl-lH4midazo[4,5-c]pyridin-
Figure imgf000063_0001
Step i): 7 -Vinyl-2,3-dihydrobenzo[b] [1 ,4] dioxin-6-amine
[00292] CH3PPh3Br (1.51 g, 4.23 mmol) was suspended in THF at 0 °C under an atmosphere of N2. NaHMDS (1 M in THF, 4.23 mL, 4.23 mmol) was added and the mixture allowed to warm to room temperature over 1 h. ((7-Nitro-2,3-dihydrobenzo[^][l,4]dioxine-6-carbaldehyde (0.68 g, 3.25 mmol) was added and the reaction mixture stirred at room temperature for 1 d. The reaction mixture was concentrated in vacuo and partitioned between DCM and water. The aqueous phase was extracted with DCM and the combined organics were dried (MgSO i), filtered and concentrated in vacuo. The crude product was purified by column chromatography using silica gel and eluting with 0 - 20% EtOAc in isohexanes to give the desired compound.
Step ii): 7-Ethyl-2,3-dihydrobenzo[b] [1 ,4] dioxin-6-amine
[00293] 7-Vinyl-2,3-dihydrobenzo[^][l,4]dioxin-6-amine (53 mg, 0.26 mmol) in MeOH (10 mL) was passed through an H-Cube (ThalesNano Nanotechnology Inc, Graphisoft Park, Zahony u. 7., H-1031 Budapest) (60°C, 60 Bar, flow rate: 1 mLmin-1) fitted with a 10 wt.% palladium on carbon CatCart® (ThalesNano Nanotechnology Inc, Graphisoft Park, Zahony u. 7., H-1031 Budapest, Catalogue n# THS 02112). The solution was passed through the H-Cube (40°C, 60 Bar, flow rate: 1 mL.min"1) twice more. The solution was concentrated in vacuo to give the desired compound.
Step Hi): N-(7-Ethyl-2, 3-dihydrobenzo[b] [ 1, 4] dioxin-6-yl)-l -methyl- lH-imidazo[ 4, 5-c]pyridin-6-amine (Compound 33)
[00294] A mixture of 6-chloro-l-methyl-lH-imidazo[4,5-c]pyridine (100 mg, 0.60 mmol), 7-ethyl- 2,3-dihydrobenzo[6][l,4]dioxin-6-amine (160 mg, 0.90 mmol), tris(dibenzylideneacetone)- dipalladium(O) (27 mg, 0.03 mmol), sodium tert-butoxide (86 mg, 0.90 mmol), 4,5- bis(diphenylphosphino)-9,9-dimethylxanthene (34 mg, 0.06 mmol) and dioxane (5 mL) was stirred at 90 °C for 16 h. The reaction mixture was allowed to cool to room temperature, filtered and concentrated in vacuo. The crude product was purified by column chromatography using silica gel and eluting with 0 - 10% MeOH in DCM to give the desired compound.
[00295] Ή NMR δ (ppm)(DMSO-d6): 8.43 (1 H, d, ArH), 7.98 (1 H, s, ArH), 7.69 (1 H, s, ArH), 6.88 (1 H, s, ArH), 6.73 (1 H, s, NH), 6.44 (1 H, d, ArH), 4.22 (4 H, s, CH2), 3.65 (3 H, s, CH3), 2.56- 2.50 (2 H, q, CH2), 1.06 (3 H, t, CH3).
[00296] LCMS (15cm_Formic_ASCENTIS_HPLC_CH3CN) tR 7.32 (min) m/z 311 (MH+).
Compound 38: N-(2-Ethyl-4-fluorophenyl)-l-methyl-lH-imidazo[4, 5-c]pyridin-6-amine
Figure imgf000064_0001
Step i): 2-Ethyl-4-fluoro-l -nitrobenzene
[00297] 2-Bromo-4-fluoro-l -nitrobenzene (1.63 g, 7.4 mmol), ethylboronic acid (0.6 g, 8.14 mmol), potassium carbonate (3.1 g, 22.2 mmol) and [l,l'-bis(diphenylphosphino)ferrocene]- dichloropalladium(II) (302 mg, 0.37 mmol) were suspended in dioxane (25 mL) and water (5 mL). The stirred reaction mixture was degassed (N2 (g)) and heated to 100 °C for 1 d. The reaction mixture was filtered through celite and partitioned between EtOAc and water. The layers were separated and the aqueous layer was extracted with EtOAc, the organics were combined, dried (MgSO i) and concentrated in vacuo. The resulting residue was purified by column chromatography using silica gel and eluting with 0-50% EtOAc in isohexanes to give the desired compound.
Step ii): 2-Ethyl-4-fluoroaniline
[00298] 2-Ethyl-4-fluoro-l -nitrobenzene (460 mg, 2.7 mmol), NH4C1 (217 mg, 4.05 mmol) and iron powder (753 mg, 13.5 mmol) were suspended in THF (3 mL), ethanol (3 mL) and water (1 mL). The stirred reaction mixture was heated to 90 °C for 1 h. The reaction mixture was filtered through celite, washed through with DCM and concentrated in vacuo. The resulting residue was partitioned between DCM and sat. aq. NaHC03 solution. The layers were separated and the aqueous layer extracted with DCM, the organics were combined, filtered through a hydrophobic filter and then concentrated in vacuo. The resulting residue was purified using column chromatography on silica gel and eluting with 0-25% EtOAc in isohexanes to give the desired compound.
[00299] Ή NMR δ (ppm)(DMSO-d6): 6.77-6.67 (2 H, m, ArH), 6.64-6.53 (1 H, m, ArH), 4.69 (2 H, s, NH2), 2.47-2.37 (2 H, m, CH2), 1.19-1.07 (3 H, m, CH3).
Step Hi): N-(2-Ethyl-4-fluorophenyl)-l-methyl-lH-imidazo[4,5-cJpyridin-6-amine (Compound 38)
[00300] To stirred degassed (N2 (g)) dioxane (3 mL) was added 6-chloro-l-methyl-lH-imidazo[4,5- c]pyridine (219 mg, 1.31 mmol), 2-ethyl-4-fluoroaniline (200 mg, 1.44 mmol), sodium-tert-butoxide (189 mg, 1.97 mmol), tris(dibenzylideneacetone)dipalladium(0) (60 mg, 0.066 mmol) and 4,5- bis(diphenylphosphino)-9,9-dimethylxanthene (76 mg, 0.131 mmol). The reaction mixture was heated to 90°C for 1 d. The reaction mixture was filtered through celite, washed through with EtOAc and partitioned between EtOAc and water. The layers were separated and the aqueous layer extracted with EtOAc, the organics were combined and dried (MgSO i), filtered and concentrated in vacuo to give the desired compound. For analytical purpose, a 100 mg sample was purified by preparative HPLC to give the desired compound.
[00301] Ή NMR δ (ppm)(DMSO-d6): 8.45 (1 H, s, NH), 8.01 (1 H, s, ArH), 7.87 (1 H, s, ArH), 7.46- 7.39 (1 H, m, ArH), 7.10-7.05 (1 H, m, ArH), 7.03-6.96 (1 H, m, ArH), 6.55 (1 H, d, ArH), 3.67 (3 H, s, CH3), 2.67-2.57 (2 H, m, CH2), 1.16-1.07 (3 H, m, CH3).
[00302] LCMS (lOcm ESCI Bicarb MeCN) tR 2.80 (min) m/z 271 (MH+).
Compound 41: [4-(4-Methoxy-benzyloxy)-2-methyl^henyl]-(l-methyl-lH-imidazo[4,5-c]pyridin-6- yl)-amine
Figure imgf000066_0001
Step i): 4-(4-Methoxybenzyloxy)-2-methylaniline
[00303] l-(Chloromethyl)-4-methoxybenzene (17 g, 0.1 mol) was added to a stirred suspension of caesium carbonate (48.9 g, 0.15 mol) and 4-amino-3-methylphenol (12.2 g, 0.1 mol) in DMF (200 mL) and the resulting suspension was stirred at room temperature for 1 d. The mixture was concentrated in vacuo and the residue was treated with water and extracted with DCM. The combined extracts were dried (MgSO i), filtered and concentrated in vacuo to give a brown oil which was purified by column chromatography using silica gel (40% to 75% EtOAc in isohexanes) to give the desired compound.
[00304] Ή NMR δ (ppm)(DMSO-d6): 7.32 (2 H, d, ArH), 6.97-6.89 (2 H, m, ArH), 6.64 (1 H, d, ArH), 6.61-6.50 (2 H, m, ArH), 4.85 (2 H, s, CH2), 4.36 (2 H, s, NH2), 3.75 (3 H, s, CH3), 2.03 (3 H, s, CH3).
Step ii): N-(4-(4-Methoxybenzyloxy)-2-methylphenyl)-l -methyl- lH-imidazo[ 4, 5-c]pyridin-6-amine (Compound 41)
[00305] 6-Chloro-l-methyl-lH-imidazo[4,5-c]pyridine (2.52 g, 0.015 mol) was stirred with 4,5- bis(diphenylphosphino)-9,9-dimethylxanthene (0.86 g, 0.0015 mol), sodium-tert-butoxide (2.16 g, 0.0225 mol) and 4-(4-methoxybenzyloxy)-2-methylaniline (3.68 g, 0.015 mol) in dry dioxane (50 mL) under N2 (g) for 30 minutes. Tris(dibenzylideneacetone)dipalladium(0) (0.68 g, 0.00075 mol) was added and the mixture was stirred at 100°C for 4.5 h. The mixture was concentrated in vacuo and the residue was treated with water (50 mL) and extracted into EtOAc (2 x 300 mL). The combined extracts were washed with brine, dried (MgSO i), filtered and concentrated in vacuo to give a solid which was recrystalised from isopropanol to give the desired compound.
[00306] Ή NMR δ (ppm)(DMSO-d6): 8.43 (1 Η, d, ArH), 7.98 (1 H, s, NH), 7.75 (1 H, s, ArH), 7.39 (2 H, d, ArH), 7.27 (1 H, d, ArH), 7.00-6.90 (3 H, m, ArH), 6.82 (1 H, dd, ArH), 6.38 (1 H, d, ArH), 5.00 (2 H, s, CH), 3.77 (3 H, s, CH3), 3.64 (3 H, s, CH3), 2.17 (3 H, s, CH3).
Compound 49: 6-Chloro-7-((l-methyl-lH-imidazo[4,5-c]pyridin-6-yl)amino)-2H-benzo[b][l,4]oxazin- 3(4H)-one
Figure imgf000066_0002
[00307] To a stirred solution of degassed dioxane (2 mL) was added 6-chloro-l -methyl- 1H- imidazo[4,5-c]pyridine (300 mg, 1.78 mmol), 7-amino-6-chloro-2H-benzo[6][l,4]oxazin-3(4H)-one (426 mg, 2.13 mmol), tris(dibenzylideneacetone)dipalladium(0) (81 mg, 0.089 mmol), 2- dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (170 mg, 0.356 mmol) and sodium teri-butoxide (258 mg, 2.67 mmol) and the resulting mixture was heated to 90°C for 6 h. The reaction mixture was cooled to room temperature, filtered through a PL-Thiol MP SPE+ column (pre-conditioned with 1 mL MeOH) using MeOH (3 x 2 mL) to wash the column. The combined organics were concentrated in vacuo and the resulting residue was purified by preparative HPLC to give the desired compound.
[00308] Ή NMR δ (ppm)(DMSO-d6): 10.72 (1 H, s, NH), 8.57 (1 H, d, ArH), 8.14 (1 H, s, ArH), 8.06 (1 H, s, NH), 7.65 (1 H, s, ArH), 7.03 (1 H, d, ArH), 6.98 (1 H, s, ArH), 4.61 (2 H, s, CH2), 3.76 (3 H, s, CH3).
[00309] LCMS (1 Ocm ESI Bicarb MeCN) tR 2.62 (min) m/z 330 (MH+).
Compound 52: 4-(4-Methoxybenzyl)-7-(l-methyl-lH midazo[4,5-c]pyridin-6-ylamino)-2H- benzo[b][l,4]oxazin-3(4H)-one
Figure imgf000067_0001
Step i): 4-(4-Methoxybenzyl)-7-nitro-2H-benzo fbj '[ 1, 4Joxazin-3(4H)-one
[00310] l-(Chloromethyl)-4-methoxybenzene (3.12 g, 0.02 mol) was added to a stirred suspension of caesium carbonate (9.78 g, 0.03 mol) plus 7-nitro-2H-benzo[6][l,4]oxazin-3(4H)-one (3.88 g, 0.02 mol) in DMF (100 mL) and the resulting suspension was stirred at room temperature for 7 h. The mixture was concentrated in vacuo and the residue was stirred with water (50 mL) and isohexanes (25 mL) and filtered to give the desired compound.
[00311] Ή NMR δ (ppm)(DMSO-d6): 7.87 (1 Η, dd, ArH), 7.81 (1 H, d, ArH), 7.31-7.20 (3 H, m, ArH), 6.94-6.86 (2 H, m, ArH), 5.17 (2 H, s, CH), 4.95 (2 H, s, CH), 3.72 (3 H, s, CH3).
Step ii): 7-Amino-4-(4-methoxybenzyl)-2H-benzo[b] [ l,4Joxazin-3(4H)-one
[00312] Iron powder (5.46 g, 0.095 mol) was added to a stirred suspension of ammonium chloride (1.52 g, 0.028 mol) plus 4-(4-methoxybenzyl)-7-nitro-2H-benzo[^][l,4]oxazin-3(4H)-one (6.15 g, 0.019 mol) in a mixture of THF (22 mL), ethanol (22 mL) and water (10 mL). The resulting suspension was stirred at 90 °C for 2.5 h and cooled to room temperature. Methanol (300 mL) was added and the mixture was filtered through cellite, the filtrate was concentrated to low volume in vacuo and the resulting solid was filtered and washed with water to give the desired compound. [00313] Ή NMR δ (ppm)(DMSO-d6): 7.18 (2 H, d, ArH), 6.87 (2 H, d, ArH), 6.73 (1 H, d, ArH), 6.21 (1 H, d, ArH), 6.14 (1 H, dd, ArH), 4.98 (4 H, m, 2CH, NH2), 4.62 (2 H, s, CH), 3.71 (3 H, s, CH3).
Step Hi): 4-(4-Methoxybenzyl)-7-(l-methyl-lH-imidazo[4,5-c]pyridin-6-ylamino)-2H- benzofbjf 1, 4] oxazin-3(4H)-one
[00314] 6-Chloro-l-methyl-lH-imidazo[4,5-c]pyridine (0.835 g, 5 mmol) was stirred with 4,5- bis(diphenylphosphino)-9,9-dimethylxanthene (0.29 g, 5 mmol), sodium fert-butoxide (0.73 g, 7.5 mol) and 7-amino-4-(4-methoxybenzyl)-2H-benzo[^][l,4]oxazin-3(4H)-one (1 .42 g, 5 mmol) in degassed dioxane (40 mL) under N2 (g) for 30 min. Tris(dibenzylideneacetone)dipalladium(0) (0.23 g, 0.25 mmol) was added and the reaction was stirred at 100 °C for 5 h. The mixture was concentrated in vacuo and the residue was treated with water (50 mL) and extracted into EtOAc (2 x 200 mL). The combined extracts were washed with brine, dried (MgSO i), filtered and concentrated in vacuo to give the desired compound. For analytical purpose, a sample of this material (100 mg) was purified by preparative HPLC to give the desired compound.
[00315] Ή NMR δ (ppm)(DMSO-d6): 8.85 (1 H, s, NH), 8.55 (1 H, d, ArH), 8.08 (1 H, s, ArH), 7.43 (1 H, d, ArH), 7.23 (2 H, d, ArH), 7.09 (1 H, dd, ArH), 6.98-6.83 (3 H, m, ArH), 6.83 (1 H, d, ArH), 5.06 (2 H, s, CH2), 4.73 (2 H, s, CH2), 3.72 (6 H, d, CH3).
[00316] LCMS (lOcm ESCI Bicarb MeCN) tR 3.16 (min) m/z 416 (MH+).
Com ound 59: 4-ethyl-N,N-dimethyl-3-(l-methyl-lH-imidazo[4,5-c]pyridin-6-ylamino)benzamide
Figure imgf000068_0001
Step i) 3-Nitro-4-vinylbenzoic acid
[00317] A mixture of 4-chloro-3-nitrobenzoic acid (5 g, 0.0248 mol), potassium vinyl trifluoroborate (3.67 g, 0.0273 mol) and cesium carbonate (20 g, 0.062 mol) was stirred in DMF (100 mL) and water (20 mL) under N2 for 20 min. At this point, l,l '-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (2.2 g, 2.69 mmol) was added and the mixture was stirred at 100°C for 18 h. The solvent was removed in vacuo and the residue was dissolved in water and filtered through Celite. The filtrate was acidified (pH 1) with 2 M HC1 (aqueous) and the resulting solid was filtered, washed with water and dried in a vacuum oven to give the desired compound.
[00318] Ή NMR δ (ppm)(DMSO-d6): 13.62 (1 H, s, OH), 8.40 (1 H, d, ArH), 8.20 (1 H, dd, ArH), 7.95 (1 H, d, ArH), 7.07 (1 H, m, CH), 6.04 (1 H, d, CH), 5.69-5.60 (1 H, m, CH).
Step ii) 3-Amino-4-ethylbenzoic acid
[00319] Palladium on carbon (10% wt, 1.5 g) was added to a solution of 3-nitro-4-vinylbenzoic acid (1.2 g , 0.0062 mol) and ammonium formate (3.78 g, 0.06 mol) in methanol (20 mL) under N2. The mixture was gently refluxed for 2 h and the cooled reaction mixture was filtered through Celite. The filtrate was concentrated in vacuo and the residue was treated with water (4 mL), filtered and dried to give the desired compound.
[00320] Ή NMR δ (ppm)(DMSO-d6): 12.40 (1 H, s, OH), 7.23 (1 H, d, ArH), 7.09 (1 H, dd, ArH), 7.01 (1 H, d, ArH), 5.07 (2 H, s, NH2), 2.53-2.44 (2 H, q, CH2), 1.13 (3 H, t, CH3).
Step Hi) 3-Amino-4-ethyl-N,N-dimethylbenzamide
[00321] Dimethylamine (1.5 mL of a 2 M solution in methanol, 0.003 mol) was added to a stirred solution of 3-amino-4-ethylbenzoic acid (0.247 g, 0.0015 mol), Et3N (0.45 g, 0.0045 mol) and (2-(7-aza- lH-benzotriazole-l -yl)-l,l,3,3-tetramethyluronium hexafluorophosphate) (0.627 g, 0.00165 mol) in DMF (5 mL). After stirring for 6 h at room temperature the reaction mixture was concentrated in vacuo and the residue was dissolved in EtOAc and washed with saturated aqueous sodium carbonate solution. The organics were dried (MgSO i) and concentrated in vacuo. The residue was dissolved in methanol and loaded onto a 10 g SCX column. Methanol was washed through and the compound was eluted with 7 N NH3 in methanol : methanol (1 :4). The eluent was concentrated in vacuo to give the desired compound which was used in the next step without further purification.
[00322] Ή NMR δ (ppm)(DMSO-d6): 6.95 (1 H, d, ArH), 6.61 (1 H, d, ArH), 6.49 (1 H, dd, ArH), 4.99 (2 H, s, NH2), 3.07-2.74 (6 H, m, CH3), 2.49-2.40 (2 H, m, CH), 1.13 (3 H, m, CH3).
Step iv) 4-Ethyl-N,N-dimethyl-3-(l -methyl- lH-imidazo[ 4, 5-c]pyridin-6-ylamino)benzamide
[00323] A mixture of 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (0.08 g), 6-chloro-l-methyl- lH-imidazo[4,5-c]pyridine (0.23 g, 0.0014 mol), 3-amino-4-ethyl-N,N-dimethylbenzamide (0.27 g, 0.0014 mol) and sodium tert-butoxide (0.20 g, 0.0021 mol) in 1,4-dioxane (15 mL) was stirred under N2 for 20 min. Tris(dibenzylideneacetone)dipalladium(0) (0.06 g) was added and the mixture was stirred at 100°C for 7 h. The reaction mixture was concentrated in vacuo and the residue was treated with water and extracted into EtOAc. The organics were washed with brine, dried (MgSO i) and concentrated in vacuo to give the desired compound. For analytical purpose, a 60 mg sample was further purified by preparative HPLC to give the desired compound.
[00324] Ή NMR δ (ppm)(CHCl3-d): 8.69 (1 H, d, ArH), 7.71 (1 H, s, ArH), 7.52 (1 H, d, ArH), 7.32 (1 H, m, ArH), 7.16 (1 H, dd, ArH), 6.63 (1 H, d, ArH), 6.36 (1 H, s, NH), 3.68 (3 H, s, CH3), 3.09 (3 H, s, CH3), 3.03 (3 H, s, CH3), 2.69 (2 H, q, CH2), 1.24 (3 H, t, CH3).
[00325] LCMS (lOcm ESCI Formic MeCN) tR 2.21 (min) m/z 324 (MH+).
Figure imgf000070_0001
Step i) (4-Ethyl-3-nitrophenyl)methanol
[00326] A solution of 4-ethyl-3-nitrobenzaldehyde (1.07 g, 0.006 mol) in methanol (15 mL) was treated with sodium borohydride (0.25 g, 0.0066 mol) and the resulting solution was stirred at room temperature for 1.5 h. The mixture was concentrated in vacuo and the residue was treated with water (20 mL) and extracted with DCM. The organics were dried (MgSO i) and concentrated in vacuo to give the desired compound.
[00327] Ή NMR δ (ppm)(CHCl3-d): 7.98-7.88 (1 H, m, ArH), 7.61-7.52 (1 H, m, ArH), 7.38 (1 H, d, ArH), 4.77 (2 H, d, CH2), 2.93 (2 H, q, CH2), 1.90 (1 H, t, OH), 1.38-1.26 (3 H, m, CH3).
Step ii) (3-Amino-4-ethylphenyl)methanol
[00328] Palladium-on-carbon (10% wt, 1.4 g) was added to a stirred solution of (4-ethyl-3- nitrophenyl)methanol (1.05 g, 0.0058 mol) and ammonium formate (2.9 g, 0.046 mol) in methanol (20 mL) under N2. The mixture was gently refluxed for 8 h before filtering through Celite and concentrating in vacuo. The residue was triturated with water (5 mL), filtered and dried to give the desired compound.
[00329] Ή NMR δ (ppm)(DMSO-d6): 6.93-6.81 (1 H, m, ArH), 6.58 (1 H, s, ArH), 6.53-6.38 (1 H, m, ArH), 4.90 (1 H, s, OH), 4.74 (2 H, s, NH2), 4.32 (2 H, s, CH2), 2.41 (2 H, q, CH2), 1.17-1.04 (3 H, m, CH3).
Step Hi): (4-ethyl-3-(l-methyl-lH-imidazo[4,5-cJpyridin-6-ylamino)phenyl)methanol
[00330] (4-ethyl-3-(l-methyl-lH-imidazo[4,5-c]pyridin-6-ylamino)phenyl)methanol was obtained by reacting (3-Amino-4-ethylphenyl)methanol with 6-chloro-l-methyl-lH-imidazo[4,5-c]pyridine according to method A" described above. Compound 64: Methyl 4-ethyl-3-(methyl(l-methyl-lH-imidazo[4, 5-c]pyridin-6-yl)amino)benzoate
Figure imgf000071_0001
Step i): 4-Ethyl-3-nitrobenzoic acid
[00331] 4-Ethyl-3-nitrobenzaldehyde (8.95 g, 0.05 mol) was added to a stirred solution of potassium permanganate (15.8 g, 0.1 mol) and sodium hydroxide (5 g, 0.0125 mol) in water (250 mL) at rt. The mixture was stirred under reflux for 2.5 h. The cooled mixture was filtered through Celite and the filtrate was acidified (pH 1) with 2 M HCl (aqueous). The resulting suspension was extracted with DCM and the organics were dried (MgSO i) and concentrated in vacuo to give the desired compound.
[00332] Ή NMR δ (ppm)(DMSO-d6): 13.52 (1 H, s, OH), 8.41-8.32 (1 H, m, ArH), 8.25-8.14 (1 H, m, ArH), 7.74-7.63 (1 H, m, ArH), 2.89 (2 H, q, CH2), 1.23 (3 H, t, CH3).
Step ii): Methyl 4-ethyl-3-nitrobenzoate
[00333] Acetyl chloride (1 mL) was added to methanol (75 mL) and the resulting solution was stirred at room temperature for 10 min. 4-Ethyl-3-nitrobenzoic acid (4.73 g) was added and the resulting cloudy solution was refluxed for 6 h. The reaction mixture was concentrated in vacuo and the residue was dissolved in diethyl ether and washed with saturated aqueous sodium bicarbonate solution. The organics were concentrated in vacuo to give the desired compound.
[00334] Ή NMR δ (ppm)(CHCl3-d): 8.57-8.50 (1 H, m, ArH), 8.41-8.16 (1 H, m, ArH), 7.49 (1 H, dd, ArH), 4.18-3.77 (3 H, s, CH3), 2.97 (2 H, q, CH2), 1.32 (3 H, t, CH3).
Step Hi) Methyl 3-amino-4-ethylbenzoate
[00335] Palladium-on-carbon (10% wt., 4.9 g) was added portionwise to a stirred solution of ammonium formate (10.2 g) and methyl 4-ethyl-3-nitrobenzoate (4.2 g, 0.021 mol) in methanol (80 mL) at room temperature under N2. The reaction mixture was stirred at gentle reflux for 1.5 h and cooled to rt. The reaction mixture was filtered through Celite and the filtrate was concentrated in vacuo. The residue was treated with water (20 mL) and extracted into ethyl acetate. The organics were dried (MgSO i) and concentrated in vacuo to give the desired compound. [00336] Ή NMR δ (ppm)(CHCl3-d): 7.43-7.39 (1 H, m, ArH), 7.27-7.26 (1 H, d, ArH), 7.21-7.07 (1 H, m, ArH), 4.09-3.68 (3 H, m, CH3), 3.72 (2 H, s, NH2), 2.62-2.50 (2 H, m, CH2), 1.29-1.23 (3 H, m, CH3).
Step iv) Methyl 4-ethyl-3-(methyl(l-methyl-lH-imidazo[4,5-cJpyridin-6-yl)amino)benzoate
[00337] 6-Chloro-l-methyl-lH-imidazo[4,5-c]pyridine (1.67 g, 0.01 mol), methyl 3-amino-4- ethylbenzoate (2.15 g, 0.012 mol) and caesium carbonate (4.56 g, 0.014 mol) were stirred together in tert-butanol (20 mL) under N2 for 20 min. Chloro[2-(dicyclohexylphosphino)-3,6-dimethoxy-2'-4'-6'- tri-i-propyl-l,l'-biphenyl][2-(2-aminoethyl)phenyl] palladium(II) (BrettPhos Palladacycle) (0.079 g, 1 mol%) and 2-dicyclohexylphosphino-2',6'-di-iso-propoxy-l,l'-biphenyl (RuPhos) (0.046 g, 1 mol%) were added and the mixture was refluxed for 5 h. The reaction mixture was evaporated in vacuo and the residue was treated with water and extracted into EtOAc. The organics were dried (MgSO i) and concentrated in vacuo to give an oily solid. The residue was dissolved in methanol and loaded onto a 70 g SCX column which was washed with methanol (150 mL) before eluting the product with 7 N N¾ in methanol : methanol (1 :4). The eluent was concentrated in vacuo and the residue was purified by flash chromatography (DCM to 5% methanol in DCM) to give the desired compound.
[00338] Ή NMR δ (ppm)(DMSO-d6): 8.51 (1 Η, s, ArH), 8.16 (1 H, d, ArH), 8.07 (2 H, d, ArH, NH), 7.58 (1 H, dd, ArH), 7.35 (1 H, d, ArH), 6.83 (1 H, s, ArH), 3.96-3.72 (3 H, m, CH3), 3.72 (3 H, s, CH3), 2.71 (2 H, q, CH2), 1.17 (3 H, t, CH3).
[00339] LCMS (1 Ocm ESCI Bicarb MeCN) tR 2.77 (min) m/z 311 (MH+).
Compound 72: N-(2-Ethyl-4-(l,2,3,6^etrahydropyridin-4-yl)phenyl)-N,l-dimethyl-lH-imidazo[4,5- cJpyridin-6-amine
Figure imgf000072_0001
Step i): tert-Butyl 4-(4-amino-3-ethylphenyl)-5,6-dihydropyridine-l(2H)-carboxylate
[00340] 4-Bromo-2-ethylaniline (4.96 mL, 35.0 mmol), tert-butyl 4-(4,4,5,5-tetramethyl-l,3,2- dioxaborolan-2-yl)-5,6-dihydropyridine-l (2H)-c a r b o x y l a t e ( 1 3 g , 4 2 . 0 m m o l ) , [ 1 , 1'- bis(diphenylphosphino)ferrocene]dichloropalladium(II) (1.43 g, 1.75 mmol) and cesium carbonate (34.2 g, 105 mmol) were heated at reflux in 1,4-dioxane (180 mL) and water (20 mL) for 18 h. The reaction mixture was cooled to room temperature and filtered through Celite, washed through with DCM. and the organics were washed with water, dried (MgSO i), filtered and concentrated in vacuo. The resulting residue was purified using column chromatography on silica gel and eluting with 10 - 20% EtOAc in isohexanes to give the desired compound.
Ή NMR δ (ppm)(DMSO-d6): 7.02-6.96 (2 H, m, ArH), 6.56 (1 H, d, ArH), 5.88 (1 H, s, CH), 4.89 (2 H, s, NH2), 3.94 (2 H, s, CH), 3.53-3.47 (2 H, m, CH), 2.44 (2 H, q, CH2), 2.38 (2 H, s, CH), 1.67-1.18 (9 H, m, CH3), 1.13 (3 H, t, CH3).
Step ii): tert-Butyl 4-(3-ethyl-4-((l-methyl-lH-imidazo[ 4,5-c]pyridin-6-yl)amino)phenyl)-5, 6- dihydropyridine-l(2H)-carboxylate
[00341] To stirred degassed (N2) 1,4-dioxane (230 mL) was added tert-butyl 4-(4-amino-3- ethylphenyl)-5,6-dihydropyridine-l(2H)-carboxylate (8.25 g, 27.3 mmol), 6-chloro-l -methyl- 1H- imidazo[4,5-c]pyridine (4.15 g, 24.8 mmol), tris(dibenzylideneacetone)dipalladium(0) (1.25 g, 1.37 mmol), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (1.30 g, 2.73 mmol) and sodium tert- butoxide (3.94 g, 30 mmol). The reaction mixture was heated to 100°C for 1.5 h, cooled to room temperature, filtered through Celite and washed through with DCM. The filtrate was washed with water, dried (MgSO i), filtered and concentrated in vacuo and the resulting residue was purified by column chromatography using silica gel and eluting with 0-3%> MeOH in DCM. The fractions containing product were combined and concentrated in vacuo to give the desired compound.
Ή NMR δ (ppm)(DMSO-d6): 8.48 (1 H, d, NH), 8.04 (1 H, s, ArH), 7.89 (1 H, s, ArH), 7.56-7.49 (1 H, m, ArH), 7.30 (1 H, d, ArH), 7.23 (1 H, dd, ArH), 6.74-6.71 (1 H, m, ArH), 6.10 (1 H, s, CH), 4.00 (2 H, s, CH), 3.73-3.63 (3 H, m, CH3), 3.58-3.52 (2 H, m, CH), 2.70-2.59 (2 H, m, CH), 2.53-2.46 (2 H, s, CH), 1.55-1.35 (9 H, m, CH3), 1.18-1.09 (3 H, m, CH3).
Compound 75: N-(4-Ethyl-6-(thiophen-2-yl)pyridin-3-yl)-N,l-dimethyl-lH midazo[4,5-c]pyrM amine
Figure imgf000073_0001
Step 1: 4-Ethyl-6-(thiophen-2-yl)pyridin-3-amine
[00343] A stirred mixture of 6-bromo-4-ethylpyridin-3-amine (200 mg, 1 mmol), 2-thiophene boronic acid (140 mg, 1.1 mmol), tetrakis(triphenylphosphine)palladiun(0) (25 mg) and sodium carbonate (1.5 mL of a 2 M solution in water) in dimethoxyethane (3 mL) was heated at 120°C for 30 min using microwave irradiation. The reaction mixture was cooled to room temperature and EtOAc and water were added. The aqueous was extracted with EtOAc, the combined organics were dried (MgSO^, filtered and concentrated in vacuo. The residue was purified by flash chromatography (0 to 10% 7N NH3 in methanol in EtOAc) to give the desired compound.
Step 2: N-(4-Ethyl-6-(thiophen-2-yl)pyridin-3-yl)-l-methyl-lH-imidazo[ 4, 5-c]pyridin-6-amine
[00344] A stirred mixture of 4-ethyl-6-(thiophen-2-yl)pyridin-3-amine (0.15 g, 0.71 mmol), 6-chloro- 1 -methyl- lH-imidazo[4,5-c]pyridine (0.12 g, 0.71 mmol), sodium tert-butoxide ( 175 mg), tris(dibenzylideneacetone)dipalladium(0) (23 mg) and 2,2'-bis(diphenylphosphino)-l,l'-binaphthyl (22 mg) was heated in 1,4-dioxane (5 mL) at 100°C in a sealed tube for 1 d. The mixture was evaporated in vacuo and the residue was partitioned between DCM and water. The organic phase was separated and the aqueous phase extracted with DCM (x 3), the combined organic phases were dried (MgSO i) and evaporated in vacuo. The residue was purified by flash chromatography (7N NH3 in methanol in DCM, 0-10%) to give the desired compound.
Compound 76: (4-Methoxy-6^hiophen-2-yl^yridin-3-yl)-(l-methyl-lH4midazo[4,5-c]pyridin-6-yl)- amine
Figure imgf000074_0001
Step i). 6-(5-Chloro-thiophen-2-yl)-4-methoxy-pyridin-3-ylamine
[00345] A mixture of 5-chlorothiophene-2-boronic acid (1.5 eq, 154 mg), 6-chloro-4-methoxy- pyridin-3-ylamine (1 eq, 100 mg), K3PO4 (3 eq, 602 mg) and tetrakis(triphenylphosphine)palladium(0) (0.1 eq, 58 mg) in 1.4-dioxane (3 mL) was heated at 140°C for 30 min in microwave. Reaction mixture was diluted with water (5 mL), extracted with EtOAc (3 x 10 mL), dried and concentrated. The residue was purified by silica chromatography (EtOAc/cyclohexane; 0:100 to 20:80) to give the desired compound.
Step ii. (4-Methoxy-6-thiophen-2-yl^yridin-3-yl)-(l-methyl-lH midazo[4,5-c]p
[00346] A degassed mixture of the amine (1 eq, 48 mg), 6-chloro-l-methyl-lH-imidazo[4,5- c]pyridine (Intermediate 2) (2 eq, 67 mg), CS2CO3 (3 eq, 326 mg), Xphos (0,1 eq, 9,6 mg) and Pd2(dba)3 (0,1 eq, 18 mg) in dry 1.4-dioxane (3 mL) was heated at 100°C for 5 h. Water (10 mL) was added and the reaction mixture was extracted with EtOAc (3 x). The combined organics were dried, concentrated and the residue was purified by silica chromatography (EtOAc/cyclohexane; 0:100 to 40:60) to give the desired compound. Compound 77: 4-Methoxy-6-(l,3,5-trimethyl-lH-pyrazol-4-yl)-pyridin-3-yl]-(l-methyl-lH- imidazo[4,5-c]pyridin-6-yl)-amine
Figure imgf000075_0001
Step i). 4-Methoxy-6-( 1, 3, 5-trimethyl-lH-pyrazol-4-yl)-pyridin-3-ylamine
[00347] A sealed vessel containing a mixture of 4-methoxy-5-nitro-2-(l,3,5-trimethyl-lH-pyrazol-4- yl)-pyridine (1 eq, 640 mg), iron powder (5 eq, 681 mg) and NH4Cl (1.5 eq, 196 mg) in THF (4.5 mL), EtOH (4.5 mL) and water (1.5 mL) was heated at 100°C for 3 h. Water was added, the reaction mixture was extracted with EtOAc (3x) and the combined organics were dried and concentrated. The residue was dissolved in MeOH and purified on a SCX column to give the desired compound.
Step ii). (4-Methoxy-6-thiophen-2-yl^yridin-3-yl)-(l-methyl-lH midazo
[00348] A degassed mixture of the amine (1 eq, 200 mg), 6-chloro-l -methyl- lH-imidazo[4,5- c]pyridine (1 eq, 144 mg), Cs2C03 (2 eq, 561 mg), Xphos (0,15 eq, 62 mg) and Pd2(dba)3 (0,1 eq, 79 mg) in dry 1.4-dioxane (5 mL) was heated at 120°C for 5 h. Water (10 mL) was added and the reaction mixture was extracted with EtOAc (3 x). The combined organics were dried, concentrated and the residue was purified by silica chromatography (EtOAc/cyclohexane; 0:100 to 50:50) to give the desired compound.
Compound 79: [3-Ethyl-4-(l-methyl-lH4midazo[4,5-c]pyridin-6-ylamino)-phenyl]-acetonitrile
Figure imgf000075_0002
Step i): (4-Amino-3-ethyl-phenyl)-acetonitrile
[0001] To a mixture of Cs2C03(6 eq, 41.3 g) and PdCl2dppf *DCM (0.1 eq, 1.93 g) in dry DMF (120 mL) under N2 was added (4-amino-3-bromophenyl) acetonitrile (1 eq, 5.0 g) and triethylborane solution (1M in THF, 1.3 eq, 30.8 mL). The reaction mixture was heated in a sealed flask at 55°C for 2 h. The reaction mixture was concentrated in vacuo to a smaller volume, filtered over a pad of Celite and washed with DCM. The filtrate was diluted with water and extracted with DCM (3x). The combined organics were dried and purified by silica chromatography (EtOAc/cyclohexane; 0:100 to 100:0) to give the desired compound. Step ii): [3-Ethyl-4-(l-methyl-lH-imidazo[4,5-c]pyridin-6-ylamino)-phenyl]-acetonitrile
[0002] A mixture of the aniline (1.1 eq, 106 mg), 6-chloro-l -methyl- lH-imidazo[4,5-c]pyridine (1 eq, 100 mg), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (0.3 eq, 86 mg), palladium(II) acetate (0.1 eq, 13.5 mg) and CS2CO3 (3 eq, 391 mg) in toluene (5 mL) was stirred at 100 °C for 16 h. The reaction mixture was filtered on Celite, the filtrate concentrated and the residue purified by silica chromatography (EtOAc /cyclohexane; 0:100 to 100:0) to give the desired compound.
Compound 80: [4-Methoxy-5-(l-methyl-lH4midazo[4,5-c]pyridin-6-ylamino)-pyridin-2-yl]-
Figure imgf000076_0001
Step (i): Cyano-(4-methoxy-5-nitro-pyridin-2-yl) -acetic acid tert-butyl ester
[0003] To a solution of 2-chloro-4-methoxy-5-nitro-pyridine (1 eq, 10 g) in THF (50mL) at room temperature was added K2CO3 (2.7 eq, 20 g), cyanoacetic acid tert-butyl ester (1.34 eq, 10 g) and 4A molecular sieves and the mixture was heated at reflux for 16 h after what the reaction mixture was concentrated in vacuo. The residue was partitioned between EtOAc and aq. sat. NaHC03 solution, the layer separated and the organic washed with brine, dried and concentrated. Purification by silica chromatography (EtOAc/cyclohexane; 0:100 to 25:75) followed by trituration with cyclohexane afforded the desired compound.
Step (ii): (5-Amino-4-methoxy-pyridin-2-yl)-cyano-acetic acid tert-butyl ester
[0004] Iron powder (6.2 eq, 2.42 g) was added to a stirred solution of cyano-(4-methoxy-5-nitro- pyridin-2-yl)-acetic acid tert-butyl ester (1 eq, 2.05 g) in AcOH (26 mL) at 50°C. The reaction mixture was then heated at 80 °C for 1 h. Once at room temperature, the reaction mixture was filtered and washed with EtOAc and concentrated. The residue was neutralized with aq. sat. NaHC03 solution and extracted with EtOAc (3x). The combined organics was washed with water, dried and concentrated. The residue was triturated with mixture of acetone and petroleum ether to give the desired compound. Step (Hi): Cyano-[4-methoxy-5-(l-methyl-lH-imidazo[4,5-c]pyridin-6-ylamino)-pyridin-2-yl] -acetic acid tert-butyl ester
[0005] A degassed mixture of the amine (1 eq, 236 mg), 6-chloro-l -methyl- lH-imidazo[4,5- c]pyridine (1 eq, 150 mg), Cs2C03 (2 eq, 583 mg), BINAP (0.3 eq, 167 mg) and Pd2(dba)3 (0.1 eq, 82 mg) in dry 1.4-dioxane (3 mL) was heated at 110°C for 16 h. Water (10 mL) was added and the reaction mixture was extracted with EtOAc (3 x). The combined organics was dried, concentrated and the residue was purified by silica chromatography (MeOH/DCM; 0:100 to 5:95) to give the desired compound.
Step (iv): [4-Methoxy-5-(l-methyl-lH-imidazo[4,5-c]pyridin-6-ylamino)-pyridin-2-yl]-acetonitrile TFA (0.56 mL) was added to a solution of the tert-butyl ester compound (1 eq, 48 mg) in DCM (1 mL) and the reaction was stirred at room temperature for 16 h. The reaction mixture was concentrated and purified with an SCX column followed by silica chromatography (MeOH/DCM; 0:100 to 5:95) to give the desired compound.
Figure imgf000077_0001
Step i) : N-[ 1 -(4-Nitro-phenyl)-meth-(E)-ylidene] -methanesulfonamide
[0006] A mixture of 4-nitro-benzaldehyde (1 eq, 10 g), methanesulfonamide (1 eq, 6.1 g) and tetraethoxysilane (1.05 eq, 15.5 mL) was heated at 120°C for 16 h. The reaction mixture was cooled to room temperature and crystallized with EtOAc/petrolether. Resulting solid was collected by filtration and dried the desired compound.
Step ii) : N-[2-Cyano-l-(4-nitro-phenyl) -ethyl] -methanesulfonamide
[0007] A mixture of N-(4-nitro-benzylidene)-methanesulfonamide (1 eq, 2 g ) , trimethylsilylacetonitrile (1.4 eq, 1.7 mL) and lithium acetate (0.1 eq, 0.06 g) was stirred at room temperature for 16 h. Aq. sat. NH4C1 solution was added and the product extracted with EtOAc (5x). The combined organics was concentrated and purified by silica chromatography (EtOAc/cyclohexane; 0:100 to 100:0) to give the desired product.
Step Hi) : N-[ Ί '-(4- Amino-phenyl) -2-cyano-ethylJ-methanesulfonamide
[0008] Iron powder (3 eq, 124 mg) was added to a stirred suspension of ammonium chloride (3 eq, 119 mg) and the nitrobenzene (1 eq, 200 mg) in a mixture of acetone (5 mL) and water (1 mL) and the resulting mixture was stirred at 65°C for 4 h. The mixture was filtered through Celite and washed with EtOAc (5 x 2 mL). The filtrate was washed with aq. sat. NaHC03 (15 mL) and the organic layer was dried and concentrated. The residue was added on a previously conditioned SCX Column (1 g), washed with MeOH (3x5 ml) and then with 7N NH3 solution in MeOH/MeOH (10 mL) to retrieve the desired compound.
Step (iv) : (E)-3-[4-(l-Methyl-lH midazo[4,5-c]pyridin-6-ylamino)-phenyl]-acrylonitrile
[0009] Synthesised following the same conditions used for compound 79 {step (ii)).
Compoun itrile
Figure imgf000078_0001
[0010] NaH (60%, 1.6 eq, 6 mg) was added to a solution of compound 79 (1 eq, 40 mg) in dry DMF (3 mL) at 0°C. After 20 min, Mel (1.6 eq, 14 x ) was added and the reaction mixture was stirred for 16 h. The reaction mixture was quenched with water, extracted with EtOAc, dried and concentrated. Purification by preparative HPLC afforded the desired compound.
Compound 83: (2-Difluoromethoxy-4-methyl-phenyl)-(l-methyl-lH-imidazo[4,5-c]pyridin-6-yl)-
Figure imgf000078_0002
Step i) : 2-Difluoromethoxy-4-methyl-l-nitro-benzene
[0011] Diethyl(bromodifluoromethyl)phosphonate (2 eq, 2.5 mL) was added to a cooled (-78 °C) solution of 5-methyl-2-nitrophenol (1 eq, 1.1 g) and KOH (20 eq, 7.8 g) in MeCN/water (72 mL; 1 :1) and the reaction mixture was allowed to warm to room temperature. After 30 minutes, the mixture was diluted with Et20, the organic phase was separated and the water phase was washed with a further amount of Et20. The combined organics were dried and concentrated. The residue was purified by silica chromatography (EtOAc/cyclohexane; 2:95 to 50:50) to give the desired product.
Step ii) : 2-Difluoromethoxy-4-methyl-phenylamine
[0012] A mixture of the nitro-aryl (1 eq, 50 mg) and palladium on carbon (10% wt., 7.5 mg) in EtOH (0.6 mL) was stirred under a hydrogen atmosphere at room temperature for 2 h. The reaction mixture was filtered through Celite and the filtrate was concentrated to afford the desired product.
Step Hi) : (2-Difluoromethoxy-4-methyl-phenyl)-(l -methyl- 1 H-imidazo[ 4, 5-cj pyridin-6-yl) -amine
[0013] A degassed mixture of the amine (1 eq, 28 mg), 6-chloro-l-methyl-lH-imidazo[4,5- c]pyridine (1 eq, 27 mg), CS2CO3 (2.5 eq, 132 mg), Xphos (0.3 eq, 23 mg) and Pd2(dba)3 (0.1 eq, 15 mg) in dry DMF (1 mL) was heated at 110°C for 18 h. Water was added and the reaction mixture was extracted with EtOAc (3 x). The combined organics were dried, concentrated and the residue was purified by silica chromatography (MeOH/EtOAc; 0:100 to 10:90) to give the desired compound.
2-[3-Ethyl-4-(l-methyl-lH-imidazo[4,5-c]pyridin-6-ylamino)-phenyl]-3-
Figure imgf000079_0001
Step i): 2-Ethyl-4-isoxazol-4-yl-phenylamine
[0014] Potassium fluoride (3 eq, 174 mg) in water (0.5 mL) was added to a mixture of 4- isoxazoleboronic acid pinacol ester (1.2 eq, 234 mg) and 4-bromo-2-ethyl-phenylamine (1.0 eq, 200 mg) in DMF (1.5 mL), purged with argon before PdCl2(dppf) (0.1 eq, 73 mg) was added and the reaction was stirred at room temperature for 90 min. The reaction mixture was diluted with DCM, washed with water, dried and concentrated. Silica chromatography (EtOAc/cyclohexane; 0:100 to 20:80) afforded the desired product.
Step ii) : 2-[ 3-Ethyl-4-(l -methyl- lH-imidazo[ 4, 5-cj pyridin-6-ylamino) -phenyl) '-3-oxo-propionitrile
[0015] A degassed mixture of the amine (1 eq, 67 mg), 6-chloro-l-methyl-lH-imidazo[4,5- c]pyridine (1 eq, 59 mg), Cs2C03 (2.5 eq, 288 mg), ΒΓΝΑΡ (0.3 eq, 66 mg) and Pd2(dba)3 (0.1 eq, 32 mg) in dry DMF (2 mL) was heated at 100°C for 18 h. Water was added and the reaction mixture was extracted with EtOAc (3 x). The combined organics were dried, concentrated and the residue was purified by prep arative HPLC give the desired compound. Compound 85: 2-[4-Ethyl-5-(l-methyl-lH-imidazo[4,5-c]pyridin-6-ylamino)-pyridin-2-yl]-3-oxo-
Figure imgf000080_0001
[0016] Synthesised following the same conditions used for compound 84.
Compound 86: [3-Ethyl-4-(l-methyl-lH-imidazo[4,5-c]pyridin-6-ylamino)-phenyl]-acetic acid methyl ester
Figure imgf000080_0002
Step (i) : (4- Amino-phenyl) -acetic acid methyl ester
[0017] To a solution of 4-aminophenylacetic acid (1 eq, 10 g) in MeOH (150 mL) was added aq. 37 % HCl (25 mL). The mixture was left to stir at room temperature over 3 days. The reaction mixture was then quenched with aq. sat. of NaHC03 solution, MeOH was evaporated under reduced pressure and the aqueous layer was extracted with EtOAc (4x). The combined organics were dried and concentrated to give the desired product.
Step (ii) : (4-Amino-3-iodo-phenyl)-acetic acid methyl ester
[0018] IC1 (1M in DCM, 1.4 eq, 15 mL) was added to a solution of the ester (1 eq, 2.0 g) in DCM (200 mL). After 4 h, aq. sat. Na2S203 solution was added and the solution was extracted with DCM (3x). The combined organics were washed with water, brine, dried and concentrated. The residue was purified by silica chromatography (EtOAc/cyclohexane; 3:97 to 30:70) to afford the desired product.
[0019]
Step (Hi) and (iv): [3-Ethyl-4-(l-methyl-lH-imidazo[4,5-c]pyridin-6-ylamino)-phenyl]-acetic acid methyl ester
[0020] Synthesised following the same conditions used for compound 79 {step (ii)).
Compound 87: l-[3-Ethyl-4-(l-methyl-lH-imidazo[4,5-c]pyridin-6-ylamino)-phenyl]-cyclopropane carbonitrile
Figure imgf000081_0001
Step (i): 4-Bromo-2-ethyl-l-nitro-benzene
[0021] TFAA (4.8 eq, 34 mL) was added to a solution of H202 (4 eq, 20 niL) in DCM (100 niL) at 0°C. After 5 min, a solution 4-bromo-2-ethyl aniline (1 eq, 7.1 mL) in DCM (200 mL) was added dropwise over 30 min and the reaction was heated to reflux for 2 h. Aq. sat NaHC03 solution was added, organic layer separated, dried and concentrated. The residue was purified by silica chromatography (EtOAc/cyclohexane) to afford the intermediate.
Step (u): (3-Ethyl-4-nitro-phenyl)-acetonitrile
[0022] An extra dry vial charged with Xanthphos (0.02 eq, 151 mg), Pd2(dba)3 (0.01 eq, 119 mg) and the bromoaryl (1 eq, 3 g) was flushed with argon and dry DMF (13 mL) was added. Trimethylsilyl acetonitrile (1.2 eq, 2.1 ml) was added, followed by ZnF2 (0.6 eq, 809 mg), the mixture was again flushed with argon and the mixture was heated in a microwave at 150°C for 5h. The reaction mixture was diluted with EtOAc, washed with water, dried and concentrated. Purification by silica chromatography (EtOAc/cyclohexane; 0:100 to 30:70) afforded the desired product.
Step (Hi): l-(3-Ethyl-4-nitro-phenyl)-cyclopropanecarbonitrile
[0023] Aq. NaOH (50%, 1 mL) was added dropwised to a solution of the arylacetonitrile (1 eq, 500 mg), TBAB (1 eq, 847 mg) and 1,2-dibromoethane (3 eq, 0.68 mL) MeCN (5 ml) after what the mixture was heated to 40°C for 2h. Water was added and the solution was extracted with DCM (3x), dried and concentrated. Purification by silica chromatography (EtOAc/cyclohexane; 0:100 to 30:70) afforded the desired product.
Step (iv): l-(4-Amino-3-ethyl-phenyl)-cyclopropanecarbonitrile
[0024] Pd/C (10%, 41 mg) was added to the nitroaryl (1 eq, 410 mg) in dry methanol (8 mL) and the mixture was stirred under hydrogen atmosphere at room temperature for 3 h. The reaction mixture was filtered through Celite to afford desired product.
Step (v): l-[3-Ethyl-4-(l-methyl-lHHmidazo[4,5-cJpyridin-6-ylamino)-phenylJ-cyclopropane carbonitrile [0025] A degassed mixture of the amine (1.1 eq, 119 mg), 6-chloro-l -methyl- lH-imidazo[4,5- c]pyridine (1 eq, 98 mg), CS2CO3 (2 eq, 378 mg), Xphos (0.3 eq, 83 mg) and palladium(II) acetate (0.1 eq, 13 mg) in dry toluene (5 mL) was heated at 110°C for 18 h. The mixture was filtered through Celite and concentrated. The residue was purified by silica chromatography (MeOH/DCM; 0:100 to 10:90) to give the desired compound.
Compound 88: 2-[3-Ethyl-4-(l-methyl-lH-imidazo[4, 5-c]pyridin-6-ylamino)-phenyl]-propionic acid methyl est
Figure imgf000082_0001
[0026] Synthesised following the same conditions used for compound 82.
Compound 89: l-(3,3-Dimethyl-azetidin-l-yl)-2-[3-ethyl-4-(l-methyl-lH-imidazo[4,5-c]pyridin-6-
Figure imgf000082_0002
Step (i): [3-Ethyl-4-(l-methyl-lH-imidazo[4,5-c]pyridin-6-ylamino)-phenyl]-acetic acid
[0027] LiOH (5 eq, 49 mg) was added to a solution of [3-ethyl-4-(l-methyl-lH-imidazo[4,5- c]pyridin-6-ylamino)-phenyl]-acetic acid methyl ester (1 eq, 133 mg) in MeOH (2.5 mL) and water (1 mL) and the mixture was heated at 45°C for 2.5 h. The reaction mixture was concentrated into a smaller volume, water was added and mixture extracted with DCM. The aqueous layer was neutralised with aq. IN HC1 solution and evaporated to dryness.
Step (u): l-(3,3-Dimethyl-azetidin-l-yl)-2-[3-ethyl-4-(l-methyl-lHHmidazo
phenyl] -ethanone
[0028] DiPEA (5 eq, 0.36 mL) was added to a mixture of the acid (1 eq, 195 mg), 3,3-dimethyl- azetidine hydrochloride (1.1 eq, 55 mg), TBTU (1.1 eq, 145 mg) in dry DMF (2 mL) at 0 °C and the reaction was stirred at room temperature for 3h. The reaction mixture was diluted with EtOAc, washed with aq. sat. NaHCC>3 solution, water and brine. The organic phase was dried and concentrated. The residue was purified by silica chromatography (MeOH/EtOAc; 0:100 to 20:80) to give the desired compound. Compound 90: l-(3,3-Dimethyl-azetidin-l-yl)-2-[3-ethyl-4-(l-methyl-lH midazo[4,5-c]pyrM
Figure imgf000083_0001
Step (i): (4-Bromo-2-ethyl-phenyl)-carbamic acid tert-butyl ester
[0029] A solution of 4-bromo-2-ethyl aniline (1 eq, 0.7 mL) and Boc-anhydride (1.5 eq, 1.6 g) in EtOH (7.7 mL) was stirred at room temperature for 18 h. The reaction mixture was concentrated and the residue was dissolved in EtOAc, washed with aq. sat. NaHCC>3 solution, dried and concentrated to give the desired compound.
Step (u) : [2-Ethyl-4-(4,4,5,5-tetramethyl-[l,3,2Jdioxaborolan-2-yl)-phenylJ-carbamic acid tert-butyl ester
[0030] To a mixture of the bromo-aryl (1 eq, 1 g), bis(pinacolato)diboron (1.1 eq, 0.94 g), KOAc (2.5 eq, 0.82 g) in dry dioxane (21 mL) was added PdC¾dppf (0.02 eq, 55 mg). The mixture was degased under inert atmosphere and heated at 80°C for 18 h. After filtration through Celite, the mixture was concentrated. Purification by silica chromatography (EtOAc/cyclohexane; 0:100 to 20:80) gave the desired compound
Step (Hi): [4-(3-Cyanomethyl-oxetan-3-yl)-2-ethyl-phenyl]-carbamic acid tert-butyl ester
[0031] 2-(Oxetan-3-ylidene)acetonitrile (1 eq, 50 mg) and the boronic ester (1.5 eq, 278 mg ) were added to a solution of chloro(l,5-cyclooctadiene)rhodium(I) dimer (0.05 eq, 13 mg) and aq. 1.5M KOH (0.46 mL) in dioxane (2 mL) and the reaction mixture was stirred at room temperature for 48 h. Brine was added and the mixture extracted with EtOAc (3x). The combined organics were dried and concentrated. Purification by silica chromatography (EtOAc/cyclohexane; 0: 100 to 30:70) gave the desired compound.
Step (iv) : [[3-(4-Amino-3-ethyl-phenyl)-oxetan-3-yl]-acetonitrile
[0032] TFA (20 eq, 0.37 mL) was added to a solution of the Boc-protected amine derivative (1 eq, 77 mg) in dry DCM (2 mL) at 0°C and the solution was stirred at room temperature for 2 h. Purification by SCX column afforded the product. Step (v) : l-(3,3-Dimethyl-azetidin-l-yl)-2-[3-ethyl-4-(l-methyl-lH-imidazo[4,5-cJpyridin-6-ylam phenyl] -ethanone
[0033] Synthesised following the same conditions used for compound 80 (step (Hi)).
Compound 91: l-[4-Ethyl-5-(l-methyl-lH midazo[4,5-c]pyridin-6-ylamino)-pyridin-2-yl]
-carboxylic acid ethyl ester
Figure imgf000084_0001
Step (i) : Azetidine- 3-carboxylic acid ethyl ester
[0034] Thionyl chloride (1.2 eq, 0.22 mL) was added dropwised to a stirred solution of azetidine- 1,3-dicarboxylic acid mono-tert-butyl ester (1 eq, 500 mg) in EtOH (7 mL) at -10°C and the reaction mixture was stirred room temperature for 16 h. Concentration afforded the desired compound.
Step (ii): l-(4-Ethyl-5-nitro-pyridin-2-yl)-azetidine-3-carboxylic acid ethyl ester
[0035] A mixture of 2-bromo-4-ethyl-5-nitro-pyridine (1 eq, 522 mg), the azetidine (1.1 eq, 321 mg) and potassium carbonate (2.5 eq, 859 mg) in dry DMF (4 mL) was stirred and then 3 hours at 70 °C. Water was added and the aqueous extracted with DCM (3x). The combined organics were dried and concentrated. Purification by silica chromatography (EtOAc/hexane; 0:100 to 30:70) gave the desired compound.
Step (Hi): l-(5-Amino-4-ethyl-pyridin-2-yl)-azetidine-3-carboxylic acid ethyl ester
[0036] Palladium on carbon (10% wt, 4.5 mg) was added to a solution of the nitroaryl (1 eq, 30 mg) in EtOH (0.5 mL) and the mixture was stirred under a hydrogen atmosphere at room temperature for 16 h.
The reaction mixture was filtered through Celite and the filtrate was concentrated to afford the desired product. Step iv : 1-f 4-Ethyl-5-(l -methyl- IH-imidazof 4, 5-c] pyridin-6-ylamino)-pyridin-2-yl] -azetidine-3- carboxylic acid ethyl ester
[0037] Synthesised following the same conditions used for compound 83 (step (Hi)).
Compound 92: [6-(3,3-Difluoro-azetidin-l-yl)-4-ethyl-pyridin-3-yl]-(l-methyl-lH-imidazo[4,5-
Figure imgf000085_0001
Step (i) and (ii) : 6-(3,3-Difluoro-azetidin-l-yl)-4-ethyl-pyridin-3-ylamine
[0038] Synthesised following the same conditions used for compound 91 (step (ii) and (Hi)).
Step (Hi) : [6-(3,3-Difluoro-azetidin-l-yl)-4-ethyl-pyridin-3-yl]-(l-methyl-lH-imidazo[4,5-c]pyridm^ yl)-amine
[0039] Synthesised following the same conditions used for compound 80 (step (Hi)).
Compound 93: 4 '-Ethyl-5 '-(l-methyl-lH-imidazo[4, 5-c]pyridin-6-ylamino)-3,4, 5, 6-tetrahydro-2H- ']bipyridinyl-3-carbonitrile
Figure imgf000085_0002
[0040] Synthesised following the same conditions used for compound 92. Compound 94: [4-Ethyl-6-((S)-3-methy^yrrolidin-l-yl)^yridin-3-yI]-(l-methyl-lH4midazo[4, c]pyridin-6-yl)-amine
Figure imgf000086_0001
[0041] Synthesised following the same conditions used for compound 92.
Compound 95: l-[4-Ethyl-5-(l-methyl-lH4midazo[4,5-c]pyrldln-6-ylamino)^yridin-2-yl]- rrolidine-3-carbonitrile
Figure imgf000086_0002
[0042] Synthesised following the same conditions used for compound 92.
Compound 96: (6-Cyclopentyl-4-ethyl^yridin-3-yl)-(l-methyl-lH4midazo[4,5-c]pyridin-6-yl)-^
Figure imgf000086_0003
Step i : 6-Cyclopentyl-4-ethyl-pyridin-3-ylamine
[0043] Bromo-cyclopentane (3 eq, 445 mg) was dissolved in THF (4 mL) and heated at 65°C. Under atmosphere of nitrogen, Rieke zinc (5 g/100 mL, 2.9 eq, 3.7 mL) was added dropwise and stirred for 2 h at 65°C under nitrogen. In another flask 6-bromo-4-ethyl-pyridin-3-ylamine (1 eq, 200 mg), copper(I) iodide (0.1 eq, 18.8 mg) and Pd(dppf)Cl2 (0.05 eq, 40.4 mg) were dissolved in THF (4 mL) and heated at 85°C under atmosphere of nitrogen. The reaction mixture from the first flask was added dropwised via a cannule to the second flask. The resulting reaction mixture was stirred at 85°C for 18 h. The reaction was then quenched with water and extracted with EtOAc (3x). The combined organics was dried and concentrated under reduced pressure. The residue was purified by column chromatography (MeOH/DCM; 0:100 to 10:90) to give the expected aniline.
Step (ii) : ( 6-Cyclopentyl-4-ethyl-pyridin-3-yl)-(l -methyl- 1 H-imidazo[ 4, 5-cj pyridin-6-yl) -amine
[0044] Synthesised following the same conditions used for compound 80 (step (Hi)).
Compound 97: 4-Ethyl-5-(l-methyl-lH-imidazo[4, 5-c]pyridin-6-ylamino)-3 ',4 ',5 ',6 '-tetrahydro-2 ΊΙ- ']bipyridinyl-l '-carboxylic acid tert-butyl ester
Figure imgf000087_0001
Step ( i) : 5-Amino-4-ethyl-3 ',4 ',5 ' 6' -tetrahydro-2 'H-f 2, 4 'Jbipyridinyl-1 '-carboxylic acid tert-butyl ester
[0045] Iodine (0.21 eq, 13 mg) was added to a suspension of Zn dust (3 eq, 48 mg) in dry DMA (0.5 mL) and the mixture was stirred for 5 min. 4-Bromo-piperidine-l -carboxylic acid tert-butyl ester (3 eq, 146 \L) was added and the reaction mixture heated at 85°C for 29 h. In another flask 6-bromo-4-ethyl- pyridin-3-ylamine (1 eq, 50 mg), copper(I) iodide (0.06 eq, 2.8 mg) and Pd(dppf)Cl2 (0.03 eq, 6.1 mg) were dissolved in dry DMA (0.5 mL) and heated at 85°C for 30 min after what the reaction mixture from the first flask was added dropwised via a cannule to the second flask. The resulting reaction mixture was stirred at 85°C for 18 h. The reaction was then quenched with aq. sat. NH4C1 and extracted with EtOAc (3x). The combined organics was dried and concentrated under reduced pressure. The residue was purified by column chromatography (MeOH/DCM; 0:100 to 3 :97) to give the expected aniline.
Step (ii): 4-Ethyl-5-(l-methyl-lH-imidazo[4,5-cJpyridin-6-ylamino)-3',4',5', 6'-tetrahydro-2'H- [2, 4J ' bipyridinyl-1 '-carboxylic acid tert-butyl ester
[0046] Synthesised following the same conditions used for Compound 80 (step (Hi)). Compound 98: [6-(3,3-Difluoro-pyrrolidin-l-yl)-4-ethyl-pyridin-3-yl]-(l-methyl-lH-imidazo[4,5-
Figure imgf000088_0001
[0047] Synthesised following the same conditions used for compound 91.
Figure imgf000088_0002
Step i): 6-Cyclohexyl-4-ethyl-pyridin-3-ylamine
[0048] Iodo-cyclohexane (1.9 eq, 150 mg) was dissolved in THF (4 mL) and heated at 65°C. Under atmosphere of nitrogen, Rieke zinc (5 g/100 mL, 2.0 eq, 0.84 mL) was added dropwise and stirred for 3 h at 65°C under nitrogen. In another flask 6-bromo-4-ethyl-pyridin-3-ylamine (1 eq, 72 mg), copper(I) iodide (0.1 eq, 6.8 mg) and Pd(dppf)Cl2 (0.05 eq, 14.6 mg) were dissolved in THF (4 mL) and heated at 85°C under atmosphere of nitrogen. The reaction mixture from the first flask was added dropwised via a cannule to the second flask. The resulting reaction mixture was stirred at 85°C for 2 h. The reaction was then quenched with water and extracted with EtOAc (3x). The combined organics was dried and concentrated under reduced pressure. The residue was purified by column chromatography (MeOH/DCM; 0:100 to 10:90) to give the expected aniline.
Step ii) : ( ( 6-Cyclohexyl-4-ethyl-pyridin-3-yl)-(l -methyl- 1 H-imidazo[ 4, 5-cj pyridin-6-yl) -amine
[0049] Synthesised following the same conditions used for compound 80 (step (iii)). Compound 100: (6-Cyclobutyl-4-ethyl-pyridin-3-yl)-(l-methyl-lH-imidazo[4,5-c]pyridin - -yl)-amine
Figure imgf000089_0001
[0050] Synthesised following the same conditions used for compound 96.
Compound 101: (4-Ethyl-5-(l-methyl-lH-imidazo[4,5-c]pyridin-6-ylamino)-phthalonitrile
Figure imgf000089_0002
Step i): 4-Bromo-5-nitro-phthalonitrile
[0051] Fe powder (2.86 eq, 317 mg) was added to a solution of 4-bromo-5-nitro-phthalonitrile (1 eq, 500mg) in MeOH (4 mL) and HCl (1.4 mL) at 60°C and the reaction was heated at reflux for 2 h. The reaction mixture was hot filtered through Celite and the filtrate was concentrated. The residue was dissolved in Hot MeOH, precipitate and collected by filtration to afford the desired product.
Step ii): (4-Ethyl-5-(l-methyl-lH-imidazo[4,5-cJpyridin-6-ylamino)-phthalonitrile
[0052] Synthesised following the same conditions used for compound 91 (step (i)) compound 80
(step (Hi)).
Com ound 102: (4-Ethyl-pyrimidin-5-yl)-(l-methyl-lH-imidazo[4,5-c]pyridin-6-yl)-amine
Figure imgf000089_0003
Step i): 4-Bromo-pyrimidin-5-ylamine
[0053] Benzyltrimethylammonium tribromide (1.1 eq, 4.5 g) was added portionwised to a stirred solution of 5-aminopyrimidine (1 eq, 1 g) in DCM (50 mL) and MeOH (10 mL) at 0°C. The mixture was warmed up to room temperature and stirred for 90 min. Aq. sat. NaHCC>3 solution was added (pH ~8), the organic was separated and the aqueous extracted with EtOAc (3x). The combined organics were dried and concentrated. Purification by silica chromatography (EtOAc/cyclohexane; 0:100 to 50:50) gave the desired compound.
Step ii): N'-(4-Bromo-pyrimidin-5-yl)-N,N-dimethyl-formamidine
[0054] A solution of 4-bromo-pyrimidin-5-ylamine (1 eq, 400 mg) and N,N-dimethylformamide dimethylacetal (1.6 mL) in MeOH (35 mL) was refluxed for 2 h. The mixture was concentrated to give the compound.
Step Hi) : N'-(4-Ethyl-pyrimidin-5-yl)-N,N-dimethyl-formamidine
[0055] Diethylzinc ( 1 M in hexanes, 2.5 eq, 5.3 mL) was added to a degassed mixture of the bromoaryl (1 eq, 480 mg), tri-o-tolylphosphine (0.61 eq, 388 mg) and Pd(OAc)2 (0.19 eq, 89 mg) in DMF (8 mL) and the reaction was heated at 90°C for 10 min. Water was then added and the resulting mixture was extracted with EtOAc (3x), dried and concentrated. Purification by silica chromatography (MeOH/DCM; 0:100 to 8:92) afforded the desired compound.
Step iv): 4-Ethyl-pyrimidin-5-ylamine
[0056] The dimethyl-formamidine (1 eq, 178 mg) and K2CO3 (2.5 eq, 345 g) in dioxane (15 mL) and water (15 mL) was heated at reflux for 72 h. The mixture was then extracted with EtOAc (3x), dried and concentrated. Purification by silica chromatography (MeOH/DCM; 0:100 to 10:90) led to the product.
Step v): (4-Ethyl^yrimidin-5-yl)-(l-methyl-lH-imidazo[4,5-cJpyridin-6-yl)-amine
[0057] Synthesised following the same conditions used for compound 80 (step (Hi)).
[0058] The compound of the invention that have been prepared according to the synthetic methods described herein are listed in Table I below. The NMR spectral data of the compound of the invention and some of the comparative examples is given in Table II.
[0059] Table I
Cpd# = Compound number
MW: Calculated molecular weight
MS Mes'd: Measured molecular weight by mass spectrometry Cpd MS
Structures Name Method MW
# Mes'd
(2-Ethyl-phenyl)-(l -methyl-
1 lH-imidazo[4,5-c]pyridin-6- Method A 252 253 yl)-amine
(2-Isopropyl-phenyl)-(l -
2 methyl- 1 H-imidazo[4,5- Method A 266 267 c]pyridin-6-yl)-amine
N-(2,3- dihydrobenzo[b] [1 ,4]dioxin-6-
3 Method A 282 282.9 yl)- 1 -methyl- 1 H-imidazo[4,5-
H \
c]pyridin-6-amine
N-(4-methoxy-2- (trifluoromethyl)phenyl)- 1 -
4 Method A 322 322.9 methyl- 1 H-imidazo[4,5- c]pyridin-6-amine l-methyl-N-(2,4,6-
5 trimethoxyphenyl)- 1 H- Method A 314 314.9 imidazo[4,5-c]pyridin-6-amine
N-(benzo[d] [l,3]dioxol-5-yl)-
6 1 -methyl- 1 H-imidazo[4,5- Method A 268 268.9 c]pyridin-6-amine
H \
N-(3-cyclopropyl-l-methyl- 1 H-pyrazol-5-yl)- 1 -methyl-
7 Method A 268 269 lH-imidazo[4,5-c]pyridin-6- amine
/ H \
N-( 1 -ethyl-3 -methyl- 1 H-
8 pyrazol-5-yl)- 1 -methyl- 1 H- Method A 256 257.1 imidazo[4,5-c]pyridin-6-amine
^ H
N-(3 -tert-butyl- 1 -methyl- 1 H-
9 pyrazol-5-yl)- 1 -methyl- 1 H- Method A 284 285 imidazo[4,5-c]pyridin-6-amine
/ H \
N-( 1 -ethyl- 1 H-pyrazol-4-yl)-
10 1 -methyl- 1 H-imidazo[4,5- Method A 242 243 c]pyridin-6-amine
H \
Figure imgf000092_0001
Figure imgf000093_0001
1 H \ c]pyridin-6-yl)-amine
Figure imgf000094_0001
Figure imgf000095_0001
c]pyridin-6-yl)-amine A"
Figure imgf000096_0001
Figure imgf000097_0001
1 H \ carbonitrile
Figure imgf000098_0001
methyl ester Cpd MS
Structures Name Method MW
# Mes'd
1 - [3 -Ethyl-4-( 1 -methyl- 1 H- imidazo[4,5-c]pyridin-6-
87 described 317.4 318.1 ylamino) -phenyl] - cyclopropanecarbonitrile
2- [3 -Ethyl-4-( 1 -methyl- 1 H- imidazo[4,5-c]pyridin-6-
88 described 338.4 339.1 ylamino)-phenyl]-propionic
acid methyl ester
1 -(3 ,3 -Dimethyl-azetidin- 1 - yl)-2- [3 -ethyl-4-( 1 -methyl- 1 H-
89 described 377.5 378.8
Τ Ν imidazo[4,5-c]pyridin-6-
1 ylamino) -phenyl] - ethanone
Ο { 3 - [3 -Ethyl-4- ( 1 -methyl- 1 H- imidazo[4,5-c]pyridin-6-
90 described 347.4 348.2 ylamino)-phenyl]-oxetan-3- yl} -acetonitrile
l-[4-Ethyl-5-(l-methyl-lH- imidazo[4,5-c]pyridin-6-
91 ylamino) -pyridin-2 -yl] - described 380.4 381.2 azetidine-3 -carboxylic acid
ethyl ester
[6-(3,3-Difluoro-azetidin-l- yl)-4-ethyl-pyridin-3-yl]-(l -
92 I l x) described 344.4 345.1 methyl- 1 H-imidazo[4,5-
1 H \ c]pyridin-6-yl)-amine
4'-Ethyl-5'-(l -methyl- 1 H- imidazo[4,5-c]pyridin-6-
93 1 1) 1 y ylamino)-3,4,5,6-tetrahydro- described 361.4 362.3
1 H \ 2H-[l,2']bipyridinyl-3- carbonitrile
[4-Ethyl-6-((S)-3-methyl- pyrrolidin- 1 -yl)-pyridin-3 -yl] -
94 described 336.4 337.3
(1 -methyl- 1 H-imidazo[4,5- c]pyridin-6-yl)-amine l-[4-Ethyl-5-(l-methyl-lH- imidazo[4,5-c]pyridin-6-
95 described 347.4 348.3 ylamino) -pyridin-2 -yl] - pyrrolidine-3 -carbonitrile
(6-Cyclopentyl-4-ethyl- pyridin-3 -yl)-( 1 -methyl- 1 H-
96 described 321.4 322.3 imidazo[4,5-c]pyridin-6-yl)-
1 H \ amine
4-Ethyl-5-(l-methyl-lH- imidazo[4,5-c]pyridin-6-
97 ylamino)-3',4',5',6'-tetrahydro- described 436.6 437.3
1 2'H-[2,4']bipyridinyl-l'- carboxylic acid tert-butyl ester
Figure imgf000100_0001
60] Table II: NMR Data of Representative Compounds of the Invention
Figure imgf000100_0002
Cpd
(δ) NMR data
#
Ή NMR δ (ppm)(DMSO- dg): 8.63 (1 H, d, ArH), 8.32 (1 H, s, ArH or NH), 8.18 (1 H, s, ArH
26 or NH), 8.03 (1 H, d, ArH), 7.56-7.50 (2 H, m, ArH), 7.24 (1 H, d, ArH), 3.78 (3 H, s, CH3), 2.74 (2 H, q, CH2), 1.20 (3 H, t, CH3).
Ή NMR δ (ppm)(CHCl3-d): 8.64 (1 H, s, ArH), 7.68 (1 H, s, ArH), 6.93 (1 H, s, ArH), 6.79 (1
29 H, m, ArH), 6.39 (1 H, s, NH), 6.34 (1 H, s, ArH), 4.26-4.29 (4 H, m, CH2), 3.67 (3 H, s, CH3), 2.17 (3 H, s, CH3).
Ή NMR δ (ppm)(CHCl3-d): 8.74 (1 H, s, ArH), 8:33 (1 H, m, ArH), 7.31 (1 H, m, ArH), 6.93 (1
30 H, s, ArH), 6.82-6.86 (2 H, m, ArH, NH), 6.57 (1 H, d, ArH), 4.32 (4 H, m, CH2), 3.75 (3 H, s, CH3).
Ή NMR δ (ppm)(CHCl3-d): 8.74 (1 H, s, ArH), 8:59 (1 H, m, ArH), 8.22 (1 H, s, ArH), 7.79 (1
31 H, s, ArH), 7.44 (1 H, m, ArH), 7.31 (1 H, s, NH), 6.84 (1 H, dd, ArH), 3.86 (3 H, s, CH3), 2.68 (2 H, q, CH2), 1.35 (3 H, t, CH3).
Ή NMR δ (ppm)(CHCl3-d): 8.68 (1 H, s, ArH), 7.75 (1 H, s, ArH), 6.84-6.90 (2 H, m, ArH),
32 6.75-6.79 (1 H, m, ArH), 6.71 (1 H, s, ArH), 6.43 (1 H, s, NH), 4.25-4.31 (4 H, m, CH2), 4.08 (2 H, q, CH2), 1.47 (3 H, t, CH3).
Ή NMR δ (ppmXDMSO-dg): 8.43 (1 H, d, ArH), 7.98 (1 H, s, ArH), 7.69 (1 H, s, ArH), 6.88 (1
33 H, s, ArH), 6.73 (1 H, s, NH), 6.44 (1 H, d, ArH), 4.22 (4 H, s, CH2), 3.65 (3 H, s, CH3), 2.56- 2.50 (2 H, q, CH2), 1.06 (3 H, t, CH3).
Ή NMR δ (ppm)(CHCl3-d): 8.67 (1 H, s, ArH), 7.63 (1 H, s, ArH), 7.18-7.25 (3 H, m, ArH),
34 6.11 (1 H, s, NH), 5.78 (1 H, s, ArH), 3.59 (3 H, s, CH3), 2.65 (2 H, q, CH2), 2.28 (3 H, s, CH3), 1.16 (3 H, t, CH3).
Ή NMR δ (ppm)(CHCl3-d): 8.64 (1 H, s, ArH), 7.75 (1 H, s, ArH), 6.84-6.89 (3 H, m, ArH),
35 6.77-6.81 (1 H, m, ArH), 6.43 (1 H, s, NH), 4.25-4.30 (4 H, m, CH2), 3.21-3.29 (1 H, m, CH), 1.07-1.15 (2 H, m, CH), 0.95-1.01 (2 H, m, CH).
Ή NMR δ (ppmXDMSO-dg): 10.63 (1 H, s, NH), 8.71 (1 H, m, ArH), 8.53 (1 H, s, ArH), 8.06
36 (1 H, s, NH), 7.24 (1 H, d, ArH), 7.07 (1 H, dd, ArH), 6.84 (1 H, d, ArH), 6.81 (1 H, m, ArH), 4.49 (2 H, s, CH2), 3.73 (3 H, s, CH3).
¾ NMR 8 (ppm)(DMSO-d6) : 10.50 (1 H, s, NH), 8.76 (1 H, s, NH), 8.55 (1 H, d, ArH), 8.08 (1
37 H, s, ArH), 7.38 (1 H, d, ArH), 7.07 (1 H, dd, ArH), 6.83 (1 H, d, ArH), 6.78 (1 H, d, ArH), 4.52 (2 H, s, CH2), 3.73 (3 H, s, CH3).
Ή NMR δ (ppmXDMSO-dg): 8.45 (1 H, s, NH), 8.01 (1 H, s, ArH), 7.87 (1 H, s, ArH), 7.46-
38 7.39 (1 H, m, ArH), 7.10-7.05 (1 H, m, ArH), 7.03-6.96 (1 H, m, ArH), 6.55 (1 H, d, ArH), 3.67 (3 H, s, CH3), 2.67-2.57 (2 H, m, CH2), 1.16-1.07 (3 H, m, CH3). Cpd
(δ) NMR data
#
Ή NMR δ (ppm)(DMSO-d6): 8.55 (1 H, d, ArH), 8.15 (1 H, s, ArH), 7.88 (1 H, s, NH), 7.78 (1
39 H, d, ArH), 7.70 (1 H, d, ArH), 7.61 (1 H, m, ArH), 7.22 (1 H, m, ArH), 7.09 (1 H, d, ArH), 3.78 (3 H, s, CH3).
Ή NMR δ (ppm)(DMSO-d6): 8.56 (1 H, d, ArH), 8.09 (1 H, s, ArH), 8.01 (1 H, s, NH), 7.59 (1
40 H, d, ArH), 7.09 (1 H, m, ArH), 6.93-6.87 (2 H, m, ArH), 3.75 (3 H, s, CH3), 2.88 (4 H, m, CH2), 2.09-1.98 (2 H, m, CH2).
Ή NMR δ (ppm)(DMSO-d6): 8.43 (1 H, d, ArH), 7.98 (1 H, s, NH), 7.75 (1 H, s, ArH), 7.39 (2
41 H, d, ArH), 7.27 (1 H, d, ArH), 7.00-6.90 (3 H, m, ArH), 6.82 (1 H, dd, ArH), 6.38 (1 H, d, ArH), 5.00 (2 H, s, CH), 3.77 (3 H, s, CH3), 3.64 (3 H, s, CH3), 2.17 (3 H, s, CH3).
Ή NMR δ (ppmXDMSO-dg): 9.23 (1 H, s, ArH), 9.14 (1 H, s, ArH), 8.68-8.64 (2 H, m, ArH),
42 8.16 (1 H, s, NH), 7.97 (1 H, d, ArH), 7.60 (1 H, dd, ArH), 7.02 (1 H, d, ArH), 3.81 (3 H, s, CH3).
Ή NMR δ (ppmXDMSO-dg): 9.39-9.31 (1 H, m, ArH), 9.16 (1 H, s, ArH), 8.68 (1 H, d, ArH),
43 8.60 (1 H, d, ArH), 8.15 (1 H, s, NH), 8.00 (1 H, d, ArH), 7.63 (1 H, dd, ArH), 7.00 (1 H, d, ArH), 3.80 (3 H, s, CH3).
Ή NMR δ (ppm)(DMSO-d6): 8.60 (1 H, d, ArH), 8.45 (1 H, s, ArH), 8.26-8.15 (2 H, m, ArH
45
and NH), 7.38-7.29 (2 H, m, ArH), 7.18 (1 H, d, ArH), 7.01 (1 H, ddd, ArH), 3.79 (3 H, s, CH3).
Ή NMR δ (ppm)(CHCl3-d): 8.73 (1 H, d, ArH), 7.73 (1 H, s, ArH), 7.36 (1 H, s, ArH), 6.97 (1
46
H, s, ArH), 6.70 (1 H, d, ArH), 6.55 (1 H, s, NH), 4.30-4.24 (4 H, m, CH2), 3.74 (3 H, s, CH3).
Ή NMR δ (ppmXDMSO-dg): 8.52 (1 H, d, ArH), 8.06 (1 H, s, NH), 7.82 (1 H, s, ArH), 7.38-
47 7.30 (3 H, m, ArH), 7.07 (1 H, d, ArH), 6.95-6.90 (2 H, m, ArH), 6.80 (1 H, d, ArH), 6.58 (1 H, dd, ArH), 4.95 (2 H, s, CH2), 3.75 (3 H, s, CH3), 3.70 (3 H, s, CH3), 2.16 (3 H, s, CH3).
Ή NMR δ (ppm)(DMSO-d6): 8.48 (1 H, d, ArH), 8.08-7.98 (1 H, s, NH), 7.94 (1 H, s, ArH), 7.41 (2 H, d, ArH), 7.14-7.05 (2 H, m, ArH), 7.01-6.93 (2 H, m, ArH), 6.81-6.76 (1 H, m, ArH),
48
6.63 (1 H, d, ArH), 5.04 (2 H, s, CH2), 3.77 (3 H, s, CH3), 3.75-3.61 (3 H, m, CH3), 2.08 (3 H, s, CH3).
Ή NMR δ (ppmXDMSO-dg): 10.72 (1 H, s, NH), 8.57 (1 H, d, ArH), 8.14 (1 H, s, ArH), 8.06 (1
49 H, s, NH), 7.65 (1 H, s, ArH), 7.03 (1 H, d, ArH), 6.98 (1 H, s, ArH), 4.61 (2 H, s, CH2), 3.76 (3 H, s, CH3).
Ή NMR δ (ppm)(DMSO-d6): 9.94 (1 H, s, NH), 8.63 (1 H, s, ArH), 8.56 (1 H, d, ArH), 8.08 (1
50 H, s, NH), 7.41 (1 H, d, ArH), 7.34 (1 H, dd, ArH), 6.86-6.78 (2 H, m, ArH), 3.75 (3 H, s, CH3), 2.88 (2 H, m, CH), 2.50-2.42 (2 H, m, CH). Cpd
(δ) NMR data
#
Ή NMR δ (ppmXDMSO-dg): 8.77 (1 H, s, ArH), 8.55 (1 H, d, ArH), 8.07 (1 H, s, ArH), 7.42 (1
51 H, d, ArH), 7.21 (1 H, dd, ArH), 6.94-6.83 (2 H, m, NH, ArH), 4.58 (2 H, s, CH2), 3.73 (3 H, s, CH3), 3.28 (3 H, s, CH3).
Ή NMR δ (ppm)(DMSO-d6): 8.85 (1 H, s, NH), 8.55 (1 H, d, ArH), 8.08 (1 H, s, ArH), 7.43 (1
52 H, d, ArH), 7.23 (2 H, d, ArH), 7.09 (1 H, dd, ArH), 6.98-6.83 (3 H, m, ArH), 6.83 (1 H, d, ArH), 5.06 (2 H, s, CH2), 4.73 (2 H, s, CH2), 3.72 (6 H, d, CH3).
Ή NMR δ (ppm)(DMSO-d6): 8.69 (1 H, s, NH), 8.47 (1 H, s, ArH), 8.20-8.15 (1 H, m, ArH),
53 8.10 (1 H, s, ArH), 8.06 (1 H, s, ArH), 7.26-7.22 (1 H, m, ArH), 6.76 (1 H, s, ArH), 3.71 (3 H, s, CH3), 2.70-2.60 (2 H, m, CH2), 1.19-1.11 (3 H, m, CH3).
Ή NMR δ (ppm)(DMSO-d6): 8.50 (1 H, s, NH), 8.17-8.15 (1 H, m, ArH), 8.08 (2 H, s, ArH),
54 8.00-7.97 (1 H, m, ArH), 7.19-7.13 (1 H, m, ArH), 6.86 (1 H, d, ArH), 3.72 (3 H, s, CH3), 2.86- 2.77 (2 H, m, CH2), 1.24-1.15 (3 H, m, CH3).
Ή NMR δ (ppmXDMSO-dg): 8.56 (1 H, s, NH), 8.10 (1 H, s, ArH), 7.99 (1 H, s, ArH), 7.65-
55 7.60 (1 H, m, ArH), 7.22-7.14 (1 H, m, ArH), 6.99 (1 H, d, ArH), 6.76-6.69 (1 H, m, ArH), 3.74 (3 H, s, CH3), 2.71-2.61 (2 H, m, CH2), 1.19-1.10 (3 H, m, CH3).
Ή NMR δ (ppm)(DMSO-d6): 8.58-8.56 (1 H, m, ArH), 8.20-8.15 (2 H, m, ArH), 8.13 (1 H, s,
56 NH), 7.40-7.31 (2 H, m, ArH), 7.03 (1 H, d, ArH), 3.76 (3 H, s, CH3), 2.75 (2 H, q, CH2), 1.18 (3 H, t, CH3).
Ή NMR δ (ppmXDMSO-dg): 8.58 (1 H, d, ArH), 8.15 (1 H, s, ArH), 8.10 (1 H, s, ArH), 7.78 (1
57 H, s, NH), 7.27 (2 H, d, ArH), 7.18 (1 H, s, ArH), 7.12 (1 H, d, ArH), 6.99-6.91 (2 H, m, ArH), 5.13 (2 H, s, CH2), 4.90-4.76 (2 H, m, CH2), 3.36 (6 H, s, CH3).
Ή NMR δ (ppm)(CHCl3-d): 8.69 (1 H, d, ArH), 7.71 (1 H, s, ArH), 7.52 (1 H, d, ArH), 7.32 (1
59 H, m, ArH), 7.16 (1 H, dd, ArH), 6.63 (1 H, d, ArH), 6.36 (1 H, s, NH), 3.68 (3 H, s, CH3), 3.09 (3 H, s, CH3), 3.03 (3 H, s, CH3), 2.69 (2 H, q, CH2), 1.24 (3 H, t, CH3).
Ή NMR δ (ppm)(DMSO-d6): 9.16-9.1 1 (3 H, m, ArH), 8.54 (1 H, s, NH), 8.1 1-8.05 (2 H, m,
60 ArH), 7.82 (1 H, d, ArH), 7.67-7.65 (1 H, m, ArH), 7.62-7.58 (1 H, m, ArH), 6.94 (1 H, s, ArH), 3.73 (3 H, s, CH3), 2.80-2.72 (2 H, m, CH2), 1.25-1.18 (3 H, m, CH3).
Ή NMR δ (ppmXDMSO-dg): 8.47 (1 H, s, NH), 8.07 (1 H, s, ArH), 8.02 (1 H, s, ArH), 7.89-
61 7.76 (2 H, m, ArH), 7.48-7.40 (2 H, m, ArH), 7.37-7.33 (1 H, m, ArH), 6.64 (1 H, s, ArH), 3.87 (3 H, s, CH3), 3.67 (3 H, s, CH3), 2.70-2.60 (2 H, m, CH2), 1.20-1.11 (3 H, m, CH3).
Ή NMR δ (ppm)(DMSO-d6): 8.91 (1 H, s, ArH), 8.53-8.50 (2 H, m, ArH), 8.09-8.05 (2 H, m, ArH), 8.01 (1 H, s, NH), 7.74 (1 H, d, ArH), 7.59-7.57 (1 H, m, ArH), 7.54-7.50 (1 H, m, ArH),
62
7.48-7.44 (1 H, m, ArH), 6.88 (1 H, s, ArH), 3.72 (3 H, s, CH3), 2.80-2.70 (2 H, m, CH2), 1.25- 1.16 (3 H, m, CH3).
Figure imgf000104_0001
Cpd
(δ) NMR data
#
Ή NMR δ (ppm) (D6-DMSO): 8.44 (d, IH, ArH), 7.99 (s, IH, ArH), 7.80 (s, IH, NH), 7.37 (d, IH, ArH), 7.07 (d, IH, ArH), 7.00 (dd, IH, ArH), 6.60 (d, IH, ArH), 3.84 (s, 2H, CH2), 3.65 (s,
89
3H, CH3), 3.52 (s, 2H, CH2), 3.35 (s, 2H, CH2), 2.59 (q, 2H, CH2), 1.21 (s, 6H, 2x CH3), 1.10 (t, 3H, CH3)
Ή NMR δ (ppm) (D6-DMSO): 8.46 (s, IH, ArH), 8.02 (s, IH, ArH), 7.89 (br. s., IH, NH), 7.53 (d, IH, ArH), 7.10 (br. s., IH, ArH), 7.03 (d, IH, ArH), 6.75 (s, IH, ArH), 4.86 (d, 2H, CH2),
90
4.68 (d, 2H, CH2), 3.68 (s, 3H, CH3), 3.27 (br. s., 2H, CH2), 2.65 (q, 2H, CH2), 1.13 (t, 3H, CH3)
Ή NMR δ (ppm) (D6-DMSO): 8.38 (s, IH, ArH), 7.96 (s, IH, ArH), 7.95 (s, IH, ArH), 7.75 (s, IH, NH), 6.35 (s, IH, ArH), 6.29 (s, IH, ArH), 4.11-4.16 (dd, 2H, CH2), 4.14 (q, 2H, CH2),
91
3.99 (dd, 2H, CH2), 3.58-3.66 (m, IH, CH), 3.62 (s, 3H, CH3), 2.47 (q, 2H, CH2), 1.21 (t, 3H, CH3), 1.07 (t, 3H, CH3)
Ή NMR δ (ppm) (D6-DMSO): 8.39 (d, IH, ArH), 8.04 (s, IH, ArH), 7.97 (s, IH, ArH), 7.81
92 (br. s, IH, NH), 6.51 (br. s, IH, ArH), 6.36 (d, IH, ArH), 4.37 (t, 4H, CH2), 3.64 (s, 3H, CH3), 2.52 (q, 2H, CH2), 1.09 (t, 3H, CH3)
Ή NMR δ (ppm) (D6-DMSO): 8.39 (s, IH, ArH), 8.02 (s, IH, ArH), 7.96 (s, IH, NH), 7.75 (s, IH, ArH), 6.83 (s, IH, ArH), 6.32 (s, IH, ArH), 3.71-3.83 (m, 2H, 2 x CH), 3.63 (s, 3H, CH3),
93
3.51-3.56 (m, IH, CH), 3.44-3.50 (m, IH, CH), 3.03-3.10 (m, IH, CH), 2.50 (q, 2H, CH2), 1.82- 1.97 (m, 2H, 2 x CH), 1.66-1.75 (m, IH, CH), 1.55-1.65 (m, IH, CH), 1.09 (t, 3H, CH3)
Ή NMR δ (ppm) (D6-DMSO): 8.37 (s, IH, ArH), 7.92 (s, IH, NH), 7.88 (s, IH, ArH), 7.62 (s, IH, ArH), 6.31 (s, IH, ArH), 6.16 (s, IH, ArH), 3.56-3.62 (m, 4H, CH + CH3), 3.47-3.54 (m,
94
IH, CH), 3.33-3.39 (m, IH, CH), 2.92 (t, IH, CH), 2.45 (q, 2H, CH2), 2.27-2.39 (m, IH, CH), 2.02-2.13 (m, IH, CH), 1.52-1.62 (m, IH, CH), 1.08 (d, 3H, CH3), 1.07 (t, 3H, CH3)
Ή NMR δ (ppm) (D6-DMSO): 8.38 (s, IH, ArH), 7.95 (d, 2H, ArH + NH), 7.70 (s, IH, ArH),
95 6.44 (s, IH, ArH), 6.23 (s, IH, ArH), 3.65-3.75 (m, 2H, 2x CH), 3.61 (s, 3H, CH3), 3.44 (m, 3H, 3x CH), 2.50 (q, 2H, CH2), 2.32-2.42 (m, IH, CH), 2.19-2.30 (m, IH, CH), 1.09 (t, 3H, CH3)
Ή NMR δ (ppm) (D6-DMSO): 8.48 (s, IH, ArH), 8.43 (d, IH, ArH), 8.02 (s, IH, ArH), 7.99 (s,
96 IH, ArH), 7.12 (s, IH, ArH), 6.63 (br. s, IH, NH), 3.69 (s, 3H, CH3), 3.11 (quint, IH, CH), 2.58 (q, 2H, CH2), 2.02 - 1.92 (m, 2H, CH2), 1.80 - 1.60 (m, 6H, CH2), 1.12 (t, 3H, CH3)
Ή NMR δ (ppm) (D6-DMSO): 8.52 (s, IH, ArH), 8.42 (d, IH, ArH), 8.02 (s, 2H, ArH), 7.13 (s, IH, ArH), 6.68 (br. s, IH, NH), 4.06 (d, 2H, CH2), 3.69 (s, 3H, CH3), 2.87 - 2.75 (m, 3H, CH2
97
and H), 2.59 (q, 2H, CH2), 1.85 - 1.75 (m, 2H, CH2), 1.65 - 1.50 (m, 2H, CH2), 1.50 (s, 9H, CH3), 1.12 (t, 3H, CH3)
Ή NMR δ (ppm) (D6-DMSO): 8.46 (d, IH, ArH), 7.97 (s, IH, ArH), 7.92 (s, IH, ArH), 6.52 (s,
98 IH, ArH), 6.16 (s, IH, ArH), 3.87 (t, 2H, CH2), 3.66 (t, 2H, CH2), 3.60 (s, 3H, CH3), 2.63 - 2.50 (m, 2H, CH2), 2.35 (q, 2H, CH2), 1.08 (t, 3H, CH3)
Ή NMR δ (ppm) (D6-DMSO): 8.50 (s, IH, ArH), 8.43 (d, IH, ArH), 8.03 (s, IH, ArH), 8.00
99 (br. s, IH, NH), 7.11 (s, IH, ArH), 6.66 (d, IH, ArH), 3.69 (s, 3H, CH3), 2.68 - 2.50 (m, IH, CH), 2.59 (q, 2H, CH2), 1.90 - 1.66 (m, 5H, CH2), 1.60 - 1.20 (m, 5H, CH2), 1.12 (t, 3H, CH3)
Ή NMR δ (ppm) (D6-DMSO): 9.04(s, IH, ArH), 8.72(s, IH, ArH), 8.52(d, IH, ArH), 8.32(s,
102 IH, ArH), 8.12(s, IH, NH), 6.98(d, IH, ArH), 3.76(s, 3H, CH3), 2.81(q, 2H, CH2), 1.22(t, 3H, CH3)
Biological Examples
Example 1 : In-vitro assays
1.1 JAK1 inhibition assay
1.1.1 JAK1 assay polyGT substrate [0061] Recombinant human JAKl catalytic domain (amino acids 850-1154; catalog number 08-144) was purchased from Carna Biosciences. 10 ng of JAKl is incubated with 12.5 μg polyGT substrate (Sigma catalog number P0275) in kinase reaction buffer (15 mM Tris-HCl pH 7.5, 1 mM DTT, 0.01% Tween-20, 10 mM MgCl2, 2 μΜ non-radioactive ATP, 0.25 μθί 33P-gamma-ATP (GE Healthcare, catalog number AH9968) final concentrations) with or without 5μΕ containing test compound or vehicle (DMSO, 1%) final concentration), in a total volume of 25 μΕ, in a polypropylene 96-well plate (Greiner, V-bottom). After 45 min at 30°C, reactions are stopped by adding of 25 μΕ/well of 150 mM phosphoric acid. All of the terminated kinase reaction is transferred to prewashed (75 mM phosphoric acid) 96 well filter plates (Perkin Elmer catalog number 6005177) using a cell harvester (Perkin Elmer). Plates are washed 6 times with 300 μΕ per well of a 75 mM phosphoric acid solution and the bottom of the plates is sealed. 40 μΕ/well of Microscint-20 is added, the top of the plates is sealed and readout is performed using the Topcount (Perkin Elmer). Kinase activity is calculated by subtracting counts per min (cpm) obtained in the presence of a positive control inhibitor (10 μΜ staurosporine) from cpm obtained in the presence of vehicle. The ability of a test compound to inhibit this activity is determined as:
[0062] Percentage inhibition = ((cpm determined for sample with test compound present - cpm determined for sample with positive control inhibitor) divided by (cpm determined in the presence of vehicle - cpm determined for sample with positive control inhibitor)) * 100.
[0063] Dose dilution series are prepared for the compounds enabling the testing of dose-response effects in the JAKl assay and the calculation of the IC5o for each compound. Each compound is routinely tested at concentration of 20μΜ followed by a 1/3 serial dilution, 8 points (20μΜ - 6.67μΜ - 2.22μΜ - 740nM - 247nM - 82nM - 27nM - 9nM) in a final concentration of 1% DMSO. When potency of compound series increased, more dilutions are prepared and/or the top concentration was lowered (e.g. 5 μΜ, 1 μΜ).
1.1.2 JAKl Ulight-JAKl peptide assay
[0064] Recombinant human JAKl (catalytic domain, amino acids 866-1 154; catalog number PV4774) was purchased from Invitrogen. 1 ng of JAKl was incubated with 20 nM Ulight- JAKl (tyr 1023) peptide (Perkin Elmer catalog number TRF0121) in kinase reaction buffer (25mM MOPS pH6.8, 0.01% Brij-35, 5mM MgCl2, 2mM DTT, 7μΜ ATP) with or without 4μΕ containing test compound or vehicle (DMSO, 1%> final concentration), in a total volume of 20 μΕ, in a white 384 Opti plate (Perkin Elmer, catalog number 6007290). After 60 min at room temperature, reactions were stopped by adding 20 μΕ/well of detection mixture (1 x detection buffer (Perkin Elmer, catalog number CR97-100C), 0.5nM Europium-anti-phosphotyrosine (PT66) (Perkin Elmer, catalog number AD0068), 10 mM EDTA). Readout is performed using the Envision with excitation at 320nm and measuring emission at 615 nm (Perkin Elmer). Kinase activity was calculated by subtracting relative fluorescence units (RFU) obtained in the presence of a positive control inhibitor (10 μΜ staurosporine) from RFU obtained in the presence of vehicle. The ability of a test compound to inhibit this activity was determined as:
[0065] Percentage inhibition = ((RFU determined for sample with test compound present - RFU determined for sample with positive control inhibitor) divided by (RFU determined in the presence of vehicle - RFU determined for sample with positive control inhibitor)) * 100.
[0066] Dose dilution series were prepared for the compounds enabling the testing of dose-response effects in the JAKl assay and the calculation of the IC5o for the compound. Each compound is routinely tested at concentration of 20μΜ followed by a 1/5 serial dilution, 10 points in a final concentration of 1% DMSO. When potency of compound series increases, more dilutions are prepared and/or the top concentration are lowered (e.g. 5 μΜ, 1 μΜ). The data are expressed as the average IC50 from the assays ± standard error of the mean.
[0067] The following compounds have been tested for their activity against JAKl and the IC5o values, as determined using the assays described herein, are given below in Table III.
[0068] TABLE III: JAKl Values of Compounds
> 1001 tiM
501-1000 nM
101-500 nM
0.01-100 nM
Cpd # JAKl Cpd # JAKl
1 *** 21 *
2 * 22 **
3 *** 23 *
4 * 24 *
5 N/A 25 *
6 *** 26 **
7 * 27 *
8 * 28 *
9 N/A 29 ****
10 * 30 *
11 * 31 N/A
12 * 32 *
13 * 33 ****
14 ** 34 *
15 ** 35 *
16 * 36 **
17 * 37 ****
18 * 38 ***
19 * 39 *
20 N/A 40 * Cpd # JAK1 Cpd # JAK1
41 *** 72 **
42 * 73 ***
43 ** 74 ***
44 *** 76 ****
45 * 77 *
46 *** 79 ****
47 *** 80 **
48 *** 81 *
49 **** 82 ****
50 *** 83 ****
51 * 84 ****
52 * 85 *
53 *** 86 ***
54 * 87 ****
55 **** 88 ***
56 **** 89 **
57 * 90 ***
58 * 91 **
59 ** 92 ****
60 **** 93 ****
61 **** 94 ****
62 **** 95 ****
63 ** 96 ***
64 *** 97 *
65 **** 98 N/A
66 **** 99 ***
67 *** 100 ****
68 **** 101 N/A
69 *** 102 *
70 *** 79 ****
71 ****
7.7.3 J47C7 Ki determination assay
[0069] For the determination of Ki, different amounts of compound are mixed with the enzyme and the enzymatic reaction is followed as a function of ATP concentration. The Ki is determined by means of double reciprocal plotting of Km vs compound concentration (Lineweaver-Burk plot). 1 ng of JAK1 (Invitrogen, PV4774) is used in the assay. The substrate was 50nM Ulight-JAK-1 (Tyrl 023) Peptide (Perkin Elmer, TRF0121) The reaction is performed in 25mM MOPS pH 6.8, 0.01%, 2 mM DTT, 5 mM MgC¾ Brij-35 with varying concentrations of ATP and compound. Phosphorylated substrate is measured using an Eu-labeled anti-phosphotyrosine antibody PT66 (Perkin Elmer, AD0068) as described in 1.1.2. Readout is performed on the envision (Perkin Elmer) with excitation at 320 nm and emission followed at 615 nm and 665 nm.
JAK2 inhibition assay
JAK2 assay polyGT substrate [0070] Recombinant human JAK2 catalytic domain (amino acids 808-1 132; catalog number PV4210) was purchased from Invitrogen. 0.025mU of JAK2 is incubated with 2.5 μg polyGT substrate (Sigma catalog number P0275) in kinase reaction buffer (5 mM MOPS pH 7.5, 9 mM MgAc, 0.3mM EDTA, 0.06% Brij and 0.6 mM DTT, 1 μΜ non-radioactive ATP, 0.25μΟ 33P-gamma-ATP (GE Healthcare, catalog number AH9968) final concentrations) with or without 5μΕ containing test compound or vehicle (DMSO, 1% final concentration), in a total volume of 25 μΕ, in a polypropylene 96-well plate (Greiner, V-bottom). After 90 min at 30°C, reactions are stopped by adding of 25 μΕ/well of 150 mM phosphoric acid. All of the terminated kinase reaction is transferred to prewashed (75 mM phosphoric acid) 96 well filter plates (Perkin Elmer catalog number 6005177) using a cell harvester (Perkin Elmer). Plates are washed 6 times with 300 μΕ per well of a 75 mM phosphoric acid solution and the bottom of the plates is sealed. 40 μΕ/well of Microscint-20 is added, the top of the plates is sealed and readout is performed using the Topcount (Perkin Elmer). Kinase activity is calculated by subtracting counts per min (cpm) obtained in the presence of a positive control inhibitor (10 μΜ staurosporine) from cpm obtained in the presence of vehicle. The ability of a test compound to inhibit this activity is determined as:
[0071] Percentage inhibition = ((cpm determined for sample with test compound present - cpm determined for sample with positive control inhibitor) divided by (cpm determined in the presence of vehicle - cpm determined for sample with positive control inhibitor)) * 100.
[0072] Dose dilution series are prepared for the compounds enabling the testing of dose-response effects in the JAK2 assay and the calculation of the IC5o for each compound. Each compound is routinely tested at concentration of 20μΜ followed by a 1/3 serial dilution, 8 points (20μΜ - 6.67μΜ - 2.22μΜ - 740nM - 247nM - 82nM - 27nM - 9nM) in a final concentration of 1% DMSO. When potency of compound series increased, more dilutions are prepared and/or the top concentration is lowered (e.g. 5 μΜ, 1 μΜ).
1.2.2. JAK2 Ulight-JAKl peptide assay
[0073] Recombinant human JAK2 (catalytic domain, amino acids 866-1 154; catalog number PV4210) was purchased from Invitrogen. 0.0125mU of JAK2 was incubated with 25 nM Ulight- JAKl (tyr 1023) peptide (Perkin Elmer catalog number TRF0121) in kinase reaction buffer (25mM HEPES pH7.0, 0.01% Triton X-100, 7.5mM MgCl2 , 2mM DTT, 7.5μΜ ATP) with or without 4μΕ containing test compound or vehicle (DMSO, 1%> final concentration), in a total volume of 20 μΕ, in a white 384 Opti plate (Perkin Elmer, catalog number 6007290). After 60 min at room temperature, reactions were stopped by adding 20 μΕ/well of detection mixture (lxdetection buffer (Perkin Elmer, catalog number CR97-100C), 0.5nM Europium-anti-phosphotyrosine (PT66) (Perkin Elmer, catalog number AD0068), 10 mM EDTA). Readout is performed using the Envision with excitation at 320nm and measuring emission at 615 nm (Perkin Elmer). Kinase activity was calculated by subtracting relative fluorescence units (RFU) obtained in the presence of a positive control inhibitor (10 μΜ staurosporine) from RFU obtained in the presence of vehicle. The ability of a test compound to inhibit this activity was determined as:
[0074] Percentage inhibition = ((RFU determined for sample with test compound present - RFU determined for sample with positive control inhibitor) divided by (RFU determined in the presence of vehicle - RFU determined for sample with positive control inhibitor)) * 100.
[0075] Dose dilution series are prepared for compound enabling the testing of dose-response effects in the JAK2 assay and the calculation of the IC5o for the compound. Each compound is routinely tested at concentration of 20μΜ followed by a 1/5 serial dilution, 10 points in a final concentration of 1% DMSO. When potency of compound series increases, more dilutions are prepared and/or the top concentration are lowered (e.g. 5 μΜ, 1 μΜ). The data are expressed as the average IC5o from the assays ± standard error of the mean.
[0076] The following compounds have been tested for their activity against JAK2 and the IC5o values, as determined using the assays described herein, are given below in Table IV.
[0077] TABLE IV: JAK2 IC50 Values of Compounds
N/A not active
# > 1001 tiM
## 501-1000 nM
### 101-500 nM
#### 0.01-100 nM
Figure imgf000110_0001
Figure imgf000111_0001
1.2.3 JAK2 Kd determination assay
[0078] JAK2 (Invitrogen, PV4210) is used at a final concentration of 5 nM. The binding experiment is performed in 50mM Hepes pH 7.5, 0.01% Brij-35, lOmM MgCl2, ImM EGTA using 25nM kinase tracer 236 (Invitrogen, PV5592) and 2 nM Eu-anti-GST (Invitrogen, PV5594) with varying compound concentrations. Detection of tracer is performed according to the manufacturer's procedure.
1.3. JAK3 inhibition assay
[0079] Recombinant human JAK3 catalytic domain (amino acids 781-1 124; catalog number PV3855) was purchased from Invitrogen. 0.5 ng JAK3 protein was incubated with 2.5 μg polyGT substrate (Sigma catalog number P0275) in kinase reaction buffer (25 mM Tris pH 7.5, 0.5 mM EGTA, lOmM MgCl2, 2.5mM DTT, 0.5 mM Na3V04, 5 mM b-glycerolphosphate, 0.01% Triton X-100, 1 μΜ non-radioactive ATP, 0.25 \.C\ 33P-gamma- ATP (GE Healthcare, catalog number AH9968) final concentrations) with or without 5μΕ containing test compound or vehicle (DMSO, 1%> final concentration), in a total volume of 25 μΕ, in a polypropylene 96-well plate (Greiner, V-bottom). After 45 min at 30°C, reactions were stopped by adding 25 μΕ/well of 150 mM phosphoric acid. All of the terminated kinase reaction was transferred to prewashed (75 mM phosphoric acid) 96 well filter plates (Perkin Elmer catalog number 6005177) using a cell harvester (Perkin Elmer). Plates were washed 6 times with 300 μΕ per well of a 75 mM phosphoric acid solution and the bottom of the plates was sealed. 40 μΕ/well of Microscint-20 was added, the top of the plates was sealed and readout was performed using the Topcount (Perkin Elmer). Kinase activity was calculated by subtracting counts per min (cpm) obtained in the presence of a positive control inhibitor (10 μΜ staurosporine) from cpm obtained in the presence of vehicle. The ability of a test compound to inhibit this activity was determined as:
[0080] Percentage inhibition = ((cpm determined for sample with test compound present - cpm determined for sample with positive control inhibitor) divided by (cpm determined in the presence of vehicle - cpm determined for sample with positive control inhibitor)) * 100.
[0081] Dose dilution series were prepared for the compounds enabling the testing of dose-response effects in the JAK3 assay and the calculation of the IC5o for each compound. Each compound was routinely tested at concentration of 20μΜ followed by a 1/5 serial dilution, 10 points in a final concentration of 1% DMSO. When potency of compound series increased, more dilutions were prepared and/or the top concentration was lowered (e.g. 5 μΜ, 1 μΜ).
[0082] The following compounds have been tested for their activity against JAK3 and the IC5o values, as determined using the assays described herein, are given below in Table V.
[0083] TABLE V: JAK3 IC50 Values of Compounds
N/A not active
+ > 1001 nM
++ 501-1000 nM
+++ 101-500 nM
++++ 0.01-100 nM
Cpd # JAK3 Cpd # JAK3
1 + 39 N/A
3 + 56 +
6 + 60 +
7 N/A 61 +
8 N/A 62 +
9 N/A 65 +
10 N/A 66 +++
11 N/A 67 ++
16 + 68 +++
17 + 70 ++
18 + 76 +
29 N/A 77 +
33 +++ 82 +++
34 N/A 83 +
37 + 87 +++
38 N/A 92 +
Figure imgf000113_0001
1.3.1. JAK3 Ki determination assay
[0084] For the determination of Ki, different amounts of compound are mixed with the enzyme and the enzymatic reaction is followed as a function of ATP concentration. The Ki is determined by means of double reciprocal plotting of Km vs compound concentration (Lineweaver-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 is performed in 25mM Tris pH 7.5 , 0.01% Triton X-100 , 0.5mM EGTA, 2.5mM DTT, 0.5mM Na3V04, 5mM b- glycerolphosphate, lOmM MgCl2 with varying concentrations of ATP and compound and stopped by addition of 150 mM phosphoric acid. Measurement of incorporated phosphate into the substrate polyGT is done by loading the samples on a filter plate (using a harvester, Perkin Elmer) and subsequent washing. Incorporated 33P in polyGT is measured in a Topcount scintillation counter after addition of scintillation liquid to the filter plates (Perkin Elmer).
1.4. TYK2 inhibition assay
[0085] Recombinant human TYK2 catalytic domain (amino acids 871-1187; catalog number 08- 147) was purchased from Carna biosciences. 5 ng of TYK2 was incubated with 12.5 μg polyGT substrate (Sigma catalog number P0275) in kinase reaction buffer (25 mM Hepes pH 7.2, 50 mM NaCl, 0.5mM EDTA, lmM DTT, 5mM MnCl2, lOmM Mg<¾ 0.1% Brij-35, 0.1 μΜ non-radioactive ATP, 0.125 iCi 33P-gamma-ATP (GE Healthcare, catalog number AH9968) final concentrations) with or without 5μΕ containing test compound or vehicle (DMSO, 1%> final concentration), in a total volume of 25 μΕ, in a polypropylene 96-well plate (Greiner, V-bottom). After 90 min at 30°C, reactions were stopped by adding 25 μΕΛνεΙΙ of 150 mM phosphoric acid. All of the terminated kinase reaction was transferred to prewashed (75 mM phosphoric acid) 96 well filter plates (Perkin Elmer catalog number 6005177) using a cell harvester (Perkin Elmer). Plates were washed 6 times with 300 μΕ per well of a 75 mM phosphoric acid solution and the bottom of the plates was sealed. 40 μΕ/well of Microscint-20 was added, the top of the plates was sealed and readout was performed using the Topcount (Perkin Elmer). Kinase activity was calculated by subtracting counts per min (cpm) obtained in the presence of a positive control inhibitor (10 μΜ staurosporine) from cpm obtained in the presence of vehicle. The ability of a test compound to inhibit this activity was determined as:
[0086] Percentage inhibition = ((cpm determined for sample with test compound present - cpm determined for sample with positive control inhibitor) divided by (cpm determined in the presence of vehicle - cpm determined for sample with positive control inhibitor)) * 100.
I l l [0087] Dose dilution series were prepared for the compounds enabling the testing of dose-response effects in the TYK2 assay and the calculation of the IC5o for each compound. Each compound was routinely tested at concentration of 20μΜ followed by a 1/3 serial dilution, 8 points (20μΜ - 6.67μΜ - 2.22μΜ - 740nM - 247nM - 82nM - 27nM - 9nM) in a final concentration of 1% DMSO. When potency of compound series increased, more dilutions were prepared and/or the top concentration was lowered (e.g. 5 μΜ, 1 μΜ).
[0088] The following compounds have been tested for their activity against TYK2; and the IC5o values, as determined using the assays described herein, are given below in Table VI.
[0089] TABLE VI: TYK2 IC50 Values of Compounds
N/A not active
> 1001 nM
501-1000 nM
101-500 nM
0.01-100 nM
Figure imgf000114_0002
Figure imgf000114_0001
1.4.1. TYK2 Kd determination assay
[0090] TYK2 (Carna Biosciences, 09CBS-0983D) is used at a final concentration of 5 nM. The binding experiment is performed in 50mM Hepes pH 7.5, 0.01% Brij-35, lOmM MgC12, ImM EGTA using 50nM kinase tracer 236 (Invitrogen, PV5592) and 2 nM Eu-anti-GST (Invitrogen, PV5594) with varying compound concentrations. Detection of tracer is performed according to the manufacturers' procedure. Example 2. Cellular assays:
2.1. JAK-STAT signalling assay:
[0091] HeLa cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% heat inactivated fetal calf serum, 100 U/mL penicillin and 100 μg/mL streptomycin. HeLa cells were used at 70 % confluence for transfection. 20,000 cells in 87 μΕ cell culture medium were transiently transfected with 40 ng p STAT l(2)-lucif erase reporter (Panomics), 8 ng of LacZ reporter as internal control reporter and 52 ng of pBSK using 0.32 μΕ Jet-PEI (Polyplus) as transfection reagent per well in 96-well plate format. After overnight incubation at 37°C, 5% C02, transfection medium was removed. 81 μΕ of DMEM + 1.5% heat inactivated fetal calf serum was added. 9 μΕ compound at 10 x concentration was added for 60 min and then 10 μΕ of human OSM (Peprotech) at 33 ng/mL final concentration.
[0092] All compounds were tested in duplicate starting from 20 μΜ followed by a 1/3 serial dilution, 8 doses in total (20 μΜ - 6.6 μΜ - 2.2 μΜ - 740 nM - 250 nM - 82 nM - 27 nM - 9 nM) in a final concentration of 0.2%> DMSO.
[0093] After overnight incubation at 37°C, 5%> C02 cells were lysed by adding 100 μΕ lysis buffer/well (PBS, 0.9 mM CaC12, 0.5 mM MgC12, 10% Trehalose, 0.05% Tergitol NP9, 0.3% BSA).
[0094] 40 μΕ of cell lysate was used to read β-galactosidase activity by adding 180 μΕ β-Gal solution (30μΕ ONPG 4mg/mL + 150 μΕ β-Galactosidase buffer (0.06 M Na2HP04, 0.04 M NaH2P04, 1 mM MgC12)) for 20 min. The reaction was stopped by addition of 50 μΕ Na2C03 1 M. Absorbance was read at 405 nm.
[0095] Luciferase activity was measured using 40 μΕ cell lysate plus 40 μΕ of Steadylite® as described by the manufacturer (Perkin Elmer), on the Envision (Perkin Elmer).
[0096] Omitting OSM was used as a positive control (100% inhibition). As negative control 0.5%> DMSO (0%> inhibition) was used. The positive and negative controls were used to calculate z' and 'percent inhibition' (PIN) values.
[0097] Percentage inhibition = ((fluorescence determined in the presence of vehicle - fluorescence determined for sample with test compound present) divided by (fluorescence determined in the presence of vehicle - fluorescence determined for sample without trigger)) * 100.
[0098] PIN values were plotted for compounds tested in dose-response and EC50 values were derived.
[0099] The following compounds have been tested for their activity in the JAK-STAT assay; and the IC50 values, as determined using the assays described herein, are given below in Table VII.
[00100] TABLE VII: JAK-STAT IC n of selected compounds of the invention
& > 1001 nM && 501-1000 nM
&&& 1-500 nM
Figure imgf000116_0002
Figure imgf000116_0001
Example 2.2 OSM/IL-Ιβ signaling Assay
[00101] OSM and IL-Ι β are shown to synergistically upregulate MMP13 levels in the human chondrosarcoma cell line SW1353. The cells are seeded in 96 well plates at 15,000 cells/well in a volume of 120 \L DMEM (Invitrogen) containing 10% (v/v) FBS and 1%> penicillin/streptomycin (InVitrogen) incubated at 37°C 5% C02. Cells are preincubated with 15 μΕ of compound in Ml 99 medium with 2% DMSO 1 h before triggering with 15 μΕ OSM and IL-Ι β to reach 25 ng/mL OSM and 1 ng/mL IL-Ι β, and MMP13 levels are measured in conditioned medium 48 h after triggering. MMP13 activity is measured using an antibody capture activity assay. For this purpose, 384 well plates (NUNC, 460518, MaxiSorb black) are coated with 35 μΕ of a 1.5 μg/mL anti-human MMP13 antibody (R&D Systems, MAB511) solution for 24 h at 4°C. After washing the wells 2 times with PBS + 0.05% Tween, the remaining binding sites are blocked with 100 μΕ 5% non-fat dry milk (Santa Cruz, sc-2325, Blotto) in PBS for 24 h at 4°C. Next, the wells are washed twice with PBS + 0.05% Tween and 35 μΕ of 1/10 dilution of culture supernatant containing MMP13 in 100-fold diluted blocking buffer is added and incubated for 4 h at rt. Next the wells are washed twice with PBS + 0.05% Tween followed by MMP13 activation by addition of 35 μΕ of a 1.5 mM 4-Aminophenylmercuric acetate (APMA) (Sigma, A9563) solution and incubation at 37°C for 1 h. The wells are washed again with PBS + 0.05% Tween and 35 μΕ MMP13 substrate (Biomol, P-126, OmniMMP fluorogenic substrate) is added. After incubation for 24 h at 37°C fluorescence of the converted substrate is measured in a Perkin Elmer Wallac EnVision 2102 Multilabel Reader (wavelength excitation: 320 nm, wavelength emission: 405 nm).
[00102] Percentage inhibition = ((fluorescence determined in the presence of vehicle - fluorescence determined for sample with test compound present) divided by (fluorescence determined in the presence of vehicle - fluorescence determined for sample without trigger)) * 100.
Example 2.3 PBL Proliferation assay [00103] Human peripheral blood lymphocytes (PBL) are stimulated with IL-2 and proliferation is measured using a BrdU incorporation assay. The PBL are first stimulated for 72 h with PHA to induce IL-2 receptor, then they are fasted for 24 h to stop cell proliferation followed by IL-2 stimulation for another 72 h (including 24h BrdU labeling). Cells are preincubated with test compounds 1 h before IL-2 addition. Cells are cultured in RPMI 1640 containing 10% (v/v) FBS.
Example 2.4 Whole blood assay (WBA)
2.4.1 IFNa stimulation protocol
[00104] To predict the potency of the test compounds to inhibit JAK1 or JAK2-dependent signaling pathways in vivo, a physiologically relevant in vitro model was developed using human whole blood. In the WBA assay, blood, drawn from human volunteers who gave informed consent, is treated ex vivo with compound (lh) and subsequently stimulated either for 30 min with interferon a (IFNa, JAK1 dependent pathway) or for 2 h with granulocyte macrophage-colony stimulating factor (GM-CSF, JAK2 dependent pathway).
2.4.1.1 Phospho - STA Tl Assay
[00105] For IFNa stimulation, increase in phosphorylation of Signal Transducers and Activators of Transcription 1 (pSTATl) by IFNa in white blood cell extracts is measured using a pSTATl ELISA assay. Phosphorylation of Signal Transducer and Activator of Transcription 1 (STAT1) after interferon alpha (IFNa) triggering is a JAK1 -mediated event. The Phospho-STATl Assay, which is used to measure Phospho-STATl levels in cellular extracts, is developed to assess the ability of a compound to inhibit JAK1 -dependent signaling pathways.
[00106] Whole human blood, drawn from human volunteers who gave informed consent, is ex vivo treated with compound (lh) and subsequently stimulated for 30 min with IFNa. The increase in phosphorylation of STAT1 by INFa in white blood cell extracts was measured using a phospho-STATl ELISA.
[00107] The ACK lysis buffer consisted of 0.15 M NH4C1, 10 mM KHC03, 0.1 mM EDTA. The pH of the buffer was 7.3.
[00108] A lOx cell lysis buffer concentrate (part of the PathScan Phospho-STATl (Tyr701) sandwich ELISA kit from Cell Signaling) is diluted 10-fold in H20. Proteinase inhibitors were added to the buffer before use.
[00109] 20 μg IFNa is dissolved in 40 μΕ H20 to obtain a 500 μg/mL stock solution. The stock solution was stored at -20°C.
[00110] A 3-fold dilution series of the compound is prepared in DMSO (highest concentration: 10 mM). Subsequently, the compound is further diluted in medium (dilution factor dependent on desired final compound concentration). 2.4.1.1.1 Incubation of blood with compound and stimulation with IFNa
[00111] Human blood is collected in heparinized tubes. The blood is divided in aliquots of 392 L. Afterwards, 4 L of compound dilution is added to each aliquot and the blood samples are incubated for 1 h at 37°C. The IFNa stock solution is diluted 1000-fold in RPMI medium to obtain a 500 ng/mL working solution. 4 L of the 500 ng/mL work solution is added to the blood samples (final concentration IFNa: 5ng/mL). The samples are incubated at 37°C for 30 min.
2.4.1.1.2 Preparation of cell extracts
[00112] At the end of the stimulation period, 7.6 mL ACK buffer is added to the blood samples to lyse the red blood cells. The samples are mixed by inverting the tubes five times and the reaction is incubated on ice for 5 min. The lysis of the RBC should be evident during this incubation. The cells are pelleted by centrifugation at 300 g, 4°C for 7 min and the supernatant is removed. 10 mL lx PBS is added to each tube and the cell pellet is resuspended. The samples are centrifuged again for 7 min at 300 g, 4°C. The supernatant is removed and the pellet resuspended in 500 μL of lx PBS. Then, the cell suspension is transferred to a clean 1.5 mL microcentrifuge tube. The cells are pelleted by centrifugation at 700 g for 5 min at 4°C. The supernatant is removed and the pellet was dissolved in 150 μL cell lysis buffer. The samples are incubated on ice for 15 min. After that, the samples are stored at - 80°C until further processing.
2.4.1.1.3 Measurement of STA Tl phosphorylation by ELISA
[00113] The Pathscan Phospho-STATl (Tyr701) Sandwich ELISA kit from Cell Signaling (Cat.n°: #7234) is used to determine Phospho-STATl levels.
[00114] The cellular extracts are thawed on ice. The tubes are centrifuged for 5 min at 16,000 g, 4°C and the cleared lysates are harvested. Meanwhile, the microwell strips from the kit are equilibrated to room temperature and wash buffer is prepared by diluting 20 x wash buffer in H20. Samples are diluted 2-fold in sample diluent and 100 L is added to the microwell strips. The strips are incubated overnight at 4°C.
[00115] The following day, the wells are washed 3 times with wash buffer. 100 L of the detection antibody is added to the wells. The strips are incubated at 37°C for 1 h. Then, the wells are washed 3 times with wash buffer again. 100 \L HRP-linked secondary antibody is added to each well and the samples are incubated at 37°C. After 30 min, the wells are washed 3 times again and 100 \L TMB substrate is added to all wells. When samples turned blue, 100 L STOP solution is added to stop the reaction. Absorbance is measured at 450 nm.
2.4.1.2 Data analysis [00116] Inhibition of phosphoSTATl induction by IFNa in cell extracts is plotted against the compound concentration and IC5o values are derived using Graphpad software. Data were retained if R2 (coefficient of determination used in statistical models to measure the proportion of variability of the model and its predictive capacity. R2 ranges from 0 (no correlation of the data: no predictive value) to 1 (full correlation: great predictive value) is larger than 0.8 and the hill slope is smaller than 3.
2.4.1.3 IL-8 ELISA
[00117] For GM-CSF stimulation, increase in interleukin-8 (IL-8) levels in plasma is measured using an IL-8 ELISA assay. Granulocyte macrophage-colony stimulating factor (GM-CSF) - induced interleukin 8 (IL-8) expression is a JAK2-mediated event. The IL-8 ELISA, which can be used to measure IL-8 levels in plasma samples, has been developed to assess the ability of a compound to inhibit JAK2-dependent signaling pathways.
[00118] Whole human blood, drawn from human volunteers who gave informed consent, is ex vivo treated with compound (lh) and subsequently stimulated for 2 h with GM-CSF. The increase in IL-8 levels in plasma is measured using an IL-8 ELISA assay.
[00119] 10 μg GM-CSF is dissolved in 100 μΕ H20 to obtain a 100 μg/mL stock solution. The stock solution is stored at -20°C.
[00120] A 3-fold dilution series of the test compound is prepared in DMSO (highest concentration: 10 mM). Subsequently, the compound is further diluted in medium (dilution factor dependent on desired final compound concentration).
2.4.1.3.1 Incubation of blood with compound and stimulation with GM-CSF
[00121] Human blood is collected in heparinized tubes. The blood is divided in aliquots of 245 μΕ. Afterwards, 2.5 μΕ test compound dilution is added to each aliquot and the blood samples are incubated for 1 h at 37°C. The GM-CSF stock solution is diluted 100-fold in RPMI medium to obtain a 1 μg/mL work solution. 2.5 μΕ of the 1 μg/mL work solution is added to the blood samples (final concentration GM-CSF: 10 ng/mL). The samples are incubated at 37°C for 2 h.
2.4.1.3.2 Preparation of plasma samples
[00122] The samples are centrifuged for 15 min at 1,000 g, 4°C. 100 μΕ of the plasma is harvested and stored at -80°C until further use.
2.4.1.3.3 Measurement of IL-8 levels by ELISA
[00123] The Human IL-8 Chemiluminescent Immunoassay kit from R&D Systems (Cat.n°: Q8000B) is used to determine IL-8 levels.
[00124] Wash buffer is prepared by diluting 10 x wash buffer in H20. Working glo reagent is prepared by adding 1 part Glo Reagent 1 to 2 parts Glo Reagent B 15 min to 4 h before use. [00125] 100 μΐ. assay diluent RD1-86 is added to each well. After that, 50 μΐ. of sample (plasma) is added. The ELISA plate is incubated for 2 h at room temperature, 500 rpm. All wells are washed 4 times with wash buffer and 200 μL· IL-8 conjugate is added to each well. After incubation for 3 h at room temperature, the wells are washed 4 times with wash buffer and 100 μΐ^ working glo reagent is added to each well. The ELISA plate is incubated for 5 min at room temperature (protected from light). Luminescence is measured (0.5 s/well read time).
2.4.2 IL-6 stimulation protocol
[00126] In addition, a flow cytometry analysis was performed to establish JAKl over JAK2 compound selectivity ex vivo using human whole blood. Therefore, blood was taken from human volunteers who gave informed consent. Blood was then equilibrated for 30 min at 37°C under gentle rocking, then aliquoted in Eppendorf tubes. Compound was added at different concentrations and incubated at 37°C for 30 min under gentle rocking and subsequently stimulated for 20 min at 37°C under gentle rocking with interleukin 6 (IL-6) for JAKl -dependent pathway stimulation or GM-CSF for JAK2-dependent pathway stimulation. Phospho-STATl and phospho-STAT5 were then evaluated using FACS analysis.
2.4.2.1 Phospho-STA Tl Assays
[00127] For IL-6-stimulated increase of Signal Transducers and Activators of Transcription 1 (pSTATl) phosphorylation in white blood cell, human whole blood, drawn from human volunteers who gave informed consent, was ex vivo treated with the compound for 30 min and subsequently stimulated for 20 min with IL-6. The increase in phosphorylation of STAT1 by IL-6 in lymphocytes was measured using anti phospho-STATl antibody by FACS.
[00128] The 5X Lyse/Fix buffer (BD PhosFlow, Cat. N°558049) was diluted 5-fold with distilled water and pre-warmed at 37°C. The remaining diluted Lyse/Fix buffer was discarded.
[00129] 10 μg rhIL-6 (R&D Systems, Cat N°206-IL) was dissolved in lmL of PBS 0.1% BSA to obtain a 10μg/mL stock solution. The stock solution was aliquoted and stored at -80°C.
[00130] A 3-fold dilution series of the compound was prepared in DMSO (10 mM stock solution).
Control-treated samples received DMSO instead of compound. All samples were incubated with a 1% final DMSO concentration.
2.4.2.1.1 Incubation of blood with compound and stimulation with IL-6
[00131] Human blood was collected in heparinized tubes. The blood was divided in aliquots of 148.5μΕ. Then, 1.5 μΕ of the test compound dilution was added to each blood aliquot and the blood samples were incubated for 30 min at 37°C under gentle rocking. IL-6 stock solution (1.5μΕ) was d added to the blood samples (final concentration lOng/mL) and samples were incubated at 37°C for 20 min under gentle rocking.
2.4.2.1.2 White blood cell preparation and CD4 labeling
[00132] At the end of the stimulation period, 3mL of IX pre-warmed Lyse/Fix buffer was immediately added to the blood samples, vortexed briefly and incubated for 15 min at 37°C in a water bath in order to lyse red blood cells and fix leukocytes, then frozen at -80°C until further use.
[00133] For the following steps, tubes were thawed at 37°C for approximately 20 min and centrifuged for 5 min at 400xg at 4°C. The cell pellet was washed with 3mL of cold IX PBS, and after centrifugation the cell pellet was resuspended in ΙΟΟμΙ. of PBS containing 3% BSA. FITC-conjugated anti-CD4 antibody or control FITC-conjugated isotype antibody were added and incubated for 20 min at room temperature, in the dark.
2.4.2.1.3 Cell permeabilization and labeling with anti Phospho-STA Tl antibody
[00134] After washing cells with IX PBS, the cell pellet was resuspended in ΙΟΟμΙ. of ice-cold IX PBS and 900μΙ. ice-cold 100% MeOH was added. Cells were then incubated at 4°C for 30 min for permeabilization.
[00135] Permeabilized cells were then washed with IX PBS containing 3% BSA and finally resuspended in 80μΕ of IX PBX containing 3% BSA.
[00136] 20μΙ of PE mouse anti-STATl (pY701) or PE mouse IgG2aK isotype control antibody (BD Biosciences, Cat. N°612564 and 559319, respectively) were added and mixed, then incubated for 30 min at 4°C, in the dark.
[00137] Cells are then washed once with IX PBS and analyzed on a FACSCanto II flow cytometer (BD Biosciences).
2.4.2.1.4 Fluorescence analysis on FA CSCanto II
[00138] 50,000 total events were counted and Phospho-STATl positive cells were measured after gating on CD4+ cells, in the lymphocyte gate. Data were analyzed using the FACSDiva software and the percentage inhibition of IL-6 stimulation calculated on the percentage of positive cells for phospho- STAT1 on CD4+ cells.
2.4.2.2 Phospho-STA T5 Assay
[00139] For GM-CSF-stimulated increase of Signal Transducers and Activators of Transcription 5 (pSTAT5) phosphorylation in white blood cell, human whole blood, drawn from human volunteers who gave informed consent, is ex vivo treated with compound for 30 min and subsequently stimulated for 20 min with GM-CSF. The increase in phosphorylation of STAT5 by GM-CSF in monocytes is measured using an anti phospho-STAT5 antibody by FACS.
[00140] The 5X Lyse/Fix buffer (BD PhosFlow, Cat. N°558049) is diluted 5-fold with distilled water and pre-warmed at 37°C. Remaining diluted Lyse/Fix buffer is discarded.
[00141] 10 μg rhGM-CSF (AbCys S.A. Cat N°P300-03) is dissolved in ΙΟΟμΙ. of PBS 0.1% BSA to obtain a 100μg/mL stock solution. The stock solution is stored aliquoted at -80°C.
[00142] A 3-fold dilution series of the compound is prepared in DMSO (10 mM stock solution). Control-treated samples receive DMSO without the test compound. All samples are incubated with a 1% final DMSO concentration.
2.4.2.2.1 Incubation of blood with compound and stimulation with GM-CSF
[00143] Human blood is collected in heparinized tubes. The blood is divided in aliquots of 148.5μΙ^. Then, 1.5 μΐ. of compound dilution is added to each aliquot and the blood samples are incubated for 30 min at 37°C under gentle rocking. GM-CSF stock solution (1.5μΕ) is added to the blood samples (final concentration 20pg/mL) and samples are incubated at 37°C for 20 min under gentle rocking.
2.4.2.2.2 White blood cell preparation and CD14 labeling
[00144] At the end of the stimulation period, 3mL of IX pre-warmed Lyse/Fix buffer is immediately added to the blood samples, vortexed briefly and incubated for 15 min at 37°C in a water bath in order to lyse red blood cells and fix leukocytes, then frozen at -80°C until further use.
[00145] For the following steps, tubes are thawed at 37°C for approximately 20 min and centrifuged for 5 min at 400xg at 4°C. The cell pellet is washed with 3mL of cold IX PBS, and after centrifugation the cell pellet is resuspended in 100μL of PBS containing 3% BSA. FITC mouse anti-CD14 antibody (BD Biosciences, Cat. N°345784) or control FITC mouse IgG2bK isotype antibody (BD Biosciences, Cat. N°555057) are added and incubated for 20 min at room temperature, in the dark.
2.4.2.2.3 Cell permeabilization and labeling with anti phospho-STA T5 antibody
[00146] After washing cells with IX PBS, the cell pellet is resuspended in 100μL of ice-cold IX PBS and 900μL of ice-cold 100% MeOH is added. Cells are then incubated at 4°C for 30 min for permeabilization.
[00147] Permeabilized cells are then washed with IX PBS containing 3%> BSA and finally resuspended in 80μL of IX PBX containing 3%> BSA.
[00148] 20μL of PE mouse anti-STAT5 (pY694) or PE mouse IgGlK isotype control antibody (BD Biosciences, Cat. N°612567and 554680, respectively) are added, mixed then incubated for 30 min at 4°C, in the dark. [00149] Cells are then washed once with IX PBS and analyzed on a FACSCanto II flow cytometer (BD Biosciences).
2.4.2.2.4 Fluorescence analysis on FA CSCanto II
[00150] 50,000 total events are counted and Phospho-STAT5 positive cells are measured after gating on CD14+ cells. Data are analyzed using the FACSDiva software and correspond to the percentage of inhibition of GM-CSF stimulation calculated on the percentage of positive cells for phosphor-STAT5 on CD 14+ cells.
Example 2.5 CTLL2 visibility assay
[00151] The CTLL-2 cell line is an IL-2 dependent mouse T cell line that constitutively express IL2 receptors and depends entirely on the presence of IL2 for its growth (Haan et ah, 2011 Chem Biol., 18(3):314-23). Since IL-2 signaling depends on the activity of the JAK-kinase family member JAK1 and JAK3 , the proliferation and survival of CTLL-2 cells can be used to test the JAK1/JAK3 inhibitory capacity of the compounds of the invention.
[00152] The protocol describes the methods to analyse the activity of compounds on their ability to sustain the IL2-dependent viability of CTLL2.
[00153] CTLL2 cells were cultured in RPMI1640 medium (Life Technologies Cat no 21875-034), with 10 % fetal bovine serum (FBS, HiClone SV30160.03, 1% penicillin/streptomycin and 10% of the IL-2 culture supplement T STIM with ConA (BD Biosciences no 354115).
[00154] CTLL cells are seeded at 1000 cells per well of a white 384 well plate (Greiner, 781080) in 20 μΕ medium RPMI1640.
[00155] To the wells, 10 L of diluted compound (or controls) was added. Negative control was a DMSO dilution, positive control was Incyte INCB018424 at 10 μΜ . Final DMSO concentration was 0.1 %.
[00156] The plates were incubated at 37°C for 24 h and then the ATP content as measured using ATP-lite (Perkin Elmer, cat no 6016739). For this, 30 μΕ ATP-lite solution was added to each well, and after 2 min shaking and another 8 min incubation at room temp in the dark, bioluminescence was measured in a PerkinElmer Envision multireader equipped for luminescence.
[00157] TABLE VIII: CTLL2 IC^ of selected compounds of the invention
* > 1001 nM
** 501-1000 nM
Figure imgf000123_0001
Cpd # CTLL2
83 *
87 *
92 *
98 *
Example 2.6 BA/F3 viability assay
[00158] The BA/F3 cell line is an IL-3 dependent mouse pro-B-cell line that undergoes cell death upon IL-3 withdrawal (Collins, Marvel et al. 1992 ). Since IL-3 signaling depends on the activity of the JAK kinase family member JAK2 only (Reddy, Korapati et al. 2000), the proliferation and survival of BA/F3 cells can be used to test the JAK2 inhibitory capacity of a compound of the invention.
[00159] BA/F3 cells were cultured in RPMI1640 medium (Life Technologies Cat no 21875-034), with 10 % fetal bovine serum (FBS, HiClone SV30160.03, 1% pen/strep and 10 ng/mL IL-3 (peprotech, no 213-13) BA/F3 cells were seeded at 1500 cells per well of a white 384 well plate (Greiner, 781080) in 20 iL medium. To each wells, 10 L of diluted compound (or controls) is added. Negative control was a DMSO dilution, positive control was Tofacitinib (CP-690550) at 10 μΜ. Final DMSO concentration was 0.1 %.
[00160] The plates were incubated at 37°C for 48 h and then the ATP content was measured using ATP-lite (Perkin Elmer, cat no 6016739). For this, 30 μΕ ATP-lite solution was added to each well, and after 2 min shaking and another 8 min incubation at room temp in the dark, bioluminescence is measured in a PerkinElmer Envision mutireader equipped for luminescence.
[00161] TABLE IX: BA/F3 IC^ of selected compounds of the invention
* > 5001 nM
** 1001-5000 nM
*** 1-1000 nM
Figure imgf000124_0001
Example 3. In vivo models
Example 3.1 CIA model
3.1.1 Materials [00162] Completed Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) were purchased from Difco. Bovine collagen type II (CII), lipopolysaccharide (LPS), and Enbrel was obtained from Chondrex (Isle d'Abeau, France); Sigma (P4252, L'Isle d'Abeau, France), Whyett (25mg injectable syringe, France) Acros Organics (Palo Alto, CA), respectively. All other reagents used were of reagent grade and all solvents were of analytical grade.
3.1.2 Animals
[00163] Dark Agouti rats (male, 7-8 weeks old) were obtained from Harlan Laboratories (Maison- Alfort, France). Rats were kept on a 12 h light/dark cycle (0700 - 1900). Temperature was maintained at 22°C, and food and water were provided ad libitum.
3.1.3 Collagen induced arthritis ( CIA)
[00164] One day before the experiment, CII solution (2 mg/mL) was prepared with 0.05 M acetic acid and stored at 4°C. Just before the immunization, equal volumes of adjuvant (IFA) and CII were mixed by a homogenizer in a pre-cooled glass bottle in an ice water bath. Extra adjuvant and prolonged homogenization may be required if an emulsion is not formed. 0.2 mL of the emulsion was injected intradermally at the base of the tail of each rat on day 1, a second booster intradermal injection (CII solution at 2 mg/mL in CFA 0.1 mL saline) was performed on day 9. This immunization method was modified from published methods (Sims et al, 2004; Jou et ah, 2005).
3.1.4 Study design
[00165] The therapeutic effects of the compounds were tested in the rat CIA model. Rats were randomly divided into equal groups and each group contained 10 rats. All rats were immunized on day 1 and boosted on day 9. Therapeutic dosing lasted from day 16 to day 30. The negative control group was treated with vehicle (MC 0.5%) and the positive control group with Enbrel (10 mg/kg, 3x week. s.c). A compound of interest was typically tested at 3 doses, e.g. 3, 10, 30 mg/kg, p.o.
3.1.5 Clinical assessment of arthritis
[00166] Arthritis is scored according to the method of Khachigian 2006, Lin et al 2007 and Nishida et al. 2004). The swelling of each of the four paws is ranked with the arthritic score as follows: 0-no symptoms; 1-mild, but definite redness and swelling of one type of joint such as the ankle or wrist, or apparent redness and swelling limited to individual digits, regardless of the number of affected digits; 2- moderate redness and swelling of two or more types of joints; 3-severe redness and swelling of the entire paw including digits; 4-maximally inflamed limb with involvement of multiple joints (maximum cumulative clinical arthritis score 16 per animal) (Nishida et al., 2004). [00167] To permit the meta-analysis of multiple studies the clinical score values were normalised as follows:
[00168] AUC of clinical score (AUC score): The area under the curve (AUC) from day 1 to day 14 was calculated for each individual rat. The AUC of each animal was divided by the average AUC obtained for the vehicle in the study from which the data on that animal was obtained and multiplied by 100 (i.e. the AUC was expressed as a percentage of the average vehicle AUC per study).
[00169] Clinical score increase from day 1 to day 14 (End point score): The clinical score difference for each animal was divided by the average clinical score difference obtained for the vehicle in the study from which the data on that animal was obtained and multiplied by 100 (i.e. the difference was expressed as a percentage of the average clinical score difference for the vehicle per study).
3.1.6 Change in body weight (%) after onset of arthritis
[00170] Clinically, body weight loss is associated with arthritis (Shelton et al., 2005; Argiles et al., 1998; Rail, 2004; Walsmith et al., 2004). Hence, changes in body weight after onset of arthritis can be used as a non-specific endpoint to evaluate the effect of therapeutics in the rat model. The change in body weight (%) after onset of arthritis was calculated as follows:
Body Weight(week6) - Body Weight(week5> χ ^
[00171] Mice: Body Weight^)
Body Weigh t(week4) - Body Weigh t(weeB) η ηη .
- - x 100%
[00172] Rats: Body Weight(week3)
3.1.7 Radiology
[00173] X-ray photos were taken of the hind paws of each individual animal. A random blind identity number was assigned to each of the photos, and the severity of bone erosion was ranked by two independent scorers with the radiological Larsen's score system as follows: 0- normal with intact bony outlines and normal joint space; 1- slight abnormality with any one or two of the exterior metatarsal bones showing slight bone erosion; 2-defmite early abnormality with any three to five of the exterior metatarsal bones showing bone erosion; 3-medium destructive abnormality with all the exterior metatarsal bones as well as any one or two of the interior metatarsal bones showing definite bone erosions; 4-severe destructive abnormality with all the metatarsal bones showing definite bone erosion and at least one of the inner metatarsal joints completely eroded leaving some bony joint outlines partly preserved; 5-mutilating abnormality without bony outlines. This scoring system is a modification from Salvemini et al., 2001; Bush et al., 2002; Sims et al., 2004; Jou et al., 2005.
3.1.8 Histology [00174] After radiological analysis, the hind paws of mice were fixed in 10% phosphate-buffered formalin (pH 7.4), decalcified with rapid bone decalcifiant for fine histology (Laboratories Eurobio) and embedded in paraffin. To ensure extensive evaluation of the arthritic joints, at least four serial sections (5 μιη thick) were cut and each series of sections were 100 μιη in between. The sections were stained with hematoxylin and eosin (H&E). Histologic examinations for synovial inflammation and bone and cartilage damage were performed double blind. In each paw, four parameters were assessed using a four-point scale. The parameters were cell infiltration, pannus severity, cartilage erosion and bone erosion. Scoring was performed according as follows: 1 -normal, 2-mild, 3-moderate, 4-marked. These four scores are summed together and represented as an additional score, namely the 'RA total score'.
3.1.9 Micro-computed tomography (pCT) analysis of calcaneus (heel bone):
[00175] Bone degradation observed in RA occurs especially at the cortical bone and can be revealed by μΟΤ analysis (Sims NA et al., Arthritis Rheum. 50 (2004) 2338-2346: Targeting osteoclasts with zoledronic acid prevents bone destruction in collagen-induced arthritis; Oste L et al., ECTC Montreal 2007: A high throughput method of measuring bone architectural disturbance in a murine CIA model by micro-CT morphometry). After scanning and 3D volume reconstruction of the calcaneus bone, bone degradation is measured as the number of discrete objects present per slide, isolated in silico perpendicular to the longitudinal axis of the bone. The more the bone is degraded, the more discrete objects are measured. 1000 slices, evenly distributed along the calcaneus (spaced by about 10.8 μιη), are analyzed.
3.1.10 Steady State PK
[00176] At day 7 or 11, blood samples were collected at the retro-orbital sinus with lithium heparin as anti-coagulant at the following time points: predose, 1 , 3 and 6 h. Whole blood samples were centrifuged and the resulting plasma samples were stored at -20°C pending analysis. Plasma concentrations of each test compound were determined by an LC-MS/MS method in which the mass spectrometer was operated in positive electrospray mode. Pharmacokinetic parameters were calculated using Winnonlin® (Pharsight®, United States) and it was assumed that the predose plasma levels were equal to the 24 h plasma levels.
Example 3.2 Septic shock model
[00177] Injection of lipopolysaccharide (LPS) induces a rapid release of soluble tumour necrosis factor (TNF-alpha) into the periphery. This model is used to analyse prospective blockers of TNF release in vivo.
[00178] Six BALB/cJ female mice (20 g) per group are treated at the intended dosing once, po. Thirty min later, LPS (15 μg/kg; E. Coli serotype 0111 :B4) is injected ip. Ninety min later, mice are euthanized and blood is collected. Circulating TNF alpha levels are determined using commercially available ELISA kits. Dexamethasone (5 g kg) is used as a reference anti-inflammatory compound.
Example 3.3 MAB model
[00179] The MAB model allows a rapid assessment of the modulation of an RA-like inflammatory response by therapeutics (Kachigian LM. Nature Protocols (2006) 2512-2516: Collagen antibody- induced arthritis). DBA/J mice are injected i.v. with a cocktail of mAbs directed against collagen II. One day later, compound treatment is initiated (vehicle: 10% (v/v) HPpCD). Three days later, mice receive an i.p. LPS injection (50 μg/mouse), resulting in a fast onset of inflammation. Compound treatment is continued until 10 days after the mAb injection. Inflammation is read by measuring paw swelling and recording the clinical score of each paw. The cumulative clinical arthritis score of four limbs is presented to show the severity of inflammation. A scoring system is applied to each limb using a scale of 0-4, with 4 being the most severe inflammation.
0 Symptom free
1 Mild, but definite redness and swelling of one type of joint such as the ankle or wrist, or apparent redness and swelling limited to individual digits, regardless of the number of affected digits
2 Moderate redness and swelling of two or more types of joints
3 Severe redness and swelling of the entire paw including digits
4 Maximally inflamed limb with involvement of multiple joints
Example 3.4 Oncology models
[00180] In vivo models to validate efficacy of small molecules towards JAK2-driven myleoproliferative diseases are described by Wernig et al. Cancer Cell 13, 311, 2008 and Geron et al. Cancer Cell 13, 321, 2008.
Example 3.5 Mouse IBD model
[00181] In vitro and in vivo models to validate efficacy of small molecules towards IBD are described by Wirtz et al. 2007.
Example 3.6 Mouse Asthma model
[00182] In vitro and in vivo models to validate efficacy of small molecules towards asthma are described by Nials et al., 2008; Ip et al. 2006; Pernis et al., 2002; Kudlacz et al., 2008.
Example 4: Pharmacokinetic, DMPK and Toxicity Assays
Example 4.1 Thermodynamic solubility [00183] A solution of 1 mg/niL of the test compound is prepared in a 0.2M phosphate buffer pH 7.4 or a 0.1M citrate buffer pH 3.0 at room temperature in a glass vial.
[00184] The samples are rotated in a Rotator drive STR 4 (Stuart Scientific, Bibby) at speed 3.0 at room temperature for 24 h.
[00185] After 24 h, 800μΙ. of the sample is transferred to an eppendorf tube and centrifuged 5 min at 14000rpm. 200 μΐ. of the supernatant of the sample is then transferred to a MultiscreenR Solubility Plate (Millipore, MSSLBPC50) and the supernatant is filtered (10-12" Hg) with the aid of a vacuum manifold into a clean Greiner polypropylene V-bottom 96 well plate (Cat no.651201). 5 μΐ. of the filtrate is diluted into 95 μΐ. (F20) of the same buffer used to incubate in the plate containing the standard curve (Greiner, Cat no.651201).
[00186] The standard curve for the compound is prepared freshly in DMSO starting from a lOmM DMSO stock solution diluted factor 2 in DMSO (5000μΜ) and then further diluted in DMSO up to 19.5μΜ. 3μΙ of the dilution series as from 5000μΜ is then transferred to a 97μΙ. acetonitrile-buffer mixture (50/50). The final concentration range is 2.5 to 150 μΜ.
[00187] The plate is sealed with sealing mats (MA96RD-04S, www.kinesis.co.uk) and samples are measured at room temperature on LCMS (ZQ 1525 from Waters) under optimized conditions using Quanoptimize to determine the appropriate mass of the molecule.
[00188] The samples are analyzed on LCMS with a flow rate of lmL/min. Solvent A is 15mM ammonia and solvent B is acetonitrile. The sample is run under positive ion spray on an XBridge CI 8 3.5μΜ (2.1 x 30mm) column, from Waters. The solvent gradient has a total run time of 2 min and ranges from 5% B to 95% B.
[00189] Peak areas are analyzed with the aid of Masslynx software package and peak areas of the samples are plotted against the standard curve to obtain the solubility of the compound.
[00190] Solubility values are reported in μΜ or μg/mL.
Example 4.2 Aqueous Solubility
4.2.1: Aqueous Solubility2%DMSO procedure
[00191] Starting from a 10 mM stock in DMSO, a serial dilution of the compound is prepared in DMSO. The dilution series is transferred to a 96 NUNC Maxisorb plate F-bottom (Cat no. 442404) and 0.2M phosphate buffer pH7.4 or 0.1M citrate buffer pH 3.0 at room temperature is added.
[00192] The final concentration ranged from 200μΜ to 2.5μΜ in 5 equal dilution steps. The final DMSO concentration does not exceed 2%. 200μΜ Pyrene is added to the corner points of each 96 well plate and served as a reference point for calibration of Z-axis on the microscope.
[00193] The assay plates are sealed and incubated for 1 h at 37°C while shaking at 230rpm. The plates are then scanned under a white light microscope, yielding individual pictures of the precipitate per concentration. The precipitate is analyzed and converted into a number which is plotted onto a graph. The first concentration at which the compound appears completely dissolved is the concentration reported, however the true concentration lies somewhere between this concentration and one dilution step higher.
[00194] Solubility values mesured according to this protocol are reported in μg/mL. 4.2.1: Aqueous Solubttity3%DMSO procedure
[00195] Starting from a l OmM stock in DMSO, a serial dilution of the compound is prepared in DMSO. The dilution series is transferred to a 96 NUNC Maxisorb plate F-bottom (Cat no. 442404) and 0.1M phosphate buffer pH7.4 or 0.1M citrate buffer pH3.0 at room temperature is added.
[00196] The final concentration ranges from 300μΜ to 18.75μΜ in 5 equal dilution steps. The final DMSO concentration does not exceed 3%. 200μΜ Pyrene is added to the corner points of each 96 well plate and serves as a reference point for calibration of Z-axis on the microscope.
[00197] The assay plates are sealed and incubated for 1 h at 37°C while shaking at 230rpm. The plates are then scanned under a white light microscope, yielding individual pictures of the precipitate per concentration. The precipitate is analyzed and converted into a number with a software tool which can be plotted onto a graph. The first concentration at which the compound appears completely dissolved is the concentration reported; however the true concentration lies somewhere between this concentration and one dilution step higher.
[00198] Solubility values mesured according to this protocol are reported in μg/mL. Example 4.3 Plasma Protein Binding (Equilibrium Dialysis)
[00199] A lOmM stock solution of the compound in DMSO is diluted with a factor 5 in DMSO. This solution is further diluted in freshly thawed human, rat, mouse or dog plasma (BioReclamation INC) with a final concentration of 10μΜ and final DMSO concentration of 0.5% (5.5μΙ. in 1094.5μΙ. plasma in a PP-Masterblock 96well (Greiner, Cat no. 780285))
[00200] A Pierce Red Device plate with inserts (Thermo Scientific, Cat no. 89809) is prepared and filled with 750μΙ. PBS in the buffer chamber and 500μΙ. of the spiked plasma in the plasma chamber. The plate is incubated for 4 h at 37°C while shaking at 230rpm. After incubation, 120μΙ. of both chambers is transferred to 360μΙ. acetonitrile in a 96-well round bottom, PP deep-well plates (Nunc, Cat no. 278743) and sealed with an aluminum foil lid. The samples are mixed and placed on ice for 30 min. This plate is then centrifuged 30 min at 1200rcf at 4°C and the supernatant is transferred to a 96 v- bottom PP plate (Greiner, 651201) for analysis on LCMS.
[00201] The plate is sealed with sealing mats (MA96RD-04S) of www.kinesis.co.uk and samples are measured at room temperature on LCMS (ZQ 1525 from Waters) under optimized conditions using Quanoptimize to determine the appropriate mass of the molecule. [00202] The samples are analyzed on LCMS with a flow rate of lmL/min. Solvent A is 15mM ammonia and solvent B is acetonitrile. The sample is run under positive ion spray on an XBridge CI 8 3.5μΜ (2.1 x 30mm) column, from Waters. The solvent gradient has a total run time of 2 min and ranges from 5% B to 95% B.
[00203] Peak area from the compound in the buffer chamber and the plasma chamber are considered to be 100% compound. The percentage bound to plasma is derived from these results and is reported as percentage bound to plasma.
[00204] The solubility of the compound in the final test concentration in PBS is inspected by microscope to indicate whether precipitation is observed or not.
Example 4.4 Microsomal stability
4.4.1 Microsomal stability lit incubation procedure
[00205] A lOmM stock solution of compound in DMSO is diluted 1000 fold in a 182 mM phosphate buffer pH7.4 in a 96 deep well plate (Greiner, Cat no.780285) and pre-incubated at 37°C.
[00206] 40μΙ of deionised water is added to a well of a polypropylene Matrix 2D barcode labelled storage tube (Thermo Scientific) and pre-incubated at 37°C.
[00207] A Glucose-6-phophate-dehydrogenase (G6PDH) working stock solution is prepared in 182mM phosphate buffer pH7.4 and placed on ice before use. A co-factor containing MgC12, glucoses- phosphate and NADP+ is prepared in deionised water and placed on ice before use.
[00208] A final working solution containing liver microsomes (Xenotech) of a species of interest (human, mouse, rat, dog), previously described G6PDH and co-factors is prepared and this mix is incubated for no longer than 20 min at rt.
[00209] 30μΙ of the pre-heated compound dilution is added to 40μΙ. of pre-heated water in the Matrix tubes and 30μΙ. of the microsomal mix is added. Final reaction concentrations are 3μΜ compound, lmg microsomes, 0.4U/mL GDPDH, 3.3mM MgCl2, 3.3mM glucose-6-phosphate and 1.3mM NADP+.
[00210] To measure percentage remaining of compound at time zero MeOH or ACN is added (1 : 1) to the well before adding the microsomal mix. The plates are sealed with Matrix Sepra sealsTM (Matrix, Cat. No.4464) and shaken for a few seconds ensure complete mixing of all components.
[00211] The samples which were not stopped are incubated at 37°C, 300rpm and after 1 h of incubation the reaction is stopped with MeOH or ACN (1 :1).
[00212] After stopping the reaction the samples are mixed and placed on ice for 30 min to precipitate the proteins. The plates are then centrifuged 30 min at 1200rcf at 4°C and the supernatant is transferred to a 96 v-bottom PP plate (Greiner, 651201) for analysis on LCMS. [00213] These plates are sealed with sealing mats (MA96RD-04S) of www.kinesis.co.uk and samples are measured at room temperature on LCMS (ZQ 1525 from Waters) under optimized conditions using Quanoptimize to determine the appropriate mass of the parent molecule.
[00214] The samples are analyzed on LCMS with a flow rate of lmL/min. Solvent A is 15mM ammonia and solvent B is MeOH or acetonitrile, depending on the stop solution used. The samples are run under positive ion spray on an XBridge CI 8 3.5μΜ (2.1 x 30mm) column, from Waters. The solvent gradient had a total run time of 2 min and ranges from 5% B to 95% B.
[00215] Peak area from the parent compound at time 0 is considered to be 100% remaining. The percentage remaining after 1 h incubation is calculated from time 0 and is calculated as the percentage remaining. The solubility of the compound in the final test concentration in buffer is inspected by microscope and results are reported.
[00216] The data on microsomal stability are expressed as a percentage of the total amount of compound remaining after 60 min.
4.4.2 Microsomal stability 30 min incubation procedure
[00217] A lOmM stock solution of compound in DMSO is diluted to 6μΜ in a 105mM phosphate buffer, pH7.4 in a 96 deep well plate (Greiner, Cat no.780285) and pre-warmed at 37°C.
[00218] A Glucose-6-phosphate-dehydrogenase (G6PDH, Roche, 10127671001) working stock solution of 700U/mL is diluted with a factor 1 :700 in a 105mM phosphate buffer, pH7.4. A co-factor mix containing 0.528M MgCl2.6H20 (Sigma, M2670), 0.528M glucose-6-phosphate (Sigma, G-7879) and 0.208M NADP+ (Sigma,N-0505) is diluted with a factor 1 :8in a 105mM phosphate buffer, pH7.4.
[00219] A working solution is made containing 1 mg/mL liver microsomes (Provider, Xenotech) of the species of interest (human, mouse, rat, dog ...), 0.8U/mL G6PDH and co-factor mix (6.6mM MgCl2, 6.6mM glucose-6-phosphate, 2.6mM NADP+). This mix is pre- incubated for 15 min, but never more than 20 min, at rt.
[00220] After pre-incubation, compound dilution and the mix containing the microsomes, are added together in equal amount and incubated for 30 min at 300 rpm. For the time point of 0 min, two volumes of MeOH are added to the compound dilution before the microsome mix is added. The final concentration during incubation are: 3μΜ test compound or control compound, 0.5 mg/mL microsomes, 0.4U/mL G6PDH, 3.3mM MgCl2, 3.3mM glucose-6-phosphate and 1.3mM NaDP+.
[00221] After 30 min of incubation, the reaction is stopped with 2 volumes of MeOH.
[00222] Of both time points, samples are mixed, centrifuged and the supernatant is harvested for analysis on LC-MS/MS. The instrument responses (i.e. peak heights) are referenced to the zero time- point samples (as 100%) in order to determine the percentage of compound remaining. Standard compounds Propanolol and Verapamil are included in the assay design. [00223] The data on microsomal stability are expressed as a percentage of the total amount of compound remaining after 30 min.
Example 4.5 Caco2 Permeability
[00224] Bi-directional Caco-2 assays are performed as described below. Caco-2 cells are obtained from European Collection of Cell Cultures (ECACC, cat 86010202) and used after a 21 day cell culture in 24-well Transwell plates (Fisher TKT-545-020B).
[00225] 2xl05 cells/well are seeded in plating medium consisting of DMEM + GlutaMAXI + 1% NEAA + 10% FBS (FetalClone II) + 1% Pen/Strep. The medium is changed every 2 - 3 days.
[00226] Test and reference compounds (propranolol and rhodaminel23 or vinblastine, all purchased from Sigma) are prepared in Hanks' Balanced Salt Solution containing 25 mM HEPES (pH7.4) and added to either the apical (125μΕ) or basolateral (600μΕ) chambers of the Transwell plate assembly at a concentration of 10 μΜ with a final DMSO concentration of 0.25%.
[00227] 50μΜ Lucifer Yellow (Sigma) is added to the donor buffer in all wells to assess integrity of the cell layers by monitoring Lucifer Yellow permeation. As Lucifer Yellow (LY) cannot freely permeate lipophilic barriers, a high degree of LY transport indicates poor integrity of the cell layer.
[00228] After a 1 h incubation at 37°C while shaking at an orbital shaker at 150rpm, 70μΕ aliquots are taken from both apical (A) and basal (B) chambers and added to ΙΟΟμΕΙ 50:50 acetonitrile:water solution containing analytical internal standard (0.5μΜ carbamazepine) in a 96 well plate.
[00229] Lucifer yellow is measured with a Spectramax Gemini XS (Ex 426nm and Em 538nm) in a clean 96 well plate containing 150μΕ of liquid from basolateral and apical side.
[00230] Concentrations of compound in the samples are measured by high performance liquid- chromatography/mass spectroscopy (LC-MS/MS).
[00231] Apparent permeability (Papp) values are calculated from the relationship:
[00232] Papp = [compound]acceptor fmai x Vacceptor / ([compound]donor initial x Vdonor) / Tinc x Vdonor / surface area x 60 x 10"6 cm/s
[00233] V = chamber volume
[00234] Tinc = incubation time.
[00235] Surface area = 0.33cm2
[00236] The Efflux ratios, as an indication of active efflux from the apical cell surface, are calculated using the ratio of Papp B>A/ Papp A>B.
[00237] The following assay acceptance criteria are used:
[00238] Propranolol: Papp (A>B) value > 20(x 10"6 cm/s)
[00239] Rhodamine 123 or Vinblastine: Papp (A>B) value < 5 (xlO 6 cm/s) with Efflux ratio >5.
[00240] Lucifer yellow permeability: <100 nm/s Example 4.6 MDCKII-MDR1 Permeability
[00241] MDCKII-MDR1 cells are Madin-Darby canine kidney epithelial cells, over-expressing human multi-drug resistance (MDRl) gene, coding for P-glycoprotein (P-gp). Cells are obtained from Netherlands Cancer Institute and used after a 3-4 day cell culture in 24-well Millicell cell culture insert plates (Millipore, PSRP010R5). Bi-directional MDCKII-MDR1 permeability assay is performed as described below.
[00242] 3xl05 cells/ml (1.2xl05 cells/well) are seeded in plating medium consisting of DMEM + 1% Glutamax-100 + 1% Antibiotic/Antimycotic + 10% FBS (Biowest, SI 810). Cells are left in C02 incubator for 3-4 days. The medium is changed 24h after seeding and on the day of experiment.
[00243] Test and reference compounds (amprenavir and propranolol) are prepared in Dulbecco's phosphate buffer saline (D-PBS, pH7.4) and added to either the apical (400μΙ.) or basolateral (800μΙ.) chambers of the Millicell cell culture insert plates assembly at a final concentration of 10 μΜ (0.5 μΜ in case of amprenavir) with a final DMSO concentration of 1%.
[00244] ΙΟΟμΜ Lucifer Yellow (Sigma) is added to the all donor buffer solutions, in order to assess integrity of the cell monolayers by monitoring Lucifer Yellow permeation. Lucifer yellow is a fluorescent marker for the paracellular pathway and it is used as an internal control in every monolayer to verify tight junction integrity during the assay.
[00245] After a 1 h incubation at 37°C while shaking at an orbital shaker at 150rpm, 15 iL aliquots are taken from both apical (A) and basal (B) chambers and added to 225μΕ acetonitrile:water solution (2:1) containing analytical internal standard (10 ng/ml warfarin) in a 96 well plate. Aliquoting is also performed at the beginning of the experiment from donor solutions to obtain initial (Co) concentration.
[00246] Concentration of compound in the samples is measured by high performance liquid- chromatography/mass spectroscopy (LC-MS/MS).
[00247] Lucifer yellow is measured with a Fluoroscan Ascent FL Thermo Scientific (Ex 485nm and Em 530nm) in a 96 well plate containing 150μΕ of liquid from all receiver wells (basolateral or apical side).
Example 4.7 Microsomal stability
4.7.1 Microsomal stability lit incubation procedure
[00248] A 1 OmM stock solution of compound in DMSO is diluted 1000 fold in a 182 mM phosphate buffer pH7.4 in a 96 deep well plate (Greiner, Cat no.780285) and pre-incubated at 37°C.
[00249] 40μΕ of deionised water is added to a well of a polypropylene Matrix 2D barcode labelled storage tube (Thermo Scientific) and pre-incubated at 37°C.
[00250] A Glucose-6-phophate-dehydrogenase (G6PDH) working stock solution is prepared in 182mM phosphate buffer pH7.4 and placed on ice before use. A co-factor containing MgC12, glucoses- phosphate and NADP+ is prepared in deionised water and placed on ice before use. [00251] A final working solution containing liver microsomes (Xenotech) of a species of interest (human, mouse, rat, dog), previously described G6PDH and co-factors is prepared and this mix is incubated for no longer than 20 min at rt.
[00252] 30μΙ of the pre-heated compound dilution is added to 40μΙ. of pre-heated water in the Matrix tubes and 30μΙ. of the microsomal mix is added. Final reaction concentrations are 3μΜ compound, lmg microsomes, 0.4U/mL GDPDH, 3.3mM MgC12, 3.3mM glucose-6-phosphate and 1.3mM NADP+.
[00253] To measure percentage remaining of compound at time zero MeOH or ACN is added (1 : 1) to the well before adding the microsomal mix. The plates are sealed with Matrix Sepra sealsTM (Matrix, Cat. No.4464) and shaken for a few seconds ensure complete mixing of all components.
[00254] The samples which were not stopped are incubated at 37°C, 300rpm and after 1 h of incubation the reaction is stopped with MeOH or ACN (1 : 1).
[00255] After stopping the reaction the samples are mixed and placed on ice for 30 min to precipitate the proteins. The plates are then centrifuged 30 min at 1200rcf at 4°C and the supernatant is transferred to a 96 v-bottom PP plate (Greiner, 651201) for analysis on LCMS.
[00256] These plates are sealed with sealing mats (MA96RD-04S) of www.kinesis.co.uk and samples are measured at room temperature on LCMS (ZQ 1525 from Waters) under optimized conditions using Quanoptimize to determine the appropriate mass of the parent molecule.
[00257] The samples are analyzed on LCMS with a flow rate of lmL/min. Solvent A is 15mM ammonia and solvent B is MeOH or acetonitrile, depending on the stop solution used. The samples are run under positive ion spray on an XBridge CI 8 3.5μΜ (2.1 x 30mm) column, from Waters. The solvent gradient had a total run time of 2 min and ranges from 5% B to 95% B.
[00258] Peak area from the parent compound at time 0 is considered to be 100% remaining. The percentage remaining after 1 h incubation is calculated from time 0 and is calculated as the percentage remaining. The solubility of the compound in the final test concentration in buffer is inspected by microscope and results are reported.
[00259] The data on microsomal stability are expressed as a percentage of the total amount of compound remaining after 60 min.
4.7.2 Microsomal stability 30 min incubation procedure
[00260] A lOmM stock solution of compound in DMSO is diluted to 6μΜ in a 105mM phosphate buffer, pH7.4 in a 96 deep well plate (Greiner, Cat no.780285) and pre-warmed at 37°C.
[00261] A Glucose-6-phosphate-dehydrogenase (G6PDH, Roche, 10127671001 ) working stock solution of 700U/mL is diluted with a factor 1 :700 in a 105mM phosphate buffer, pH7.4. A co-factor mix containing 0.528M MgCl2.6H20 (Sigma, M2670), 0.528M glucose-6-phosphate (Sigma, G-7879) and 0.208M NADP+ (Sigma,N-0505) is diluted with a factor 1 :8in a 105mM phosphate buffer, pH7.4. [00262] A working solution is made containing 1 mg/mL liver microsomes (Provider, Xenotech) of the species of interest (human, mouse, rat, dog ...), 0.8U/mL G6PDH and co-factor mix (6.6mM MgCl2, 6.6mM glucose-6-phosphate, 2.6mM NADP+). This mix is pre- incubated for 15 min, but never more than 20 min, at rt.
[00263] After pre-incubation, compound dilution and the mix containing the microsomes, are added together in equal amount and incubated for 30 min at 300 rpm. For the time point of 0 min, two volumes of MeOH are added to the compound dilution before the microsome mix is added. The final concentration during incubation are: 3μΜ test compound or control compound, 0.5 mg/mL microsomes, 0.4U/mL G6PDH, 3.3mM MgCl2, 3.3mM glucose-6-phosphate and 1.3mM NaDP+.
[00264] After 30 min of incubation, the reaction is stopped with 2 volumes of MeOH.
[00265] Of both time points, samples are mixed, centrifuged and the supernatant is harvested for analysis on LC-MS/MS. The instrument responses (i.e. peak heights) are referenced to the zero time- point samples (as 100%) in order to determine the percentage of compound remaining. Standard compounds Propanolol and Verapamil are included in the assay design.
[00266] The data on microsomal stability are expressed as a percentage of the total amount of compound remaining after 30 min.
Example 4.8 Pharmacokinetic study in rodents
4.8.1 Animals
[00267] Sprague-Dawley rats (male, 5-6 weeks old) are obtained from Janvier (France). Rats are acclimatized for at least 7 days before treatment and are kept on a 12 h light/dark cycle (0700 - 1900). Temperature is maintained at approximately 22°C, and food and water are provided ad libitum. Two days before administration of the test compounds, rats underwent surgery to place a catheter in the jugular vein under isoflurane anesthesia. After the surgery, rats are housed individually. Rats are deprived of food for at least 16 h before oral dosing and 6 h after. Water is provided ad libitum.
4.8.2 Pharmacokinetic study
[00268] Compounds are formulated in PEG200/physiological saline (60/40) for the intravenous route and in 0.5%> methylcellulose and 10%> hydroxylpropyl-P-cyclodextrine pH 3 for the oral route. Test compounds are orally dosed as a single esophageal gavage at 5 mg/kg under a dosing volume of 5 mL/kg and intravenously dosed as a bolus via the caudal vein at 1 mg/kg under a dosing volume of 5 mL/kg. Each group consisted of 3 rats. Blood samples are collected via the jugular vein with lithium heparin as anti-coagulant at the following time points: 0.05, 0.25, 0.5, 1, 3, 5 and 8 h (intravenous route), and 0.25, 0.5, 1 , 3, 5, 8 and 24 h (oral route). Alternatively, blood samples are collected at the retro- orbital sinus with lithium heparin as anti-coagulant at the following time points 0.25, 1, 3 and 6 h (oral route). Whole blood samples are centrifuged at 5000 rpm for 10 min and the resulting plasma samples are stored at -20°C pending analysis.
4.8.3 Quantification of compound levels in plasma
[00269] Plasma concentrations of each test compound are determined by an LC-MS/MS method in which the mass spectrometer is operated in positive electrospray mode.
4.8.4 Determination of pharmacokinetic parameters
[00270] Pharmacokinetic parameters are calculated using Winnonlin® (Pharsight®, United States). Example 4.9 7-Day rat toxicity study
[00271] A 7-day oral toxicity study with test compounds is performed in Sprague-Dawley male rats to assess their toxic potential and toxicokinetics, at daily doses of 100, 300 and 500 mg/kg/day, by gavage, at the constant dosage- volume of 5 mL/kg/day.
[00272] The test compounds are formulated in 30% (v/v) ΗΡβΟϋ in purified water. Each group included 5 principal male rats as well as 3 satellite animals for toxicokinetics. A fourth group is given 30%) (v/v) HP CD in water only, at the same frequency, dosage volume and by the same route of administration, and acted as the vehicle control group.
[00273] The goal of the study is to determine the lowest dose that resulted in no adverse events being identified (no observable adverse effect level - NOAEL).
Example 4.10 Hepatocyte stability
[00274] Models to evaluate metabolic clearance in hepatocyte are described by McGinnity et al. Drug Metabolism and Disposition 2008, 32, 11, 1247.
Example 4.11 Liability for QT prolongation
[00275] Potential for QT prolongation is assessed in the hERG patch clamp assay. Example 4.10 Conventional whole-cell patch-clamp
[00276] 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 ΜΩ and compensated by greater than 60%>, recordings are not leak subtracted. Electrodes are manufactured from GC150TF pipette glass (Harvard).
[00277] The external bathing solution contained: 135 mM NaCl, 5 mM KC1, 1.8 mM CaCl2, 5 mM Glucose, 10 mM HEPES, pH 7.4. [00278] The internal patch pipette solution contained: lOOmM Kgluconate, 20 mM KC1, lmM CaCl2, 1 mM MgCl2, 5mM Na2ATP, 2mM Glutathione, 11 mM EGTA, 10 mM HEPES, pH 7.2.
[00279] Drugs are perfused using a Biologic MEV-9/EVH-9 rapid perfusion system.
[00280] All recordings are performed on HEK293 cells stably expressing hERG channels. Cells are cultured on 12 mm round coverslips (German glass, Bellco) anchored in the recording chamber using two platinum rods (Goodfellow). hERG currents are evoked using an activating pulse to +40 mV for 1000 ms followed by a tail current pulse to -50 mV for 2000 ms, holding potential is -80 mV. Pulses are applied every 20s and all experiments are performed at rt.
[00281] It will be appreciated by those skilled in the art that the foregoing descriptions are exemplary and explanatory in nature, and intended to illustrate the invention and its preferred embodiments. Through routine experimentation, an artisan will recognise apparent modifications and variations that may be made without departing from the spirit of the invention. Thus, the invention is intended to be defined not by the above description, but by the following claims and their equivalents.
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FINAL REMARKS
[00282] All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth.
[00283] From the foregoing description, various modifications and changes in the compositions and methods of this invention will occur to those skilled in the art. All such modifications coming within the scope of the appended claims are intended to be included therein. [00284] It should be understood that factors such as the differential cell penetration capacity of the various compounds can contribute to discrepancies between the activity of the compounds in the in vitro biochemical and cellular assays.
[00285] At least some of the chemical names of compounds of the invention as given and set forth in this application, may have been generated on an automated basis by use of a commercially available chemical naming software program, and have not been independently verified. Representative programs performing this function include the Lexichem naming tool sold by Open Eye Software, Inc. and the Autonom Software tool sold by MDL, Inc. In the instance where the indicated chemical name and the depicted structure differ, the depicted structure will control.
[00286] Chemical structures shown herein were prepared using either ChemDraw® or ISIS® /DRAW. Any open valency appearing on a carbon, oxygen or nitrogen atom in the structures herein indicates the presence of a hydrogen atom. Where a chiral center exists in a structure but no specific stereochemistry is shown for the chiral center, both enantiomers associated with the chiral structure are encompassed by the structure.

Claims

WHAT IS CLAIMED IS:
1. A compound according to Formul
Figure imgf000141_0001
wherein
R1 is C3.7 cycloalkyl, or C1.4 alkyl optionally substituted with one or more halo;
Cy is
Figure imgf000141_0002
Cyi Cy2 Cy3
wherein
each of w, x, y, and z, is independantly N, CH or is absent;
the ring A is a fused 5-6-membered saturated or 5-membered unsaturated ring, each of which optionally comprises 1 or 2 heteroatoms independently selected from N, O, and S; to afford a 8-10 member ed bicyclic group;
R2a is
halo,
cyclopropyl,
C alkyl optionally substituted with one or more halo, or
C alkoxy optionally substituted with one or more halo;
each R2b is independently selected from:
- CN,
halo,
- OH,
amino optionally substituted with one or two groups independently selected from Ci_4 alkyl,
Figure imgf000141_0003
alkyl, and -S(0)2C alkyl,
-C(=0)NH2 optionally substituted with one or two independently selected CM alkyl, -S(0)2NH2 optionally substituted with one or two independently selected CM alkyl,
-
Figure imgf000141_0004
alkoxy,
CM alkyl optionally substituted with one or more groups independently selected from halo, -CN, -C(=0)H,
Figure imgf000141_0005
alkyl, -OH, C6 o aryl (optionally substituted with one or more groups independently selected from CM alkoxy), - C(=0)-(4-6 membered heterocycloalkyl comprising 1 to 3 heterotaoms independently selected from N, S, or O) (which heterocycloalkyl is optionally substituted with one or more oxo, or CM alkyl) and amino (optionally substituted with one or more CM alkyl),
C2-4 alkenyl comprising 1 double bond (optionally substituted with one CN group) C2-6 alkoxy,
Ci_6 alkoxy substituted with one or more groups independently selected from OH, CN, halo, C alkoxy, C6-10 aryl (optionally substituted with one or more groups independently selected from CM alkoxy), C3.7 cycloalkyl, -C(=0)-CM alkoxy, -
Figure imgf000142_0001
alkyl, and 5-10 membered heteroaryl (optionally substituted with one or more CM alkyl),
- -O-Ce-io aryl,
4-10 membered heterocycloalkyl (optionally substituted with oxo, halo, CM alkyl (optionally substituted with one or more -CN), -C(=0)0-CM alkyl, or -SO2-C1.4 alkyl),
phenyl optionally substituted with one or more groups independently selected from CN, halo, OH, CM alkyl, CM alkoxy, and NR3aR¾
4- 7 membered heterocycloalkenyl comprising 1 double bond, and comprising 1 or 2 heteroatoms independently selected from N, O, and S, optionally substituted with one or more groups independently selected from:
o C alkyl,
o C alkoxy,
o C3.7 cycloalkyl (optionally substituted with one or more -CN), o
Figure imgf000142_0002
alkyl optionally substituted with one or more independently selected CN, OH, or halo,
o
Figure imgf000142_0003
alkyl,
o -C(=0)NR4aR4b,
5- 10 membered heteroaryl optionally substituted with one or more groups independently selected from CN, halo, OH, CM alkyl, CM alkoxy, and NR3aR¾ C3.7 cycloalkyl,
C thioalkyl,
Figure imgf000142_0004
alkyl (optionally substituted with CN)
-C(=0)-(4-6 membered heterocycloalkyl), which heterocycloalkyl is optionally substituted with one or more C alkyl (optionally substituted with one or more CN));
each R2c is independently selected from oxo, CM alkyl and halo; - H,
halo,
CM alkyl optionally substituted with one or more independently selected halo, or CM alkoxy optionally substituted with one or more independently selected halo; each R3a, R3b, R4a, and R4b is independently selected from H, or CM alkyl;
the subscript m is 0, 1 , 2, 3, or 4; and
the subscript p is 0, 1, or 2; or
a pharmaceutically acceptable salt, or a solvate, or a solvate of the pharmaceutically acceptable salts, provided that
o when the subscript m is 0, R2a is not CI, Me or OMe
o no more than two of w, x, y, or z are simultaneously N; and
o only one of w, x, y, or z may be absent.
A compound or pharmaceutically acceptable salt thereof according to claim 1 , wherein R1 is Me.
A compound or pharmaceutically acceptable salt thereof according to claim 1 , wherein Cy is Cyi, and wherein the subscript m is 0.
A compound or pharmaceutically acceptable salt thereof according to claim 1 , wherein Cy is Cyi, and wherein the subscript m is 1.
A compound or pharmaceutically acceptable salt thereof according to claim 4, wherein R2b is
• CN,
• halo,
• OH,
• CM alkyl optionally substituted with CN, OH, or Ce-io aryl (optionally substituted with one or more groups independently selected from C alkoxy),
• C2-6 alkoxy,
• Ci_6 alkoxy substituted with one or more groups independently selected from OH, halo, Ce. io aryl (optionally substituted with one or more groups independently selected from CM alkoxy), -C(=0)-CM alkyl, and C3.7 cycloalkyl ,
• -C(=0)NH2 optionally substituted with one or two independently selected C alkyl,
• alkoxy, or
• C thioalkoxy.
A compound or pharmaceutically acceptable salt thereof according to claim 4, wherein R2b is 4- 7 membered heterocycloalkenyl comprising 1 double bond, and comprising 1 or 2 heteroatoms independently selected from N, O, and S, optionally substituted with one or more groups independently selected from C alkyl, alkyl (optionally substituted with one or more independently selected CN, OH, or halo), or
Figure imgf000144_0001
alkyl.
A compound or pharmaceutically acceptable salt thereof according to claim 4, wherein R2b is phenyl, thienyl, pyrrazolyl, or pyrimidyl, each of which is optionally substituted with one or more independently groups selected from CN, halo, OH, C alkyl, and CM alkoxy.
A compound or pharmaceutically acceptable salt thereof according to claim 1 , wherein the compound is accordin to Formula Ila or lib:
Figure imgf000144_0002
I la Mb
wherein R a is halo, cyclopropyl, CM alkyl optionally substituted with one or more independently selected halo , or CM alkoxy optionally substituted with one or more independently selected halo;
R2b is CN, halo, OH, CM alkyl (optionally substituted with CN, OH, or Ce-ιο aryl (optionally substituted with one or more independently selected CM alkoxy)), C2-6 alkoxy, Ci_6 alkoxy substituted with one or more groups independently selected from halo, C6-10 aryl (optionally substituted with one or more independently selected CM alkoxy), -C(=0)-CM alkyl, C3.7 cycloalkyl and OH, -C(=0)NH2 optionally substituted with one or two independently selected CM alkyl, -C(=0)-CM alkoxy, or CM thioalkoxy.
9. A compound or pharmaceutically acceptable salt thereof according to claim 8, wherein R2a is F, CI, Me, Et, or -OCHF2.
10. A compound or pharmaceutically acceptable salt thereof according to claim 1 , wherein Cy is selected from Cy2, and Cy3; and the ring A is a fused 5-6 membered saturated ring optionally comprising 1 or 2 heteroatoms independently selected from N, O, and S; to afford a 8-10 membered bicyclic group.
1 1. A compound or pharmaceutically acceptable salt thereof according to claim 1 , wherein Cy is selected from Cy2, and Cy3; and the ring A is a fused 5-6 membered saturated ring optionally comprising 1 or 2 heteroatoms independently selected from N, O, and S; to afford a 9-10 membered bicyclic group.
12. A compound or pharmaceutically acceptable salt thereof according to claim 1 1 , wherein Cy is Cy2.
13. A compound or pharmaceutically acceptable salt thereof according to claim 12, wherein the subscript p is 2, and R2c is CM alkyl or halo.
14. A compound or pharmaceutically acceptable salt thereof according to claim 12, wherein the subscript p is 1 , and R2c is oxo.
15. A compoun 2, wherein Cy is:
Figure imgf000145_0001
wherein RZd is H, CI, Me or Et.
16. A compound or pharmaceutically acceptable salt thereof according to claim 1 , wherein Cy is selected from Cy2, and Cy3; and the ring A is a fused 5 membered unsaturated saturated ring optionally comprising 1 or 2 heteroatoms independently selected from N, O, and S; to afford a
8- 10 membered bicyclic group.
17. A compound or pharmaceutically acceptable salt thereof according to claim 1 , wherein Cy is selected from Cy2, and Cy3 ; and the ring A is a fused 5 membered unsaturated saturated ring optionally comprising 1 or 2 heteroatoms independently selected from N, O, and S; to afford a
9- 10 membered bicyclic group.
18. A compound or pharmaceutically acceptable salt thereof according to claim 17, wherein Cy is Cy2 and Cy2 is
Figure imgf000145_0002
wherein R is H, C alkyl, or halo; and R e and R are independently selected from H, and C alkyl.
19. A compound or pharmaceutically acceptable salt thereof according to claim 1 8, wherein R2D is H, Me, Et, or CI.
20. A compound or pharmaceutically acceptable salt thereof according to claim 18, wherein each R2E and R2F are independently selected from H, and Me
21. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a pharmaceutically effective amount of a compound according to any one of claims 1 -20.
22. The pharmaceutical composition according to claim 21 comprising a further therapeutic agent.
23. The compound or pharmaceutically acceptable salt thereof, according to any one of claims 1 -20, or the pharmaceutical composition according to claim 21 or 22, for use in medicine.
24. A compound according to any one of claims claims 1 -20, or the pharmaceutical composition according to claim 21 or 22, for use in the treatment, or prophylaxis of inflammatory conditions, autoimmune diseases, proliferative diseases, transplantation rejection, diseases involving impairment of cartilage turnover, congenital cartilage malformations, and/or diseases associated with hypersecretion of IL6.
25. A method for the treatment, or prophylaxis of inflammatory conditions, autoimmune diseases, proliferative diseases, transplantation rejection, diseases involving impairment of cartilage turnover, congenital cartilage malformations, and/or diseases associated with hypersecretion of IL6, comprising administering an amount of a compound according to any one of claims claims 1-20, or the pharmaceutical composition according to claim 21 or 22, sufficient to effect said treatment, or prophylaxis.
26. The method according to claim 25, wherein the compound according to any one of claims claims 1-20, or the pharmaceutical composition according to claims 21 or 22, is administered in combination with a further therapeutic agent.
27. The pharmaceutical composition according to claim 22, or the method according to claim 26, wherein the further therapeutic agent is an agent for the treatment, or prophylaxis of inflammatory conditions, autoimmune diseases, proliferative diseases, transplantation rejection, diseases involving impairment of cartilage turnover, congenital cartilage malformations, and/or diseases associated with hypersecretion of IL6.
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