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1. (WO2009135885) FUSED PYRAZINE COMPOUNDS USEFUL FOR THE TREATMENT OF DEGENERATIVE AND INFLAMMATORY DISEASES
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FUSED PYRAZINE COMPOUNDS USEFUL FOR THE TREATMENT OF DEGENERATIVE

AND INFLAMMATORY DISEASES

FIELD OF THE INVENTION [0001] The present invention relates to a class of fused pyrazine compounds capable of binding to the active site of a serine/threonine kinase, the expression of which is involved in the pathway resulting in the degradation of extra-cellular matrix (ECM), joint degeneration and diseases involving such degradation and/or inflammation. [0002] Diseases involving the degradation of extra-cellular matrix include, but are not limited to, psoriatic arthritis, juvenile arthritis, early arthritis, reactive arthritis, osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, osteoporosis, muskulo skeletal diseases like tendonitis and periodontal disease, cancer metastasis, airway diseases (COPD, asthma), renal and liver fibrosis, cardio-vascular diseases like atherosclerosis and heart failure, and neurological diseases like neuroinflammation and multiple sclerosis. Diseases involving primarily joint degeneration include, but are not limited to, psoriatic arthritis, juvenile arthritis, early arthritis, reactive arthritis, rheumatoid arthritis, osteoarthritis, and ankylosing spondylitis.

[0003] 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 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)). [0004] Although it is widely accepted that RA is an auto-immune disease, there is no consensus concerning the precise mechanisms driving the 'initiation stage' of the disease. What is known is that the initial trigger(s) does mediate, in a predisposed host, a cascade of events that leads to the activation of various cell types (B-cells, T-cells, macrophages, fibroblasts, endothelial cells, dendritic cells and others). Concomitantly, an increased production of various cytokines is observed in the joints and tissues surrounding the joint (e.g. TNF-α, IL-6, IL-I, IL-15, IL-18 and others). When the disease progresses, the cellular activation and cytokine production cascade becomes self-perpetuating. At this early stage, the destruction of joint structures is already very clear. Thirty percent of the patients have radiographic evidence of bony erosions at the time of diagnosis and this proportion increases to 60 percent after two years.

[0005] Histological analysis of the joints of RA patients clearly evidences the mechanisms involved in the RA-associated degradative processes. This analysis shows that the main effector responsible for RA-associated joint degradation is the pannus, where the synovial fibroblast, by producing diverse proteolytic enzymes, is the prime driver of cartilage and bone erosion. A joint classically contains two adjacent bones that articulate on a cartilage layer and are surrounded by the synovial membrane and joint capsule. In the advanced RA patient, the synovium of the joint increases in size to form the pannus, due to the proliferation of the synovial fibroblasts and the infiltration of mononuclear cells such as T-cells, B-cells, monocytes, macrophages and neutrophils. The pannus mediates the degradation of the adjacent cartilage, leading to the narrowing of the joint space, and has the potential to invade adjacent bone and cartilage. As bone and cartilage tissues are composed mainly of collagen type I or II, respectively, the pannus destructive and invasive properties are mediated by the secretion of collagenolytic proteases, principally the matrix metallo proteinases (MMPs). The erosion of the bone under and adjacent to the cartilage is also part of the RA process, and results principally from the presence of osteoclasts at the interface of bone and pannus. Osteoclasts are multinucleated cells that, upon adhesion to the bone tissue, form a closed compartment, within which the osteoclasts secrete proteases (Cathepsin K, MMP9) that degrade the bone tissue. The osteoclast population in the joint is abnormally increased by osteoblast formation from precursor cells induced by the secretion of the receptor activator of NFKB ligand (RANKL) by activated SFs and T-cells. [0006] Various collagen types have a key role in defining the stability of the extracellular matrix (ECM). Collagen type I and collagen type II, for example, are the main components of bone and cartilage, respectively. Collagen proteins typically organise into multimeric structures referred to as collagen fibrils. Native collagen fibrils are very resistant to proteolytic cleavage. Only a few types of ECM-degrading proteins have been reported to have the capacity to degrade native collagen: MMPs and Cathepsins. Among the Cathepsins, cathepsin K, which is active mainly in osteoclasts, is the best characterised. Among the MMPs, MMPl, MMP2, MMP8 MMP 13 and MMP 14 are known to have collagenolytic properties. The correlation between an increased expression of MMPl by synovial fibroblasts (SFs) and the progression of the arthritic disease is well-established and is predictive for joint erosive processes (Cunnane et al., 2001). In the context of RA, therefore, MMPl represents a highly relevant collagen degrading protein. In vitro, the treatment of cultured SFs with cytokines relevant in the RA pathology (e.g. TNF-α and ILlB) will increase the expression of MMPl by these cells (Andreakos et al., 2003). Monitoring the levels of MMPl expressed by SFs therefore is a relevant readout in the field of RA as it is indicative for the activation of SFs towards an erosive phenotype that, in vivo, is responsible for cartilage degradation. Inhibition of the MMPl expression by SFs represents a valuable therapeutic approach towards the treatment of RA. [0007] The activity of the ECM-degrading proteins can also be causative or correlate with the progression of various diseases different from RA, as e.g. other diseases that involve the degradation of the joints. These diseases include, but are not limited to, psoriatic arthritis, juvenile arthritis, early arthritis, reactive arthritis, osteoarthritis, and ankylosing spondylitis. Other diseases that may be treatable with compounds identified according to the present invention and using the targets involved in the expression of MMPs as described herein are osteoporosis, muscular skeletal diseases like tendonitis and periodontal disease (Gapski et al., 2004), cancer metastasis (Coussens et al., 2002), airway diseases (COPD, asthma) (Suzuki et al., 2004), lung, renal fibrosis (Schanstra et al., 2002), liver fibrosis associated with chronic hepatitis C (Reiff et al., 2005), cardiovascular diseases like atherosclerosis and heart failure (Creemers et al., 2001), and neurological diseases like neuroinflammation and multiple sclerosis (Rosenberg, 2002). Patients suffering from such diseases may benefit from stabilizing the ECM (by protecting it from degradation). [0008] The 471 -amino acid serine/threonine kinase identified as Mitogen- Activated Protein

Kinase- Activated Protein Kinase 5 (MAPKAPK5 or PRAK) is expressed in a wide panel of tissues. The protein contains its catalytic domain at the N-terminal end and both a nuclear localization signal (NLS) and nuclear export signal (NES) at its C-terminal end. Endogenous MAPKAPK5 is predominantly present in the cytoplasm, but stress or cytokine activation of the cells mediates its translocation into the nucleus (New et al., 2003). This event is dependent on phosphorylation of MAPKAPK5. Thrl 82 is the regulatory phosphorylation site of MAPKAPK5. Although the p38α kinase is able to phosphorylate MAPKAPK5 in an overexpression setting, experiments with endogenous MAPKAPK5 do not support this hypothesis (Shi et al., 2003). MAPKAPK5 knock-out mice have been generated that are viable and fertile. The phenotype of these mice is quite different from that of mice deficient for MAPKAPK2, a MAPKAPK5 related kinase that is regulated by p38α (Shi et al., 2003). This indicates that the function of each protein is distinct and that neither kinase can compensate for the other's activity. Taken together, MAPKAPK5 and MAPKAPK2 represent distinct targets with a non-redundant role. MAPK6 (also referred to as ERK3) has recently been identified as a physiologically relevant substrate for MAPKAPK5, defining a novel signal transduction pathway (Seternes et al., 2004).

BACKGROUND OF THE INVENTION

[0009] NSAIDS (Non-steroidal anti-inflammatory drugs) are used to reduce the pain associated with RA and improve life quality of the patients. These drugs will not, however, put a brake on the RA-associated joint destruction. [0010] Corticosteroids were found to decrease the progression of RA as detected radiographically and are used at low doses to treat part of the RA patients (30 to 60%). Serious side effects, however, are associated with long corticosteroid use (skin thinning, osteoporosis, cataracts, hypertension, and hyperlipidemia). [0011] Synthetic DMARDs (Disease-Modifying Anti-Rheumatic Drugs) (e.g. methotrexate, leflunomide, sulfasalazine) mainly tackle the immuno-inflammatory component of RA. As a main disadvantage, these drugs only have a limited efficacy (joint destruction is only slowed down but not blocked by DMARDs such that disease progression in the long term continues). The lack of efficacy is indicated by the fact that, on average, only 30% of the patients achieve an ACR50 score after 24 months treatment with methotrexate. This means that, according to the American College of Rheumatology, only 30% of the patients do achieve a 50% improvement of their symptoms (O'Dell et al., 1996). In addition, the precise mechanism of action of DMARDs is often unclear.

[0012] Biological DMARDs (Infliximab, Etanercept, Adalimumab, Rituximab, Abatacept) are therapeutic proteins that do inactivate cytokines (e.g. TNF-α) or cells (e.g. B-cells or T-cells) that have an important role in the RA pathophysiology (Kremer et al., 2003; Edwards et al., 2004). Although the TNF-α-blockers (Infliximab, Etanercept, Adalimumab) and methotrexate combination therapy is the most effective RA treatment currently available, it is striking that even this therapy only achieves a 50% improvement (ACR50) in disease symptoms in 50-60% of patients after 12 months therapy (St Clair et al., 2004). Some adverse events warnings for anti-TNF-α drugs exist, shedding a light on the side effects associated to this type of drugs. Increased risk for infections (tuberculosis), hematologic events and demyelinating disorders have been described for the TNF-α blockers (see also Gomez-Reino et al., 2003). Besides the serious side effects, the TNF-α blockers do also share the general disadvantages of the biological class of therapeutics, which are the unpleasant way of administration (frequent injections accompanied by infusion site reactions) and the high production cost. Newer agents in late development phase target cytokines such as IL-6, T-cell co-stimulatory molecules and B-cells. The efficacy of these agents is expected to be similar to that of the TNF-α blockers. The fact that a variety of targeted therapies have similar but limited efficacies, suggests that there is a multiplicity of pathogenic factors for RA. This is also indicative for the deficiencies in our understanding of pathogenic events relevant to RA. [0013] The current therapies for RA are not satisfactory due to a limited efficacy (no adequate therapy exists for 30% of the patients). This calls for additional strategies to achieve remission. Remission is required since residual disease bears the risk of progressive joint damage and thus progressive disability. Inhibiting the immuno-inflammatory component of the RA disease, which represents the main target of drugs currently used for RA treatment, does not result in a blockade of joint degradation, the major hallmark of the disease.

[0014] US 2005/0009832 describes substituted imidazo[l,2-a]pyrazine-8-yl-amines as modulators of protein kinases, including MAPKAPK5. WO02/056888 describes inhibitors of MAPKAPK5 as TNF modulators able to regulate the expression of certain cytokines. Neither of these prior art references discloses any compound within the scope of the class of compounds described herein below.

SUMMARY OF THE INVENTION [0015] The present invention is based on the discovery that MAPKAPK5 functions in the pathway that results in the expression of MMPl, and that inhibitors of MAPKAPK5 activity, such as the compounds of the present invention, are useful for the treatment of diseases involving the abnormally high expression of MMP activity.

[0016] The compounds of the invention may be described generally as [1.2.4]triazolo[l,5-a]pyrazines and imidazo[l,2-a]pyrazines substituted in the 5-position by an aryl or heteroaryl group, and an in the 8-position by a heteroarylamino group.

[0017] The compounds of the invention may show less toxicity, good absorption, good half-life, good solubility, low protein binding affinity, less drug-drug interaction, and good metabolic stability. In a particular aspect, the compounds of the present invention exhibit unexpected significant improvements in pharmacological properties, in particular improved efficacy and improved tolerability. [0018] More particularly, the present invention relates to a compound of the invention according to Formula (Ia) or (Ib):


wherein each of W, W, Y, and Y' is independently CR a or N; provided that no more than two of W,

W, Y, and Y' can be N at the same time;

X is N or CH;

L is selected from a single bond, -CO-, -SO-, -SO2-, -N(R2c)CO-, and -N(R2c)SO2-; the ring P is substituted or unsubstituted:


R1 is H, or substituted or unsubstituted Ci-C6 alkyl; each R2a is independently selected from H, substituted or unsubstituted Ci-C6 alkyl, Ci-C6 alkoxy, cyano, and halo; each R2c is selected from H and Ci-C6 alkyl;

R2d is H, C3-C8 cycloalkyl, or Ci-C6 alkyl optionally substituted with halo, amido, or C3-C8 cycloalkyl; each ml, m2 and m3 is independently 1 or 2; and R3 is selected from substituted or unsubstituted Ci-C6 alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.

[0019] In one embodiment, with respect to a compound of the invention according to Formula I or Ib, R1 is H, Me, Et, i-Pr or CF3.

[0020] In one embodiment, with respect to a compound of the invention according to Formula Ia or Ib, R1 is H. [0021] In another embodiment, with respect to a compound of the invention according to

Formula Ia or Ib, R3 is selected from substituted or unsubstituted phenyl.

[0022] In another embodiment, with respect to a compound of the invention according to

Formula Ia or Ib, R3 is selected from substituted or unsubstituted pyridyl.

[0023] In another embodiment, with respect to a compound of the invention according to Formula Ia or Ib, R3 is selected from phenyl, indolyl, isoinolyl, pyrrolyl, furanyl, thienyl, pyrazolyl, oxazolyl, and thiazolyl, each of which may be substituted or unsubstituted.

[0024] In a preferred embodiment, with respect to a compound of the invention according to

Formula Ia or Ib, R is substituted or unsubstituted furanyl.

[0025] In a further aspect, the present invention provides pharmaceutical compositions comprising a compound of the invention, and a pharmaceutical carrier, excipient or diluent. In this aspect of the invention, the pharmaceutical composition can comprise one or more of the compounds of the invention described herein. Moreover, the compounds of the invention useful in the pharmaceutical compositions and treatment methods disclosed herein, are all pharmaceutically acceptable as prepared and used. [0026] Another aspect of this invention relates to the use of a compound of the invention in a therapeutic method, a pharmaceutical composition, and the manufacture of such composition, useful for the treatment of diseases involving inflammation, collagen degradation, and in particular, diseases characteristic of abnormal matrix metallo protease (MMPl) and/or Mitogen-Activated Protein-Kinase

Activated Protein Kinase 5 (MAPKAP K5) activity, of which rheumatoid arthritis (RA) is a particular such disease. This invention also relates to processes for the preparation of the compounds of the invention.

[0027] Other objects and advantages will become apparent to those skilled in the art from a consideration of the ensuing detailed description, considered in conjunctin with the following illustrative drawings.

BRIEF DESCRIPTION OF THE DRAWINGS [0028] Figure 1. This diagram shows the striking histological differences between a healthy joint and that of a RA patient.

[0029] Figure 2. This chart shows the increased expression of MMPl in synovial fibroblasts triggered with cytokines involved in rheumatoid arthritis pathology. [0030] Figure 3. This graph shows the dose-dependent inhibition of the "TNF-α-based trigger"-induced expression of MMPl by SFs by a known anti-inflammatory compound.

[0031] Figure 4. This gel shows the reduction, at the protein level, of the expression of

MAPKAPK5 in SFs by infection of the cells with Ad-siRNA virus targeting MAPKAPK5.

[0032] Figure 5. This chart shows the reduction of 'complex trigger' induced levels of

MMPl expression by SFs by an Ad-siRNA virus targeting MAPKAPK5.

[0033] Figure 6A This graph shows the results of tolerability study conducted with a compound of the invention, where the measured effect was against total body weight. [0034] Figure 6B This graph shows the results of tolability study against a comparative compound.

[0035] Figure 7 This graph shows the percentage inhibition of TNF alpha release obtained with a compound of the invention, after injection of LPS (bacterial lipopolysaccharides) which is known to cause cholestasis in sepsis. The benchmark here is the well known dexamethasone (100% inhibition) (DEX).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0036] 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.

[0037] 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. [0038] 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.

[0039] 'Acyl' or 'Alkanoyl' refers to a radical -C(O)R20, where R20 is hydrogen, Ci-C8 alkyl, C3- Cio cycloalkyl, C3-Ci0 cycloalkylmethyl, 4-10 membered heterocycloalkyl, aryl, arylalkyl, 5-10 membered heteroaryl or heteroarylalkyl as defined herein. Representative examples include, but are not limited to, formyl, acetyl, cyclohexylcarbonyl, cyclohexylmethylcarbonyl, benzoyl and benzylcarbonyl. Exemplary 'acyl' groups are -C(O)H, -C(O)-Ci-C8 alkyl, -C(O)-(CH2)t(C6-Ci0 aryl), -C(O)-(CH2)t(5-10 membered heteroaryl), -C(O)-(CH2X(C3-Ci0 cycloalkyl), and -C(O)-(CH2)t(4-10 membered heterocycloalkyl), wherein t is an integer from 0 to 4. [0040] 'Substituted Acyl' or 'Substituted Alkanoyl' refers to a radical -C(O)R21, wherein R21 is independently

• Ci-Cg alkyl, substituted with halo or hydroxy; or • C3-Ci0 cycloalkyl, 4-10 membered heterocycloalkyl, C6-Ci0 aryl, arylalkyl, 5-10 membered heteroaryl or heteroarylalkyl, each of which is substituted with unsubstituted Ci-C4 alkyl, halo, unsubstituted Ci-C4 alkoxy, unsubstituted Ci-C4 haloalkyl, unsubstituted Ci-C4 hydroxyalkyl, or unsubstituted Ci-C4 haloalkoxy or hydroxy. [0041] 'Acylamino' refers to a radical -NR22C(O)R23, where R22 is hydrogen, Ci-C8 alkyl, C3- Cio cycloalkyl, 4-10 membered heterocycloalkyl, C6-CiO aryl, arylalkyl, 5-10 memberd heteroaryl or heteroarylalkyl and R23 is hydrogen, Ci-Cg alkyl, C3-CiO cycloalkyl, 4-10 membered heterocycloalkyl, C6-Cio aryl, arylalkyl, 5-10 membered heteroaryl or heteroarylalkyl, as defined herein. Exemplary 'acylamino' include, but are not limited to, formylamino, acetylamino, cyclohexylcarbonylamino, cyclohexylmethyl-carbonylamino, benzoylamino and benzylcarbonylamino. Exemplary 'acylamino' groups are -NR21 C(O)-Ci-C8 alkyl, -NR21'C(O)-(CH2)t(C6-Ci0 aryl), -NR2rC(O)-(CH2)t(5-10 membered heteroaryl), -NR21'C(0)-(CH2)t(C3-Cio cycloalkyl), and -NR21 C(O)-(CH2)t(4-10 membered heterocycloalkyl), wherein t is an integer from 0 to 4, each R21 independently represents H or CpC8 alkyl. [0042] 'Substituted Acylamino' refers to a radical -NR24C(O)R25, wherein:

R24 is independently

• H, CpC8 alkyl, substituted with halo or hydroxy; or

• C3-CiO cycloalkyl, 4-10 membered heterocycloalkyl, C6-CiO aryl, arylalkyl, 5-10 membered heteroaryl or heteroarylalkyl, each of which is substituted with unsubstituted Ci-C4 alkyl, halo, unsubstituted Ci-C4 alkoxy, unsubstituted Ci-C4 haloalkyl, unsubstituted Ci-C4 hydroxyalkyl, or unsubstituted Ci-C4 haloalkoxy or hydroxy; and R25 is independently

• H, Ci-C8 alkyl, substituted with halo or hydroxy; or

• C3-CiO cycloalkyl, 4-10 membered heterocycloalkyl, C6-CiO aryl, arylalkyl, 5-10 membered heteroaryl or heteroarylalkyl, each of which is substituted with unsubstituted Ci-C4 alkyl, halo, unsubstituted Ci-C4 alkoxy, unsubstituted Ci-C4 haloalkyl, unsubstituted Ci-C4 hydroxyalkyl, or unsubstituted Ci-C4 haloalkoxy or hydro xyl; provided at least one of R24 and R25 is other than H.

[0043] 'Alkoxy' refers to the group -OR26 where R26 is Ci-Cs alkyl. 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.

[0044] ' Substituted alkoxy' refers to an alkoxy group substituted with one or more of those groups recited in the definition of "substituted" herein, and particularly refers to an alkoxy group having 1 or more substituents, for instance from 1 to 5 substituents, and particularly from 1 to 3 substituents, in particular 1 substituent, selected from the group consisting of amino, substituted amino, C6-Ci0 aryl, -O-aryl, carboxyl, cyano, C3-Ci0 cycloalkyl, 4-10 membered heterocycloalkyl, halogen, 5-10 membered heteroaryl, hydroxyl, nitro, thioalkoxy, thio-O-aryl, thiol, alkyl-S(O)-, aryl-S(O)-, alkyl-S(O)2- and aryl-S(O)2-. Exemplary 'substituted alkoxy' groups are -0-(CH2)t(C6-Cio aryl), -O-(CH2)t(5-10 membered heteroaryl), -0-(CH2X(Cs-CiO cycloalkyl), and -O-(CH2)t(4-10 membered heterocycloalkyl), wherein t is an integer from 0 to 4 and any aryl, heteroaryl, cycloalkyl or heterocycloalkyl groups present, may themselves be substituted by unsubstituted C1-C4 alkyl, halo, unsubstituted C1-C4 alkoxy, unsubstituted C1-C4 haloalkyl, unsubstituted C1-C4 hydroxyalkyl, or unsubstituted C1-C4 haloalkoxy or hydroxy. Particular exemplary 'substituted alkoxy' groups are OCF3, OCH2CF3, OCH2Ph, OCH2-cyclopropyl, OCH2CH2OH, OCH2CH2NMe2. [0045] 'Alkoxycarbonyl' refers to a radical -C(O)-OR27 where R27 represents an Ci-Cs alkyl, C3-Cio cycloalkyl, C3-Ci0 cycloalkylalkyl, 4-10 membered heterocycloalkylalkyl, aralkyl, or 5-10 membered heteroarylalkyl as defined herein. Exemplary "alkoxycarbonyl" groups are C(O)O-Ci-Cs alkyl, -C(O)O-(CH2)t(C6-Cio aryl), -C(O)O-(CH2)t(5- 10 membered heteroaryl), -C(O)O-(CH2WC3-Ci0 cycloalkyl), and — C(O)O-(CH2)t(4-10 membered heterocycloalkyl), wherein t is an integer from 1 to 4. [0046] 'Substituted Alkoxycarbonyl' refers to a radical -C(O)-OR28 where R28 represents: • CpC8 alkyl, C3-Ci0 cycloalkyl, C3-Ci0 cycloalkylalkyl, or 4-10 membered heterocycloalkylalkyl, each of which is substituted with halo, substituted or unsubstituted amino, or hydroxy; or

• C6-Ci0 aralkyl, or 5-10 membered heteroarylalkyl, each of which is substituted with unsubstituted CpC4 alkyl, halo, unsubstituted CpC4 alkoxy, unsubstituted CpC4 haloalkyl, unsubstituted CpC4 hydroxyalkyl, or unsubstituted CpC4 haloalkoxy or hydroxyl.

[0047] '-O-arylcarbonyl' refers to a radical -C(O)-OR29 where R29 represents an C6-Ci0 aryl, as defined herein. Exemplary "-O-arylcarbonyl" groups is -C(O)O-(C6-Ci0 aryl).

[0048] ' Substituted -O-arylcarbonyl' refers to a radical -C(O)-OR30 where R30 represents a

• C5-Ci0 aryl, substituted with unsubstituted CpC4 alkyl, halo, unsubstituted CpC4 alkoxy, unsubstituted CpC4 haloalkyl, unsubstituted CpC4 hydroxyalkyl, or unsubstituted CpC4 haloalkoxy or hydroxyl.

[0049] 'Hetero-O-arylcarbonyl' refers to a radical -C(O)-OR31 where R31 represents a 5-10 membered heteroaryl, as defined herein.

[0050] 'Substituted Hetero-O-arylcarbonyl' refers to a radical -C(O)-OR32 where R32 represents a:

• 5-10 membered heteroaryl, substituted with unsubstituted CpC4 alkyl, halo, unsubstituted Cp C4 alkoxy, unsubstituted CpC4 haloalkyl, unsubstituted CpC4 hydroxyalkyl, or unsubstituted CpC4 haloalkoxy or hydroxyl.

[0051] 'Alkyl' means straight or branched aliphatic hydrocarbon having 1 to 20 carbon atoms. Particular alkyl has 1 to 12 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.

[0052] 'Substituted alkyl' refers to an alkyl group as defined above substituted with one or more of those groups recited in the definition of "substituted" herein, and particularly refers to an alkyl group having 1 or more substituents, for instance from 1 to 5 substituents, and particularly from 1 to 3 substituents, in particular 1 substituent, selected from the group consisting of acyl, acylamino, acyloxy (-O-acyl or -OC(O)R20), alkoxy, alkoxycarbonyl, alkoxycarbonylamino (-NR -alkoxycarbonyl or -NH-C(O)-OR27), amino, substituted amino, aminocarbonyl (carbamoyl or amido or -C(O)-NR 2), aminocarbonylamino (-NR -C(O)-NR 2) , aminocarbonyloxy (-0-C(O)-NR 2), aminosulfonyl, sulfonylamino, aryl, -O-aryl, azido, carboxyl, cyano, cycloalkyl, halogen, hydroxy, heteroaryl, nitro, thiol, -S-alkyl, -S-aryl, -S(O)-alkyl,-S(O)-aryl, -S(O)2-alkyl, and -S(O)2-aryl. In a particular embodiment 'substituted alkyl' refers to a CpC8 alkyl group substituted with halo, cyano, nitro, trifluoromethyl, trifluoromethoxy, azido, -NR"'SO2R", -SO2NR"R'", -C(O)R", -C(O)OR", -OC(O)R", -NR'"C(O)R", -C(0)NR"R ", -NR"R'", or -(CR"'R"")mOR"'; wherein each R" is independently selected from H, C1-C8 alkyl, -(CH2)t(C6-Ci0 aryl), -(CH2)t(5-10 membered heteroaryl), -(CH2),(C3-Cio cycloalkyl), and -(CH2)t(4-10 membered heterocycloalkyl), wherein t is an integer from O to 4 and any aryl, heteroaryl, cycloalkyl or heterocycloalkyl groups present, may themselves be substituted by unsubstituted Ci-C4 alkyl, halo, unsubstituted Ci-C4 alkoxy, unsubstituted Ci-C4 haloalkyl, unsubstituted Ci-C4 hydroxyalkyl, or unsubstituted Ci-C4 haloalkoxy or hydroxy. Each of R and R independently represents H or Ci-C8 alkyl.

[0053] 'Amino' refers to the radical -NH2.

[0054] 'Substituted amino' refers to an amino group substituted with one or more of those groups recited in the definition of 'substituted' herein, and particularly refers to the group -N(R33)2 where each R33 is independently selected from: • hydrogen, Ci-C8 alkyl, C6-Ci0 aryl, 5-10 membered heteroaryl, 4-10 membered heterocycloalkyl, or C3-Ci0 cycloalkyl; or

• Ci-C8 alkyl, substituted with halo or hydroxy; or

• -(CH2)t(C6-Cio aryl), -(CH2)t(5-10 membered heteroaryl), -(CH2)t(C3-Ci0 cycloalkyl) or - (CH2)t(4-10 membered heterocycloalkyl) wherein t is an integer between O and 8, each of which is substituted by unsubstituted Ci-C4 alkyl, halo, unsubstituted Ci-C4 alkoxy, unsubstituted Ci-C4 haloalkyl, unsubstituted Ci-C4 hydroxyalkyl, or unsubstituted Ci-C4 haloalkoxy or hydroxy; or

• both R groups are joined to form an alkylene group.

When both R33 groups are hydrogen, -N(R33)2 is an amino group. Exemplary 'substituted amino' groups are -NR33'-Ci-C8 alkyl, -NR33'-(CH2)t(C6-Ci0 aryl), -NR33'-(CH2)t(5-10 membered heteroaryl), -NR33'-(CH2)t(C3-Ci0 cycloalkyl), and -NR33'-(CH2)t(4-10 membered heterocycloalkyl), wherein t is an integer from 0 to 4, each R33 independently represents H or Ci- Cg alkyl; and any alkyl groups present, may themselves be substituted by halo, substituted or unsubstituted amino, or hydroxy; and any aryl, heteroaryl, cycloalkyl or heterocyclo alkyl groups present, may themselves be substituted by unsubstituted C1-C4 alkyl, halo, unsubstituted C1-C4 alkoxy, unsubstituted C1-C4 haloalkyl, unsubstituted C1-C4 hydroxyalkyl, or unsubstituted C1-C4 haloalkoxy or hydroxy. For the avoidance of doubt the term "substituted amino" includes the groups alkylamino, substituted alkylamino, alkylarylamino, substituted alkylarylamino, arylamino, substituted arylamino, dialkylamino and substituted dialkylamino as defined below. [0055] 'Alkylamino' refers to the group -NHR34, wherein R34 is CpC8 alkyl.

[0056] 'Substituted Alkylamino' refers to the group -NHR35, wherein R35 is CpCs alkyl; and the alkyl group is substituted with halo, substituted or unsubstituted amino, hydroxy, C3-Ci0 cycloalkyl, 4-10 membered heterocycloalkyl, C6-Ci0 aryl, 5-10 membered heteroaryl, aralkyl or heteroaralkyl; and any aryl, heteroaryl, cycloalkyl or heterocycloalkyl groups present, may themselves be substituted by unsubstituted Ci-C4 alkyl, halo, unsubstituted Ci-C4 alkoxy, unsubstituted Q-C4 haloalkyl, unsubstituted C1-C4 hydroxyalkyl, or unsubstituted Ci-C4 haloalkoxy or hydroxy. [0057] 'Alkylarylamino' refers to the group -NR36R37, wherein R36 is C6-Ci0 aryl and R37 is C1- C8 alkyl.

[0058] 'Substituted Alkylarylamino' refers to the group -NR38R39, wherein R38 is C6-C10 aryl and

R39 is C1-C8 alkyl; and the alkyl group is substituted with halo, substituted or unsubstituted amino, hydroxy, C3-C10 cycloalkyl, 4-10 membered heterocycloalkyl, C6-C10 aryl, 5-10 membered heteroaryl, aralkyl or heteroaralkyl; and any aryl, heteroaryl, cycloalkyl or heterocycloalkyl groups present, may themselves be substituted by unsubstituted C1-C4 alkyl, halo, cyano, unsubstituted C1-C4 alkoxy, unsubstituted Ci-C4 haloalkyl, unsubstituted C1-C4 hydroxyalkyl, or unsubstituted Ci-C4 haloalkoxy or hydroxy. [0059] 'Arylamino' means a radical -NHR40 where R40 is selected from C6-C10 aryl and 5-10 membered heteroaryl as defined herein.

[0060] 'Substituted Arylamino' refers to the group -NHR41, wherein R41 is independently selected from C6-C10 aryl and 5-10 membered heteroaryl; and any aryl or heteroaryl groups present, may themselves be substituted by unsubstituted C1-C4 alkyl, halo, cyano, unsubstituted C1-C4 alkoxy, unsubstituted Ci-C4 haloalkyl, unsubstituted C1-C4 hydroxyalkyl, or unsubstituted Ci-C4 haloalkoxy or hydroxy.

[0061] 'Dialkylamino' refers to the group -NR42R43, wherein each of R42 and R43 are independently selected from Ci-C8 alkyl.

[0062] 'Substituted Dialkylamino' refers to the group -NR44R45, wherein each of R44 and R45 are independently selected from Ci-C8 alkyl; and the alkyl group is independently substituted with halo, hydroxy, C3-Ci0 cycloalkyl, 4-10 membered heterocycloalkyl, C6-Ci0 aryl, 5-10 membered heteroaryl, aralkyl or heteroaralkyl; and any aryl, heteroaryl, cycloalkyl or heterocycloalkyl groups present, may themselves be substituted by unsubstituted C1-C4 alkyl, halo, unsubstituted C1-C4 alkoxy, unsubstituted C1-4 haloalkyl, unsubstituted C1-C4 hydroxyalkyl, or unsubstituted C1-C4 haloalkoxy or hydroxy. [0063] 'Diarylamino' refers to the group -NR46R47, wherein each of R46 and R47 are independently selected from C6-CiO aryl. [0064] "Aminosulfonyl" or "Sulfonamide" refers to the radical -S(O2)NH2.

[0065] "Substituted aminosulfonyl" or "substituted sulfonamide" refers to a radical such as - S(O2)N(R48)2 wherein each R48 is independently selected from:

• H, Ci-Cg alkyl, C3-C10 cycloalkyl, 4-10 membered heterocycloalkyl, C6-CiO aryl, aralkyl, 5-10 membered heteroaryl, and heteroaralkyl; or • Ci-Cg alkyl substituted with halo or hydroxy; or

• C3-Ci0 cycloalkyl, 4-10 membered heterocycloalkyl, C6-Ci0 aryl, aralkyl, 5-10 membered heteroaryl, or heteroaralkyl, substituted by unsubstituted Ci-C4 alkyl, halo, unsubstituted Ci-C4 alkoxy, unsubstituted C1-C4 haloalkyl, unsubstituted C1-C4 hydroxyalkyl, or unsubstituted C1-C4 haloalkoxy or hydroxy; provided that at least one R48 is other than H.

[0066] Exemplary 'substituted aminosulfonyl' or 'substituted sulfonamide' groups are - S(O2)N(R48')-Ci-C8 alkyl, -S(O2)N(R48')-(CH2)t(C6-Ci0 aryl), -S(O2)N(R48')-(CH2)t(5-10 membered heteroaryl), -S(O2)N(R48')-(CH2)t(C3-Ci0 cycloalkyl), and -S(O2)N(R48')-(CH2)t(4-10 membered heterocycloalkyl), wherein t is an integer from 0 to 4; each R48 independently represents H or Ci-Cg alkyl; and any aryl, heteroaryl, cycloalkyl or heterocycloalkyl groups present, may themselves be substituted by unsubstituted Ci-C4 alkyl, halo, unsubstituted Ci-C4 alkoxy, unsubstituted Ci-C4 haloalkyl, unsubstituted CpC4 hydroxyalkyl, or unsubstituted CpC4 haloalkoxy or hydroxy.

[0067] 'Aralkyl' or 'arylalkyl' refers to an alkyl group, as defined above, substituted with one or more aryl groups, as defined above. Particular aralkyl or arylalkyl groups are alkyl groups substituted with one aryl group.

[0068] 'Substituted Aralkyl' or 'substituted arylalkyl' refers to an alkyl group, as defined above, substituted with one or more aryl groups; and at least one of any aryl group present, may themselves be substituted by unsubstituted Q-C4 alkyl, halo, cyano, unsubstituted CpC4 alkoxy, unsubstituted CpC4 haloalkyl, unsubstituted CpC4 hydroxyalkyl, or unsubstituted CpC4 haloalkoxy or hydroxy. [0069] '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, mono-cyclic or poly-cyclic that includes from 5 to 12 ring members, more usually 6 to 10. Where the aryl group is a monocyclic ring system it preferentially contains 6 carbon atoms. Typical aryl groups include, but are not limited to, groups derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene and trinaphthalene.

Particularly aryl groups include phenyl, naphthyl, indenyl, and tetrahydronaphthyl.

[0070] 'Substituted Aryl' refers to an aryl group substituted with one or more of those groups recited in the definition of 'substituted' herein, and particularly refers to an aryl group that may optionally be substituted with 1 or more substituents, for instance from 1 to 5 substituents, particularly 1 to 3 substituents, in particular 1 substituent. Particularly, 'Substituted Aryl' refers to an aryl group substituted with one or more of groups selected from halo, Ci-Cs alkyl, Ci-Cs haloalkyl, Ci-Cs haloalkoxy, cyano, hydroxy, CpCg alkoxy, and amino.

[0071] Examples of representative substituted aryls include the following


[0072] In these formulae one of R49 and R50 may be hydrogen and at least one of R49 and R50 is each independently selected from Ci-C8 alkyl, 4-10 membered heterocycloalkyl, alkanoyl, Ci-C8 alkoxy, hetero-O-aryl, alkylamino, arylamino, heteroarylamino, NR51COR52, NR51SOR52NR51SO2R52, COOalkyl,

COOaryl, CONR51R52, CONR51OR52, NR51R52, SO2NR51R52, S -alkyl, SOalkyl, SO2alkyl, Saryl, SOaryl,

SO2aryl; or R49 and R50 may be joined to form a cyclic ring (saturated or unsaturated) from 5 to 8 atoms, optionally containing one or more heteroatoms selected from the group N, O or S. R51, and R52 are independently hydrogen, Ci-C8 alkyl, C1-C4 haloalkyl, C3-C10 cycloalkyl, 4-10 membered heterocycloalkyl, Cβ-Cio aryl, substituted aryl, 5-10 membered heteroaryl.

[0073] 'Arylalkyloxy' refers to an -O-alkylaryl radical where alkylaryl is as defined herein.

[0074] 'Substituted Arylalkyloxy' refers to an -O-alkylaryl radical where alkylaryl is as defined herein; and any aryl groups present, may themselves be substituted by unsubstituted C1-C4 alkyl, halo, cyano, unsubstituted C1-C4 alkoxy, unsubstituted C1-4 haloalkyl, unsubstituted C1-C4 hydroxyalkyl, or unsubstituted C1-C4 haloalkoxy or hydroxy.

[0075] 'Azido' refers to the radical -N3.

[0076] 'Carbamoyl or amido' refers to the radical -C(O)NH2.

[0077] 'Substituted Carbamoyl or substituted amido' refers to the radical -C(O)N(R53)2 wherein each R53 is independently

• H, Ci-C8 alkyl, C3-C10 cycloalkyl, 4-10 membered heterocycloalkyl, Cβ-Cio aryl, aralkyl, 5-10 membered heteroaryl, and heteroaralkyl; or

• Ci-C8 alkyl substituted with halo or hydroxy; or

• C3-C10 cycloalkyl, 4-10 membered heterocycloalkyl, Cβ-Cio aryl, aralkyl, 5-10 membered heteroaryl, or heteroaralkyl, each of which is substituted by unsubstituted C1-C4 alkyl, halo, unsubstituted C1-C4 alkoxy, unsubstituted C1-C4 haloalkyl, unsubstituted C1-C4 hydroxyalkyl, or unsubstituted C1-C4 haloalkoxy or hydroxy;

provided that at least one R is other than H.

Exemplary 'Substituted Amido / Carbamoyl' groups are -C(O) NR3 -Ci-C8 alkyl, -C(O)NR -(CH2)t(C6-Cio aryl), -C(O)N53'-(CH2)t(5-10 membered heteroaryl), -C(0)NR53'-(CH2)t(C3-Cio cycloalkyl), and -C(O)NR53 -(CH2)t(4-10 membered heterocycloalkyl), wherein t is an integer from O to 4, each R independently represents H or Ci-Cs alkyl and any aryl, heteroaryl, cycloalkyl or heterocycloalkyl groups present, may themselves be substituted by unsubstituted C1-C4 alkyl, halo, unsubstituted C1-C4 alkoxy, unsubstituted C1-C4 haloalkyl, unsubstituted C1-C4 hydroxyalkyl, or unsubstituted C1-C4 haloalkoxy or hydroxy. [0078] 'Carboxy' refers to the radical -C(O)OH. [0079] 'Cycloalkyl' refers to cyclic non-aromatic hydrocarbyl groups having from 3 to 10 carbon atoms. Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

[0080] 'Substituted cycloalkyl' refers to a cycloalkyl group as defined above substituted with one or more of those groups recited in the definition of 'substituted' herein, and particularly refers to a cycloalkyl group having 1 or more substituents, for instance from 1 to 5 substituents, and particularly from 1 to 3 substituents, in particular 1 substituent. [0081] 'Cyano' refers to the radical -CN.

[0082] 'Halo' or 'halogen' refers to fluoro (F), chloro (Cl), bromo (Br) and iodo (I). Particular halo groups are either fluoro or chloro. [0083] '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, cycloalkenyl, e.g. cycloheteroalkenyl, and the like having from 1 to 5, and particularly from 1 to 3 heteroatoms. [0084] 'Heteroaryl' means an aromatic ring structure, mono-cyclic or polycyclic, that includes one or more heteroatoms and 5 to 12 ring members, more usually 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.

[0085] Examples of representative aryl having hetero atoms containing substitution include the following:


wherein each W is selected from C(R54)2, NR54, O and S; and each Y is selected from carbonyl, NR54, O and S ; and R54 is independently hydrogen, Ci-C8 alkyl, C3-Ci0 cycloalkyl, 4-10 membered heterocycloalkyl, C6-Ci0 aryl, and 5-10 membered heteroaryl. [0086] Examples of representative heteroaryls include the following:



wherein each Y is selected from carbonyl, N, NR55, O and S; and R55 is independently hydrogen, Ci-Ce alkyl, C3-Ci0 cycloalkyl, 4-10 membered heterocycloalkyl, C6-Ci0 aryl, and 5-10 membered heteroaryl.

[0087] As used herein, the term 'heterocycloalkyl' refers to a 4-10 membered, stable heterocyclic non-aromatic ring and/or including rings containing one or more heteroatoms independently selected from N, O and S, fused thereto. A fused heterocyclic ring system may include carbocyclic rings and need only 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 (2H-pyran or 4H-pyran), dihydrothiophene, dihydropyran, dihydrofuran, dihydrothiazole, 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 heterocycloalkyl groups are shown in the following illustrative examples:


wherein each W is selected from CR56, C(R56)2, NR56, O and S; and each Y is selected from NR56, O and S; and R56 is independently hydrogen, Ci-C8 alkyl, C3-Ci0 cycloalkyl, 4-10 membered heterocycloalkyl, Cβ-Cio aryl, 5-10 membered heteroaryl, These heterocycloalkyl rings may be optionally substituted with one or more groups selected from the group consisting of acyl, acylamino, acyloxy (-O-acyl or -OC(O)R20), alkoxy, alkoxycarbonyl, alkoxycarbonylamino (-NR -alkoxycarbonyl or -NH-C(O)-OR27), amino, substituted amino, aminocarbonyl (amido or -C(O)-NR 2), aminocarbonylamino (-NR -C(O)-NR 2), aminocarbonyloxy (-O-C(O)-NR 2), aminosulfonyl, sulfonylamino, aryl, -O-aryl, azido, carboxyl, cyano, cycloalkyl, halogen, hydroxy, nitro, thiol, -S-alkyl, -S-aryl, -S(O)-alkyl,-S(O)-aryl, -S(O)2-alkyl, and -S(0)2-aryl. Substituting groups include carbonyl or thiocarbonyl which provide, for example, lactam and urea derivatives.

[0088] 'Hydroxy' refers to the radical -OH.

[0089] 'Nitro ' refers to the radical -NO2.

[0090] 'Substituted' refers to a group in which one or more hydrogen atoms are each independently replaced with the same or different substituent(s). Typical substituents may be selected from the group consisting of: halogen, -R57, -CT, =0, -OR57, -SR57, -S\ =S, -NR57R58, =NR57, -CCl3, -CF3, -CN, -OCN, -SCN, - NO, -NO2, =N2, -N3, -S(O)2O; -S(O)2OH, -S(O)2R57, -OS(O2)O; -OS(O)2R57, -P(O)(O )2, - P(O)(OR57XO-), -OP(O)(OR57XOR58), -C(O)R57, -C(S)R57, -C(O)OR57, -C(O)NR57R58, -C(O)O; -C(S)OR57, -NR59C(O)NR57R58, -NR59C(S)NR57R58, -NR60C(NR59)NR57R58 and -C(NR59)NR57R58; wherein each R57, R58, R59 and R60 are independently:

• hydrogen, Ci-Cg alkyl, C6-Ci0 aryl, arylalkyl, C3-CiO cycloalkyl, 4-10 membered heterocycloalkyl, 5-10 membered heteroaryl, heteroarylalkyl; or

• CrCe alkyl substituted with halo or hydroxy; or • C6-Ci0 aryl, 5-10 membered heteroaryl, C6-Ci0 cycloalkyl or 4-10 membered heterocycloalkyl substituted by unsubstituted Ci-C4 alkyl, halo, unsubstituted Ci-C4 alkoxy, unsubstituted Ci-C4 haloalkyl, unsubstituted Ci-C4 hydroxyalkyl, or unsubstituted Ci-C4 haloalkoxy or hydroxy.

In a particular embodiment, substituted groups are substituted with one or more substituents, particularly with 1 to 3 substituents, in particular with one substituent group.

[0091] In a further particular embodiment the substituent group or groups are selected from: halo, cyano, nitro, trifluoromethyl, trifluoromethoxy, azido, -NR"SO2R , -SO2NR R", -C(O)R , -C(O)OR", -OC(O)R", -NR'"C(O)R", -C(0)NR"R ", -NR"R", -(CR'"R'")mOR'", wherein, each R" is independently selected from H, Ci-Cs alkyl, -(CH2X(C6-C io aryl), -(CH2)t(5- 10 membered heteroaryl), -(CH2)t(C3-Cio cycloalkyl), and -(CH2)t(4-10 membered heterocycloalkyl), wherein t is an integer from O to 4; and

• any alkyl groups present, may themselves be substituted by halo or hydroxy; and

• any aryl, heteroaryl, cycloalkyl or heterocycloalkyl groups present, may themselves be substituted by unsubstituted C1-C4 alkyl, halo, unsubstituted C1-C4 alkoxy, unsubstituted C1-C4 haloalkyl, unsubstituted C1-C4 hydroxyalkyl, or unsubstituted C1-C4 haloalkoxy or hydroxy. Each R independently represents H or Ci-C6alkyl. [0092] 'Substituted sulfanyP refers to the group -SR51, wherein R51 is selected from:

• Ci-Cg alkyl, C3-CiO cycloalkyl, 4-10 membered heterocycloalkyl, C6-CiO aryl, aralkyl, 5-10 membered heteroaryl, and heteroaralkyl; or • Ci-C8 alkyl substituted with halo, substituted or unsubstituted amino, or hydroxy; or

• C3-Ci0 cycloalkyl, 4-10 membered heterocycloalkyl, C6-Ci0 aryl, aralkyl, 5-10 membered heteroaryl, or heteroaralkyl, each of which is substituted by unsubstituted Ci-C4 alkyl, halo, unsubstituted Ci-C4 alkoxy, unsubstituted Ci-C4 haloalkyl, unsubstituted Ci-C4 hydroxyalkyl, or unsubstituted Ci-C4 haloalkoxy or hydroxy. [0093] Exemplary 'substituted sulfanyF groups are -S-(Ci-C8 alkyl) and -S-(C3-Ci0 cycloalkyl),

-S-(CH2X(C6-Ci0 aryl), -S-(CH2)t(5- 10 membered heteroaryl), -S-(CH2X(C3-Ci0 cycloalkyl), and -S- (CH2)t(4-10 membered heterocycloalkyl), wherein t is an integer from 0 to 4 and any aryl, heteroaryl, cycloalkyl or heterocycloalkyl groups present, may themselves be substituted by unsubstituted C1-C4 alkyl, halo, unsubstituted C1-C4 alkoxy, unsubstituted C1-C4 haloalkyl, unsubstituted C1-C4 hydroxyalkyl, or unsubstituted C1-C4 haloalkoxy or hydroxy. The term 'substituted sulfanyl' includes the groups 'alkylsulfanyl' or 'alkylthio', 'substituted alkylthio' or 'substituted alkylsulfanyP, 'cycloalkylsulfanyl' or 'cycloalkylthio', 'substituted cycloalkylsulfanyl' or 'substituted cycloalkylthio',, 'arylsulfanyl' or 'arylthio' and 'heteroarylsulfanyl' or 'heteroarylthio' as defined below.

[0094] 'Alkylthio' or 'Alkylsulfanyl' refers to a radical -SR62 where R62 is a CpC8 alkyl or group as defined herein. Representative examples include, but are not limited to, methylthio, ethylthio, propylthio and butylthio.

[0095] 'Substituted Alkylthio'or 'substituted alkylsulfanyl' refers to the group -SR63 where R63 is a CpC8 alkyl, substituted with halo, substituted or unsubstituted amino, or hydroxy. [0096] 'Cycloalkylthio' or 'Cycloalkylsulfanyl' refers to a radical -SR64 where R64 is a C3-Ci0 cycloalkyl or group as defined herein. Representative examples include, but are not limited to, cyclopropylthio, cyclohexylthio, and cyclopentylthio.

[0097] 'Substituted cycloalkylthio' or 'substituted cycloalkylsulfanyl' refers to the group -SR65 where R65 is a C3-Ci0 cycloalkyl, substituted with halo, substituted or unsubstituted amino, or hydroxy. [0098] 'Arylthio' or 'Arylsulfanyl' refers to a radical -SR66 where R66 is a Ce-Ci0 aryl group as defined herein. [0099] 'Heteroarylthio' or 'Heteroarylsulfanyl' refers to a radical -SR67 where R67 is a 5-10 membered heteroaryl group as defined herein. [00100] 'Substituted sulfmyl' refers to the group -S(O)R68, wherein R68 is selected from:

• Ci-C8 alkyl, C3-Ci0 cycloalkyl, 4-10 membered heterocycloalkyl, C6-Ci0 aryl, aralkyl, 5-10 membered heteroaryl, and heteroaralkyl; or • Ci-C8 alkyl substituted with halo, substituted or unsubstituted amino, or hydroxy; or

• C3-C10 cycloalkyl, 4-10 membered heterocycloalkyl, Cβ-Cio aryl, aralkyl, 5-10 membered heteroaryl, or heteroaralkyl, substituted by unsubstituted C1-C4 alkyl, halo, unsubstituted C1-C4 alkoxy, unsubstituted C1-C4 haloalkyl, unsubstituted C1-C4 hydroxyalkyl, or unsubstituted C1-C4 haloalkoxy or hydroxy. [00101] Exemplary 'substituted sulfinyl' groups are -S(O)-(CpC8 alkyl) and -S(O)-(C3-Ci0 cycloalkyl), -S(0)-(CH2)t(C6-Cio aryl), -S(O)-(CH2)t(5- 10 membered heteroaryl), -S(O)-(CH2MC3-C10 cycloalkyl), and -S(O)-(CH2)t(4-10 membered heterocycloalkyl), wherein t is an integer from 0 to 4 and any aryl, heteroaryl, cycloalkyl or heterocycloalkyl groups present, may themselves be substituted by unsubstituted CpC4 alkyl, halo, unsubstituted CpC4 alkoxy, unsubstituted CpC4 haloalkyl, unsubstituted CpC4 hydroxyalkyl, or unsubstituted CpC4 haloalkoxy or hydroxy. The term substituted sulfinyl includes the groups 'alkylsulfinyl', 'substituted alkylsulfmyF, 'cycloalkylsulfinyl', 'substituted cycloalkylsulfinyl', 'arylsulfinyP and 'heteroarylsulfinyP as defined herein.

[00102] 'Alkylsulfinyl' refers to a radical -S(O)R69 where R59 is a Ci-C8 alkyl group as defined herein. Representative examples include, but are not limited to, methylsulfinyl, ethylsulfinyl, propylsulfinyl and butylsulfmyl.

[00103] 'Substituted Alkylsulfinyl' refers to a radical -S(O)R70 where R70 is a Ci-C8 alkyl group as defined herein, substituted with halo, substituted or unsubstituted amino, or hydroxy.

[00104] 'Cycloalkylsulfmyl' refers to a radical -S(O)R71 where R71 is a C3-Ci0 cycloalkyl or group as defined herein. Representative examples include, but are not limited to, cyclopropylsulfinyl, cyclohexylsulfinyl, and cyclopentylsulfinyl.

[00105] 'Substituted cycloalkylsulfmyl' refers to the group -S(O)R72 where R72 is a C3-C10 cycloalkyl, substituted with halo, substituted or unsubstituted amino, or hydroxy.

[00106] 'ArylsulfmyP refers to a radical -S(O)R73 where R73 is a C6-Ci0 aryl group as defined herein.

[00107] 'Heteroarylsulfinyl' refers to a radical -S(O)R74 where R74 is a 5-10 membered heteroaryl group as defined herein. [00108] 'Substituted sulfonyF refers to the group -S(O)2R75, wherein R75 is selected from:

• CpC8 alkyl, C3-Ci0 cycloalkyl, 4-10 membered heterocycloalkyl, C5-Ci0 aryl, aralkyl, 5-10 membered heteroaryl, and heteroaralkyl; or

• CpC8 alkyl substituted with halo, substituted or unsubstituted amino, or hydroxy; or

• C3-CiO cycloalkyl, 4-10 membered heterocycloalkyl, Ce-Ci0 aryl, aralkyl, 5-10 membered heteroaryl, or heteroaralkyl, each of which is substituted by unsubstituted C1-C4 alkyl, halo, unsubstituted C1-C4 alkoxy, unsubstituted C1-C4 haloalkyl, unsubstituted C1-C4 hydroxyalkyl, or unsubstituted C1-C4 haloalkoxy or hydroxy.

[00109] Exemplary 'substituted sulfonyl' groups are -S(O)2-(CrC8 alkyl) and -S(O)2-(C3-Ci0 cycloalkyl), -S(O)2-(CH2)t(C6-Ci0 aryl), -S(O)2-(CH2)t(5- 10 membered heteroaryl), -S(O)2-(CH2WC3-Ci0 cycloalkyl), and -S(O)2-(CH2)t(4-10 membered heterocycloalkyl), wherein t is an integer from 0 to 4 and any aryl, heteroaryl, cycloalkyl or heterocycloalkyl groups present, may themselves be substituted by unsubstituted CpC4 alkyl, halo, unsubstituted CpC4 alkoxy, unsubstituted CpC4 haloalkyl, unsubstituted CpC4 hydroxyalkyl, or unsubstituted CpC4 haloalkoxy or hydroxy. The term substituted sulfonyl includes the groups alkylsulfonyl, substituted alkylsulfonyl, cycloalkylsulfonyl, substituted cycloalkylsulfonyl, arylsulfonyl and heteroarylsulfonyl.

[00110] 'Alkylsulfonyl' refers to a radical -S(O)2R76 where R76 is an CpC8 alkyl group as defined herein. Representative examples include, but are not limited to, methylsulfonyl, ethylsulfonyl, propylsulfonyl and butylsulfonyl. [00111] 'Substituted Alkylsulfonyl' refers to a radical -S(O)2R77 where R77 is an CpC8 alkyl group as defined herein, substituted with halo, substituted or unsubstituted amino, or hydroxy.

[00112] 'Cycloalkylsulfonyl' refers to a radical -S(O)2R78 where R78 is a C3-Ci0 cycloalkyl or group as defined herein. Representative examples include, but are not limited to, cyclopropylsulfonyl, cyclohexylsulfonyl, and cyclopentylsulfonyl.

[00113] 'Substituted cycloalkylsulfonyl' refers to the group -S(O)2R79 where R79 is a C3-Ci0 cycloalkyl, substituted with halo, substituted or unsubstituted amino, or hydroxy.

[00114] 'Arylsulfonyl' refers to a radical -S(O)2R80 where R80 is an C6-Ci0 aryl group as defined herein.

[00115] 'Heteroarylsulfonyl' refers to a radical -S(O)2R81 where R81 is an 5-10 membered heteroaryl group as defined herein. [00116] 'Sulfo' or 'sulfonic acid' refers to a radical such as -SO3H.

[00117] 'Substituted sulfo' or 'sulfonic acid ester' refers to the group -S(O)2OR82, wherein R82 is selected from:

• CpC8 alkyl, C3-Ci0 cycloalkyl, 4-10 membered heterocycloalkyl, C5-Ci0 aryl, aralkyl, 5-10 membered heteroaryl, and heteroaralkyl; or • Ci-Cg alkyl substituted with halo, substituted or unsubstituted amino, or hydroxy; or

• C3-Ci0 cycloalkyl, 4-10 membered heterocycloalkyl, C6-Ci0 aryl, aralkyl, 5-10 membered heteroaryl, or heteroaralkyl, each of which is substituted by unsubstituted C1-C4 alkyl, halo, unsubstituted Ci-C4 alkoxy, unsubstituted Ci-C4 haloalkyl, unsubstituted Ci-C4 hydroxyalkyl, or unsubstituted Ci-C4 haloalkoxy or hydroxy. [00118] Exemplary 'Substituted sulfo' or 'sulfonic acid ester' groups are -S(O)2-O-(Ci-Cs alkyl) and -S(O)2-O-(C3-Ci0 cycloalkyl), -S(O)2-O-(CH2X(C6-Ci0 aryl), -S(O)2-O-(CH2)t(5-10 membered heteroaryl), -S(O)2-O-(CH2MC3-Ci0 cycloalkyl), and -S(O)2-O-(CH2)t(4-10 membered heterocycloalkyl), wherein t is an integer from 0 to 4 and any aryl, heteroaryl, cycloalkyl or heterocycloalkyl groups present, may themselves be substituted by unsubstituted Ci-C4 alkyl, halo, unsubstituted Ci-C4 alkoxy, unsubstituted Ci-C4 haloalkyl, unsubstituted Ci-C4 hydroxyalkyl, or unsubstituted Ci-C4 haloalkoxy or hydroxy.

[00119] 'Thiol' refers to the group -SH.

[00120] 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.

[00121] '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.

[00122] '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.

[00123] 'Pharmaceutically acceptable vehicle' refers to a diluent, adjuvant, excipient or carrier with which a compound of the invention is administered. [00124] '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- alky lmorpho line esters and the like. [00125] '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.

[00126] 'Subject' includes humans. The terms 'human', 'patient' and 'subject' are used interchangeably herein.

[00127] 'Therapeutically effective amount' means the amount of a compound of the invention 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.

[00128] '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.

[00129] 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.

[00130] '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. [00131] As used herein the term 'compound(s) of the invention', and equivalent expressions, are meant to embrace compounds according to any one of the Formula(e) as herein described, which expression includes the pharmaceutically acceptable salts, the solvates of the compounds, and the solvates of the pharmaceutically acceptable salts, e.g., hydrates, 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.

[00132] When ranges are referred to herein, for example but without limitation, Ci-Cs alkyl, the citation of a range should be considered a representation of each member of said range. [00133] Other derivatives of a compound of the invention may 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 to Cs alkyl, C2-Cg alkenyl, aryl, C7-C i2 substituted aryl, and C7-Ci2arylalkyl esters of the compounds of the invention.

[00134] 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 11C, 18F, 15O and 13N, and would be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. [00135] All isotopic variants of a compound of the invention provided herein, radioactive or not, are intended to be encompassed within the scope of the invention.

[00136] 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'.

[00137] 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'. [00138] '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. Such tautomers, as appropriate, are encompassed within the compounds of the invention as disclosed herein. [00139] Tautomeric forms may be relevant to the attainment of the optimal chemical reactivity and biological activity of a compound of interest.

[00140] A compound of the invention may possess one or more asymmetric centers; such a compound can therefore be produced as an individual (R)- or (S)- stereoisomer or as a mixture thereof. [00141] 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.

THE COMPOUNDS [00142] The present invention is based on the discovery that MAPKAPK5 functions in the pathway that results in the expression of MMPl, and that inhibitors of MAPKAPK5 activity, such as the compounds of the invention, are useful for the treatment of diseases involving the abnormally high expression of MMP activity. [00143] The compounds of the invention may be described generally as [1.2.4]triazolo[l,5-a]pyrazines and imidazo[l,2-a]pyrazines substituted in the 5-position by an aryl or heteroaryl group, and an in the 8-position by a heteroarylamino group.

[00144] The compounds of the invention may show less toxicity, good absorption, good half- life, good solubility, low protein binding affinity, less drug-drug interaction, and good metabolic stability. In a particular aspect, the compounds of the invention exhibit unexpected significant improvements in pharmacological properties, in particular improved efficacy and improved tolerability.

[00145] More particularly, the present invention relates to a compound of the invention according to Formula Ia or Ib:


(Ia) (Ib) wherein each of W, W, Y, and Y' is independently CR2a or N; provided that no more than two of W,

W, Y, and Y' can be N at the same time;

X is N or CH;

L is selected from a single bond, -CO-, -SO-, -SO2-, -N(R2c)C0-, and -N(R2c)SO2-; the ring P is substituted or unsubstituted:


R1 is H, or substituted or unsubstituted Ci-C6 alkyl; each R2a is independently selected from H, substituted or unsubstituted Ci-C6 alkyl, Ci-C6 alkoxy, cyano, and halo; each R2c is selected from H and Ci-C6 alkyl;

R2d is H, C3-C8 cycloalkyl, or Ci-C6 alkyl optionally substituted with halo, amido, or C3-C8 cycloalkyl; each ml, m2 and m3 is independently 1 or 2; and

R3 is selected from substituted or unsubstituted Ci-C6 alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.

[00146] In one embodiment, with respect to a compound of the invention according to Formula Ia or Ib, R1 is H, Me, Et, i-Pr or CF3.

[00147] In one embodiment, with respect to a compound of the invention according to Formula Ia or Ib, R1 is H.

[00148] In one embodiment, with respect to a compound of the invention according to Formula Ia or Ib, L is a single bond, -CO-, or -N(R2c)CO-.

[00149] In one particular embodiment, with respect to a compound of the invention according to

Formula Ia or Ib, L is a single bond. [00150] In one embodiment, with respect to a compound of the invention according to Formula Ia or Ib, R1 is Me, Et, n-Pr or i-Pr.

[00151] In one embodiment, with respect to a compound of the invention according to Formula Ia or Ib, each of W, W, Y, and Y' is independently CR2a.

[00152] In one embodiment, with respect to a compound of the invention according to Formula Ia or Ib, one of W, W, Y, and Y' is N and the rest are independently CR2a.

[00153] In one embodiment, with respect to a compound of the invention according to Formula Ia or Ib, two of W, W, Y, and Y' is N and the rest are independently CR2a.

[00154] In one particular embodiment, with respect to a compound of the invention according to

Formula Ia or Ib, each of W, W, Y, and Y' is independently CH.

[00155] In one embodiment, with respect to a compound of the invention according to Formula Ia or Ib, each of W, W, and Y is independently CH; and Y' is N.

[00156] In one embodiment, with respect to s compound of the invention according to Formula Ia or Ib, each of W, and W is independently CH; and each of Y and Y' is N.

[00157] In one embodiment, with respect to a compound of the invention according to Formula Ia, the compound is according to Formula Ha, Hb, Hc, or lid:


wherein X, L and the ring P are as defined for Formula Ia; each R2a is independently selected from H, substituted or unsubstituted Ci-C6 alkyl, substituted or unsubstituted Ci-C6 alkoxy, cyano, and halo; and R3 is independently selected from substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl.

[00158] In a further embodiment, with respect to a compound of the invention according to

Formula Ib, the compound is according to Formula He, Hf, or Hg:


wherein L and the ring P are defined for Formula Ib; each R a is independently selected from H, substituted or unsubstituted Ci-C6 alkyl, substituted or unsubstituted Ci-C6 alkoxy, cyano, and halo; and R3 is independently selected from substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl. [00159] In one embodiment, with respect to a compound of the invention according to any one of Formulae Ia-IIg, each R2a is H.

[00160] In one embodiment, with respect to a compound of the invention according to any one of

Formulae I-IIg, each R2a is selected from Me, Et, Pr, iso-Pr, Cl, F, CN, OMe, and CF3.

[00161] In one embodiment, with respect to a compound of the invention according to any one of

Formulae Ia-IIg, L is a single bond.

[00162] In one embodiment, with respect to a compound of the invention according to any one of

Formulae Ia-IIg, L is -CO- or -NHCO-.

[00163] In one embodiment, with respect to a compound of the invention according to any one of

Formulae Ia-IIg, the ring P is substituted or unsubstituted:


and wherein R2d and ml are as defined for formula I.

[00164] In one embodiment, with respect to a compound of the invention according to any one of

Formulae Ia-IIg, R2a is H, unsubstituted Ci-C6 alkyl, unsubstituted Ci-C6 haloalkyl, unsubstituted Ci-C6 alkoxy, cyano, or halo. [00165] In another further embodiment, with respect to a compound of the invention according to any one of Formulae Ia-IIg, one R a is selected from Me, Et, Pr, iso-Pr, Cl, F, CN, OMe, and CF3, and the rest are H.

[00166] In one embodiment, with respect to a compound of the invention according to any one of

Formulae Ia-IIg, each of W, W, Y, and Y', X, L, the ring P, R1, R2a, R2c and R2d are described in any one of the preceding paragraphs, and R3 is selected from substituted or unsubstituted aryl. [00167] In one embodiment, with respect to a compound of the invention according to any one of

Formulae Ia-IIg, each of W, W, Y, and Y', X, L, the ring P, R1, R2a, R2c and R2d are described in any one of the preceding paragraphs, and R3 is phenyl optionally substituted with halo, cyano, unsubstituted Ci-C6 alkoxy or amido optionally substituted with unsubstituted Ci-C6 alkyl. [00168] In a further embodiment, with respect to a compound of the invention according to any one of Formulae Ia-IIg, each of W, W, Y, and Y', X, L, the ring P, R1, R2a, R2c and R2d are described in any one of the preceding paragraphs, and R3 is phenyl optionally substituted with F, Cl, Br, cyano, OMe, OEt, On-Pr, 0/-Pr or amido optionally substituted with Me, Et, n-Pr, or i'-Pr.

[00169] In another embodiment, with respect to a compound of the invention according to any onf of Formulae Ia-IIg, each of W, W, Y, and Y', X, L, the ring P, R1, R2a, R2c and R2d are described in any one of the preceding paragraphs, and R3 is selected from substituted or unsubstituted heteroaryl.

[00170] In further embodiment, with respect to a compound of the invention according to any one of Formulae Ia-IIg, each of W, W, Y, and Y', X, L, the ring P, R1, R2a, R2c and R2d are described in any one of the preceding paragraphs, and R3 is selected from phenyl, pyridyl, indolyl, isoindolyl, pyrrolyl, furanyl, thienyl, pyrazolyl, oxazolyl, and thiazolyl, each of which may be unsubstituted or substituted with hydroxyl, cyano, halo, or amido optionally substituted with unsubstituted Ci-Ce alkyl. [00171] In another further embodiment, with respect to a compound of the invention according to any one of Formulae Ia-IIg, each of W, W, Y, and Y', X, L, the ring P, R1, R2a, R2c and R2d are described in any one of the preceding paragraphs, and R is selected from phenyl, pyridyl, indolyl, isoindolyl, pyrrolyl, furanyl, thienyl, pyrazolyl, oxazolyl, and thiazolyl, each of which may be unsubstituted or substituted with hydroxyl, cyano, F, Cl, Br, or amido optionally substituted with Me, Et, n-Pr, j'-Pr. [00172] In one embodiment, with respect to a compound of the invention according to any one of

Formulae Ia-IIg, each of W, W, Y, and Y', X, L, the ring P, R1, R2a, R2c and R2d are described in any one of the preceding paragraphs, and R3 is


and each of A1, A2 and A3 is independently selected from S, O, N, NR3a, and CR3a; each of R3a is independently H or substituted or unsubstituted Ci-C6 alkyl; and R3b is CONH2, CONHMe, or CN. [00173] In one embodiment, with respect to a compound of the invention according to any one of

Formulae Ia-IIg, each of W, W, Y, and Y', X, L, the ring P, R1, R2a, R2c and R2d are described in any one of the preceding paragraphs, and R3 is


[00174] In one embodiment, with respect to a compound of the invention according to any one of

Formulae Ia-IIg, each of W, W, Y, and Y', X, L, the ring P, R1, R2a, R2c and R2d are described in any one of the preceding paragraphs, and R3 is


[00175] In one embodiment, with respect to a compound of the invention according to any one of

Formulae Ia-IIg, each of W, W, Y, and Y', X, L, the ring P, R1, R2a, R2c and R2d are described in any one of the preceding paragraphs, and R3 is selected from


[00176] In one embodiment, with respect to a compound of the invention according to any one of

Formulae Ia-IIg, each of W, W, Y, and Y', X, L, the ring P, R1, R2a, R2c and R2d are described in any one of the preceding paragraphs, and R3 is


and wherein the subscript m is selected from 0, 1, 2, 3, and 4 and each R3d is independently substituted or unsubstituted Ci-Ce alkyl or halo.

[00177] In one embodiment, with respect to a compound of the invention according to any one of

Formulae Ia-IIg, each of W, W, Y, and Y', X, L, the ring P, R1, R2a, R2c and R2d are described in any one of the preceding paragraphs, and R3 is


and wherein the subscript m is selected from 0, 1, 2, 3, and 4 and each R 3d ; is independently substituted or unsubstituted Ci-C6 alkyl or halo.

[00178] In one embodiment, with respect to a compound of the invention according to any one of

Formulae Ia-IIg, each of W, W, Y, and Y', X, L, the ring P, R1, R2a, R2c and R2d are described in any one of the preceding paragraphs, and R3 is


or and wherein the subscript m is selected from O, 1, 2, or 3 and each R3d is independently substituted or unsubstituted Ci-Ce alkyl or halo.

[00179] In one embodiment, with respect to a compound of the invention according to any one of

Formulae Ia-IIg, each of W, W, Y, and Y', X, L, the ring P, R1, R2a, R2c, R2d and R3 are as described in any one of the preceding paragraphs; m is 1 or 2; and each R3d is Me, Cl or F.

[00180] In certain embodiments, with respect to a compound of the invention according to any one of Formulae Ia-IIg, each of W, W, Y, and Y', X, L, the ring P, R1, R2a, R2c, R2d and R3 are as described in any one of the preceding paragraphs and R3a is CpC6 alkyl. In another embodiment, R3a is Cp

C4 alkyl. [00181] In certain embodiments, with respect to a compound of the invention according to any one of Formulae Ia-IIg, each of W, W, Y, and Y', X, L, the ring P, R1, R2a, R2c, R2d and R3 are as described in any one of the preceding paragraphs and R3d is CpC6 alkyl. In another embodiment, R3d is

C1-C4 alkyl.

[00182] In one embodiment, a Ci-C6 alkyl group is optionally substituted by one or more groups (such as 1 to 3 substituents, in particular 1 substituent group) independently selected from halo, cyano, nifro, trifluoromethyl, trifluoromethoxy, azido, -NR10SO2R9, -SO2NR9R10, -C(O)R9, -C(O)OR9, -OC(O)R9,

-NR10C(O)R9, -C(O)NR9R10, -NR9R10, -(CR10R11XnOR10 and wherein m is an integer from 1 to 5.

[00183] In one embodiment, each R9 is independently selected from H, Ci-C8 alkyl, -(CH2X(C6-Ci0 aryl), -(CH2)t(C5-Ci0 heteroaryl), -(CH2)f(C3-C10 cycloalkyl), and -(CH2X(C5-Ci0 heterocycloalkyl) wherein t is an integer from 0 to 4.

[00184] In one embodiment, each R9 is as described above and the Ci-C6 alkyl group may optionally be substituted by halo and optionally contains 1 or 2 hetero moieties selected from O, S and - N(R12)- with the proviso that two O atoms, two S atoms, or an O and S atom are not attached directly to each other. [00185] In one embodiment, each R9 is as described above and any of which aryl, heteroaryl, cycloalkyl or heterocycloalkyl groups may themselves be substituted by Ci-C4alkyl, halo, Ci-C4alkoxy,

Ci 4haloalkyl, Ci-C4hydroxyalkyl or Ci-C4haloalkoxy or hydroxy.

[00186] In one embodiment, each R9 is as described above and each of R10 and R11 independently represents H or Ci-C6 alkyl; [00187] In one embodiment, each R9 is as described above and each of R12 and R13 independently represents H or C1-C4 alkyl;

[00188] In one embodiment, each of R10 and R11 independently represents H or Ci-C6 alkyl

[00189] In one embodiment, each R9 is other than H.

[00190] In certain embodiments, with respect to a compound of the invention according to any one of Formulae Ia-IIg, R2a is Ci-C6 alkoxy; and the alkoxy group is -OR9; and R9 is as described in any one of the above embodiments; provided that R9 is other than H.

[00191] In one embodiment, with respect to a compound of the invention according to Formula Ia, the compound is according to formula Ilia, IHb, or IHc:


Ilia , 1Mb or INc and X is CH or N; L is a single bond, -CO-, or -NHCO-; the ring P is


and R > 2d . is H, C3-C8 cycloalkyl, or Ci-C6 alkyl optionally substituted with halo, amido, or C3-C8 cycloalkyl.

[00192] In a further embodiment, with respect to a compound of the invention according to

Formula Ib, the compound is according to Formula IHd, or HIe:


IMd , IHe and X is CH or N; L is a single bond, -CO-, or -NHCO-; the ring P is


and R2d is H, C3-C8 cycloalkyl, or Ci-C6 alkyl optionally substituted with halo, amido, or C3-C8 cycloalkyl. [00193] In one particular embodiment, with respect to a compound of the invention according to any one of Formula HIa-IIIe, L is a single bond.

[00194] In one embodiment, with respect to a compound of the invention according to Formula Ia, the compound is according to Formula IVa, IVb, or IVc:


IVa IVb IVc

and X is CH or N; L is a single bond, -CO-, or -NHCO-; the ring P is


and R is H, C3-C8 cycloalkyl, or Ci-Cβ alkyl optionally substituted with halo, amido, or C3-C8 cycloalkyl.

[00195] In one embodiment, with respect to a compound of the invention according to Formula Ib, the compound is according to Formula IVd, or IVe:


IVd , IVe and X is as in claim 1 ; L is a single bond, -CO-, or -NHCO-; the ring P is


and R2d is H, CrCg cycloalkyl, or Ci-Ce alkyl optionally substituted with halo, amido, or C3-C8 cycloalkyl. [00196] In one particular embodiment, with respect to a compound of the invention according to

Formula IVa-IVe, L is a single bond.

[00197] In one embodiment, with respect to a compound of the invention according to Formula Ia, the compound is according to Formula Va, Vb, or Vc:


Va Vb Vc

and X is CH or N; L is a single bond, -CO-, or -NHCO-; the ring P is


and R2d is R2d is H, C3-C8 cycloalkyl, or Ci-Ce alkyl optionally substituted with halo, amido, or C3-C8 cycloalkyl.

[00198] In one embodiment, with respect to a compound of the invention according to Formula Ib, the compound is according to Formula Vd, or Ve:


Vd , Ve and X is CH or N; L is a single bond, -CO-, or -NHCO-; the ring P is


and R2d is R2d is H, C3-C8 cycloalkyl, or Ci-Ce alkyl optionally substituted with halo, amido, or

C3-C8 cycloalkyl.

[00199] In one particular embodiment, with respect to a compound of the invention according to any one of Formula Va-Ve, L is a single bond. [00200] In one embodiment, with respect to a compound of the invention according to Formula Ia, the compound is according to Formula Via, VIb, or VIc:


Via VIb VIc

and X is CH or N; L is a single bond, -CO-, or -NHCO-; the ring P is


and R2d is R2d is H, C3-C8 cycloalkyl, or CpCβ alkyl optionally substituted with halo, amido, or

C3-C8 cycloalkyl.

[00201] In one embodiment, with respect to a compound of the invention according to Formula Ib, the compound is according to Formula VId, or VIe:


and X is CH or N; L is a single bond, -CO-, or -NHCO-; the ring P is
and R2d is R2d is H, C3-C8 cycloalkyl, or Ci-C6 alkyl optionally substituted with halo, amido, or C3-C8 cycloalkyl.

[00202] In one particular embodiment, with respect to a compound of the invention according to any one of Formula VIa-VIe, L is a single bond.

[00203] In one embodiment, with respect to a compound of the invention according to any one of

Formula Ia-VIe, L is a single bond.

[00204] In one embodiment, with respect to a compound of the invention according to any one of

Formula Ia-VIe, L is -CO-. [00205] In one embodiment, with respect to a compound of the invention according to any one of

Formula Ia-VIe, L is -NHCO-.

[00206] In one embodiment, with respect to a compound of the invention according to any one of

Formula Ia-VIe, R2d is H, Me, Et, i-Pr, t-Bu, cyclopropylmethyl or CH2CF3.

[00207] In one embodiment, with respect to a compound of the invention according to any one of Formula I-VIe, R2d is H, Me, i-Pr, t-Bu, CH2CONH2, cyclopropylmethyl, or CH2CF3.

[00208] In one embodiment, with respect to a compound of the invention according to any one of

Formula Ia-VIe, R2d is H.

[00209] In one embodiment, with respect to a compound of the invention according to any one of

Formula Ia-VIe, R2d is i-Pr. [00210] In one embodiment, with respect to a compound of the invention according to any one of

Formula Ia-VIe, R2d is t-Bu.

[00211] In one embodiment, with respect to a compound of the invention according to any one of

Formula Ia-VIe, R2d is cyclopropylmethyl.

[00212] In one embodiment, with respect to a compound of the invention according to any one of Formula Ia-VIe, the ring P is


[00213] In one embodiment, with respect to a compound of the invention according to any one of

Formula Ia-VIe, the ring P is


or [00214] In one embodiment, with respect to a compound of the invention according to any one of

Formula Ia-VIe, the ring P is


[00215] In one embodiment, with respect to a compound of the invention according to any one of Formula Ia-VIe, X is CH.

[00216] In one embodiment, with respect to a compound of the invention according to any one of

Formula Ia-VIe, X is N.

[00217] In one embodiment, with respect to a compound of the invention according to Formula Ia or Ib, the compound is selected from the compounds listed in Table 1. [00218] In one embodiment, with respect to a compound of the invention according to Formula Ia, the compound is selected from:

5-(8-(4-((l S,4S)-5-isopropyl-2,5-diazabicyclo[2.2.1 ]heptan-2-yl)phenylamino)- [l,2,4]τriazolo[l,5-a]pyrazin-5-yl)isoindolin-l-one;

4-(8-(4-((l S,4S)-5-isopropyl-2,5-diazabicyclo[2.2.1 ]heptan-2-yl)phenylamino)- [l,2,4]τriazolo[l,5-a]pyrazin-5-yl)furan-2-carboxamide;

4-(8-(4-((lS,4R)-5-tert-butyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)phenylamino)- [1 ,2,4]τriazolo[l ,5-a]pyrazin-5-yl)furan-2-carboxarnide;

4-(8-(4-((lS,4S)-5-isopropyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)phenylamino)imidazo[l,2- a]pyrazin-5-yl)furan-2-carboxamide; 5- {8-(4-((l S,4S)-5-isopropyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)phenyl-amino)- [l,2,4]τriazolo[l,5-a]pyrazin-5-yl}-lH-pyrazole-3- carboxylic acid amide;

4- {8-(4-((l S,4S)-5-tert-butyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)-phenyl-amino)imidazo[l,2- a]pyrazin-5-yl}-furan-2 -carboxylic acid amide;

4- {8-(6-((l S,4S)-5-isopropyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)pyridin-3-ylamino)- [l,2,4]τriazolo[l,5-a]pyrazin-5-yl}-furan-2-carboxylic acid amide;

4- {8-(4-((l S,4S)-5-isopropyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)phenyl-amino)imidazo[l,2- a]pyrazin-5-yl} - 1 H-pyridin-2-one;

4- {8-(4-(5-isopropyl-2,5-diazabicyclo[2.2.2]octan-2-yl)phenylamino)[l,2,4]-triazolo[l,5- a]pyrazin-5-yl}-furan-2 -carboxylic acid amide; 4- {8-(4-(5-isopropyl-2,5-diazabicyclo[2.2.2]octan-2-yl)phenylamino)-imidazo[l,2-a]pyrazin- 5-yl}-furan-2-carboxylic acid amide;

4- {8-(4-(3-isopropyl-3,8-diazabicyclo[3.2.1]octan-8-yl)phenylamino)-imidazo[l,2-a]pyrazin- 5-yl}-furan-2-carboxylic acid amide;

4-{8-(4-(8-isopropyl-3,8-diazabicyclo[3.2.1]octan-3-yl)phenylamino)[l,2,4]-triazolo[l,5- a]pyrazin-5-yl}-:furan-2 -carboxylic acid amide;

4-{8-(4-(3-isopropyl-3,8-diazabicyclo[3.2.1]octan-8-yl)phenylamino)[l,2,4]-triazolo[l,5- a]pyrazin-5-yl}-:furan-2 -carboxylic acid amide; 4-{8-(4-(8-isopropyl-3,8-diazabicyclo[3.2.1]octan-3-yl)phenylamino)-imidazo[l,2-a]pyrazin- 5-yl}-furan-2-carboxylic acid amide;

5-{8-(6-((lS,4S)-5-isopropyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)pyridin-3-ylamino)- [l,2,4]triazolo[l,5-a]pyrazin-5-yl}-lH-pyrazole-3- carboxylic acid amide;

5-{8-(4-((lS,4S)-5-tert-butyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)phenylamino) - [l,2,4]triazolo-[l,5-a]pyrazin-5-yl}-2,3-dihydro-isoindol-l-one;

5-{8-(4-((lS,4S)-5-tert-butyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)phenyl-amino)- [l,2,4]triazolo[l,5-a]pyrazin-5-yl}-lH-pyrazole-3- carboxylic acid amide; and

5-{8-(6-((lS,4S)-5-isopropyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)pyridin-3-ylamino)- [l,2,4]triazolo[l,5-a]pyrazin-5-yl}-2,3-dihydro-isoindol-l-one. [00219] In another embodiment, with respect to a compound of the invention according to

Formula Ib, the compound is selected from:

4-(8-(3-((l S,4S)-5-isopropyl-2,5-diazabicyclo[2.2.1 ]heptan-2-yl)phenylamino)- [1 ,2,4]triazolo[l ,5-a]pyrazin-5-yl)furan-2-carboxamide;

4-(8-(3-((lS,4S)-5-isopropyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)phenylamino)imidazo[l,2- a]pyrazin-5-yl)furan-2-carboxamide;

5-(8-(3-((lS,4S)-5-isopropyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)phenylamino)- [l,2,4]triazolo[l,5-a]pyrazin-5-yl)isoindolin-l-one.

[00220] In one aspect a compound of the invention according to any one of the embodiments herein described is present as the free base. [00221] In one aspect a compound of the invention according to any one of the embodiments herein described is a pharmaceutically acceptable salt.

[00222] In one aspect a compound of the invention according to any one of the embodiments herein described is a solvate of the compound of the invention.

[00223] 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 of the invention.

[00224] 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.

[00225] In certain aspects, the present invention provides prodrugs and derivatives of the compounds of the invention 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. A prodrug may be inactive when administered to a subject but is converted in vivo to an active compound of the invention. "Pharmaceutically acceptable prodrugs" as used herein refers to those prodrugs of the compounds useful in the present invention, which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients with undue toxicity, irritation, allergic response commensurate with a reasonable benefit/risk ratio, and effective for their intended use of the compounds of the invention. The term 'prodrug' means a compound that is transformed in vivo to yield an effective compound useful in the present invention or a pharmaceutically acceptable salt, hydrate or solvate thereof. The transformation may occur by various mechanisms, such as through hydrolysis in blood. The compounds bearing metabolically cleavable groups have the advantage that they may exhibit improved bioavailability as a result of enhanced solubility and/or rate of absorption conferred upon the parent compound by virtue of the presence of the metabolically cleavable group, thus, such compounds act as pro-drugs. A thorough discussion is provided in Design of Prodrugs, H. Bundgard, ed., Elsevier (1985); Methods in Enzymology; K. Widder et al, Ed., Academic Press, 42, 309-396 (1985); A Textbook of Drug Design and Development, Krogsgaard-Larsen and H. Bundgard, ed., Chapter 5; "Design and Applications of Prodrugs" 113-191 (1991); Advanced Drug Delivery Reviews, H. Bundgard, 8 , 1-38, (1992); J. Pharm. Sci., 77,285 (1988); Chem. Pharm. Bull., N. Nakeya et al, 32, 692 (1984); Prodrugs as Novel Delivery Systems, T. Higuchi and V. Stella, 14 A. C. S. Symposium Series, and Bioreversible Carriers in Drug Design, E. B. Roche, ed., American Pharmaceutical Association and Pergamon Press, 1987, which are incorporated herein by reference. Such examples include, but are not limited to, choline ester derivatives and the like, N-alkylmorpholine esters and the like. [00226] Other derivatives of the compounds of the 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 de sirab le to prep are double ester type pro drugs such as (acyloxy) alkyl esters or ((alkoxycarbonyl)oxy)alkylesters. Preferred are the Ci to Cg alkyl, C2-Cg alkenyl, aryl, C7-C12 substituted aryl, and C7-C12 arylalkyl esters of the compounds of the invention.

PHARMACEUTICAL COMPOSITIONS [00227] When employed as pharmaceuticals, 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. [00228] Generally, a compound of the 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. [00229] The pharmaceutical compositions of this invention can be administered by a variety of routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal. Depending on the intended route of delivery, the compounds of this invention are preferably formulated as either injectable or oral compositions or as salves, as lotions or as patches all for transdermal administration. [00230] 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. 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 furansulfonic acid compound 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. [00231] 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.

[00232] 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. [00233] 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. [00234] 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. [00235] 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. [00236] 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.

[00237] 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

[00238] 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 is added as a lubricant. The mixture is formed into 240-270 mg tablets (80-90 mg of active amide compound per tablet) in a tablet press. Formulation 2 - Capsules

[00239] 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 is filled into 250 mg capsules (125 mg of active amide compound per capsule).

Formulation 3 - Liquid [00240] 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 are diluted with water and added with stirring. Sufficient water may then be added to produce a total volume of 5 mL.

Formulation 4 - Tablets

[00241] 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 is added as a lubricant. The mixture is formed into 450-900 mg tablets (150-300 mg of active amide compound) in a tablet press. Formulation 5 - Injection

[00242] 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 [00243] Stearyl alcohol (250 g) and a white petrolatum (250 g) may be melted at about 750C and then a mixture of a 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 would be stirred until it congeals.

METHODS QF TREATMENT [00244] 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 MMPl and / or MAPKAPK5. Accordingly, the compounds of the invention and pharmaceutical compositions thereof find use as therapeutics for preventing and/or treating inflammatory diseases in mammals including humans. Thus, and as stated earlier, the present invention includes within its scope, and extends to, the recited methods of treatment, as well as to the compounds for use in such methods, and for the preparation of medicaments useful for such methods.

[00245] In a method of treatment aspect, this invention provides a method of treating a mammal susceptible to or afflicted with a condition associated with extra-cellular matrix (ECM) degradation, in particular arthritis, and more particularly, rheumatoid arthritis which method comprises administering an effective amount of a compound of the invention or a pharmaceutical composition thereof. [00246] In another method of treatment aspect, the invention provides a method of treating a mammal sucepible to or afflicted with a condition associated with an abnormal cellular expression of MMPl, which comprises administering a therapeutically effective amount of a compound of the invention, or a pharmaceutical composition thereof. [00247] In another method of treatment aspect, the present invention provides a method of treatment or prophylaxis of a condition characterized by abnormal matrix metallo proteinase activity, which comprises administering a therapeutically effective matrix metallo proteinase inhibiting amount of a compound of the invention, or pharmaceutical composition thereof.

[00248] In yet another method of treatment aspect, this invention provides methods of treating a mammal susceptible to or afflicted with diseases and disorders which are mediated by or result in inflammation such as, for example rheumatoid arthritis and osteoarthritis, myocardial infarction, various autoimmune diseases and disorders, uveitis and atherosclerosis; itch / pruritus such as, for example psoriasis; and renal disorders method comprises administering an effective condition-treating or condition-preventing amount of a compound of the invention or pharmaceutical compositions thereof. [00249] This invention also relates to the use of a compound of the invention in the manufacture of a medicament for treatment or prophylaxis of a condition prevented, ameliorated or eliminated by administration of an inhibitor of Mitogen- Activated Protein Kinase- Activated Protein Kinase 5, or a condition characterised by abnormal collagenase activity, or a condition associated with ECM degradation or a condition selected from diseases involving inflammation, most preferably in for the treatment of rheumatoid arthritis. [00250] As a further aspect of the invention there is provided a compound of the invention for use as a pharmaceutical especially in the treatment or prevention of the aforementioned conditions and diseases. Also provided herein is the use of a compound of the invention in the manufacture of a medicament for the treatment or prevention of one of the aforementioned conditions and diseases. [00251] In a further aspect the present invention provides a compound of the invention for use in the prevention or treatment of conditions in mammals that are causally related or attributable to aberrant activity of MMPl and / or MAPKAPK5. In particular, the present invention provides a compound of the invention and/or pharmaceutical compositions thereof for use in the treatment or prevention of inflammatory diseases in mammals including humans.

[00252] In a further aspect, this invention provides a compound of the invention for use in the prevention or treatment of a condition associated with extra-cellular matrix (ECM) degradation, in particular arthritis, and more particularly, rheumatoid arthritis.

[00253] In a further aspect, this invention provides a compound of the invention for use in the prevention or treatment of a condition associated with an abnormal cellular expression of MMPl . [00254] In a further aspect, this invention provides a compound of the invention for use in the prevention or treatment of a condition characterized by abnormal matrix metallo proteinase activity. [00255] In a further aspect, this invention provides a compound of the invention for use in the prevention or treatment of diseases and disorders which are mediated by or result in inflammation such as, for example rheumatoid arthritis and osteoarthritis, myocardial infarction, various autoimmune diseases and disorders, uveitis and atherosclerosis; itch / pruritus such as, for example psoriasis; and renal disorders. [00256] A preferred regimen of the present method comprises the administration to a subject in suffering from a disease condition characterized by extracellular matrix degradation, with an effective matrix metallo-protease inhibiting amount of a compound of the invention for a period of time sufficient to reduce the abnormal levels of extracellular matrix degradation in the patient, and preferably terminate, the self-perpetuating processes responsible for said degradation. A special embodiment of the method comprises administering of an effective matrix metallo-protease inhibiting amount of a compound of the present 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, collagen and bone degradation in the joints of said patient, and preferably terminate, the self-perpetuating processes responsible for said degradation.

[00257] The compounds of the invention may show less toxicity, good absorption, good half- life, good solubility, low protein binding affinity, less drug-drug interaction, and good metabolic stability. In a particular aspect, a compound of the invention exhibits unexpected significant improvements in pharmacological properties, in particular improved efficacy and improved tolerability. Where the compounds exhibit any one or more of these improvements, this may have an effect on their use in the conditions described herein. For example, where the compounds exhibit an improved efficacy it would be expected that the compounds could be administered at a lower dose, thus reducing the occurrence of any possible undesired side effects. Similarly, where the compounds exhibit increased tolerability, this might allow the compounds to be dosed at a higher concentration without causing unwanted side effects. Such alterations in efficacy or tolerability might be expected to result in an improved therapeutic window for said compounds of the invention. Similarly, improvements in the other properties listed above will also confer advantages in the potential uses of the compounds. [00258] Injection dose levels range from about 0.1 mg/kg/hour to at least 10 mg/kg/hour, all for from about 1 to about 120 hours and especially 24 to 96 hours. 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. [00259] For the prevention and/or treatment of long-term conditions, such as inflammatory and autoimmune conditions, the regimen for treatment usually extends over many months or years, and accordingly 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 the compound of the invention, with preferred doses each providing from about 0.1 to about 10 mg/kg and especially about 1 to about 5 mg/kg.

[00260] Transdermal doses are generally selected to provide similar or lower blood levels than are achieved using injection doses.

[00261] When used to prevent the onset of an inflammatory condition, the compounds of this 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. [00262] 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.

[00263] In one embodiment, a compound of the invention is co-administered with another therapeutic agent for the treatment and/or prevention of a disease involving inflammation; particular agents include, but are not limited to, immunoregulatory agents e.g. azathioprine, corticosteroids, cyclophosphamide, cyclosporin A, FK506, Mycophenolate Mofetil, OKT-3 and ATG. [00264] In one embodiment, a compound of the invention is co-administered with another therapeutic agent for the treatment and/or prevention of 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). [00265] 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 [00266] The triazolopyrazine and imidazopyrazine compounds 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.

[00267] 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. [00268] The following methods are presented with details as to the preparation of representative bicycloheteroaryls that have been listed hereinabove. The compounds of the invention may be prepared from known or commercially available starting materials and reagents by one skilled in the art of organic synthesis.

[00269] 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 μm). Thin layer chromatography was carried out using pre-coated silica gel F-254 plates (thickness 0.25 mm). 1H 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. Column Used for all LCMS analysis: Waters Acquity UPLC BEH C18 1.7μm, 2.1mm ID x 50mm L (Part No.186002350)). Preparative HPLC: Waters XBridge Prep Cl 8 5μm ODB 19mm ID x 100mm L (Part No.186002978). All the methods are using MeCWH2O gradients. H2O contains either 0.1% TFA or 0.1% NH3. List of abbreviations used in the experimental section

DCM: Dichloromethane dichloropalladium(II)

DiPEA: N, N-dUs opropylethylamine TEA Triethylamine

MeCN Acetonitrile AIBN 2,2'-azobisisobutyronitrile

BOC tert- Butyloxy-carbonyl IPA /yo-Propyl Alcohol

DMF N, Ν-dimethylformamide BINAP 2,2 '-bis(diphenylphosphino)- 1,1'- TFA Trifluoroacetic acid binaphthyl

THF Tetrahydrofuran MTBE Methyl tert-Butyl Ether

NMR Nuclear Magnetic Resonnance 2-MeTHF 2-Methyl Tetrahydrofuran

DMSO Dimethylsulfoxide EDTA Ethylenediaminetetraacetic acid

DPPA Diphenylphosphorylazide ATP Adenosine triiphosphate

LC-MS Liquid Chromatography-Mass EGTA Ethylene Glycol Tetraacetic Acid

Spectrometry BSA Bovine Serum Albumine

Ppm part-per-million DTT Dithiothreitol

EtOAc ethyl acetate FBS Fetal bovine serum

APCI atmospheric pressure chemical PBST Phosphate buffered saline with ionization Tween 3.2 mM Na2HPO4, 0.5

Rt retention time mM KH2PO4, 1.3 mM KCl, 135

S singlet mM NaCl, 0.05% Tween 20, pH br s broad singlet 7.4 m multiplet MMP Matrix Metallo Proteinase d doublet shRNA short hairpin RNA

PdCl2dppf [U'- RNA Ribonucleic acid

Bis(diphenylphosphino)ferrocene] Ad-Si RNA Adenoviral encoded siRNA DMEM Dulbecco's Modified Eagle RRIIPPAA buffer Radioimmunoprecipitation assay

Medium buffer

APMA 4-aminophenylmercuric acetate MMAAPPEKAPK5 Mitogen-activated protein kinase-hCAR human cellular adenovirus activated protein kinase 5 receptor PBMC Peripheral Blood Mononuclear dNTP deoxyribonucleoside triphosphate Cell

QPCR quantitative polymerase chain TNFα Tumor Necrosis Factor alpha reaction LPS Lip op o lys accharide cDNA copy deoxyribonucleic acid ip Intra-peritoneal

GAPDH Glyceraldehyde phosphate iv Intraveinous dehydrogenase

PVDF Polyvinylidene Fluoride

Synthetic Preparation of Compounds of the Invention Synthesis of Intermediates

Intermediate Ia: Preparation of 3,6-Dibromo-pyrazin-2-ylamine

General reaction scheme:

LiOH, THF 1. DPPA, t-BuOH MeOH, H2O TEA, reflux IWBr

Br N CO2Me Br N CO2H 2. TFA/DCM Br N N H2 4:1

(A) (B) (C)

Step 1: Synthesis of compound (B) as described in the general reaction scheme; 3, 6-dibromo-pyrazine-2-carboxylic acid.

[00270] LiOH (655 mg, 27 mmol) is added to a solution of 3,6-dibromo-pyrazine-2-carboxylic acid methyl ester (A) (J. Med. Chem. 1969, 12, 285-87) (2.7 g, 9 mmol) in THF:water:MeOH (18:4.5:4.5 mL). The reaction is stirred at 5 0C for 30 min, concentrated in vacuo, taken up in DCM and washed with IN HCl. The organic phase is dried over anhydrous MgSU4 and concentrated in vacuo to afford compound (B). 1H NMR (250MHz, CDCl3) δ (ppm) 8.70 (s, IH).

Step 2: Synthesis of compound (C) as described in the general reaction scheme; 3,6-Dιbromo-pyrazιn-2-ylamine.

[00271] Diphenylphosphorylazide (2.59 mL, 12 mmol) and triethylamine (1.67 mL, 12 mmol) are added to a solution of 3,6-dibromo-pyrazin-2-carboxylic acid (3.52 g, 12 mmol) in t-butanol (90 mL). The reaction is heated at reflux for 18 hours. The reaction is quenched with water, then concentrated in vacuo and taken up in DCM. The organic solution is washed with water and IN NaOH, dried over MgSO4 and concentrated in vacuo. The resultant solid is filtered through a pad of silica using EtOAc, then concentrated and TFA:DCM (4.1, 12 mL) is added to the solid and stirred for 30 min. The solution is concentrated in vacuo then neutralised with IN NaOH and extracted with DCM. The organic layer is dried over anhydrous MgSO4 and concentrated in vacuo to give the product. 1H NMR (250MHz, DMSO-d6) δ(ppm) 7.25 (br s, 2H), 7 68 (s, IH); m/z (APCI) 254 (M+H)+; m.p 135-139°C.

Intermediate Ib: Preparation of 3-Choro-6-bromo-pyrazin-2-yl-amine

[00272] Alternatively 3-chloro-6-bromopyrazin-2-yl-amine can be used in place of 3,6-dibromo-pyrazin-2-yl amine and is prepared according to the following scheme:

8SOO C



A' B'

Step / Synthesis of compound (A ') as described in the general reaction scheme; 2-chloro-3,5-dιbromo-pyrazine

[00273] To a well stirred solution of 2-amino-3,5-dibromopyrazine (3.21 g, 12.692 mmol) in

DCM (20 mL) cooled to 00C is added TiCl4 (2 41 g, 12 692 mmol, 1.00 equiv.) in one portion, thus giving a dark red slurry. t-Butylnitrite (2.62 g, 25.385 mmol, 2.00 equiv.) is then added dropwise, causing the solution to turn bright yellow. The ice bath is then removed and the reaction is then allowed to proceed at room temperature. More TiCl4 (1.50 g, 1.2 equiv.) is added and the mixture is stirred further for one hour. At that point an orange solution has formed and LC-MS shows full conversion of the starting material to the desired product which ionises very poorly. Water (100 mL) is added to the reaction, forming an emulsion. DCM (50 mL) is added, and the DCM layer is separated, and the aqueous layer is further extracted with DCM (3 x 50 mL) until the DCM layer is colorless. The DCM layers are gathered, washed with brine and dried over anhydrous Na2SO4, to yield after solvent removal, compound A' (2.81g, 82%) as an orange oil, which is used as such in the following step.

Step 2: Synthesis of compound (B') as described in the general reaction scheme; 3-chloro-6-bromopyrazιn-2-yl amine

[00274] Compound A' described in the previous step (9.5 g, 37.55 mmol) is suspended in concentrated NH4OH (60 mL) and the resulting mixture is heated in a pressure autoclave to 80° C, typically overnight. The vessel is then allowed to cool down to room temperature slowly, and is then further cooled in an ice bath, causing the precipitation of the desired material. The solid is separated by filtration, washed with cyclohexane, to afford after drying, the title compound B' (5 g) as a 83/17 mixture of regiosiomers. The mxiture is then purified by column chromatography. M+H+, m/z = 209 Intermediate 2: 5,8-Dibromo-imidazo[l,2-alpyrazine


[00275] Bromoacetaldehyde diethyl acetal (49 mL, 326 mmol) and 48% hydrobromic acid is heated to reflux for 1.5 h, then poured into propan-2-ol (600 mL) and quenched with NaHCθ3. After filtering, 3,6-dibromopyrazin-2-yl amine (41.34 g, 163 mmol) is added to the solution and heated at reflux overnight. The reaction is cooled and solvents removed in vacuo, followed by addition of aq. NaHCO3 and extraction with EtOAc. The organic phase is dried over anhydrous MgSOzi, filtered, and concentrated in vacuo to afford a brown solid. 1H NMR (250MHz, CDCl3) δ(ppm) 7.86 (s, IH), 7.93-7.94 (d, IH), 7.98-7.99 (d, IH); m/z (APCI) 278 (M+H)+; rap 132-135 0C.

Intermediate 3: 5,8-Dibromo-[l,2,41triazolo[l,5-alpyrazine


General scheme:


(D) (E (F)

Step 1: N'-(3, 6-Dibromo-pyrazin-2-yl)-N,N-dimethylformamidine(D)


[00276] A mixture of 3,6-dibromo-pyrazin-2-ylamine (15.37 g, 60.80 mmol) and N,N-dimethylformamide dimethyl acetal (10.1 mL, 76.00 mmol), suspended in ethanol (150 mL), is refluxed for 2 hours. The reaction mixture is evaporated in vacuo affording the title compound. 1H-NMR (400MHz, CDCl3) δ(ppm) 3.20 (s, 3H), 3.21 (s, 3H), 7.93 (s, IH), 8.48 (s, IH). LCMS: Rt 3.81 min (99.1%), m/z (APCI) 307 (M+H)+.

Step 2: N-β, 6-Dibromo-pyrazιn-2-yl)-N'-hydroxyformamidine (E)


[00277] To a solution of Λ^-(3,6-dibromo-pyrazin-2-yl)-N,N-dimethylformamidine (18.6 g, 60.80 mmol) in methanol (200 niL) is added hydroxylamine hydrochloride (5.91 g, 85.12 mmol) in one portion. The reaction is stirred at room temperature for 16 hours. The solvent is evaporated and the solid residue is treated with cold (ice cooling) water and collected by filtration. The precipitate is washed twice with water and petroleum ether and dried in vacuo yielding the title compound. 1H-NMR (400MHz, DMSO-d6) δ(ppm) 7.82 (br s,lH), 8.21 (s, IH), 8.34 (m, IH), 11.17 (br s, IH). LCMS: Rt 3.17 min (98.7 %), m/z (APCI) 295 (M+H)+.

Step 3: 5 ,8-Dibromo-[ 1 ,2,4] triazolof 1 , 5-a] pyrazine (F)


[00278] W-(3,6-dibromo-pyrazin-2-yl)-A^-hydroxyformamidine (17.4 mg, 58.80 mmol) is treated with polyphosphoric acid (150 g) for one hour at 500C and then for 1.75 hours at 70°C. After cooling to room temperature, water is added to the reaction mixture. The resultant suspension is brought to pH 8 by careful addition of solid NaHCC>3 in small portions. The precipitate formed is collected by filtration, washed once with IN NaOH, three times with water and dried in vacuo. The residue is partitioned between ethyl acetate and IN NaOH and the organic phase is washed one more time with IN NaOH and once with brine. The organic phase is dried over anhydrous MgSO4, filtered and evaporated to give the title compound (10.15 g) as a white solid. 1H-NMR (400MHz, DMSO- d6) δ (ppm) 8.43 (s, IH), 8.92 (s, IH). LCMS: Rt 2.73 min (94.2 %), m/z (APCI) 277 (M+H)+.

Intermediate 4: 5-(4,4,5,5-Tetramethyl-[l,3-,21dioxaborolan-2-yl)-2,3-dihydro-isoindol-l-one


Step 1: 4-Bromo-2-bromomethyl-benzoic acid methyl ester

[00279] 4-Bromo-2-methyl-benzoic acid (4.6 g, 21.39 mmol) is dissolved in 2M HCl in MeOH and refluxed for 3 hours. The solvent is evaporated to give the 4-bromo-2-methyl-benzoic acid methyl ester (4.24 g, 86 %). This intermediate (18.51 mmol) is dissolved in carbon tetrachloride (100 mL) and N-bromosuccinirnide (5.57 g, 24.06 mmol) is added. AIBN (122 mg, 740 μmol) is then added and the mixture purged with nitrogen for 5 minutes. The reaction mixture is then refluxed for four hours. After cooling to room temperature the reaction mixture is filtered and the filtrate is evaporated. The residue is purified by flash chromatography (silica gel, 2: 1 petroleum ether/ethyl acetate) to give the title compound (3.42g, 60 %).

Step 2: 5-Bromo-2,3-dihydro-ιsoindol-l-one


[00280] 4-Bromo-2-bromomethyl-benzoic acid methyl ester (0.5g, 16.2mmol) is treated with methanolic ammonia (1OmL, 7 N NH3 in MeOH) for 5 minutes at 9O0C. After cooling to room temperature the precipitate formed is filtered off and washed with a small amount of methanol affording the title compound (224mg, 65%) as a colourless solid.

[00281] 1H-NMR (400MHz, DMSO-c/<5) δ (ppm) 4.41 (s, 2H, CH2), 7.64 (d, IH, H»), 7.70 (d, IH,

Ha1), 7.87 (s, IH, H211), 8.67 (bs, IH, NH). LCMS 99.6 %, R4 = 2.49 min, m/z 212 (M+H, AP+ formic acid).

Step 3: 5-(4, 4, 5, 5-Tetrαmethyl-[l, 3, 2]dioxαborolαn-2-yl)-2, 3-dihydro-isoindol-l-one


[00282] 5-Bromo-2,3-dihydro-isoindol-l-one (230 mg, 1.08 mmol), bis(pinacolato)diboron (300 mg, 1.18 mmol), PdCl2dppf (25 mg, 31 μmol) and KOAc (320 mg, 3.26 mmol) are suspended in dioxane (4 ml), purged with nitrogen for 5 minutes and then heated at 85°C overnight. The solvent is removed in vacuo and the residue partitioned between ethyl acetate and water. The aqueous layer is extracted three times with ethyl acetate and the combined organic phases are washed once with brine, filtered through anhydrous MgSO4 and evaporated. The solid residue is triturated with hexane and dried in vacuo to furnish the title compound (185mg, 66 %) as a grey solid.

[00283] 1H-NMR (400MHz, CDCl3) δ (ppm) 1.37 (s, 12H, 4xCH3), 4.45 (s, 2H, CH2), 6.38 (bs,

IH, NH), 7.87 (d, IH, H31), 7.93 (m, 2H, H31).

Intermediate 5: 4-(4,4,5,5-Tetramethyl-[l,3,21dioxaborolan-2-yl)-furan-2-carboxylic acid amide

NH2


Step 1: 4-Bromo-furan-2-carboxylιc acid amide


[00284] To a cooled (using a cold water bath) solution of 4,5-dibromo-furan-2-carboxylic acid

(12.5 g, 46.32 mmol) in NH4OH (100 niL) is added zinc dust (activated, powdered (washed with 2M HCl, water, MeOH, CH2CI2) 4.54 g, 65.39 mmol) in small portions. The reaction mixture is stirred at room temperature for 10 minutes then filtered over celite and washed with water. The filtrate is cooled to -10 0C (ice/salt bath) and acidified slowly to pH 1 using cone. HCl. The aq layer is immediately extracted with ethyl acetate (4x). The organic phase is washed with brine, dried over anhydrous MgSO4, filtrated and concentrated in vacuo to give an oil (4.96 g) which solidifies on standing to give a white solid, which is used without further purification.

[00285] The solid (4.93 g, 25.81 mmol) is dissolved in thionyl chloride (44.2 niL) and refluxed for

1 hour. After removing the solvent in vacuo the residue is dissolved in dichloromethane (75mL) and a solution of 0.5 M NH3 in dioxane (52 mL) is added. The reaction mixture is stirred at room temperature for 1 hour, then 33% aq. NH3 (5 mL) is added and the reaction stirred for additional 2 hours. The solvent is removed in vacuo and the residue taken-up with a solution of sat. NaHCO3. The basic solution is extracted using ethyl acetate (3x), the combined organic layers are dried over anhydrous MgSO4 and concentrated in vacuo. Purification by silica gel column chromatography eluting with a mixture of (50:49: 1) ethyl acetate: petroleum ether: acetic acid, affords the title compound (1.2 g, 22 %).

Step 2: 4-(4,4,5,5-Tetramethyl-[l,3,2]dioxaborolan-2-yl)-furan-2-carboxylic acid amide

[00286] 4-Bromo-furan-2-carboxylic acid amide (1.2 g, 6.32 mmol), bis(pinacolato)diboron (1.76 g, 6.94 mmol), PdCl2dppf (0.154 g, 0.189 mmol) and KOAc (1.85 g, 18.94 mmol) are suspended in dioxane (20 mL), purged with nitrogen for 5 minutes and then heated at 85 0C overnight. The solvent is removed in vacuo and the residue partitioned between ethyl acetate and brine. The aqueous layer is extracted four times with ethyl acetate, filtered through anhydrous MgSC>4 and evaporated. The solid residue is triturated with hexane and dried in vacuo to afford the title compound as a solid (0.984 g, 66 %). N.B. compound is usually 50-60% pure by 1H-NMR.

Alternative route to intermediate 5:


[00287] 3-(4,4,5,5-Tetramethyl-[l,3,2]dioxaborolan-2-yl)-furan (5.Og, 25.77 mmol) is dissolved in dry acetonitrile (30 mL). Chlorosulfonylisocyanate (5.47 g, 38.65 mmol, 1.5 equiv.) in solution in dry acetonitrile (20 mL) is added in one portion at room temperature to the furan producing a pink solution that subsides overnight to turn yellow. The resulting solution is cooled with an ice bath and water (5mL) is added, causing an exotherm. The resulting mixture is partitionned between DCM (100 mL) and water (30 mL). The aqueous layer is extracted 3 more times with DCM (50 mL), then the organic layers are gathered, washed with brine (10 mL), dried over anhydrous Na2SC^ and finally the solvent is removed under vacuum. The oily residue is dissolved in DCM (3mL), sonicated to give a suspension of a crystalline solid. The solid is separated by filtration, and the cake is washed with a very small amount of DCM, then diethyl ether and dried under suction to afford 3g of the title compound as a white powder.

Intermediate 6: 2-Ethoxypyridyl-4-boronic acid pinacol ester

[00288] 2-Ethoxy-4-bromopyridine (2.5 g, 12.4 mmol), έω-pinacolatodiboron (3.4 g, 13.7 mmol) and potassium acetate (3.64 g, 37 20 mmol) are dissolved in 1,4-dioxane (40 ml) and degassed with nitrogen for 15 minutes PdC^dppf (3 mol%, 0.37 mmol, 0.3 g) is then added and the mixture was heated in a sealed vessel at 900C for 16 hours. Water is added and the mixture was extracted with EtOAc. The organics are washed with brine and dried over anhydrous MgSO4 then concentrated in vacuo. The crude product is purified by flash chromatography on silica (petrol to 10% EtOAc in petrol) to give 2-ethoxypyridyl-4-boronic acid pinacol ester as a pale oil (2.58 g, 83%).

[00289] NMR δ 1H (400 MHz, DMSO-Λ5): 8.16 (IH, m); 7.15 (IH, m); 7.11 (IH, s); 4.33 (2H, q); 1.38 (3H, t); 1.34 (12H, s).

Intermediate 7: 4-((lS,4S)-5-Isopropyl-2,5-diaza-bicvclo[2.2.11hept-2-yl)-phenylamine

Step 1 ((lS,4S)-5-(4-Nιtro-phenyl)-2,5-dιaza-bιcyclo[2.2 lJheptane-2-carboxylιc acid tert-butyl ester


[00290] 4-Fluoronitrophenyl (4.0Og, 28.348 mmol), DiPEA (5.89 mL, 60.667 mmol, 2.14 equiv.) and (7S,4S>2-BOC-2,5-diazabicyclo[2.2.1]heptane (6.02 g, 30.333 mmol, 1.07 equiv.) are mixed in acetonitrile (20 mL). The resulting solution is heated to reflux overnight, after which full conversion has occurred. The solvent is removed under vacuum, and the solid yellow residue is stirred in cyclohexane (50 mL) for 0.25h, then allowed to settle, the supernatant is discarded, and the process is repeated twice. On the third time, the solid is separated by filtration, allowed to dry under suction, to afford the title compound clean as a yellow solid (8.8 g).

Step 2: (IS, 4S)-2-(4-Nιtro-phenyl)-2, 5-dιaza-bιcyclo[2.2.1] heptane


[00291] The solid obtained in the previous step (8.2 g) is dissolved in a mixture of DCM (12 mL) and TFA (12 mL) The reaction is allowed to proceed at RT for 2 h, at which point full deprotection has occurred. The volatiles are removed under vacuum and the crude resulting solid is used as such without further treatment.

Step 3: (IS, 4S)-2-Isopropyl-5-(4-nitro-phenyl)-2, 5-diaza-bicyclo[2.2.1] heptane


[00292] The crude compound obtained in the previous step (6.22 g, 28.356 mmol) is dissolved in acetonitrile (70 mL). K2CO3 (19.59 g, 141.770 mmol, 5.00 equiv.) is added, followed by i-propyl iodide (9.64 g, 56.708 mmol, 2.00 equiv.) and the resulting suspension is heated to reflux with stirring, for 3 h, at which point full conversion has occurred. The reaction mixture is partitioned between DCM (100 mL) and water (50 mL). The organic layer is washed with water (50 mL), brine (25 mL), dried on anhydrous Na2SO/|, filtered and evaporated in vacuo to yield the title compound (9.90 g) as a yellow solid.

Step 4: 4-((lS, 4S)-5-Isopropyl-2, 5-diaza-bicyclo[2.2. l]hept-2-yl)-phenylamine


[00293] The compound obtained in the previous step (3.3Og, 9.35 mmol) is dissolved in EtOH

(107 mL). The system is degassed and placed under nitrogen. Pd/C 10% (0.50 g, 5 mol%) is added followed by hydrazine, 35% in water (4.3 mL, 46.75 mmol, 5 equiv.), and the reaction is allowed to proceed at 1000C until full conversion has occurred (typically 1 h). The reaction is then allowed to cool down, filtered on celite and the filtrate is evaporated in vacuo to afford the title compound (2.07 g) as pink oil.

Intermediate 8: ((lS,4S)-5-tert-Butyl-2,5-diaza-bicvclo[2.2.11hept-2-yl)-phenylamine

Step 1: (2S,4R)-4-Hydroxy-l-(4-nitro-phenyl)-pyrrolidine-2-carboxylic acid methyl ester


[00294] (25,4R)-L-PrOIiIiOl methyl ester (4.7 g, 25 878 mmol) is dissolved in acetonitrile (10 mL) and DiPEA (13.5 mL, 77.635, 3 equiv.). 4-fluoronitrobenzene is then added to the reaction mixture which is heated to 5O0C overnight. After solvent removal, under vacuum, the orange oily residue is partitioned between DCM (50 mL) and water pH 4 (50 mL). The aqueous layer is further extracted with DCM (4x50 mL), the combined organic layers are washed with brine and dried over anhydrous MgSO/i, to afford the title compound as an orange oil.

Step 2: (3R, 5S)-5-Hydroxymethyl-l-(4-nιtro-phenyl)-pyrrolιdιn-3-ol

OH


[00295] The compound obtained in the previous step (6.89 g, 25 878 mmol) is dissolved in THF

(50 mL), and LiBH4 (51.756 mmol, 1.13 g, 2.00 equiv.) is added portionwise to the resulting solution causing effervescence. The resulting mixture is allowed to react at room temperature until full conversion has occurred. The reaction is then quenched with IM HCl, to pH 7, and the resulting solution is partitioned between DCM (100 mL) and water (50 mL). The aqueous layer is then extracted with DCM (4 x 5OmL). The organic layers are gathered, washed with brine and dried over anhydrous Na2SO4, to afford the title compound as a yellow oil (3.2 g) used as such in the next step.

Step 3: (3R, 5S)-3-Tosyloxy-5-Tosyloxymethyl-l-(4-nιtw-phenyl)-pyrrohdιne

PTos


[00296] The diol obtained in the previous step (6.2 g, 25.878 mmol) is solubilised in pyridine (31 mL), and cooled to 00C Tosyl chloride (14 8 g, 77 734 mmol, 3 equiv.) is then added in one portion and the mixture is stirred at that temperature, and placed in the freezer until needed.

Step 4- (lS,4S)-2-tert-Butyl-5-(4-nιtro-phenyl)-2,5-dιaza-bιcyclo[22.1] heptane


[00297] The di-tosylated material (1 g, 1.829 mmol) obtained in the previous step is dissolved in toluene (3 mL) in a pressure tube, and t-butyl amine (0.67 g, 9.147 mmol, 5 equiv.) is added thus giving a burgundy solution. The tube is sealed and heated to 11O0C overnight, at which point full conversion of the starting material has occurred. The crude mixture is allowed to cool down to room temperature, and is diluted in DCM (20 mL), then extracted with 3 M HCl (2 x 10 mL). The acidic aqueous layers are gathered, washed with DCM (5 mL), then basicified to pH = 12-13 by addition of 10% NaOH. The resulting basic layer is extracted with DCM (4 x 20 mL), the organic layers are gathered, washed with brine and dried over anhydrous Na2SC^ to afford after solvent removal and silica chromatography using DCM/ MeOH 96/4 as the eluent, the title compound as a yellow oil.

Step 5: 4-((1S, 4S)-5-tert-Butyl-2, 5-diaza-bicyclo[2.2. l]hept-2-yl)-phenylamine


[00298] This compound is prepared according to the same procedure as described for Intermediate

7, Step 4.

Intermediate 9 : 4-(5-isopropyl-2.,5-diazabicvclo [2.2.21 octan-2-vDaniline


[00299] This compound is prepared according to the same procedure as described for Intermediate

7 using 2,5-diazabicyclo[2.2.2]octane dihydrochloride (J. Heterocycl. Chem, 1974, 11, 449-451).

Intermediate 10; 4-(8-isopropyl-3,8-diazabicyclo[3.2.11octan-3-yl)aniline


[00300] This compound is prepared according to the same procedure as described for Intermediate

7 using 8-(tert-butoxycarbonyl)-3,8-diazabicyclo[3.2.1]octane (J. Med.Chem., 1998, 41, 674-681).

Intermediate 11: 4-(8-isopropyl-3,8-diazabicvclo[3.2.11octan-3-yl)aniline

Step 1 : 3-(tert-butoxycarbonyl) -8-ιsopropyl-3, 8-dιazabιcyclo[3.2.1] octane


[00301] To a solution of 8-(tert-butoxycarbonyl)-3,8-diazabicyclo[3.2.1]octane (J. Med. Chem.,

1998, 41, 674-681) (1.54 g, 7.25 mmol) in methanol (25 niL) is added sodium acetate (595.0 mg, 7.25 mmol), acetic acid (415μL, 7.25 mmol) and acetone (2.66 mL, 36.25 mmol). The reaction mixture is stirred at 40 0C during one hour, and then sodium cyanoborohydride (912.0 mg, 14.50 mmol) is added. The reaction is heated at 40 0C for 18 hours, concentrated in vacuo, taken up in DCM and washed with aq. NaHCO3. The organic phase is dried over anhydrous MgSO4 and concentrated in vacuo to afford 3-(tert-butoxycarbonyl)-8-isopropyl-3,8-diazabicyclo[3.2.1]octane (1.5Ig, 82 %).

Step 2- 8-ιsopropyl-3,8-dιazabιcyclo[3.2.1Joctane

N NH

[00302] This compound is prepared according to the same procedure as described for Intermediate

7, Step 2.

Step 3: 8-ιsopropyl-3-(4-nιtrophenyl)-3, 8-dιazabιcyclo[3.2.1] octane


[00303] This compound is prepared according to the same procedure as described for Intermediate

7, Step 3.

Step 4: 4-(8-ιsopropyl-3, 8-dιazabιcyclo[3.2. l]octan-3-yl) aniline


[00304] This compound is prepared according to the same procedure as described for Intermediate

7, Step 4.

Intermediate 12: 6-((lS,4S)-5-isopropyl-2,5-diazabicvclo[2.2.11heptan-2-vI)pyridin-3-amine


[00305] This compound is prepared according to the same procedure as described for Intermediate

7 using 2-chloro-5-nitropyridine.

Intermediate 13: 3-((lS,4S)-5-isopropyl-2,5-diazabicvclo[2.2.11heptan-2-yl)phenylamine

Step 1: (lS,4S)-2-ιsopropyl-5-(3-nιtrophenyl)-2,5-dιazabιcyclo[2.2.1]heptane


[00306] A solution of BINAP (0.47 g, 0.75 mmol) and tris(dibenzylideneacetone)dipalladium (0)

(0.34g, 0 37 mmol) in toluene (120 mL) was heated to 90 0C during 15 minutes under nitrogen The reaction mixture was cooled to 40 0C before adding l-bromo-3-nitrobenzene (1.89 g, 9.37 mmol), (lS,4S)-2-isopropyl-2,5-diazabicyclo[2.2.1]heptane dihydrochloride (2.0 g, 9.37 mmol) and sodium tert-butoxide (3 14 g, 32.74 mmol). The reaction was heated to 90 0C during 18 hours under nitrogen. After return to room temperature, the reaction was diluted with ethyl acetate and filtered through celite. The filtrate was concentrated under reduced pressure The residue was purified by flash chromatography on silica gel eluting with a mixture of DCM/7N NH3 in methanol (96/4) to afford the title compound (1.92 g, 78 %).

Step 2: 3-((1S, 4S)-5-ιsopropyl-2, 5-dιazabιcyclo[2.2. l]heptan-2-yl)phenylamιne


The compound obtained in the previous step (1.92 g, 7.34 mmol) was dissolved in EtOH (50 mL). The system was degassed and placed under nitrogen. Pd(OH)2 on activated charcoal (10%, 0.2 g) was added and the resulting suspension was stirred at room temperature during 18 hours under hydrogen atmosphere. The reaction was then filtered on celite and the filtrate was evaporated in vacuo to afford the title compound (1.89g, 98 %) as a brown oil.

Intermediate 14: 2-((lS.,4S)-5-Isopropyl-2,5-diaza-bicvclo[2.2.11hept-2-yl)-Pyrimidin-5-ylamine


[00307] Intermediates of the type above can be produced by the methods described by DiMauro et al (J. Med. Chem.,2008, 51, 1681-1694). Initial reaction of 2-chloro-5-nitropyrimidine with (\S,4S)-2-BOC-2,5-diazabicyclo[2.2.1]heptane, followed by application of the procedures for intermediate 7 would give an intermediate suitable for inclusion in compounds of the invention.

I n t e r m e d i a t e 1 5 : ( 4-Amino-phenylH(lS,4S)-5-isopropyl-2,5-diaza-bicvclo[2.2.11hept-2-yl)-methanone


[00308] Intermediates of the type above can be produced using methods described in WO

2007/138072. Initial reaction of 4-nitrobenzoic acid with (lS^^-isopropyl^S-diazabicyclo[2.2.1]heptane, followed by reduction of the nitro group would give an intermediate suitable for inclusion in compounds of the invention.

Intermediate 16: (lS,4S)-5-Isopropyl-2,5-diaza-bicvclo[2.2.11heptane-2-carboxylic acid (4-amino-phenvD-amide.


[00309] Intermediates of the type above can be produced using methods described in Bioorg. Med

Chem Lett, 2008; 4838-4843. Initial reaction of l-isocyanato-4-nitrobenzene with (lS^S^-isopropyl-2,5-diazabicyclo[2.2.1]heptane, followed by reduction of the nitro group would give an intermediate suitable for inclusion in compounds of the invention.

Intermediate 17: (lR,4R)-2-Isopropyl-2,5-diaza-bicylo[2.2.11heptane dihydrobromide TsO, AcO

HO, HO,

SOCI2 EtOH TsCI, Et3N I \ DMF, 600C

O COOEt COOEt lux, 2hr O COOEt pyridine

N , 'fCOOH ref N , T1 KOAc O- H Ts Ts

2 3


HBr in acetic acid acetic acid 2HBr

Step 1: (2R,4R)-4-Hydroxy-pyrrohdine-2-carboxylic acid ethyl ester (1)

[00310] To a stirred solution of cis-4-hydroxy-pyrrolidine-2-carboxylic acid (Ig, 7.6mmol) in absolute ethanol (2OmL) is added dropwise thionyl chloride (0.67mL, 9.15mmol) at O 0C under nitrogen atmosphere. The reaction mixture is then refluxed under nitrogen for about 2h. The mixture is cooled to room temperature, and all solvent is removed under reduced pressure. The white precipitate is filtered and washed with diethyl ether (lχ25mL), to obtain compound (1) as a white solid.

1H-NMR (400 MHz, DMSO-J6): δ 1.23 (t, 3H), 2.12 (d, IH), 2.27 (t, IH), 3.19 (q, 2H), 4.20 (m, 2H),

4.35 (s, IH), 4.47 (d, IH). Mass (M+l): m/z 160.

Step 2: (2R,4R)-l-(Toluene-4-sulfonyl)-4-(toluene-4-sulfonyloxy)-pyrrolidine-2-carboxylic acid ethyl ester (2)

[00311] To a cold solution of 4-hydroxy-pyrrolidine-2-carboxylic acid ethyl ester (1) (1.4g,

7.1mmol) and triethyl amine (0.998mL, 7.1mmol) in pyridine (14mL) at -5 0C was added portion-wise 4-toluenesulfonyl chloride (3.42g, 17.9mmol). The cold solution was then stirred for Ih at O 0C and stored overnight in the refrigerator. Then the mixture was further stirred at room temperature for 5h and poured into ice water (1OmL). The precipitate separated out was filtered, washed with water (2><5mL), and dried to give compound (2) as a white solid.

[00312] 1H-NMR (400 MHz, DMSO-J6): δ 1.13 (t, 3H), 2.05 (d IH ), 2.16 (m, IH), 2.41 (d, 6H),

4.05 (q, 2H), 4.47 (d, IH), 5.00 (s, IH), 7.45 (dd, 4H), 7.72 (d, 4H). Mass (M+l): w/z 468.

Step 3: (2R,4S)-4-Acetoxy-l-(toluene-4-sulfonyl)-pyrrolidine-2-carboxylιc acid ethyl ester (3)

[00313] To a stirred solution of l-(toluene-4-sulfonyl)-4-(toluene-4-sulfonyloxy)-pyrrolidine-2-carboxylic acid ethyl ester (2) (Ig, 2.14mmol) in dry DMF (25mL) is added potassium acetate (0.314g, 3 21mmol) in one portion The reaction mixture is then heated at 60 0C for 4h Water (5OmL) is added to the reaction mixture and extracted with ethyl acetate (2><75mL), combined organic layer are washed with water (2χ50mL), dried (Na2SO/!), filtered, and concentrated under reduced pressure to give the crude compound Crude compound is purified by column chromatography over silica gel (100-200 mesh) using 15% ethyl acetate-hexane as eluent to give compound (3).

[00314] 1H-NMR (400 MHz, CDCl3). δ 1 26 (t, 3H), 1.68 (s, 3H), 2.20 (m, IH), 2.29 (m, IH),

2.41 (s, 3H), 3 52 (d, IH), 3 67 (d, IH), 4 22 (m, 2H), 4.30 (t, IH), 5.11 (s, IH), 7.33 (d, 2H), 7 74 (d, 2H). Mass (M+l): w/z 356.

Step 4- (3S,5R)-5-Hydroxymethyl-l-(toluene-4-sulfonyl)-pyrrohdιn-3-ol (4)

[00315] To an ice-cold solution of 4-acetoxy-l-(toluene-4-sulfonyl)-pyrrolidine-2-carboxyhc acid ethyl ester (3) (0.65g, 1 83mmol) in THF (1OmL) is added L1AIH4 (0.135g, 3.66mmol) portion wise. Then the reaction mixture is stirred at room temperature for Ih. After completion of the reaction, it is cooled to 00C and then pH of reaction mixture is adjusted to 3 by adding 6N HCl (0.65mL) The mixture is concentrated, and the residue is triturated with water (8mL), the solid precipitated out is filtered, washed with cold water (2><4mL), and dried under reduced pressure to give compound (4) as white solid. [00316] 1H-NMR (400 MHz, CDCl3): δ 1.14 (s, IH), 1 82 (m, IH), 1.91 (m, IH), 2.42 (s, 3H)

3.39 (d, IH), 3.55 (d, IH), 3 60 (m, IH), 3.77 (m, IH), 3.85 (d, IH), 4.30 (s, IH), 7.31 (d, 2H), 7 76 (d, 2H). Mass (M+l): w/z 272.

Step 5 (2R, 4S)-I -(4-Tolylsulfonyl)-2-[[(4-tolylsulfonyl)oxy] -methyl] -4-[(4-tolylsulfonyl)oxy]-pyrrohdine (5)

[00317] To an ice-cold solution of 5-hydroxymethyl-l-(toluene-4-sulfonyl)-pyrrolidm-3-ol (4)

(0.45g, 1.66mmol) in pyridine (3mL) is added 4-toluylsulfonyl chloride (1.1 Ig, 5 81mmol) in one portion.

The temperature rises to 500C, then the reaction mixture is cooled to 100C, kept at that temperature for an additional 2h and then left at room temperature overnight The mixture is poured into 2N HCl (13mL) On cooling the compound precipitates out and separated by filtration, washed with cold water (2χ5mL), and dried under reduced pressure to give compound (5) as white solid

[00318] 1H-NMR (400 MHz, CDCl3) δ 2 02 (m, 2H), 2 42(s, 9H), 3 50 (d, 2H), 3 80 (d, IH) 4 10

(q,lH), 4 30(d, IH), 4 76 (t, IH), 7 27 (dd, 4H), 7 35 (d, 2H), 7 56 (d, 2H), 7 62 (d, 2H), 7 78 (d, 2H)

Step 6 (lR,4R)-2-Isopropoyl-5-(toluene-4-sulfonyl)-2,5-dιaza-bιcyclo [2.2.1] heptane (6)

[00319] To a mixture of (2R,4S)-l-(4-tolylsulfonyl)-2-[[(4-tolylsulfonyl)oxy]-methyl]-4-[(4-tolylsulfonyl)oxy]pyrrolidine (5) (0.5g, 0.93mmol) and isopropyl amine (0.3mL, 0.33mmol) m dry toluene (3mL) is heated to HO0C for 1 Oh. The mixture is then cooled to room temperature, a solid separates out and is separated by filtration and washed with toluene (1 >< 15mL). The combined organic layer is dried over (Na2SO4), filtered and concentrated under reduced pressure to give crude product which is purified by column chromatography over silica gel (100-200 mesh) using 1% triethylamine- ethyl acetate as eluent to afford compound (6) as white solid.

[00320] 1H-NMR (400 MHz, CDCl3): δ 0.72 (d, IH), 0.89 (t, 6H), 1.46 (d, IH), 2.32 (s, IH), 2.38

(s,3H), 2.46 (t, IH), 2.85 (d, 2H), 3.51 (s, IH), 4.18 (s, IH), 7.44 (d, 2H), 7.71 (d, 2H). Mass (M+l): m/z

295.

Step 7: (lR,4R)-2-Isopropyl-2,5-dιaza-bιcylo[2.2.1]heptane dihydrobromide.

[00321] To a solution of 33% HBr (0.5mL) and acetic acid (3mL) at 70 0C is added (lR,4R)-2-isopropoyl-5-(toluene-4-sulfonyl)-2,5-diaza-bicyclo[2.2.1]heptane (6) (0.24g, 0.000816 mol). The mixture is then stirred for 12h at same temperature. The mixture is cooled to 100C to yield a white precipitate which is filtered and washed with diisopropyl ether (l x5mL) and ethyl acetate (l χ5mL), dried under reduced pressure to afford intermediate 17 as a white solid.

[00322] 1H-NMR (400 MHz, D2O): δ 1.33 (d, 3H), 1.39 (d, 3H), 2.29 (d, IH), 2.41 (d, IH), 3.57

(m,3H), 3.73 (s, 2H), 4.61 (s, IH), 4.75 (s, IH). Mass (M+l): m/z 141.

Intermediate 18: 2-Carboxamido-4-furanboronic acid.


[00323] 15 g 3-Furanboronic acid pinacol ester (77.3 mmol, 1.0 eq) is dissolved in 120 mL acetonitrile, 10.2 mL chlorsulfonyl isocyanate (116 mmol, 1.5 eq) is added in one portion. Stirring is continued over night. Full conversion is determined by LCMS Reaction is quenched by slowly adding 3OmL H2O The solution is concentrated and 100 mL IPA is added. This procedure is repeated and the reaction mixture is diluted by adding 120 mL H2O and 8 mL IPA and stirred for 2 hours. The precipitate formed during the process is filtered off and washed with 30 mL H2O. After drying, the solid is recrystallised from 120 mL IPA and 6 mL H2O. The crystals are washed with 30 mL IPA and isolated in 99+% purity.

Specific Examples of Compounds of the Invention

Compound 1: 5-{8-[4-((lS,4S)-5-Isopropyl-2,5-diaza-bicyclo[2.2.1]hept-2-yl)-phenylamino]-[l,2,4]triazolo[l,5-a]pyrazin-5-yl}-2,3-dihydro-isoindol-l-one


[00324] This compound may be prepared according to the same procedure as described for

Compound 2 using the corresponding intermediates described above.

[00325] 1H NMR (400 MHz, OMSO-d6): δ 9.81 (s, IH); 8.65 (m, 2H); 8.19 (s, IH); 8.07-8.05

(m, IH); 7.93 )s, IH); 7.82-7.79 (m, 2H); 7.75-7.73 (m, 2H); 6.60 (m, 2H); 4.47 (s, 2H); 4.25 (s, IH); 3.69

(s, IH); 3.18 (m, IH); 2.99 (m, IH); 2.39-2.36 (m, 2H); 1.80 (s, 2H); 0.93 (d, 6H); m/z: M+H+ (481.1;

100%).

Compound 2: 4-{8- [4-((l S,4S)-5-Isopropyl-2,5-diaza-bicyclo [2.2.1 ] hept-2-yl)-phenylamino]-[l,2,4]triazolo[l,5-a]pyrazin-5-yl}-furan-2-carboxamide

Step 1: (5-Bromo-fl , 2, 4]triazolo[l, 5-a]pyrazin-8-yl)-[4-((lS, 4S)-5-ιsopropyl-2, 5-diaza-bιcyclo[2.2. l]hept-2-yl) -phenyl] -amine


[00326] 5,8-Dibromo-[l,2,4]triazolo[l,5-a]pyrazine (2.26 g, 8.14 mtnol), 4-((lS,4S)-5-Isopropyl- 2,5-diaza-bicyclo[2.2.1]hept-2-yl)-phenylamine (2.07 g, 8.95 mmol, 1.10 equiv.) and DiPEA (4.3 mL, 24.42 mmol, 3.00 equiv.) are mixed in isopropanol (28 mL) under nitrogen. The reaction is heated to 850C until completion of the reaction (typically 5 h). The solvent is removed under vacuum and the residue is partitioned between 60 mL aqueous sodium phosphates buffer (pH 7) and 200 mL DCM, the organic layer is washed with 60 mL satd. NaCl, dried on anhydrous Na2SO4, filtered and evaporated in vacuo to yield the title compound (3.67 g) as a green-black foamy solid.

Step 2 4-{8-[4-((lS, 4S)-5-Isopropyl-2, 5-dιaza-bιcyclo[2.2. l]hept-2-yl)-phenylamιno]-[1, 2, 4]ϊrιazolo[l, 5-a]pyrazιn-5-yl}-furan-2-carboxamιde


[00327] The compound obtained in the previous step (3.25g, 7.59 mmol) is mixed with 4-(4,4,5,5- Tetramethyl-[l,3,2]dioxaborolan-2-yl)-furan-2-carboxylic acid amide (2.70 g, 11.40 mmol, 1.50 equiv.), PdCl2dppf.DCM (0.31Og, 0.38 mmol, 5 mol%), DiPEA (2.65 mL, 15.20 mmol, 2.00 equiv.) in 1,4-Dioxane (51 mL) and water (13 mL). The system is sealed, purged by vacuum/N2 and heated to 1100C for 6h, at which point full conversion has occurred. The reaction mixture is diluted with DCM (60 mL) and MeOH (60 mL) and filtered on celite. The filtrate is evaporated to yield a muddy brown residue. This residue is treated with EtOH (50 mL), MeOH (25 mL) and DCM (20 mL), and evaporated to dryness, then left in vacuo at 4O0C for another 1 h to try and eliminate as much moisture and alcohols as possible. The dry residue is suspended in DCM (100 mL) and sonicated for about 1 h, to disperse all the solid bits. A suspension of fine solid is obtained. It is cooled to 00C, filtered on Buchner, and the solid was washed with DCM (30 mL) and dried under vacuum.

[00328] The residue is treated with IM KOH (40 mL), sonicated until the solid is well dispersed, and filtered on a sintered glass funnel. Finally, the solid is dissolved m DCM (450 mL) and MeOH (50 mL), washed with a mixture of saturated aqueous NaF (250 mL), water (500 mL) and iPrOH (250 mL). The organic layer was dried on anhydrous Na2SO4, filtered and evaporated in vacuo to yield 4-{8-[4-((lS,4S)-5-Isopropyl-2,5-diaza-bicyclo[2.2.1]hept-2-yl)-phenylamino]-[l,2,4]triazolo[l,5-a]pyrazin-5-yl} -furan-2-carboxylic acid amide (2.34 g) as a yellow-brown solid.

1H NMR (400 MHz, OMSO-d6): δ 9.76 (s, 1 H); 8.74 (s, 1 H); 8.69 (d. 1 H); 8.13 (s, 1 H); 7.93 (broad s, 1 H); 7.86 (d, 1 H); 7.72 (d, 2 H); 7.54 (broad s, 1 H); 6.59 (d, 2 H); 4.31 (d, 1 H (iPrOH)); 4.25 (s, 1.1 H); 3.78 (m, 1 H (iPrOH)); 3.69 (s, 1 H); 3.30 (H2O); 3.16 (d(d), 1 H); 3.00 (d(d), 1 H); 2.50 (DMSO); 2.42-2.37 (m, 2 H); 1 81 (s, 2 H); 1.04 (d, 7 H) (i-PrOH); 0.95 (2 d, 6 H)

Compound 3: 4-{8-[4-((lS,4S)-5-tert-Butyl-2,5-diaza-bicyclo[2.2.1]hept-2-yl)-phenylamino]-[l,2,4]triazolo[l,5-a]pyrazin-5-yl}-furan-2-carboxamide


[00329] This compound may be prepared according to the same procedure as described for

Compound 2 above using the corresponding intermediates described above.

1H NMR (400 MHz, OMSO-d6): δ 9.27 (s, IH), 8.72 (s, IH), 8.68 (d, IH), 8.11 (s, IH), 7.91 (broad s, IH), 7.84 (d, IH), 7.68 (d, 2H), 7.58 (broad s, IH), 6.55 (d, 2H), 3.40-3.36 (m, 2H), 2.89-2.80 (m, 4H), 1.72-1.70 (d, IH), 1.63-1.60 (d, IH), 0.97 (s, 9H); m/z: M+H+ (473.1; 100%).

Compound 4 : 4-{8- [4-((lS,4S)-5-Isopropyl-2,5-diaza-bicyclo [2.2.1 ] hept-2-yl)-phenyl amino]-imidazo[l,2-a]pyrazin-5-yl}-furan-2-carboxamide


[00330] This compound may be prepared according to the same procedure as described for

Compound 2 using the corresponding intermediates described above.

[00331] H NMR (400 MHz, DMSO-J6): δ 9.33 (s, IH), 8.45 (s, IH), 8.17 (s, IH), 7.94 (brs,

IH), 7.75-7.72 (m, 3H), 7.62 (d, 2H), 7.59 (brs, IH), 6.58 (d, 2H), 4.24 (s, IH), 3.69 (s, IH), 3.32 (d, IH), 3.15 (d, IH), 2.98 (d, IH), 2.43-2.38 (m, 2H), 1.81 (s, 2H), 0.98 (d, 3H), 0.92 (d, 3H); m/z: M+H+ (458; 100%).

Compound 5 : 5-{8-(4-((lS,4S)-5-isopropyl-2,5-diazabicyclo [2.2.1 ] heptan-2-yl)phenyl-amino)-[l,2,4]triazolo[l,5-a]pyrazin-5-yl}-lH-pyrazole-3- carboxamide

Step 1: Ethyl 5-(8-(4-((lS,4S)-5-ιsopropyl-2,5-dιazabιcyclo[2.2.1]heptan-2-yl)phenylamιno)-[1, 2, 4]tnazolo[l, 5-a]pyrazin-5-yl)-lH-pyrazole-3-carboxylate


[00332] A mixture of ethyl 5-(tributylstannyl)-lH-pyrazole-3-carboxylate (109.0 mg, 0.25 mmol)

(Heterocyles, 1992, 813-818), (5-bromo-[l,2,4]triazolo[l,5-a]pyrazin-8-yl)-[4-((lS,4S)-5-isopropyl-2,5-diazabicyclo[2.2.1]hept-2-yl)-phenyl]amine (163.0 mg, 0 38 mmol), and Pd(PPh3)2Cl2 (27 0 mg, 0 04 mmol) in THF (3 mL) is refluxed for 18 hours. After return to room temperature, solvent is removed under reduced pressure Purification of the residue by silica gel column chromatography eluting with a mixture of DCM/7N NH3 in methanol (97/3) affords the title compound (35.0 mg, 28 %).

Step 2: 5-{8-(4-((lS, 4S)-5-ιsopropyl-2, 5-dιazabιcyclo[2.2. l]heptan-2-yl)phenylamιno)-[l, 2, 4J-triazolofl, 5-a]pyrazιn-5-yl}-lH-pyrazole-3- carboxamide


H2N [00333] A mixture of the compound obtained in the previous step (30.0 mg, 0.06 mmol), ammonium chloride (200 mg) and ammonium hydroxide (2 mL) in methanol (12 mL) is heated at 85 0C for 18 hours. After return to room temperature, solvents are removed under reduced pressure. Purification by silica gel column chromatography eluting with a mixture of DCM/7N NH3 in methanol (95/5) affords the title compound (20.0 mg, 71 %).

NMR 1H (400 MHz, OMSO-d6): δ 14.01 (broad s, IH); 9.83 (s, IH); 8.73 (s, IH); 8.23 (s, IH); 8.14 (broad s, IH); 7.78-7.75 (m, 3H); 7.32 (broad s, IH); 6.64-6.62 (m, 2H); 4.65 (s, IH); 4.35 (s, IH); 3.21-3.19 (m, IH); 3.04-3.02 (m, IH); 2.54 (s, IH); 2.45-2.41 (m, 2H); 1.84-1.82 (m, 2H); 1.01 (2d, 6H); m/z: 459 (M+H)+.

Compound 6: 4-{8-(4-((lS,4S)-5-tert-butyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)-phenyl-amino)imidazo[l,2-a]pyrazin-5-yl}-furan-2-carboxamide


[00334] This compound may be prepared according to the same procedure as described for

Compound 2 using the corresponding intermediates described above.

1H NMR (400 MHz, OMSO-d6): δ 9.33 (s, IH); 8.46 (s, IH); 8.11 (s, IH); 7.99 (broad s, IH); 7.72-7.70 (m, 3H); 7.62-7.59 (m, 3H); 6.56-6.54 (m, 2H); 4.28 (s, IH); 4.67 (s, IH); 3.91-3.76 (m, 4H); 1.73 (d, IH); 1.65 (d, IH); 0.98 (s, 9H); m/z: 472 (M+H)+.

Compound 7: 4-{8-(6-((lS,4S)-5-isopropyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)pyridin-3-ylamino)-[l,2,4]triazolo[l,5-a]pyrazin-5-yl}-furan-2-carboxamide

[00335] This compound may be prepared according to the same procedure as described for

Compound 2 using the corresponding intermediates described above.

1H NMR (400 MHz, DMSO-J6): δ 9.94 (s, IH); 8.80 (s, IH); 8.74 (s, IH); 8.58 (s, IH); 8.17 (s, IH); 8.03-7.99 (m, 2H), 7.90 (s, IH); 7.59 (broad s, IH); 6.58 (d, IH); 4.58 (s, IH); 3.76 (s, IH), 3.49 (d, IH); 3.31-3.29 (m, IH); 3.06 (d, IH); 2.51-2.48 (m, IH); 2.39 (d, IH); 1.86-1.83 (m, 2H); 0.98 (2d, 6H); m/z: 460 (M+H)+.

Compound 8: 4-{8-(4-((lS,4S)-5-isopropyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)phenyl-amino)imidazo[l,2-a]pyrazin-5-yl}-lH-pyridin-2-one

Step 1: 5-(2-ethoxypyndin-4-yl)-N-(4-((lS,4S)-5-isopropyl-2,5-diazabicyclo[2.2.1]heptan-2-yl) phenyl) ιmιdazo[ 1 ,2-a] 'pyrazιn-8-amιne


[00336] This compound may be prepared according to the same procedure as described for

Compound 2 using the corresponding intermediates described above.

Step 2: 4-{8-(4-((l S, 4S)-5-ιsopropyl-2, 5-dιazabιcyclo[2.2. l]heptan-2-yl)pheny-amιno)ιmιdazo-[l, 2-aJ-pyrazιn-5-yl}-lH-pyndιn-2-one


[00337] A mixture of the compound obtained in the previous step (135.0 mg, 0.29 mmol) and pyridine hydrochloride (332.0 mg, 2.90 mmol) is heated to 120 0C for 18 hours. After return to room temperature, the crude is dissolved in methanol, dry loaded onto silica. Purification by silica gel column chromatography eluting with a mixture of DCM/7N NH3 in methanol (96/4) affords the title compound (46.0 mg, 36%).

NMR 1H (400 MHz, CDCl3): δ 12.51 (broad s, IH); 8.00 (s, IH); 7.82 (s, IH); 7.64-7.59 (m, 4H); 7.50-7.48 (m, IH); 6.86 (s, IH); 6.60-6.59 (m, 2H); 6.55-6.53 (m, IH); 4.20 (s, IH); 3.81 (s, IH); 3.49-3.41 (m, 2H); 3.21-3.18 (m, IH); 2.51-2.49 9m, 2H); 2.05-2.03 (m, IH); 1.97-1.95 (m, IH); 1.07 (d, 3H); 1.02 (d, 3H); m/z: 442 (M+H)+.

Compound 9: 4-{8-(4-(5-isopropyl-2,5-diazabicyclo[2.2.2]octan-2-yl)phenylamino)[l,2,4]-triazolo[l,5-a]pyrazin-5-yl}-furan-2-carboxamide


[00338] This compound is prepared according to the same procedure as described for Compound

2 using the corresponding intermediates described above.

1H NMR (400 MHz, DMSO-J6): δ 9.80 (s, IH); 8.78 (s, IH); 8.73 (s, IH); 8.17 (s, IH); 7.98 (broad s, IH); 7.89 (s, IH); 7.76 (d, 2H); 7.59 (broad s, IH); 6.68 (d, 2H); 3.92-3.91 (m, IH); 3.58 (d, IH); 3.22- 3.19 (m, 2H); 2.96-2.94 (m, 2H); 2.71-2.68 (m, IH); 1.94-1.76 (m, 3H); 1.60-1.59 (m, IH); 1.04 (2d, 6H); m/z: 473 (M+H)+.

Compound 10: 4-{8-(4-(5-isopropyl-2,5-diazabicyclo[2.2.2]octan-2-yl)phenylamino)-iniidazo[l,2-a]pyrazin-5-yl}-furan-2-carboxamide


[00339] This compound is prepared according to the same procedure as described for Compound

2 using the corresponding intermediates described above.

1H NMR (400 MHz, OMSO-d6): δ 9.23 (s, IH); 8.38 (s, IH); 8.02 (s, IH); 7.86 (broad s, IH); 7.68-7.64 (m, 3H); 7.55-7.52 (m, 2H); 7.47 (broad s, IH); 6.59 (m, 2H); 3.79 (s, IH); 3.48-3.45 (m, IH); 3.11-3.08 (m, 2H); 2.84-2.82 (m, 2H); 2.59-2.57 (m, IH); 1.83-1.74 (m, 3H); 1.67-1.65 (m, IH); 0.94 (2d, 6H); m/z: 472 (M+H)+.

Compound 11: 4-{8-(4-(3-isopropyl-3,8-diazabicyclo[3.2.1]octan-8-yl)phenylamino)-imidazo[l,2-a]pyrazin-5-yl}-furan-2-carboxamide


[00340] This compound is prepared according to the same procedure as described for Compound

2 using the corresponding intermediates described above.

1H NMR (400 MHz, DMSO-J6): δ 9.29 (s, IH); 8.38 (s, IH); 8.03 (s, IH); 7.86 (broad s, IH); 7.72 (d, 2H); 7.69 (s, IH); 7.55 (s, IH); 7.54 (s, IH); 7.47 (broad s, IH); 6.73 (d, 2H); 4.13-4.11 (m, 2H); 2.47-2.36 (m, 5H); 1.78-1.73 (m, 4H); 0.84 (2d, 6H); m/z: 472 (M+H)+.

Compound 12: 4-{8-(4-(8-isopropyl-3,8-diazabicyclo[3.2.1]octan-3-yl)phenylamino)[l,2,4]-triazolo[l,5-a]pyrazin-5-yl}-furan-2-carboxamide


[00341] This compound is prepared according to the same procedure as described for Compound

2 using the corresponding intermediates described above.

1H NMR (400 MHz, OMSO-d6): δ 9.79 (s, IH); 8.69 (s, IH); 8.64 (s, IH); 8.09 (s, IH); 7.93-7.91 (broad s, IH); 7.71 (s, IH); 7.69 (d, 2H); 7.53-7.51 (broad s, IH); 6.74 (d, 2H); 4.07-4.04 (m, IH); 3.51-5.48 (m,

2H); 3.10-3.09 (m, 2H); 2.77-2.75 (m, 2H); 1.76-1.74 (m, 2H); 1.59-1.57 (m, 2H); 0.96 (2d, 6H); m/z: 473

(M+H)+.

Compound 13: 4-{8-(4-(3-isopropyl-3,8-diazabicyclo[3.2.1]octan-8-yl)phenylamino)[l,2,4]-triazolo[l,5-a]pyrazin-5-yl}-furan-2-carboxamide

[00342] This compound is prepared according to the same procedure as described for Compound

2 using the corresponding intermediates described above.

1H NMR (400 MHz, OMSO-d6): δ 9.84 (s, IH); 8.76 (d, 2H); 8.19 (s, IH); 7.99 (broad s, IH); 7.90 (s, IH); 7.79 (d, 2H); 7.59 (broad s, IH); 6.86 (d, 2H); 4.22-4.25 (m, 2H); 2.56-2.47 (m, 5H); 1.89-1.84 (m, 4H); 0.95 (2d, 6H); m/z: 473 (M+H)+.

Compound 14 : 4-{8-(4-(8-isopropyl-3,8-diazabicyclo[3.2.1]octan-3-yl)phenylamino)-imidazo[l,2-a]pyrazin-5-yl}-furan-2-carboxamide


[00343] This compound is prepared according to the same procedure as described for Compound

2 using the corresponding intermediates described above.

1H NMR (400 MHz, OMSO-d6): δ 9.42 (s, IH); 8.47 (s, IH); 8.11 (s, IH); 7.96 (broad s, IH); 7.81-7.79 (m, 2H); 7.73 (s, IH); 7.63-7.61 (m, 2H); 7.57 (broad s, IH); 6.80-6.78 (m, 2H); 3.55-3.53 (m, 2H); 3.34-3.31 (m, 2H); 2.83-2.80 (m, 2H); 2.53-2.51 (m, IH); 1.82-1.81 (m, 2H); 1.66-1.64 (m, 2H); 1.04 (2d, 6H); m/z: 472 (M+H)+.

Compound 15: S-JS-Cό-^lS^SJ-S-isopropyl^S-diazabicycloP^.llheptan^-ylJpyridin-S-ylamino)-[l,2,4]triazolo[l,5-a]pyrazin-5-yl}-lH-pyrazole-3- carboxylic acid amide

[00344] This compound is prepared according to the same procedure as described for Compound

2 using the corresponding intermediates described above.

1H NMR (400 MHz, OMSO-d6): δ 14.04 (broad s, IH); 10.06 (s, IH); 8.79 (s, IH); 8.68 (s, IH); 8.22 (s, IH); 8.04-8.02 (m, 2H); 7.74 (broad s, IH); 7.69 (broad s, IH); 6.59-6.57 (m, IH); 4.58 (s, IH); 3.76 (s, IH); 3.49-3.42 (m, IH); 3.35-3.31 (m, IH); 3.08-3.06 (m, IH); 2.49-2.42 (m, 2H); 1.84-1.83 (m, 2H); 0.98 (d, 3H); 0.79 (d, 3H); m/z: 460 (M+H)+.

Compound 16: 5-{8-(4-((lS,4S)-5-tert-butyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)phenylamino) [l,2,4]triazolo-[l,5-a]pyrazin-5-yl}-2,3-dihydro-isoindol-l-one


[00345] This compound is prepared according to the same procedure as described for Compound

2 using the corresponding intermediates described above.

1H NMR (400 MHz, CDCl3): δ 8.38 (s, IH); 8.09 (s, IH); 8.02-7.95 (m, 2H); 7.84 (s, IH); 7.73 (s, IH); 7.52 (d, 2H); 6.62 (d, 2H); 6.25 (broad s, IH); 4.56 (s, 2H); 4.27 (s, IH); 3.71 (s, IH); 3.50-3.48 (m, IH); 3.15-3.10 (m, 2H); 2.88-2.86 (m, IH); 1.83-1.81 (m, 2H); 1.05 (s, 9H); m/z: 495 (M+H)+.

Compound 17: 5-{8-(4-((lS,4S)-5-tert-butyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)phenyl-amino)-[l,2,4]triazolo[l,5-a]pyrazin-5-yl}-lH-pyrazole-3- carboxylic acid amide


[00346] This compound is prepared according to the same procedure as described for Compound

2 using the corresponding intermediates described above.

1H NMR (400 MHz, OMSO-d6): δ 13.82 (broad s, IH); 9.70 (s, IH); 8.66 (s, IH); 8.11 (s, IH); 7.65 (broad s, IH); 7.65-7.62 (m, 3H); 7.37 (broad s, IH); 6.51-6.49 (m, 2H); 4.24 (s, IH); 3.62 (s, IH); 3.36-3.35 (m, IH); 2.83-2.77 (m, 3H); 1.64-1.57 (m, 2H); 0.92 (s, 9H); m/z: 473 (M+H)+.

Compound 18: S-JS^δ-^lS^SJ-S-isopropyl^^-diazabicyclo^^.llheptan^-y^pyridin-S-ylamino)-[l,2,4]triazolo[l,5-a]pyrazin-5-yl}-2,3-dihydro-isoindol-l-one


[00347] This compound is prepared according to the same procedure as described for Compound

2 using the corresponding intermediates described above.

1H NMR (400 MHz, OMSO-d6): δ 9.89 (s, IH); 8.61 (s, IH); 8.59 (broad s, IH); 8.49-8.47 (m, IH); 8.11 (s, IH); 7.99-7.95 (m, 2H); 7.85 (s, IH); 7.74-7.72 (m, IH); 6.49-6.47 (m, IH); 4.48 (s, IH); 4.41 (s, 2H); 3.65 (s, IH); 3.41-3.39 (m, IH); 3.20-3.18 (m, IH); 2.97-2.95 (m, IH); 2.40-2.38 (m, IH); 2.28-2.26 (m, IH); 1.74-1.72 (m, 2H); 0.93 (d, 3H); 0.86 (d, 3H); m/z: 482 (M+H)+.

Compound 19 : 5-(2-ethoxypyridin-4-yl)-N-(4-((l S,4S)-5-isopropyl-2,5-diazabicyclo [2.2.1 ] heptan-2-yl)phenyl)-[l,2,4]triazolo[l,5-a]pyrazin-8-amine


[00348] This compound is prepared according to the same procedure as described for Compound

2 using the corresponding intermediates described above.

1H NMR (400 MHz, OMSO-d6): δ 8.37 (s, IH); 8.28 (d, IH), 7.92 (s, IH); 7.79 (s, IH); 7.61 (d, 2H),

7.42-7.38 (m, 2H), 6.63 (d, 2H); 4.46-4.42 (q, 2H), 4.22 (s, IH); 3.85 (s, IH); 3.43-3.40 (m, 2H); 3.24- 3.22 (m, IH); 2.55-2.53 (m, 2H); 2.03-1.99 (m, 2H); 1.42 (t, 3H); 1.08 (d, 3H); 1.04 (d, 3H); m/z: 471

(M+H)+.

Compound 20 : 4-(8-(4-((lS,4S)-5-isopropyl-2,5-diazabicyclo [2.2.1 ] heptan-2-yl)phenylamino)-[l,2,4]triazolo[l,5-a]pyrazin-5-yl)pyridin-2(lH)-one


[00349] This compound is prepared according to the same procedure as described for Compound

2 using the corresponding intermediates described above.

NMR 1H (400 MHz, CDCl3): δ 12.99 (broad s, IH); 8.37 (s, IH); 7.95-7.93 (m, 2H); 7.63 (d, 2H); 7.49 (d, IH); 7.30 (s, IH); 6.92 (d, IH); 6.63 (d, 2H); 4.21 (s, IH); 3.82 (s, IH); 3.42-3.37 (m, 2H); 3.22-3.19 (m, IH); 2.56-2.50 (m, 2H); 2.06-2.04 (m, IH); 1.87-1.76 (m, IH); 1.07 (d, 3H); 1.03 (d, 3H); m/z: 443

(M+H)+.

Compound 21: Ethyl 5-(8-(4-((lS,4S)-5-isopropyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)phenylamino)-[l,2,4]triazolo[l,5-a]pyrazin-5-yl)-lH-pyrazole-3-carboxylate


[00350] The prepration of this compound is described in step 1 towards compound 5 using the corresponding intermediates described above.

NMR 1H (400 MHz, CDCl3): δ 8.53 (s, IH); 8.20 (s, IH); 7.79 (s, IH); 7.64 (d, 2H); 7.39 (s, IH); 6.62 (d, 2H); 4.47 (q, 2H); 4.21 (s, IH); 3.81 (s, IH); 3.41-3.36 (m, 2H); 3.20-3.18 (m, IH); 2.54-2.49 (m, 2H); 2.04-2.02 (m, IH); 1.96-1.94 (m, IH); 1.42 (t, 3H); 1.07 (d, 3H); 1.03 (d, 3H); m/z: 488 (M+H)+

Compound 22: 4-{8-(4-((lS,4S)-5-isopropyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)phenyl-amino)imidazo[l,2-a]pyrazin-5-yl}-lH-pyridin-2-one.


[00351] The prepration of this compound is described in step 1 towards compound 8 using the corresponding intermediates described above.

1H NMR (400 MHz, OMSO-d6): δ 9.50 (s, IH); 8.33 (d, IH); 8.07 (s, IH); 7.78-7.70 (m, 2H); 7.57 (s, IH); 7.33 (d, 2H); 7.10 (s, IH); 6.62 (d, 2H); 4.42 (q, 2H); 4.27 (s, IH), 3.68 (s, IH), 3.34 (s, IH), 3.18- 3 16 (m, IH), 3 01-2 98 (m, IH), 2 42-2 96 (m, 2H), 1 84 (s, 2H), 1 41 (t, 3H); 1 04 (d, 3H), 0 96 (d, 3H); m/z 470 (M+H)+

Compound 23 : 3-(8-(4-((lS,4S)-5-isopropyl-2,5-diazabicyclo [2.2.1 ] heptan-2-yl)phenylamino)-[l,2,4]triazolo[l,5-a]pyrazin-5-yl)-l,2,4-oxadiazole-5-carboxamide

Step 1 8-(4-((1 S, 4S)-5-ιsopropyl-2, 5-dιazabιcyclo[2 2 1]heptan-2-yl)phenylammo)-[1, 2, 4]trιazolo-[1 , 5-a]pyrazιne-5-carbonιtπle


[00352] A sealed tube is charged with (5-bromo-[l,2,4]triazolo[l,5-α]pyrazin-8-yl)-[4-((lS,4S)-5-isopropyl-2,5-diazabicyclo[2 2 l]hept-2-yl)phenylamine (compound 2, stepl) (0 30 g, 0 70 mmol), potassium ferrocyanide (0 13 g, 0.35 mmol), sodium carbonate (0.037 g, 0 35 mmol), potassium iodide (0.058 g, 0.35 mmol), copper(II) tetrafluoroborate hydrate (0.36 g, 1 05 mmol), DMA (5 niL) and N, N-dimethylethylenediamine (340 μL, 3 15 mmol) The reaction mixture is heated at 85 °C during 18 hours After return to room temperature, the reaction is partitioned between ethyl acetate and water Aqueous phase is extracted twice with ethyl acetate Combined organic phases are washed with water, dried over MgSO4, filtered and evaporated Purification of the residue by silica gel column chromatography eluting with a mixture of DCM/7Ν NH3 in methanol (99/1) affords the title compound

Step 2 S-tδ-μ-tfiSASj-δ-isopropyl^δ-diazabicycloP 2 1]heptan-2-yl)phenylamιno)-[1 ,2,4]trιazolo[1 ,5-a]pyrazιn-5-yl)-1 , 2, 4-oxadιazole-5-carboxamιde

[00353] A mixture of the compound obtained in the previous step (26.0 mg, 0.07 mmol) , DIPEA

(12 μL, 0.07 mmol) and hydroxylamine hydrochloride (5 mg, 0.07 mmol) in ethanol (300 μL) is heated at 85 0C during 18 hours. After return to room temperature, solvent is evaporated to dryness and the residue is dissolved in pyridine (400 μL). The solution is cooled down to 0 0C and ethyl oxalyl chloride (24 μL, 0.21 mmol) is added then the reaction is heated to 70 0C for 2 hours. After return to room temperature, iced water is added and left to stir for 30 min. DCM (5 mL) is added and the aqueous phase is extracted twice. Combined organic phases are dried over MgSO4, filtered and evaporated. Purification of the residue by silica gel column chromatography eluting with a mixture of DCM/7N NH3 in methanol (99/1 to 98/2) affords the title compound.

NMR 1H (400 MHz, CDCl3): δ 8.57 (s, IH); 8.51 (s, IH); 8.11 (s, IH); 7.64 (d, 2H); 7.10 (s, IH); 6.63 (d, 2H); 6.08 (s, IH); 4.22 (s, IH); 3.83 (s, IH); 3.42-3.39 (m, 2H); 3.22-3.19 (m, IH); 2.54-2.51 (m, 2H); 2.04-2.02 (m, IH); 1.95-1.90 (m, IH); 1.08 (d, 3H); 1.03 (d, 3H); m/z: 461 (M+H)+.

Compound 24: 4-(8-(3-((l£,4^-5-isopropyl-2,5-diazabicyclo[2.2.1]heptan-2-y])phenyl amino)-[l,2,4]triazolo[l,5-α]pyrazin-5-yl)furan-2-carboxamide


[00354] This compound was prepared according to the same procedure as described for

Compound 2 using the corresponding intermediates described above.

NMR δ 1H (400 MHz, DMSO-dδ): 9.71 (s, IH), 8.72 (s, IH), 8.65 (s, IH), 8.17 (s, IH), 7.92 (brs, IH), 7.73 (s, IH), 7.51 (brs, IH), 7.32 (d, IH), 7.29 (s, IH), 7.08 (m, IH), 6.27 (d, IH), 4.18 (s, IH), 3.62 (s, IH), 3.24 (d, IH), 3.16 (d, IH), 2.98 (d, IH), 2.44-2.39 (m, 2H), 1.76 (s, 2H), 0.92 (d, 3H), 0.89 (d, 3H);

Compound 25: 4-(8-(3-((lιSr,4S)-5-isopropyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)phenylamino)imidazo[l,2-α]pyrazin-5-yl)furan-2-carboxamide


[00355] This compound was prepared according to the same procedure as described for

Compound 2 using the corresponding intermediates described above.

NMR δ 1H (400 MHz, DMSOd6): 9.23 (s, IH), 8.42 (s, IH), 8.07 (s, IH), 7.87 (brs, IH), 7.69 (s, IH), 7.61 (s, IH), 7.58 (s, IH), 7.48 (brs, IH), 7.38 (m, IH), 7.21 (s, IH), 7.02 (t, IH), 6.21 (d, IH), 4.15 (s, IH), 3.64 (s, IH), 3.32 (d, IH), 3.14 (d, IH), 2.95 (d, IH), 2.36-2.32 (m, 2H), 1.75 (s, 2H), 0.92 (d, 3H), 0.85 (d, 3H);

Compound 26: S-CS-β-^lS^S^-S-isoprOpyl^S-diazabicyclo [2.2.1 ] heptan-2-yl)phenylamino)-[l,2,4]triazolo[l,5-α]pyrazin-5-yl)isoindolin-l-one


[00356] This compound was prepared according to the same procedure as described for

Compound 2 above using the corresponding intermediates described above.

NMR δ 1H (400 MHz, DMSO-d6): 9.87 (s, IH), 8.71 (m, 2H); 8.24 (m, IH), 8.12 (m, IH), 8.01 (m, IH), 7.81 (m, IH), 7.43 (m, IH), 7.24 (m, IH), 7.12 (m, IH), 6.37 (m, IH); 4.50 (s, 2H); 4.25 (s, IH); 3.74 (s, IH); 3.44 (m, IH), 3.21 (m, IH); 3.03 (m, IH); 2.43-2.46 (m, 2H); 1.85 (s, 2H); 0.96 (d, 6H);

Compound 27: 4-[8-({4-[(lR,4R)-5-isopropyl-2,5-diazabicyclo[2.2.1]hept-2-yl]phenyl}amino)[l,2,4]triazolo[l,5-a]pyrazin-5-yl]-2-furamide

2-MeTHF

H2 Pd /C

[5-(Amιnocaronyl)- 3-furyl]boronιc acid

Compound 27

Pd(dppf)CI2.CH2CI2 Et3N, 2-MeTHF, H2O, heat


Step 1 - (lR,4R)-2-Isopropyl-5-(4-nιtrophenyl)-2,5-dιazabιcyclo[2.2.1]heptane (3)

[00357] To a mixture of (lR,4R)-5-Isopropyl-2,5-diazabicyclo[2.2 l]heptane dihydrobromide (2);

3.0 g, 9.9 mmol), 4-chloronitrobenzene (1.7 g, 11 mmol), dimethylsulfoxide (6.2 mL), and tap water (2.5 mL) is added solid K2CO3 (1.7 g; mmol; gas evolution). The resulting suspension is heated to 500C, after which 2-MeTHF (0.5 mL) and more solid K2CO3 (1.9 g; mmol) are added. Reaction temperature is increased to 125°C and the reactor contents are held at this temperature overnight. The reaction mixture is cooled down to ambient temperature, after which tap water (25 mL) is added. Extraction of the aqueous mixture with ethyl acetate (3x30 mL) and concentration of the combined organic extracts in vacuo give a solid residue, which is redissolved in hot MTBE (300 mL). The hot solution is filtered to remove residual solids and concentrated in vacuo to furnish crude (2). Purification is accomplished by partitioning the crude material between ethyl acetate (70 mL) and dilute hydrochloric acid (pH 1 ; 250 mL), separating the layers, washing of the aqueous phase with ethyl acetate (2x50 and 2x100 mL), extraction of the combined organic layers with dilute hydrochloric acid (pH 1; 100 mL), basification of the combined aqueous layers with IO N aqueous NaOH to pH 10, extraction of the alkaline aqueous layer with MTBE (2x200 mL) and ethyl acetate (2x200 mL), drying over Na2SO/!, and concentration in vacuo to give a white solid. This solid is reslurried in heptane/MTBE 1.1 v/v (20 mL), the resulting suspension filtered and the filter cake washed with heptane/MTBE 1 : 1 v/v (20 mL) and air dried to give (3) as a white solid. LC-purity: 99.3 area-%.

Step 2: 4-[(IR, 4R)-5-Isopropyl-2,5-dιazabιcyclo[2.2. lJhept-2-ylj 'aniline (4)

[00358] A solution of 3 (1.6 g; 6.1 mmol) in 2-MeTHF (25 mL) is stirred under a 1 bar hydrogen atmosphere in the presence of Pd/C catalyst (10% Degussa type ElOl NE/W; 0.1 g) at 3O0C for a period of 3 h. The catalyst is filtered off over a bed of Dicalite 478 and the filter cake washed with 2-MeTHF (2x10 mL). The filtrate is concentrated in vacuo to a volume of 25 mL and the resulting solution is used as such in the next step.

Step 3: 5-Bromo-N-{4-[(lR, 4R)-5-ιsopropyl-2, 5-dιazabιcyclo[2.2. lJhept-2-yl] phenyl} [1,2, 4]trιazolo[l, 5-a] -pyrazιn-8-amιne (5)

[00359] To the solution obtained from step 2 is added 5,8-dibromo[l,2,4]triazolo[l,5-a]-pyrazine

(1.6 g; 5.8 mmol) and tπethylamine (3.4 mL). The resulting mixture was heated at reflux for 70 h, after which the reactor contents were cooled down and filtered to remove solids. The filter cake was washed with 2-MeTHF (2x5 mL) and the filtrate used as such in the next step.

Step 4: Compound 27 4-[8-({4-[(lR,4R)-5-Isopropyl-2,5-dιazabιcyclo[2.2.1]hept-2-ylj 'phenyl} amino) [1, 2, 4]trιazolo[l, 5-a] -pyrazιn-5-yl] -2-furamιde (6)

[00360] To the solution obtained from step 3 is added 2-MeTHF (5 mL), tap water (6.5 mL), [5- (aminocarbonyl)-3-furyl]boronic acid (1.3 g; 8.6 mmol), and Pd(dppf)2Cl2 (0.26 g; 0.3 mmol). The resulting mixture is degassed 5 times by means of a vacuum/nitrogen purge cycle and heated at 800C for 6h. The reactor contents are then cooled down to ambient temperature, 1,2-diamino-propane (2 mL) is added, the resulting suspension filtered (slow!), and the filter cake washed with 2-MeTHF (6x5 mL) to obtain crude compound 27 as a green solid. The crude material is reslurried in methanol (15 mL), filtered, and the filter cake washed with methanol (5 mL). The filter cake is then mixed with water/acetic acid (pH 1; approx. 50 mL), the resulting suspension filtered until a clear filtrate is obtained, and the filter cake washed with water until the washing liquid turned colorless. The combined filtrate and washing liquids are concentrated in vacuo at 500C to remove water, the residue stripped with 2-propanol (twice) and toluene (three times) and subsequently taken up in methanol (70 mL) and toluene (5 mL). To the resulting suspension is added 1,2-diaminopropane (2 mL) and dppe (0.08 g; 0.2 mmol), after which stirring is continued overnight. The purified product is isolated by filtration, washing of the filter cake with methanol until the washing liquid turns pale yellow, followed by washing with ethyl acetate, and dried at

400C in vacuo to compound 27 as a yellow solid.

LC-purity 98.6 area-%.

LC-MS: m/z = 459 (100) [M+H]+.

Purification Conditions and Characterization

[00361] Routinely, post-synthesis all compounds may be purified using reverse phase HPLC using a Gilson preparative HPLC system (322 pump, 155 UV/VIS detector, 215 liquid handler). The Gilson 215 acts as both auto-sampler and fraction collector. Compounds can also be purified by flash chromatography on silica gel.

[00362] Compounds are characterised by mass spectrometry using single quadrupole instrumentation with an electrospray source.

Biological Examples Example 1 : MAPKAP-K5 Assay

[00363] MAPKAP-K5 reactions are performed in FlashPlate format using 0.1 or 0.2 μCi 33P- ATP; 0.6μM ATP; ImU MAPKAP-K5 ; 3μM MAPKAP-K5 peptide substrate, incubated at room temperature for 30 minutes. Flashplate assay:

[00364] The MAPKAP-K5 kinase reaction is performed in a 384 well polypropylene plate (Matrix

Technologies) and then transferred to a streptavidin-coated 384 well flashplate (Perkin-Elmer). To wells containing 2μL test compound or standard inhibitor, 13μL Enzyme mix or diluent are added using a Hydra (Robbins Scientific). Reactions are started by addition of lOμL of [2.5x] substrate cocktail using a Multidrop (Thermo-Labsystems), to give final concentrations in the assay of: lmU MAPKAP-K5

3μM MAPKAP-K5 peptide substrate

0.6μM ATP

0.004μCi [33P]-γ-ATP/μL

Ix reaction buffer

[00365] Plates are incubated at room temperature for 30 minutes. Reactions are terminated by the addition of 25μL EDTA (5OmM) to each well using a Micro-fill (Biotek). Reactions are transferred to a streptavidin-coated flashplate using a Zymark robotic system. Plates are incubated for 60 minutes at room temperature. All wells are washed 3 times with lOOμl phosphate buffered saline using a Tecan plate washer. Radioactivity is determined by scintillation counting of the flashplate (empty wells) on a Packard TopCount.

Enzyme Mix:

Enzyme 5OmM Tris HcI (pH 7.5) 0. ImM EGTA 2mM DTT lmg/mL BSA Reaction Buffer:

5OmM Tris HcI (pH 7.5)

0. ImM EGTA

1 OmM Magnesium acetate

2mM DTT

Example 2. Development of an assay for the identification of regulators of the expression of MMPl by activated primary synovial fibroblasts.

[00366] To identify compounds that decrease the ECM-degrading activity of cells, the ECM-degrading activity of cells may be induced to allow proper detection of this activity, and to achieve a clearer read-out. In the context of RA, the cells of choice are mammalian synovial fibroblasts and the triggers that may be used to induce the ECM-degrading activity are cytokines relevant in the field of arthritis: for instance TNF-α, ILlB, IL6, OSM, ILl 7, and MIFl-α. This list is not comprehensive due to the plethora of cytokines potentially involved in the RA pathogenesis (Smolen and Steiner, 2003). To set up an in vitro assay that is as close as possible to the complexity of the pathology, the trigger applied should be a mixture of factors generated by contacting cytokine-producing cells relevant in the field of arthritis, such as monocytes, macrophages, T-cells, and B-cells, with a trigger. The cytokine-producing cells will respond to the contact by producing a complex and unbiased mixture of factors. If the cytokine-producing cell used is also found in a pannus, and the cytokine applied to produce this trigger is found in the synovial fluid of rheumatoid arthritis patients, the mixture of factors ultimately produced will contain part of the factors that are present in the joints of arthritis patients. Principle of the 'MMP assay'

[00367] Matrix Metallo Proteases (MMPs) possess various physiological roles, as e.g. the maturation of other proteases, growth factors, and the degradation of extra-cellular matrix components. MMPl is one of the members of the MMP family that is able to degrade native collagen, the main component of bone and cartilage. An increased expression of MMPl by synovial fibroblasts (SFs) is diagnostic for the progression of the arthritic disease and is predictive for erosive processes in the joint (Cunnane et al., 2001). The expression of MMPl by SFs can be increased by the activation of SFs with triggers relevant for rheumatoid arthritis, as cytokines like TNF-α or ILlB (Andreakos et al., 2003). Taken together, measurement of the levels of MMPl produced by activated SFs is a readout that is highly relevant in the context of RA as this event reflects the level of activation of SFs towards an erosive phenotype as it is seen in the pannus. If a reduced expression of a candidate drug target in activated SFs leads to the reduction of MMPl expression by these cells, the drug target is then proven to be involved in the regulation of MMPl expression and thus considered relevant for the development of therapeutic strategies for the treatment of RA.

[00368] In the following examples, the development of an assay, further referred to as 'MMP assay', monitors the MMPl production by synovial fibroblasts (SFs) in response to diverse activating triggers (Example 2.1). The use of this assay is then described for the validation of gene products that are considered drug targets for the development of RA therapies (Example 2.2). The validation of drug targets is performed using recombinant adenoviruses, further referred to as knock-down viruses or Ad-siRNAs, that mediate the expression in cells of shRNA's which reduce the expression levels of targeted genes by a RNAi (RNA interference)-based mechanism (see WO 03/020931). The identification of compounds modulating the activity of the validated drug targets is then described in Table 3. The use of the 'MMP assay' for the testing of compounds that modulate the activity of the drug targets identified is described further below.

Assay Examples

Control viruses used:

[00369] The control viruses used in these studies are listed below. dEl/dE2A adenoviruses are generated from these adapter plasmids by co-transfection of the helper plasmid pWEAd5AflII-rITR.dE2A in PER.E2A packaging cells, as described in WO99/64582. Negative control viruses:

Ad5-eGFP_KD: Target sequence: GCTGACCCTGAAGTTCATC (SEQ ID NO: 1). Cloned using Sapl-sites into vector and virus generated as described in WO03/020931.

Ad5-Luc vl3 KD: Target sequence GGTTACCTAAGGGTGTGGC (SEQ ID NO: 2). Cloned using Sap 1 -sites into vector and virus generated as described in WO03/020931.

Ad5-M6PR_vl_KD: Target sequence CTCTGAGTGCAGTGAAATC (SEQ ID NO: 3). Cloned using Sap 1 -sites into vector and virus generated as described in WO03/020931.

Positive control viruses:

Ad5-MMPl_vlO_KD: Target sequence ACAAGAGCAAGATGTGGAC (SEQ ID NO: 4). Cloned using Sap 1 -sites into vector and virus generated as described in WO03/020931.

Viruses used for target validation:

Ad5-MAPKAPK5 vl3 KD: Target sequence CGGCACTTTACAGAGAAGC (SEQ ID NO: 5). Cloned using Sapl-sites into vector and virus generated as described in WO03/020931.

Ad5-MAPKAPK5_vl2_KD: Target sequence ATGATGTGTGCCACACACC (SEQ ID NO: 6). Cloned using Sapl-sites into vector and virus generated as described in WO03/020931.

Example 2.1: Development of the MMP assay

[00370] A 384-well format ELISA for measurement of MMPl is developed. Various primary antibodies are tested, as well as various ELISA protocols. The following protocol is developed and validated to measure MMPl levels in SF supernatant in 384 well plates: white Lumitrac 600 384 well plates (Greiner) are coated with 2 μg/mL anti-MMPl antibody MAB 1346 (Chemicon). The antibody is diluted in buffer 40 (1.21 g Tris base (Sigma), 0.58 g NaCl (Calbiochem) and 5 ml 10% NaN3 (Sigma) in

1 L milliQ water and adjusted to pH 8.5). After overnight incubation at 4°C, plates are washed with PBS (80 g NaCl, 2g KCl (Sigma), 11.5 g Na2HPO4.7H2O and 2 g KH2PO4 in 10 L milliQ; pH 7.4) and blocked with 100 μL/well Casein buffer (2% Casein (VWR International) in PBS). Next day, casein buffer is removed from ELISA plates and replaced by 50 μL/well EC buffer (4 g casein, 2.13 g Na2HPO4 (Sigma),

2 g bovine albumin (Sigma), 0.69 g NaH2PO4-H2O (Sigma), 0.5 g CHAPS (Roche), 23.3 g NaCl, 4 ml 0,5 M EDTA pH 8 (Invitrogen), 5 ml 10% NaN3 in 1 L milliQ and adjusted to pH 7.0). 0.25 mM DTT (Sigma) is added to the thawed samples plates. After removal of the EC buffer, 20 μL of sample is transferred to the ELISA plates. After overnight incubation at 4°C plates are washed twice with PBS and once with PBST (PBS with 0.05% Tween-20 (Sigma)) and incubated with 35 μL/well biotinylated anti-MMPl antibody solution (R&D). This secondary antibody is diluted in buffer C (0.82 g NaH2PO4-H2O, 4.82 g Na2HPO4, 46.6 g NaCl, 20 g bovine albumin and 4 mL 0,5M EDTA pH 8 in 2 L milliQ and adjusted to pH 7.0) at a concentration of 5 μg/mL. After 2 h of incubation at RT, plates are washed as described above and incubated with 50 μL/well streptavidin-HRP conjugate (Biosource). Streptavidin-HRP conjugate is diluted in buffer C at a concentration of 0.25 μg/mL. After 45 min, plates are washed as described above and incubated for 5 min with 50 μL/well BM Chem ELISA Substrate (Roche). Readout is performed on the Luminoscan Ascent Luminometer (Labsystems) with an integration time of 200 msec or with an Envision reader (Perkin Elmer).

[00371] The increase of MMPl expression by SFs upon treatment with cytokines relevant in the field of RA (TNF-α, ILlB and OSM) or a combination thereof is shown in Figure 2 as white bars. For this experiment, SFs are seeded in 96 well plates, 3,000 cells/well. 24 h later, the medium is changed to Ml 99 medium supplemented with 1% FBS. One day after the medium change, cytokines or combinations thereof are added to the cultures, each cytokine being added to a final concentration of 25 ng/niL. 72 h after cytokine addition, the supernatant is collected and processed in the MMPl ELISA as described in the protocol given above. In parallel with this experiment, SFs are triggered, using the same protocol, with the supernatant of THPl cells (2-fold diluted in Ml 99 + 1% FBS) treated with the same cytokines or combinations of cytokines for 48 h in M199 medium + 1% FBS. MMPl levels for these samples are shown in Figure 2 as grey bars. The induction of the MMPl expression by SFs triggered with the supernatants of TNF-α-treated THPl cells is stronger (>4.5 fold induction) as compared to the SFs triggered with recombinant TNF-α alone (3-fold induction) and almost equals the 5-fold induction obtained by a mixture of 3 purified cytokines (TNF-α, ILlBb, OSM) This result indicates that the supernatant of TNF-α-induced THPl cells contains, besides TNF-α, additional pro-inflammatory factors that activate SFs towards MMPl expression. As the role of TNF-α in the RA pathogenesis is validated (TNF-α-blockers such as Infliximab and Etanercept show some efficacy in the treatment of RA patients) and the THP-I cells are representative for monocytes / macrophages present m the joint of RA patients, the TNF-α-based trigger mixture prepared by contacting THP-I cells with TNF-α will contain factors present in the joints of RA patients and subsequently is relevant to RA. This TNF-α-based complex trigger, further referred to as the 'complex trigger', will further be used as basis for the 'MMP assay' . [00372] Inhibition of the activation of SF by the 'complex trigger' is shown using dexamethasone, a potent anti- inflammatory agent that also strongly reduces collagen-induced arthritis in rodents (Yang et al , 2004) (Figure 3). Dexamethasone is shown to dose-dependently reduce amounts of MMPl produced by complex trigger activated SFs. SFs are seeded at a density of 3000 cells/well in 96 well plates. 24hrs after seeding, increasing concentrations of dexamethasone are added to the cells. After overnight incubation, medium of every well is refreshed to supernatant of THP-I cells treated with TNF-α (50% diluted m M199 + 0.5%FBS), and the same concentration of dexamethasone as added the day before. 48hrs after treatment, the supernatant is collected and subjected to the MMPl ELISA described above. The addition of dexamethasone clearly reduced the MMPl expression by SFs, with an IC50 value of about InM (see Figure 3). These data show that the MMPl expression by activated SFs can be reduced by the addition of a physiologically relevant inhibitor and represent a proof of principle for the 'MMP assay' .

Example 2 2. MAPKAPK5 Modulates SF 'Complex Trigger '-induced MMPl Expression (A) Ad-siRNA Virus Functions to Knock Down MAPKAPK5 Expression

[00373] Recombinant adenoviruses mediating the expression of siRNA's targeting MAPKAPK5 and eGFP are generated according to the procedure described in WO03/020931. The target sequence used in the recombinant adenovirus is CGGCACTTTACAGAGAAGC (SEQ ID NO. 5) as well as ATGATGTGTGCCACACACC (SEQ ID NO. 6). The target sequence within the eGFP mRNA used in the recombinant adenovirus is. GCTGACCCTGAAGTTCATC (SEQ ID NO 1). These sequences are cloned into the adapter plasmid using Sapl sites dEl/dE2A adenoviruses are generated from these adapter plasmids by co-transfection of the helper plasmid pWEAd5AfHI-rITR dE2A m PER E2A packaging cells, as described in WO99/64582.

[00374] The functionality of an adenovirus targeting MAPKAPK5 is tested as follows These adenoviruses are used to infect primary human SFs cultured in petri dishes as follows. On day 1, 500 000 SFs are seeded per petri dish. One day later, the cells are infected with Ad5-MAPKAPK5-vl 3 KD (1.6E9 VP/mL) or Ad5-eGFP-v5_KD (1.3E10 VP/mL) at an MOI of 4000 (based on the titers (number of virus particles per mL) defined for the viruses by Q-rt-PCR) On day 7, cells are detached from the petri dish according to standard procedure using a trypsin EDTA solution. The trypsin is then neutralized by addition of DMEM growth medium supplemented with 10%FBS. The cells are then collected by a centrifugation step (1000 rpm, 5 min). The pellet is lysed in lOOμL of fresh RIPA buffer (5OmM Tris pH7.5, 15OmM NaCl, 1% deoxycholate, 1% Triton XlOO, 0.1% SDS). The samples are then sonicated for lOsec. The protein concentration of the samples is then determined using the BCA kit (Pierce, Cat N° 23227) as described by the provider, using BSA as a standard. To 30μg of cell lysate diluted to 19.5μl in RIPA buffer, 3.5μL of reducing agent (NuPage reducing agent N0IO, Invitrogen NP0004) and 7.5μL of sample buffer (NuPage LDS sample buffer, Invitrogen NP0007) are added. The 30μL sample is then boiled for 5min and loaded on a 10% polyacrylamide gel (Invitrogen NP0301). To allow the estimation of the level of protein knock-down, 15μg, 7.5μg and 3.75μg of the lysate of the Ad5-eGFP-v5_KD infected cells are also loaded onto the gel. The gel is then run for 2 hours at 100V in Ix MOPS/SDS NuPage running buffer (Invitrogen NPOOl). lOμl of Seablue Plus Prestained standard (Invitrogen LC5925) is used to estimate protein size on the gel. The proteins on the gel are then transferred onto a PVDF membrane (Invitrogen LC2002) by a wet blotting procedure using a transfer buffer prepared by mixing 100ml Nupage Transfer buffer 20* (NP0006-1), 40OmL methanol and 150OmL Milli Q water. Before the transfer, the membrane is first soaked in methanol and in transfer buffer. The transfer is performed at 100V for 90 minutes. The membrane is then blocked by 30 min soaking in blocking buffer (2% blocking blocking powder (Amersham, RPN 2109) prepared in PBST (PBS supplemented with 0,1% Tween 20 (Sigma, Pl 379)). After blocking, the immunodetection is performed using a mouse monoclonal antibody against MAPKAPK5 (BD Biosciences, Cat N°612080) diluted 250 fold in blocking buffer. After overnight incubation with this primary antibody, the membrane is washed 3 times with PBST and incubated 1 hr with the secondary antibody ((Polyclonal goat anti-mouse Ig, HRP conjugated (DAKO P0447) diluted 50000 fold in blocking buffer. The blot is then washed 3 times in PBST and the detection is performed with ECL advance (RPN2109, Amersham) on a Kodakimager according to the manufacturers instructions. The Western Blotting revealed a lower expression level of MAPKAPK5 in the Ad5-MAPKAPK5-vl3_KD infected cells compared to the cells infected with the Ad5-eGFP-v5_KD negative control virus. Comparison with the diluted Ad5-eGFP-v5_KD infected samples allowed to estimate the reduction in expression to be 2-fold. Equal loading of the 30μg samples is demonstrated by immunodetection of β-actin after removal of the MAPKAPK5 antibody by a 'stripping procedure' (5 minutes boiling of the membrane in PBST). Immunodetection of β-actin is performed according to the method described for MAPKAPK5 detection, but using a goat polyclonal antibody against β-actin (Santa Cruz, Cat N° SC-1615) at a 1000 fold dilution as primary antibody and a rabbit anti goat antibody at a 50000 fold dilution as a secondary antibody. Results of this experiment are given in Figure 4. Taken together, this experiment demonstrated the functionality of the Ad-siRNA virus produced to reduce the MAPKAPK5 expression levels in primary human SFs.

(B) MAPKAPK5 knock-down Ad-siRNA Reduces SF-induced MMPl Expression [00375] The efficacy of Ad5-MAPKAPK5-vl 3 KD virus in the 'MMP assay' is tested as follows.

Day 1 , SFs (passage 9 to 10) are seeded in 96 well plates at a density of 3000 cells per well in complete synovial growth medium (Cell Applications). One day later, the cells are infected with increasing amounts (3 , 6; 9, 12 or 1 5 μl) of following viruses: Ad5-eGFP-v5_KD, Ad5-MAPKAPK5-vl2_KD, Ad5-MAPKAPK5-vl3_KD, Ad5-MMPl-vl O_KD. The virus load is corrected by addition of the neutral virus Ad5-Luc-vl 3_KD to bring the final virus volume on the cells to 15μL in every well. This correction guarantees that the effects observed do not result from the virus load applied to the cells. The cells are then incubated for 5 days before the activation step. This step involves the replacement, in every well, of the growth medium by 75μL of Ml 99 medium supplemented with 25μL of 'complex trigger'. 48 hrs after the activation step, the supernatant is collected and subjected to the MMPl ELISA as described in Example 1. The results of the experiment are shown in Figure 5. The quality of the experiment is demonstrated by the efficacy of the Ad-siRNA virus targeting MMPl itself. This positive control virus strongly reduces the MMPl expression by SFs, whereas the negative control virus, designed to target the expression of luciferase, does not influence the levels of MMPl expression. Two viruses used to validate the MAPKAPK5 target (Ad5-MAPKAPK5-vl2 KD and Ad5-MAPKAPK5-vl3) do also lead to a clear reduction of the complex trigger induced MMPl expression by primary human SFs. It can be concluded, from this experiment, that MAPKAPK5 represents a valuable drug target that is shown to modulate MMP l expression in SFs. Similarly, the inhibition of MAPKAPK5 enzymatic activity by a small molecule compound is expected to reduce the 'complex cytokine' induced MMPl expression in the 'MMP assay'. The inhibition of MAPKAPK5 enzymatic activity by a small molecule compound is also predicted to reduce the degradation of the joint associated with RA.

(C) In vitro 'MMP assay' Testing of Compounds Inhibiting MAPKAPK5

[00376] Compounds inhibiting the MAPKAPK5 activity in a biochemical assay (i.e. cell free, using purified enzyme), are tested in the 'MMP assay' according to following protocol. [00377] The compound master stocks (all at 1 OmM concentration in 100% DMSO) are diluted 10-fold in water (Distilled water, GIBCO, DNAse and RNAse free) to obtain a ImM intermediate work stock in 10% DMSO. This intermediate work stock is further diluted either 3-fold (or 10-fold) in 10%DMSO to obtain an intermediate work stock of 333μM (or lOOμM) concentration, respectively, in 10% DMSO. The ImM as well as 333 μM (or lOOμM) intermediate work stocks are then further diluted 10-fold in 1.1% DMSO to obtain the 1Ox workstocks at lOOμM and 33.3μM (or lOμM) concentration in 2% DMSO. This 1Ox work stock is then diluted 10-fold in M199 medium supplemented with 1%FBS to obtain the final ' Ix compound preparation' containing the compounds at lOμM and 3.33μM (or l μM) as well as 0.2% DMSO. These are the final conditions at which the compounds are tested on the cells. In parallel, the 1Ox work stock is diluted 10-fold in 'complex trigger' (i.e. the supernatant of TNF-α treated THPl cells produced as described in Example 1) that is diluted 2-fold in Ml 99 supplemented with 1% FBS to produce the ' Ix compound in 50% complex trigger preparation' .

[00378] At day 1, RASFs are seeded in 96 well plates (Flat bottom, tissue culture treated, Greiner) at a density of 3000 cells/ well m complete synovial growth medium (Cell Applications) Day 5, the compounds are added to the cultured cells as follows Medium is completely removed from the cells and replaced by 75μL of the 'Ix compound preparations' containing the compounds at either lOμM or 3.33μM (or lμM) in M199 medium supplemented with 1%FBS and 0.2% DMSO. After an incubation period of 2 hours, which allows the compounds to equilibrate and enter the cells, 25μL of the ' Ix compound m 50% complex trigger preparations' are added to the wells on top of the 'Ix compound preparation', in the wells containing the corresponding compounds at corresponding concentration In this way, an 8-fold diluted complex trigger is ultimately applied to the cells. An incubation of 48 hrs is then performed and 20μl of the cell supernatant is then processed in the MMPl ELISA as described above, delivering raw data (RLU: relative luminescence units). Following controls are included in the experiments. A maximal signal control, in which the cells are activated by the complex trigger but only the 0.2% DMSO vehicle (and thus, no compound) is added. This control indicates the maximal level of MMPl that can be achieved in the test. A minimal signal control is also included m these experiments. Here, cells are not triggered. The medium of the cells is then changed to lOOμl M199 medium supplemented with 1% FBS at day 5. This control returns the basal MMPl levels produced by the RASFs. The percent inhibition of the MMPl expression achieved by the compounds is then calculated based on the RLU data returned by the ELISA with following formula:

[[(maximal MMPl levels - minimal MMPl levels) - (MMPl level compound X at concentration Y-mmimal MMPl levels)]/(maximal MMPl levels - minimal MMPl levels)]xlOO.

[00379] Toxicity of the compounds is assessed as follows. Day 1, SFs are seeded m white, tissue culture treated 96 well plates at a density of 3000 cells per well in lOOμL complete synovial growth medium. The compound handling, compound addition to the cells as well as activation of the cells is further performed as described above in this example for the determination of the MMPl levels. After the 48hrs incubation period, the medium is removed from the wells, replaced by 50μL fresh M199 medium supplemented with 1% FBS 50μL of substrate (Promega Celltiter Glow cell viability kit) is then added to the wells After an incubation period of 10 min, luminescence signal is measured A reduction of the luminescence signal by more than 50% as compared to the maximal control wells is considered to reflect significant toxicity No toxicity is observed for the compounds tested in the 'MMP assay' [00380] 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 MMP assays

Example 3: Assay to assess effect of compounds on cytokine release by human PBMCs

[00381] Human peripheral blood mononuclear cells (PBMCs) are isolated from "buffy coats" prepared from the blood of healthy volunteers, isolated essentially according to method of Bøyum (1984).

In brief, buffy coat is diluted 1 1 with Ix PBS (Gibco) and 30 mL is carefully put on top of 20 mL Lymphoprep™ (Lucron Bioproducts) in 50 mL Falcon tubes. After centrifugation (35 min, 400 g, 18°C) the mononuclear cells are collected from the white interphase and washed 3 times with Ix PBS by resuspending and centrifugation (10 min, 200 g). Isolated PBMCs are finally resuspended in RPMI 1640 (Cat.No. 21875, Gibco) that is supplemented with 10% heat-inactivated FBS (Hyclone). [00382] For the assay PBMCs are seeded at 2.5E6 cells/mL in 160 μL in 96-well plates (Nunc).

Serial dilution of the test compounds are made first in DMSO (Sigma) and then diluted 50-fold in Ml 99 medium (Gibco) containing 1% heat-inactivated FBS. Compounds are further 1/10 diluted in the assay plates to obtain final DMSO concentration of 0.2%. Cells are preincubated with the compounds for 1 hr at 37°C, 5% CO2. Then, cells are stimulated with LPS {Escherichia coll serotype 026:B6, Cat.No. L2654, Sigma) that is added in a volume of 20 μL to a final concentration of 1 μg/mL and cells are further cultured for 24 hr. The plates are centrifuged and the supernatant is collected and stored at -8O0C until analysis of appropriate dilutions in ELISAs.

[00383] The following 384-well chemiluminescent ELISA protocol was developed to measure

TNFα levels in the supernatant : White Lumitrac 600 384-well plates (Greiner) are coated with (40 μL/well) anti-TNFα capture antibody (Cat.No. 551220, BD Pharmingen) that is diluted to 1 μg/mL in Ix PBS (Gibco). After overnight incubation at 4°C, plates are washed with Ix PBS (80 g NaCl, 2g KCl (Sigma), 11.5 g Na2HPO4.7H2O and 2 g KH2PO4 in 10 L milliQ; pH 7.4) and blocked with 100 μL/well buffer B (Ix PBS containing 1% BSA (Sigma), 5% sucrose (Sigma) and 0.05% NaN3 (Sigma)). After 4 hr incubation at RT, blocking buffer is removed and plates are washed once with PBST (Ix PBS with 0.05% Tween-20 (Sigma)). Then, 40 μL of sample is transferred to the ELISA plates and plates are incubated at 4°C. Next day, plates are washed 3 times (twice with PBST and once with PBS) and 35 μL/well biotinylated anti-TNFα antibody (Cat.No. 554511, BD Pharmingen) diluted first to a concentration of 250 ng/ml in buffer D (Ix PBS with 1% BSA) is added. After 2 h of incubation at RT, plates are washed as described above and 35 μL/well of a 1/2000 dilution of streptavidin-HRP conjugate (Cat.No. SNN2004, Biosource) in buffer D is added. After 45 min, plates are washed as described above and incubated for 5 min with 50 μL/well BM Chemiluminescence ELISA Substrate POD (Roche). Readout is performed on the Luminoscan Ascent Luminometer (Labsystems) with an integration time of 100 msec delivering raw data (RLU: relative luminescence units). The following controls are included in the experiments, a maximal signal control, in which the cells are activated by LPS but only the 0.2% DMSO vehicle (and thus no compound) is added. This control indicates the maximal level of TNFα that can be achieved in the test. A minimal signal control is also included in these experiments. Here, cells are not triggered. This control returns the basal TNFα levels produced by the PBMCs. The percent inhibition (PIN) of the TNFα release, achieved by the compounds is then calculated based on the RLU data returned by the ELISA with following formula: 100 - [((TNFα level compound X at concentration Y- minimal TNFα levels)/(maximal TNFα levels - minimal TNFα levels))xlOO]. Where compounds are tested at 8 concentrations (1/3 serial dilution), EC50-values can be calculated by curve fitting of the means of the PIN data achieved for a compound at each test concentration.

[00384] To assay the effect of compounds on the release of ILl and IL6 by LPS stimulated PBMC cultures, appropriate dilutions of the supernatant can be measured using the same ELISA protocol as described above. Matched pair antibodies for ILl and IL6 ELISA (all from R&D Systems) may be used as follows: anti-ILl capture antibody (Cat.No. MAB601) used at 0.5 μg/mL , biotinylated anti-ILl detection antibody (Cat.No. BAF201) used at 50 ng/mL; anti-IL6 capture antibody (Cat.No. MAB206) used at 1 μg/mL; biotinylated anti-IL6 detection antibody (Cat.No. BAF206) used at 50 ng/mL.

Example 4: MMP13 Assay

[00385] The protocol of the MMP 13 ELISA is described in section 4.1, then testing of compounds on release of IL-1/OSM-driven MMP13 expression in SW1353 chondrosarcoma cell line is described in section 4.2.

4.1 MMPl 3 ELISA protocol

[00386] A 384 well format ELISA for measurement of MMP 13 was developed. Various primary antibodies were tested, as well as various ELISA protocols. The following protocol is developed and validated to measure MMP13 levels in supernatant of cell cultures in 384 well plates. [00387] Black maxisorb 384 well plates (Nunc 460518) are coated with 35 μl of a buffered solution containing 1.5 μg/mL anti-MMP13 antibody MAB511 (R&D systems). The antibody is diluted in carbonate-bicarbonate coating buffer (1.59 g Na2CO3 (Sigma S-7795) and 2.93 g NaHCO3 (Sigma S-5761) in 1 L MiIIiQ water, adjusted to pH 9.6). After overnight incubation at 4°C, wells are washed twice with lOOμL PBST (80 g NaCl, 2g KCl (Sigma), 11.5 g Na2HPO4.7H2O and 2 g KH2PO4 in 10 L milliQ water; pH 7.4 + 0.05% Tween-20 (Sigma)) and blocked with 100 μL/well blocking buffer (5% non fat dry milk in PBS). After overnight incubation at 4°C, wells are washed twice with lOOμL PBST. The PBST is removed and 35 μL of sample is transferred to the ELISA plates. After 4 hr incubation at RT, plates are washed twice with PBST and incubated for 1 hr at 37°C with 35 μL/well 1.5 mM APMA solution (a 10 mM APMA stock solution is prepared one day before (35.18 mg APMA (Sigma A-9563) in 1 OmL 0.1 M NaOH (Merck 1.06469.1000) and stored at 4°C. Before use, the 10 mM APMA stock solution is diluted to 1.5 mM in IXAPMA buffer (10X APMA buffer: 500 mM Tris (Roche 708976), 50 mM CaCl2 (Sigma C-5080), 500 μM ZnCl2 (Sigma Z-0173), 1.5 M NaCl (Calbiochem 567441), 0.5% Brij35 (Sigma 430 AG-6) and adjust to pH 7.0). After activation of MMP13 by APMA, plates are washed again two times with lOOμL PBST/well and 35 μL of substrate solution is added to each well. Substrate solution is prepared as follows: OmniMMP Fluorescent substrate (Biomol P- 126) stock solution (2mM in DMSO, stored at -200C) is diluted in IX OmniMMP buffer (10X OmniMMP buffer: 500 mM Hepes (Sigma H4034), 100 mM CaCl2 (Sigma C5080), 0.5% Brij35 (Sigma 430 AG-6; adjusted to pH 7.0) to a final concentration of 0 OlmM After an overnight incubation at 37°C, the active MMP13 in the sample has cleaved the substrate and released fluorescence Readout is performed on the En Vision (Perkm Elmer) using 320nm excitation/405nm emission filters

4 2 Assessing effect of compounds on cytokine driven MMPl 3 expression in SWl 353 cells

[00388] Human chondrosarcoma cell line SWl 353 was acquired from ATCC and grown in

DMEM supplemented with 10% heat-mactivated FBS and Ix penicillin/streptomycin (Invitrogen) in a humidified 5% CO2 incubator at 370C Ahquots of the cells were frozen and cryopreserved in liquid nitrogen Starting from a cryopreserved aliquot, cells are further grown by sub-culturmg at a 1/5-1/8 ratio twice a week by trypsinisation

[00389] Starting from the compound master stocks (all at 10 mM concentration in 100% DMSO) a 3-fold serial dilution is made m 96-well plates m 100% DMSO Then, plates are futher diluted 50-fold m M199 medium supplemented with 1% heat-inactivated FBS to obtain an intermediate work stock [00390] At day 1, SW1353 cells are seeded m 96-well plates (flat bottom, tissue culture treated,

Gremer) at a density of 15000 cells/well m 120 μL growth medium The next day, 15 μL compound out of the intermediate work stock is added After an incubation period of 60 minutes, which allows the compounds to equilibrate and enter the cells, cells are stimulated with a mixture of IL l β and OSM, added in a volume of 15 μL to obtain final concentrations of 1 ng/ml IL lβ and 25 ng/ml OSM For that, stocks of IL-I (10 μg/ml) and OSM (25 μg/ml) (both PeproTech) were diluted to 10 ng/ml and 250 ng/ml respectively, m M199 medium supplemented with 1 % FBS After incubation for 48hr, the cell supernatant was harvested and an appropriate dilution was processed in the MMP 13 ELISA as described above, delivering raw data (RFU relative fluorescence units) The following controls are included m the experiments a maximal signal control, m which the cells are activated by the ILl-β/OSM cytokine mixture but only the 0 2% DMSO vehicle (and thus no compound) is added This controls indicated the maximal MMP 13 levels that can be achieved m the test A minimal signal control, m which cells only receive the 0 2% DMSO vehicle and no trigger, is also included This control returns the basal MMP 13 levels produced by the SW1353 cells The percent inhibition of the MMP13 expression achieved by the compounds is then calculated based on the RFU data returned by the ELISA with the following formula [[(maximal MMP13 levels - minimal MMP13 levels) - (MMP13 level compound X at concentration Y -minimal MMP13 levels)] / (maximal MMP13 levels - minimal MMP13 levels)] x 100 Based on a plot of percent inhibition vs Log (molar concentration) and curve fitting, IC50 values of a particular compound can be calculated

[00391] The following compounds have been or can be prepared according to the synthetic methods described above For the purpose of Table 1 below, activity of each compound, which can be determined using the MAPKAPK5 assay method described m Example 1, is expressed as follows

++++ compound exhibited MAPKAPK5 IC50 0 01 - 100 nM

+++ compound exhibited MAPKAPK5 IC50 101 -500 nM ++ compound exhibited MAPKAPK5 IC50 501 - 1000 nM + compound exhibited MAPKAPK5 IC50 >1000 nM

[00392] TABLE 1










MMPl data

§ § compound exhibited MMP 1 IC50 1 - 1000 nM § compound exhibited MMPl IC50 >1000 nM



MMP13 data

**# compound exhibited MMP 13 IC50 1-500 nM compound exhibited MMP 13 IC50 501-1000 nM compound exhibited MMP13IC50 >1000 nM

[00393] TABLE 3



In Vivo Studies

Example 5: Tolerability of compounds

[00394] This protocol is designed to assess the tolerability of the compounds of the invention m healthy DBA/U mice to determine the "therapeutic window" as defined by the dosing range between efficacious (mouse therapeutic Collagen Induced Arthritis model) and toxic doses 5 1 Animals

[00395] DBA/1 J nude mice are used (CERJ (France)), the mice were 10-11 weeks old, and had a body weight of approx 2Og

5 2 Compounds Preparation

[00396] Compounds are prepared for a dosmg regimen of 100 mg/kg/d, po, free base, m a standard volume of injection of 0 1 mL/ 10 g of mice (equivalent to 10 mg/ 1 mL) For solution preparations, compounds were dissolved in 0 5% methyl-cellulose and 1% DMSO, once a week

5 3 Experimental groups

[00397] Groups are randomized based on body weight and treated for up to two weeks Each group contained 5 mice and received either test compound at 100 mg/kg, a comparison compound at 100 mg/kg/d (Compound A) or vehicle on a daily basis m a dosing volume of 200 μL per mouse


Compound A - 5 4 Animal Monitoring (Clinical signs and Body Weight) [00398] Potential drug toxicity is monitored by observation every day and by recording body weights three times a week for weight loss All statistical analyses were performed using Student's t test [00399] To assess the general tolerability of the treatments, the total body weight is followed throughout the course of the study Figure 6A shows the effect of Compound 1 and Compound 2 on the total body weight, Figure 6B shows the effect of a comparator compound A (see below for structure) The data indicates that the bridged compounds are tolerated better than the non-bridged compounds. In particular there was no statistically significant difference between the weight of the animals treated with compounds 1 or 2 at 100 mg/kg/d compared to the vehicle.

Example 6: CIA Model

[00400] Complete Freund Adjuvant / Collagen II (CFA/Coll II, bovine) is injected (1 mg/mL, lOOμL per animal) into the tail (intradermic) at the start of the experiment. Incomplete Freund Adjuvant /

Collagen II (IFA/Coll II) is injected into the tail at the same level (1 mg/mL, lOOμL per animal) 21 days after CFA/Coll II injection.

[00401] Animals are then randomized based on score and assigned to treatment groups assuring an equal distribution of score in the different groups.

[00402] Treatment with either the test compound (1 mg/kg/d, 3 mg/kg/d or 30 mg/kg/d), positive control (Enbrel, 10 mg/kg/3 x week, ip) or vehicle (Methyl Cellulose, 1%DMSO) starts at day 8 post

IFA/Coll II-boost (i.e. day 28 of the experiment).

[00403] Animals are dosed daily with the test compound, positive control or vehicle for 14 days.

The animals are scored daily for clinical symptoms, scoring is reported for the individual paws. During the treatment period the body weight of the animals is monitored. Bone protection is analysed by x-ray imaging.

[00404] Selected compounds of the invention were efficacious at 3 or 30 mg/kd/d.

Example 7: Septic shock model

[00405] Injection of lipopolysaccharide (LPS) induces a rapid release of soluble tumour necrosis factor (TNF-α) into the periphery. This model is used to analyse prospective blockers of TNF release in

VIVO.

[00406] Six BALB/cJ female mice (20 g) per group are treated at the intended dosing once, po.

Thirty minutes later, LPS (15 μg/kg; E. coll serotype 0111 :B4) is injected ip. Ninety minutes later, mice are euthanized and blood is collected. Circulating TNF alpha levels are determined using commercially available ELISA kits. Dexamethasone (5 μg/kg) is used as a reference anti-inflammatory compound. Selected compounds of the invention were efficacious at 3, 10 and 20 mg/kg, po.

Example 8: Mouse Collagen Antibody Induced Arthritis (CAIA) model (also called mouse Monoclonal AntiBody (MAB) model)

[00407] Eight BALB/cJ female mice (20 g) per group are treated at the intended dosing once, po.

The same day, 2 mg/mouse of a cocktail of four monoclonal antibodies (MDBiosciences; ref. CIA-MAB-50) is injected i.v. LPS (50 μg/mouse; E. CoIi serotype 55:B5) is administered i.p., three days later. Treatment with either the test compound, positive control (Enbrel, 10 mg/kg/3 Xweek, i.p. or dexamethasone, 1 mg/kg, daily, p.o.) or vehicle (Methyl Cellulose, 1%DMSO) starts the day of the antibodies injection. Animals are dosed daily with the test compound, positive control or vehicle for up to 10 days. The animals are scored for clinical symptoms and scoring is reported for the individual paws. During the treatment period, the body weight of the animals is monitored.

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[00409] It will be appreciated by those skilled in the art that the foregoing description is exemplary and explanatory in nature, and is 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. [00410] From the foregoing description, various modifications and changes in the compositions and methods of this invention will occur to those skilled m the art. All such modifications coming within the scope of the appended claims are intended to be included therein

[00411] 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.

[00412] 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. [00413] 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.

[00414] 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.