PAR2 RECEPTOR ANTAGONISTS

Introduction

This invention relates to compounds that are PAR2 receptor antagonists, to compositions containing them, to processes for their preparation, and to their use in medicine, in particular for the treatment of conditions which respond to antagonism of the PAR2 receptor, such as inflammation including intestinal inflammation, ulcerative colitis, Chrohn's disease, inflammatory bowel disease and irritable bowel syndrome, fibrosis, arthritis and inflammatory skin diseases including psoriasis, atopic dermatitis and itch, pain and cancer.

Background to the invention

Protease activated receptors (PARs) are a family of seven transmembrane domain G- protein-coupled receptors that are activated by cleavage of their extracellular N-terminal domain by proteolytic enzymes. The newly exposed N-terminal sequence acts as a tethered ligand that binds to the extracellular face of the receptor and activates it. Four PARs have been described that are selectively cleaved by different enzymes; PARI , PAR3 and PAR4 are cleaved by thrombin, PAR2 and PAR4 predominantly by trypsin and tryptase and PAR4 also cleaved by cathepsin G.

PAR-2 in the GI tract

The GI tract and pancreas are particularly exposed to a large array of proteases which can activate PAR2 receptors. Trypsin is released into the lumen of the pancreatic duct and the upper GI tract, for physiological digestive purposes. Other proteases abundant in the GI tract include those derived from enteric bacteria and those generated during disease processes. On mucosal surfaces, a balance between proteolytic activity and the presence of protease inhibitors such as pancreatic secretory trypsin inhibitor (PSTI) is constantly present. PAR2 receptors are expressed throughout the GI tract specifically on mast cells, smooth muscle cells, myenteric neurons and endothelial cells, and on both the apical and basolateral sides of enterocytes (Kong et al., 1997). Since trypsin present in the GI lumen could activate PAR2 on apical surfaces, this receptor may provide a means by which the epithelium "senses" luminal processes.

APC00482WO In the gut, motility and secretion are regulated by neurons of the submucosal and myenteric plexi of the gastrointestinal tract. These neurones express PARI, PAR2 and PAR4. PAR2 is expressed by secretomotor neurons in the submucosal plexus of the small intestine, where brief activation of PAR2 by agonists such as SLIGRL-NH ₂ or trypsin results in a prolonged depolarisation that is often accompanied by increased excitability. Tryptase also induces a transient depolarization and a sustained increase in neuronal excitability (Linden et al., 2001). These observations indicate that PAR2 excites a proportion of myenteric neurons, which may contribute to dysmotility during intestinal inflammation.

A recent report concluded that activation of PAR2 in GI epithelial cells could trigger pro-mflammatory signaling including release of IL-8 via two independent pathways, MEK/ERK and PBK/Akt. PAR2 would thus be confirmed to be a therapeutic target for treatment of inflammatory diseases of the GI tract (Tanaka et al, 2008). Histological studies in fibrotic intestine from patients with Crohn's disease indicated that (myo)fibroblasts are expanded in number and are the major cell types at sites of fibrosis in all layers of the intestinal wall (Pucilowska et al., 2000). Recent analysis of inflammatory tissues from patients with Crohn's Disease showed PAR2 over-expression in all cell types analyzed, including fibroblasts (Ketabchi et al., 2007). These results are in line with observations showing PAR2 over expression during fibrosis of lung and kidney (Cederqvist et al., 2005; Grandaliano et al., 2003). In these studies, PAR2 was identified as a potentially crucial receptor for the pathogenesis and sustainability of fibrosis. This hypothesis has recently been substantiated by a report linking normal activation of PAR2 by the protease Factor X resulting in tissue regeneration following injury, to the fibrotic response seen following repeated stimulation of this system as a consequence of chronic inflammatory bowel disease (IBD, Borensztajn et al, 2008).

In conclusion, PAR2 activation is important in the establishment, maintenance, and progression of intestinal inflammation and of fibrosis.

Itch associated with psoriasis and atopic dermatitis

Itch in human skin can be induced by both histamine and proteases. The ability of the PAR2 agonist SLIGRL-NH ₂ to cause scratching behaviour in mice was not antagonised by antihistamine treatment (Shimada et al., 2006). The authors concluded that PAR2 was a histamine independent mediator of itch. Such an interpretation has been strengthened

APC00482WO by the identification of the receptor on the terminals of sensory nerve fibres which transduce the itch sensation (Steinhoff et al, 2000). In non-GI tissues such as the skin, "tissue trypsins" are secreted which can activate PAR2 receptors, as can other proteases including tryptase, Factor X, Factor Vila and Tissue Factor (Bunnett, 2006). In patients with atopic dermatitis PAR-2 has also been strongly implicated as a major cause of itch (Steinhoff et al., 2003).

Psoriasis is a common skin condition which typically develops as patches ('plaques') of red, scaly skin. People with psoriasis have a faster turnover of skin cells associated with changes in the blood supply of the skin (redness) which causes local inflammation. Psoriasis is not due to an infection and is not infectious, nor is it cancerous.

Itch in psoriasis is a significant but often unrecognized problem in dermatology. A recent study found that itching was the most frequent complaint (64%) among patients hospitalised for psoriasis, (Sampogna et al., 2004) and several other studies confirm that itch is a principal symptom of psoriasis (Van de Kerkhof et al., 1998, 2000). Interestingly PAR2 receptors are highly expressed in the skin of psoriatic patients (Steinhoff et al, 1999), as are numerous tryptase-positive cells. These are found in the dermis and at the dermal-epidermal border in atopic dermatitis and psoriasis, and occasionally in the epidermis of psoriasis lesions. Tryptase released from such cells activates PAR2 in keratinocytes which may induce local inflammatory changes and thereby contribute to the pathophysiology of atopic dermatitis and psoriasis.

Furthermore it is hypothesised that other types of itch such as neuropathic itch are linked to an activation of PAR2 receptors by proteases (Binder et al., 2008).

For the forgoing reasons it is expected that a PAR2 antagonist will be effective in the treatment of inflammatory skin diseases including psoriasis and itch In particular, it is expected that topical or systemic administration of a PAR2 antagonist would reduce the itch caused by local inflammation in psoriasis, and therefore would constitute a targeted treatment for this unchallenged symptom of psoriasis. It is also expected that a PAR2 antagonist will be effective in the treatment of arthritis due to inflammation in or around the joint.

Pain

APC00482WO The transmission of pain and/or unpleasant sensation is also enhanced by activation of PAR2 receptors as application of activating peptide excites C fibres and sensitises them to heat (Ding-Pfenmgdorf et al., 2004). Cancer

PAR-2 has been implicated in cellular proliferation, invasion and metastasis. There is increasing evidence that PAR2 is an important mediator of tumour progression, with trypsin levels being elevated in gastric, colon, ovarian and lung tumours (Ducroc et al., 2002). In addition PAR-2 is expressed in cancers of the lungs, liver, prostate, thyroid, breast, gastrium, colon, pancreas, gallbladder, melanoma and glioblastoma (see Jahan et al., 2007 and references therein).

Tissue factor (TF) is a primary component of the clotting cascade which with Factor Vila or Factor Xa can initiate clotting. Cancer patients are frequently in a pro- thrombotic state, apparently partly due to the release of TF containing microparticles (small membranous fragments perhaps released on apoptosis). TF is expressed at high levels in vessel wall fibroblasts but may also be expressed on endothelial and smooth muscle cells (Kasthuri et al., 2009). TF is also heavily implicated in cancer, its expression generally increasing with cancer stage (Kakkar et al., 1995; Kasthuri et al., 2009) and appears to be involved in metastasis (Belting et al., 2005). Indeed TF may play a role in forming the fibrinous clot around metastatic cells which serves to protect them from K cells and to maintain them in the vasculature (Palumbo et al., 2005, 2007). TF Factor Vila Factor Xa complexes stimulate breast carcinoma cell migration and invasion through activation of PAR2 (Hjortoe et al., 2004; Morris et al., 2006). In addition in other cancers including colon and gastric carcinomas, activated PAR2 stimulates EGFR activity and thus cellular proliferation (Caruso et al., 2006); Darmoul et al., 2004). Indeed in ovarian cancer increase in PAR-2 was seen with progression of the cancer irrespective of the histopathological classification of the tumour type, and high cancer cell PAR-2 expression was associated with a significantly worse prognosis (Jahan et al, 2007). Similarly patients with lymph node metastases of uterine cancers with high levels of PAR-2 had significantly worse prognosis than those with lower levels (Jahan et al., 2008). PAR-2 has also been implicated in tumour angiogenesis in cancers of the

APC00482WO breast, colon, gastrium, pancreas, lungs, prostate, melanoma and glioblastoma (see Jahan et al., 2007).

Inflammation

PAR2 receptor activation has been shown to be important in inflammatory disorders. Based on in vivo studies in models of inflammatory disorders (Kelso et al, JPET, 2006, 316, 1017-1024, Sevigny, PNAS, 2011, 108, 20, 8491-8496 and Cenac et al, JDR, 2010, 89, 10, 1123-1128) it is expected that antagonism of the PAR2 receptor will be effective in the treatment of inflammatory disorders.

Pancreatitis

Pancreatitis is an inflammatory condition understood to be the result of undesirable trypsin activity within the pancreas. The biological effects of trypsin in the pancreas have been shown to act through PAR2, which is strongly expressed on the luminal surface of acinar and ductal cells (Ceppa et al., 2011). Antagonism of the effects of trypsin at PAR2, within the pancreas, can be expected to be an effective treatment for pancreatitis. '

For the above reasons, the PAR2 receptor is regarded as a target for intervention in the treatment of the conditions referred to above. There are few antagonists of PAR-2 available which are suitable for therapeutic treatment. Accordingly a small molecule antagonist is desirable for therapy.

Brief Description of the Invention

This invention makes available a class of compounds having surprisingly high antagonist activity at the PAR2 receptor, and their use in indications which respond to the antagonism of the PAR2 receptor such as those mentioned above.

Detailed Description of the Invention

In a first aspect the present invention makes available a compound of formula (I) or a pharmaceutically acceptable salt, solvate or hydrate thereof

APC00482WO

for use in the treatment of a disease or condition selected from inflammation, intestinal inflammation, ulcerative colitis, Chrohn's disease, inflammatory bowel disease and irritable bowel syndrome, fibrosis, arthntis and inflammatory skin diseases including psoriasis, atopic dermatitis and itch, pain and cancer

wherein ring P is a monocyclic aryl or heteroaryl ring having 5 or 6 ring atoms, which aryl or heteroaryl ring is optionally substituted with one or more R ¹ groups; ring Q is a monocyclic aryl or heteroaryl ring having 5 or 6 ring atoms, which aryl or heteroaryl ring is optionally substituted with one or more R ² groups; X is -(CH ₂ )- _n; n is 1, 2, 3, or 4;

Y is -C(O)- or -S(0) ₂ -;

R ¹ is one or more optional substituents selected from: hydrogen, halo, hydroxyl, oxo, cyano, d _-4 -alkyl, fluoro-Ci _-4 -alkyl, C^-alkoxy, -S0 ₂ R ⁴ , -NR ⁵ R ⁶ , -NR ⁷ S0 ₂ R ⁴ , - C(0)NR ⁵ R ⁶ , - R ⁷ C(0)OR ⁵ , - R ⁷ C(0)R ⁵ , -NR ⁷ C(0)NR ⁵ R ⁶ , -C(0)R ⁵ , -C(0)OR ⁵ , - NR ⁷ S(0) ₂ R ⁵ , and wherein any Ci _-4 -alkyl or Ci _-4 -alkoxy residue is optionally substituted with one or more substituents selected from halogen, cyano, hydroxyl, NR ⁵ R ⁶ , Ci _-4 - alkoxy, fluoro-Ci _-4 -alkoxy, -S0 ₂ R ⁴ , -NR ⁷ S0 ₂ R ⁴ , -C(0)NR ⁵ R ⁶ , -NR ⁷ C(0)OR ⁵ , - NR ⁷ C(0)R ⁵ , -NR ⁷ C(0)NR ⁵ R ⁶ , -C(0)NR ⁵ R ⁶ , -C(0)R ⁵ , -C(0)OR ⁵ , -NR ⁷ S(0) ₂ R ⁵ ;

R ² is one or more optional substituents selected from: hydrogen, halo, hydroxyl, oxo, cyano, C _M -alkyl, fluoro-Ci _-4 -alkyl, C _M -alkoxy, -S0 ₂ R ⁴ , -NR ⁵ R ⁶ , -NR ⁷ S0 ₂ R ⁴ , - C(0)NR ⁵ R ⁶ , - R ⁷ C(0)OR ⁵ , - R ⁷ C(0)R ⁵ , -NR ⁷ C(0)NR ⁵ R ⁶ , -C(0)R ⁵ , -C(0)OR ⁵ , - NR ⁷ S(0) ₂ R ⁵ , and wherein any Ci _-4 -alkyl or Ci _-4 -alkoxy residue is optionally substituted

APC00482WO with one or more substituents selected from halogen, cyano, hydroxyl, NR ⁵ R ⁶ , C ₁ -4- alkoxy, fluoro-Ci _-4 -alkoxy, -S0 ₂ R ⁴ , -NR ⁷ S0 ₂ R ⁴ , -C(0)NR ⁵ R ⁶ , -NR ⁷ C(0)OR ⁵ , - NR ⁷ C(0)R ⁵ , -NR ⁷ C(0)NR ⁵ R ⁶ , -C(0)NR ⁵ R ⁶ , -C(0)R ⁵ , -C(0)OR ⁵ , - R ⁷ S(0) ₂ R ⁵ ; R ³ is one or more optional substituents selected from hydrogen, halogen, Ci-4-alkyl, fluoro-Ci-4-alkyl, C3-6-cycloalkyl, C3-6-cycloalkyl-Ci-4-alkyl;

R ⁴ is independently selected from hydrogen, NR ⁵ R ⁶ , Ci-4-alkyl, fluoro-Ci-4-alkyl, C3-6- cycloalkyl or C3.6-cycloalkyl-Ci-4-alkyl;

R ⁵ and R ⁶ are independently selected from hydrogen, Ci-4-alkyl, fluoroCi-4-alkyl, C3-6- cycloalkyl or C3-6-cycloalkyl-Ci-4-alkyl, or hydroxyCi-4-alkyl; or

R ⁵ and R ⁶ together with the nitrogen atom to which they are attached join together to form a 5 or 6-membered heterocyclic ring, the heterocyclic ring optionally further comprising a further heteroatom selected from nitrogen, oxygen or sulphur wherein the heterocyclic ring is optionally substituted with one or more substituents selected from halo, hydroxyl, oxo, cyano, Ci-4-alkyl, and fluoro-Ci.4- alkoxy;

R ⁷ is hydrogen, d-4-alkyl, C3_6-cycloalkyl, or C3.6-cycloalkyl-C ₁ .4- alkyl;

R ¹⁰ , R ¹¹ , and R ¹² are independently selected from hydrogen, Ci-4-alkyl, fluoro-Ci.4-alkyl, C3-6-cycloalkyl, or C3-6-cycloalkyl-Ci-4-alkyl.

In a second aspect of the invention, the applicant makes available a compound of formula (I) or a pharmaceutically acceptable salt, solvate or hydrate thereof wherein P, Q, X, Y, R ¹ , R ² , R ³ , Rio, Rn, and R _i2 are as defined above, with the proviso that R ² is not oxo, hydroxyl or -NR ⁵ R ⁶

Compounds of formula (I) above may be prepared in the form of salts, especially pharmaceutically acceptable salts, N-oxides, hydrates, solvates and polymorphic forms thereof. Any claim to a compound herein, or reference herein to "compounds of the invention", "compounds with which the invention is concerned", "compounds of

APC00482WO formula (I)" and the like, includes salts, N-oxides, hydrates, solvates and polymorphs of such compounds;

Although the above definition potentially includes molecules of high molecular weight, it is preferable, in line with general principles of medicinal chemistry practice, that the compounds with which this invention is concerned should have molecular weights of no more than 600.

The compounds of the invention are antagonists of the PAR2 receptor. Therefore, in another broad aspect the invention provides the use of a compound of the invention in the treatment of, or in the preparation of a composition for treatment of, diseases or conditions responsive to the reduction of PAR2 mediated activity.

Examples of diseases or conditions which are responsive to the reduction of PAR2 mediated activity include inflammation including intestinal inflammation (such as ulcerative colitis, Chrohn's disease, inflammatory bowel disease and irritable bowel syndrome), fibrosis, arthritis and inflammatory skin diseases (such as psoriasis, atopic dermatitis and itch), pain and cancers including cancers of the breast, colon, gastrium, pancreas, lungs, prostate, melanoma and glioblastoma.

The compounds with which the invention is concerned may be used for the reduction of PAR2 mediated activity, ex vivo or in vivo.

The compounds of the invention may be used in the preparation of a composition for the treatment of diseases or conditions selected from inflammation including intestinal inflammation (such as ulcerative colitis, Chrohn's disease, inflammatory bowel disease and irritable bowel syndrome), fibrosis, arthritis and inflammatory skin diseases (such as psoriasis, atopic dermatitis and itch), pain and cancers including cancers of the breast, colon, gastrium, pancreas, lungs, prostate, melanoma and glioblastoma.

The invention provides a method for the treatment of the foregoing disease types, which comprises administering to a subject suffering such disease an effective amount of a compound of the invention.

APC00482WO According to the invention there is also provided a pharmaceutical composition comprising a compound of the invention together with one or more pharmaceutically acceptable carriers and/or excipients. The compounds of the invention may be administered in a variety of dosage forms. Thus, they can be administered orally, for example as tablets, capsules, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules. The compounds can be administered in a sublingual formulation, for example a buccal formulation. The compounds of the invention may also be administered parenterally, whether subcutaneously, intravenously, intramuscularly, intrasternally, transdermally, by inhalation, intranasally, or by infusion techniques. The compounds may also be administered as suppositories. The compounds may also be administered topically. Thus, the compounds of the invention are administered orally, or by inhalation, topically, or intranasally. In a preferred embodiment, the compounds of the invention are administered orally and more preferably, the compounds of the invention are administered as a tablet or capsule. In the latter connection, administration of the compounds in a hard gelatine capsule form, or in one of the many sustained release formulations known in the art will often be preferred. In an alternative preferred embodiment the compounds of the invention are administered as a topical treatment.

The present invention further provides a pharmaceutical composition containing a compound of the invention or a pharmaceutically acceptable salt thereof, as defined above, and a pharmaceutically acceptable carrier. The compounds of the invention are typically formulated for administration with a pharmaceutically acceptable carrier or diluent. For example, solid oral forms may contain, together with the active compound, diluents, e.g. lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g. starches, arabic gums, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents, e.g. starch, algmic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents, such as lecithin, polysorbates, laurylsulphates; and, in general, non toxic and pharmacologically inactive substances used in pharmaceutical formulations. Such pharmaceutical preparations may

APC00482WO be manufactured in known manner, for example, by means of mixing, granulating, tableting, sugar coating, or film coating processes.

Liquid dispersions for oral administration may be syrups, emulsions and suspensions. The syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol. Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. The suspension or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.

Since the compounds of the invention are preferably administered orally, the present invention further provides a pharmaceutical composition containing a compound of the invention or a pharmaceutically acceptable salt thereof, as defined above, and a pharmaceutically acceptable carrier in the form of a capsule or tablet.

Alternatively, the compounds of the invention are preferable administered topically. The compounds may be formulated in any form suitable for topical administration including semi-solid, spray, medicated powders, solution, and medicated adhesive systems. Once formulated, the compounds of the invention may be administered as external topicals that are spread, sprayed, or otherwise dispersed on to cutaneous tissues to cover the affected area. Topical drug delivery is especially effective in the fields of psoriasis, itch, and pain management.

Solutions for injection or infusion may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.

It will be understood that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing treatment. Optimum dose levels and frequency of dosing will be determined by clinical trial, as is required in the art. However, it is expected that a typical dose will be in the range from about 0.001 to 50 mg per kg of body weight.

APC00482WO Terminology

The following definitions shall apply throughout the specification and the appended claims, unless otherwise stated or indicated. Where elements present in the compounds of the invention exist as different isotopes, for example carbon (C ¹³ and C ¹⁴ ) nitrogen (N ¹⁴ and N ¹⁵ ) and hydrogen (H ¹ and H ² i.e. deuterium), such compounds form part of the invention irrespective of the isotopic form of the element present in the compound. In particular, where a compound of the invention has a hydrogen atom in any position, that hydrogen may be replaced by deuterium. It is known in the art that deuterium substitution can increase the metabolic stability of biologically active molecules.

The term "halo" means -F, -CI, -Br, or -I. For example "halo" includes -F, -CI, -Br. The term "hydroxyl" means -OH.

The term "cyano" means -CN.

The term "oxo" means =0 where the symbol "=" is a double bond.

The term "C _a-b -alkyl" wherein a and b are integers denotes a straight or branched alkyl group having from a to b carbon atoms. For example "Ci-4-alkyl" includes methyl, ethyl, n-propyl, isopropyl, n-butyl, /so-butyl, sec-butyl and tert-b tyl and "Ci-6-alkyl" includes the foregoing and straight- and branched-chain pentyl and hexyl.

The term "fluoro-C _a-t ,-alkyl" wherein a and b are integers denotes a straight or branched C _a-b -alkyl group substituted by one or more fluorine atoms. For example fluoro-Ci-4- alkyl includes fluoromethyl, trifluoromethyl, 2-fluoroethyl and 2,2,2-trifluoroethyl.

The term "hydroxyl-C _a _ _b -alkyl" wherein a and b are integers denotes a straight or branched C _a-b -alkyl group substituted by one or more hydroxyl groups. For example hydroxyl-Ci-4-alkyl includes 1 -hydroxyl ethyl (-CH ₂ CH ₂ OH) and 1 -hydroxypropyl (- CH ₂ CH ₂ CH ₂ OH).

The term "C _a-b -alkoxy" wherein a and b are integers refers to a straight or branched C _a - _b - alkyl group which is attached to the remainder of the molecule through an oxygen atom. For example includes methoxy, ethoxy, «-propoxy, wo-propoxy, «-butoxy, wo-butoxy, seobutoxy and feri-butoxy.

APC00482WO The term "fluoro-C _a-b -alkoxy" wherein a and b are integers denotes a fluoro-C _a-b -alkyl group which is attached to the remainder of the molecule through an oxygen atom. For example "fluoro-Ci-4-alkoxy" groups include trifluoromethoxy and 2,2,2-trifluoroethoxy.

The term "Ca- _b -alkoxy-Cc-d-alkyl" wherein a, b, c and d are integers denotes a straight or branched alkoxy group having from a to b carbon atoms connected to a straight or branched alkyl group having from c to d carbon atoms. For example "Ci-4-alkoxy-Ci.4- alkyl" includes methoxymethyl, methoxyethyl, ethoxyethyl, ώο-propoxyethyl, n- butoxy ethyl and tert-butoxy ethyl.

The term fluoro-C _a-b -alkoxy-C _c- d-alkyl wherein a, b, c and d are integers denotes a C _a-b - alkoxy-C _c- d-alkyl group substituted by one or more fluorine atoms. For example "fluoro- Ci_4-alkoxy-Ci-4-alkyl" includes trifluoromethoxymethyl and trifluoromethoxyethyl.

The term "C _a-b -cycloalkyl" wherein a and b are integers denotes a saturated monocyclic hydrocarbon ring having from a to b carbon atoms. For examples "C3-5-cycloalkyl" includes cyclopropyl, cyclobutyl and cyclopentyl. The term "C _a-b -cycloalkyl-C _c -d-alkyl" wherein a, b, c and d are integers denotes a saturated monocyclic hydrocarbon ring having from a to b carbon atoms connected to a straight or branched alkyl group having from c to d carbon atoms. For example "C3-5- cycloalkyl-Ci-4-alkyl" includes cyclopropylmethyl and cyclobutylmethyl. As used herein the term "carbocyclic" refers to a mono-, bi- or tricyclic radical having up to 16 ring atoms, all of which are carbon, and includes aryl and cycloalkyl.

Unless otherwise particularised, the term "heterocyclyl" or "heterocyclic ring" denotes a saturated, monocyclic ring having from 4 to 7 ring atoms with at least one heteroatom such as O, N, or S, and the remaining ring atoms are carbon. Examples of heterocyclic rings include piperidinyl, tetrahydropyranyl, tetrahydrofuranyl, oxetanyl, azetidmyl, pyrrolidinyl, morpholmyl, thiomorpholmyl, dioxanyl, piperazmyl and homopiperazinyl. When present, the sulfur atom may be in an oxidized form (i.e., S=0 or 0=S=0). Exemplary heterocyclic groups containing sulfur in oxidized form are 1,1-dioxido- thiomorpholinyl and 1,1-dioxido-isothiazolidinyl.

APC00482WO Unless otherwise particularised the term "heterocyclyl-C _a- -alkyl" wherein a and b are integers denotes a heterocyclic ring as defined above that is directly attached to a straight or branched C _a- b-alkyl group via a carbon or nitrogen atom of said ring. For example "heterocyclyl-Ci-4-alkyl" groups include piperidin-l -ylmethyl, piperidin-4-ylmethyl and morpholin-4-ylmethyl.

Unless otherwise particularised the term "heteroaryl" denotes a monocyclic or fused bicyclic heteroaromatic ring system comprising 5 to 10 ring atoms in which one or more of the ring atoms are other than carbon, such as nitrogen, sulphur or oxygen. Only one ring need be aromatic and said heteroaryl moiety can be linked to the remainder of the molecule via a carbon or nitrogen atom in any ring. Examples of heteroaryl groups include furyl, pyrrolyl, thienyl, oxazolyl, isoxazolyl, imidazolyl, thiazolyl, isothiazolyl, pyridinyl, pyrimidinyl, tetrazolyl, quinazolinyl, indolyl, indolinyl, isoindolyl, isoindolinyl, pyrazolyl, pyridazinyl, pyrazinyl, quinolinyl, quinoxalinyl, oxadiazolyl, thiadiazolyl, benzofuranyl, 2,3-dihydrobenzofuranyl, 1,3-benzodioxolyl, 1 ,4- benzodioxmyl, benzothiazolyl, benzimidazolyl, azabenzimidazole, benzotriazolyl and chromanyl. Unless otherwise particularised the term "C _a- b-aryl" wherein a and b are integers denotes a monocyclic or fused bicyclic hydrocarbon ring system comprising a to b ring atoms and wherein at least one ring is an aromatic ring. For example "C6-io-aryl" groups include phenyl, indenyl, 2,3-dihydroindenyl (indanyl), 1-naphthyl, 2-naphthyl or 1,2,3,4- tetrahydronaphthyl.

Unless otherwise particularised the term "C _a- b-aryl-C _c- d-alkyl" wherein a, b, c and d are integers refers to a C _a _b-aryl group that is directly linked to a straight or branched C _c _d_ alkyl group. For example "C6-io-aryl-Ci-4-alkyl"groups include phenylmethyl (i.e. , benzyl) and phenylethyl.

Unless otherwise particularised the term "heteroaryl-C _a- b-alkyl" wherein a and b are integers denotes a heteroaryl ring as defined above that is directly linked to a straight or branched C _a- b-alkyl group via a carbon or nitrogen atom of said ring. For examples "heteroaryl-Ci.4-alkyl" groups include 2-(pyridin-2-yl)-ethyl and l ,2,4-oxadiazol-5- ylmethyl.

APC00482WO Unless otherwise specified in the context in which it occurs, the term "substituted" as applied to any moiety herein means substituted with up to four compatible substituents, each of which independently may be, for example, (Ci-Ce)alkyl, (Ci-C6)alkoxy, hydroxy, hydroxy(Ci-C6)alkyl, mercapto, mercapto(Ci-C6)alkyl, (Ci-C6)alkylthio, phenyl, halo (including fluoro, bromo and chloro), trifluoromethyl, trifluoromethoxy, nitro, nitnle (-CN), oxo, -COOH, -COOR ^A , -COR ^A , -S0 ₂ R ^A ,

-CONH ₂ , -SO ₂ NH ₂ , -CONHR ^A , -S0 ₂ NHR ^A , -CONR ^A R ^B , -S0 ₂ NR ^A R ^B , - H ₂ , -NHR ^A , -NR ^A R ^B , -OCONH ₂ , -OCONHR ^A , -OCONR ^A R ^B , -NHCOR ^A , -NHCOOR ^A ,

-NR ^B COOR ^A , -NHS0 ₂ OR ^A , -NR ^B S0 ₂ OH, - R ^B S0 ₂ OR ^A , -NHCON¾, -NR ^A CON¾, -NHCONHR ^B -NR ^A CONHR ^B , -NHCONR ^A R ^B _, or -NR ^A CONR ^A R ^B wherein R ^A and R ^B are independently a (Ci-Ce)alkyl, (C3-C6) cycloalkyl , phenyl or monocyclic heteroaryl having 5 or 6 ring atoms, or R ^A and R ^B when attached to the same nitrogen atom form a cyclic amino group(for example morpholino, piperidinyl, piperazinyl, or tetrahydropyrrolyl). An "optional substituent" may be one of the foregoing substituent groups.

Compounds of the invention may exist in one or more geometrical, optical, enantiomeric, diastereomeric and tautomeric forms, including but not limited to cis- and trans -ioims, E- and Z-forms, R-, S- and w¾ero-forms, keto-, and enol-forms. Unless otherwise stated a reference to a particular compound includes all such isomeric forms, including racemic and other mixtures thereof. Where appropriate such isomers can be separated from their mixtures by the application or adaptation of known methods (e.g. chromatographic techniques and recrystallisation techniques). Where appropriate such isomers may be prepared by the application of adaptation of known methods (e.g. asymmetric synthesis).

As used herein the term "salt" includes base addition, acid addition and ammonium salts. As briefly mentioned above compounds of the invention which are acidic can form salts, including pharmaceutically acceptable salts, with bases such as alkali metal hydroxides, e.g. sodium and potassium hydroxides; alkaline earth metal hydroxides e.g. calcium, barium and magnesium hydroxides; with organic bases e.g. N-methyl-D-glucamine, choline tris(hydroxymethyl)amino-methane, L-arginine, L-lysine, N-ethyl piperidine, dibenzylamine and the like. Those compounds of the invention which are basic can form salts, including pharmaceutically acceptable salts with inorganic acids, e.g. with

APC00482WO hydrohalic acids such as hydrochloric or hydrobromic acids, sulphuric acid, nitric acid or phosphoric acid and the like, and with organic acids e.g. with acetic, trifluoroacetic, tartaric, succinic, fumaric, maleic, malic, salicylic, citric, methanesulphonic, p- toluenesulphonic, benzoic, benzenesulfonic, glutamic, lactic, and mandelic acids and the like. Those compounds (I) which have a basic nitrogen can also form quaternary ammonium salts with a pharmaceutically acceptable counter-ion such as chloride, bromide, acetate, formate, p-toluenesulfonate, succinate, hemi-succinate, naphthalene- bis sulfonate, methanesulfonate, trifluoroacetate, xinafoate, and the like. For a review on salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002).

It is expected that compounds of the invention may be prepared in the form of hydrates, and solvates. Any reference herein, including the claims herein, to "compounds with which the invention is concerned" or "compounds of the invention" or "the present compounds", and the like, includes reference to salts, hydrates, and solvates of such compounds. The term 'solvate' is used herein to describe a molecular complex comprising the compound of the invention and a stoichiometric amount of one or more pharmaceutically acceptable solvent molecules, for example, ethanol. The term 'hydrate' is employed when said solvent is water.

Individual compounds of the invention may exist in an amorphous form and /or several polymorphic forms and may be obtained in different crystal habits. Any reference herein, including the claims herein, to "compounds with which the invention is concerned" or "compounds of the invention" or "the present compounds", and the like, includes reference to the compounds irrespective of amorphous or polymorphic form.

Some compounds of the invention, having a nitrogen atom in an aromatic ring, may form N-oxides, and the invention includes compounds of the invention in their N-oxide form. The Ring P

As defined above, ring P is a monocyclic aryl or heteroaryl ring having 5 or 6 ring atoms, which aryl or heteroaryl ring is optionally substituted with one or more R ¹ groups. Any such monocyclic aryl or heteroaryl ring is contemplated. In an embodiment the ring P is selected from

APC00482WO

where the bond marked * is directly connected to Q, and ring P is optionally substituted with one or more R ¹ groups. The ring P may be connected to ring Q via any P ring atom.

and wherein one or more of the ring P ring atoms is optionally substituted with one or more R ¹ groups. In an embodiment ring P is a 5-membered monocyclic heteroaryl ring optionally substituted with one or more R ¹ groups as defined in claims 1. In an alternative embodiment ring P is phenyl or pyridyl ring optionally substituted with one or more R ¹ groups.

The Ring Q

Ring Q is a monocyclic aryl or heteroaryl ring having 5 or 6 ring atoms, optionally substituted with one or more R ² groups. Any such monocyclic aryl or heteroaryl ring is contemplated herein.

In an embodiment the ring Q is selected from:

APC00482WO

wherein the bond marked ** is connected to ring P, and the bond marked *** is connected to X, and wherein one or more of the ring Q ring atoms is optionally substituted with one or more R ² groups.

In an embodiment ring Q is connected to X via a Q ring carbon atom.

In an embodiment ring Q is a monocyclic aryl or heteroaryl ring having 6 ring atoms selected from:

Q is a monocyclic heteroaryl ring having 5 ring atoms selected from:

APC00482WO

wherein the bond marked ** is connected to ring P, and the bond marked *** is connected to X, and wherein ring Q is optionally substituted with one or more R ² groups. In an embodiment ring Q is phenylene or pyridmylene either of which is optionally substituted with one or more R ² groups.

In an embodiment the bonds connecting ring P and X to ring Q are ortho to one another In other words the ring Q atoms connecting ring Q to X and ring Q to P are adjacent. For example, in the case where ring Q is a phenylene group, ring Q is substituted in the 1 -position by a direct bond to P and in the 2-position by a direct bond to X.

In an alternative embodiment the bonds connecting ring P and X to ring Q are meta to one another. In other words the ring Q atoms connecting ring Q to X and P are separated by one intermediate ring Q atom. For example, in the case where ring Q is a phenylene group, ring Q is substituted in the 1 -position by a direct bond to P and in the 3 -position by a direct bond to X.

The group X

X is -(CH ₂ )-n, wherein n is 1, 2, 3, or 4. Examples of X include straight chain methylene, ethylene, propylene, or butylene. In an embodiment n = 1. In another embodiment n = 2, in another embodiment n = 3, in a yet further embodiment n = 4. For the avoidance of doubt, compounds of formula (I) where n = 0 (in other words where X = a direct bond) do not fall within the scope of the claimed invention. Compounds having n = 0 are included for reference only, and are referred to herein as Reference Examples.

The groups R to R

R ¹ is one or more optional substituents selected from: halogen such as fluoro, chloro, and bromo, hydroxyl, oxo, cyano (-CN), Ci-4-alkyl such as methyl, ethyl, propyl, butyl, iso- propyl, sec-butyl, and tert-butyl, fluoro-Ci-4-alkyl such as trifluoromethyl or trifluoroethyl, Ci-4-alkoxy such as methoxy, ethoxy, isopropoxy, -SO ₂ R ⁴ such as - S0 ₂ NH ₂ , and -S0 ₂ CH ₃ , -NR ⁵ R ⁶ such as -NH ₂ , -NHC¾ and N(CH ₃ ) ₂ , -NR ⁷ S0 ₂ R ⁴ such as -NHSO ₂ NH ₂ , -NHSO ₂ CH3, -NHSOi'propyl, -C(0)NR ⁵ R ⁶ such as -C(0)NH ₂ , -

APC00482WO C(0)NHCH2-cyclopropyl, and -C(0)NHCH2-cyclohexyl, -NR ⁷ C(0)OR ⁵ , -NR ⁷ C(0)R ⁵ , -NR ⁷ C(0)NR ⁵ R ⁶ , -C(0)R ⁵ , -C(0)OR ⁵ , -NR ⁷ S(0) ₂ R ⁵ , and wherein any Ci _-4 -alkyl or C _M - alkoxy residue is optionally substituted with one or more substituents selected from halogen, cyano, hydroxyl, NR ⁵ R ⁶ , Ci _-4 -alkoxy, fluoro-Ci _-4 -alkoxy, -S0 ₂ R ⁴ , -NR ⁷ S0 ₂ R ⁴ , - C(0)NR ⁵ R ⁶ , -NR ⁷ C(0)OR ⁵ , -NR ⁷ C(0)R ⁵ , -NR ⁷ C(0)NR ⁵ R ⁶ , -C(0)NR ⁵ R ⁶ , -C(0)R ⁵ , - C(0)OR ⁵ , -NR ⁷ S(0) ₂ R ⁵ ,

In an embodiment R ¹ is selected from hydrogen, Ci-4-alkyl such as methyl or isopropyl, fluoro-Ci-4-alkyl such as trifluoromethyl, Ci-4-alkoxy such as methoxy, halo such as fluoro or chloro, cyano, -S0 ₂ R ⁴ such as -S0 ₂ H ₂ , S0 ₂ N(CH ₃ ) ₂ , _, S0 ₂ NHC¾ _, S0 ₂ NHCH(CH ₃ ) ₂ , and -S0 ₂ CH ₃ , -NHS0 ₂ R ⁴ such as -NHS0 ₂ NH ₂ , -NHS0 ₂ N(CH ₃ ) ₂ ,- NHS0 ₂ NHCH ₃ , -NHS0 ₂ NHCH(CH ₃ ) ₂ , and -NHS0 ₂ CH ₃ , -C(0)NR ⁵ R ⁶ such as - C(0)NH ₂ , -C(0)NHCH ₂ -cyclopropyl, and -C(0)NHCH ₂ -cyclohexyl, -NR ⁷ C(0)R ⁵ such as -NHC(0)NH ₂ , -NHC(0)NHCH ₂ -cyclopropyl, and -NHC(0)NHCH ₂ -cyclohexyl, and Ci-4-alkyl substituted with -C(0)NR ⁵ R ⁶ such as -CH ₂ C(0)NH ₂ , -CH ₂ C(0)NHCH ₂ - cyclopropyl, and -CH ₂ C(0)NHCH ₂ -cyclohexyl, and Ci-4-alkyl substituted with - NR ⁷ C(0)R ⁵ such as -CH ₂ NR ⁷ C(0)R ⁵ .

In an embodiment according to the first aspect of the invention R ² is one or more optional substituents selected from: halogen such as fluoro, chloro, and bromo, hydroxyl, oxo, cyano (-CN), Ci-4-alkyl such as methyl, ethyl, propyl, butyl, iso-propyl, sec-butyl, and tert-butyl, fluoro-Ci _-4 -alkyl such as trifluoromethyl or trifluoroethyl, Ci-4-alkoxy such as methoxy, ethoxy, isopropoxy, -S0 ₂ R ⁴ such as -S0 ₂ NH ₂ , and -S0 ₂ CH ₃ , -NR ⁵ R ⁶ such as -NH ₂ , -NHCH ₃ and N(CH ₃ ) ₂ , -NR ⁷ S0 ₂ R ⁴ such as -NHS0 ₂ NH ₂ , -NHS0 ₂ CH ₃ , - NHSOj'propyl, -C(0)NR ⁵ R ⁶ such as -C(0)NH ₂ , -C(0)NHCH2-cyclopropyl, and - C(0)NHCH2-cyclohexyl, -NR ⁷ C(0)OR ⁵ , -NR ⁷ C(0)R ⁵ , -NR ⁷ C(0)NR ⁵ R ⁶ , -C(0)R ⁵ , - C(0)OR ⁵ , -NR ⁷ S(0) ₂ R ⁵ , and wherein any Ci_4-alkyl or Ci_4-alkoxy residue is optionally substituted with one or more substituents selected from halogen, cyano, hydroxyl, NR ⁵ R ⁶ , Ci _-4 -alkoxy, fluoro-Ci _-4 -alkoxy, -S0 ₂ R ⁴ , -NR ⁷ S0 ₂ R ⁴ , -C(0)NR ⁵ R ⁶ , - NR ⁷ C(0)OR ⁵ , -NR ⁷ C(0)R ⁵ , - R ⁷ C(0)NR ⁵ R ⁶ , -C(0)NR ⁵ R ⁶ , -C(0)R ⁵ , -C(0)OR ⁵ , - NR ⁷ S(0) ₂ R ⁵

In an embodiment according to the second aspect of the invention R ² is one or more optional substituents selected from: halogen such as fluoro, chloro, and bromo, cyano (- CN), Ci-4-alkyl such as methyl, ethyl, propyl, butyl, iso-propyl, sec-butyl, and tert-butyl,

APC00482WO fluoro-Ci-4-alkyl such as trifluoromethyl or trifluoroethyl, Ci _-4 -alkoxy such as methoxy, ethoxy, isopropoxy, -S0 ₂ R ⁴ such as -S0 ₂ NH ₂ , and -S0 ₂ C¾, -NR ⁷ S0 ₂ R ⁴ such as - NHS0 ₂ H ₂ , -NHS0 ₂ CH ₃ , -NHSCVpropy -C(0)NR ⁵ R ⁶ such as -C(0)NH ₂ , - C(0)NHCH2-cyclopropyl, and -C(0)NHCH2-cyclohexyl, -NR ⁷ C(0)OR ⁵ , -NR ⁷ C(0)R ⁵ , -NR ⁷ C(0)NR ⁵ R ⁶ , -C(0)R ⁵ , -C(0)OR ⁵ , -NR ⁷ S(0) ₂ R ⁵ , and wherein any Ci _-4 -alkyl or C _M - alkoxy residue is optionally substituted with one or more substituents selected from halogen, cyano, hydroxy., NR ⁵ R ⁶ , Ci_ ₄ -alkoxy, fluoro-Ci_ ₄ -alkoxy, -S0 ₂ R ⁴ , -NR ⁷ S0 ₂ R ⁴ , - C(0)NR ⁵ R ⁶ , -NR ⁷ C(0)OR ⁵ , -NR ⁷ C(0)R ⁵ , -NR ⁷ C(0)NR ⁵ R ⁶ , -C(0)NR ⁵ R ⁶ , -C(0)R ⁵ , - C(0)OR ⁵ , -NR ⁷ S(0) ₂ R ⁵

In another embodiment according to the first and/or or the second aspects of the invention R ² is selected from hydrogen, halo such as chloro or fluoro, Ci-4-alkyl such as methyl or isopropyl, Ci _-4 -alkoxy such as methoxy, cyano, or -C(0)NR ⁵ R ⁶ such as such as -C(0)NH ₂ , -C(0)NHCH ₂ -cyclopropyl, and -C(0)NHCH ₂ -cyclohexyl, and Ci-4-alkyl substituted with -C(0)NR ⁵ R ⁶ such as -CH ₂ C(0)NH ₂ , -CH ₂ C(0)NHCH ₂ -cyclopropyl, and -CH ₂ C(0) HCH ₂ -cyclohexyl, and d _-4 -alkyl substituted with -NR ⁷ C(0)R ⁵ such as -CH ₂ R ⁷ C(0)R ⁵ .

R ³ is one or more optional substituents selected from hydrogen, halogen such as fluoro, chloro, and bromo, Ci _-4 -alkyl such as methyl, ethyl, propyl, butyl, iso-propyl, sec-butyl, and tert-butyl, fluoro-Ci _-4 -alkyl, C3_6-cycloalkyl such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl or C3.6-cycloalkyl-Ci _-4 -alkyl such as cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethy and cyclohexylmethyl, R ⁴ is independently selected from hydrogen, NR ⁵ R ⁶ , Ci _-4 -alkyl such as methyl, ethyl, propyl, butyl, iso-propyl, sec-butyl, and tert-butyl, fluoro-Ci _-4 -alkyl such at trifluoromethyl or trifluoroethyl, C3_6-cycloalkyl such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl or C3.6-cycloalkyl-Ci _-4 -alkyl such as cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethy and cyclohexylmethyl,

R ⁵ and R ⁶ are independently selected from hydrogen, Ci _-4 -alkyl such as methyl, ethyl, propyl, butyl, iso-propyl, sec-butyl, and tert-butyl, fluoro-Ci _-4 -alkyl such at trifluoromethyl or trifluoroethyl, C3-6-cycloalkyl such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl or C3.6-cycloalkyl-Ci _-4 -alkyl such as cyclopropylmethyl,

APC00482WO cyclobutylmethyl, cyclopentylmethy and cyclohexylmethyl, hydroxyCi-4-alkyl such as hydroxylethyl or hydroxylpropyl; or

R ⁵ and R ⁶ together with the nitrogen atom to which they are attached join together to form a 5 or 6-membered heterocyclic ring such as piperidine, the heterocyclic ring optionally further comprising a further heteroatom selected from nitrogen, oxygen or sulphur to form a ring such as morpholine, or piperazine wherein the heterocyclic ring is optionally substituted with one or more substituents selected from halo, hydroxyl, oxo, cyano, Ci-4-alkyl such as methyl, ethyl, propyl, butyl, iso-propyl, sec-butyl, and tert- butyl, and Ci-4-alkoxy such as methoxy or ethoxy, and such as trifluoromethyl, fluoro-Ci-4-alkoxy such as trifluoromethoxy;

R ⁷ is hydrogen, such as methyl, ethyl, propyl, butyl, iso-propyl, sec-butyl, and tert-butyl, fluoro-Ci-4-alkyl such as trifluoromethyl, C3_6-cycloalkyl such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl or such as cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethy and cyclohexylmethyl,.

R ¹⁰ , R ¹¹ , and R ¹² are independently selected from hydrogen, Ci-4-alkyl, fluoro-Ci-4-alkyl, C3-6-cycloalkyl, or In an embodiment R ¹⁰ , R ¹¹ , and R ¹² are independently selected from hydrogen, methyl, isopropyl and cyclopropyl. In an embodiment R ¹⁰ , R ¹¹ , and R ¹² are hydrogen.

The group Y

In an embodiment Y is -C(O)-.

Unless specified to the contrary, the groups P, Q, X, Y, R ¹ , R ² , R ³ , Rio, Rn, and R12 as defined above may be present in any combination in the compounds of formula (I) according to the first and/or the second aspects of the invention. Specific Examples of compounds according to the invention include:

N-[(4-Carbamimidoylphenyl)methyl]-2-(3-phenylphenyl)aceta mide,

N-[(4-Carbamimidoylphenyl)methyl]-3-[3-(4-sulfamoylphenyl )phenyl]propanamide, N-[(4-Carbamimidoylphenyl)methyl]-2-[3-(4-sulfamoylphenyl)ph enyl]acetamide, N-[(4-Carbamimidoylphenyl)methyl]-2-[3-(3-sulfamoylphenyl)ph enyl]acetamide, N-[(4-Carbamimidoylphenyl)methyl]-2-(3-{4-[(2-hydroxyethyl)s ulfamoyl]phenyl}

APC00482WO phenyl)acetamide,

N-[(4-Carbamimidoylphenyl)methyl]-2-[3-(2-methyl-4-sulfamoyl phenyl)phenyl] acetamide,

and a pharmaceutically acceptable salt, solvate or hydrate thereof.

Reference Examples of compounds having n = 0 are listed below:

N-[(4-Carbamimidoylphenyl)methyl]-3-phenylbenzamide hydrochloride,

N-[(4-Carbamimidoylphenyl)methyl]-3-[3-(acetamidomethyl)p henyl]benzamide, N-[(4-Carbamimidoylphenyl)methyl]-2-phenylbenzamide,

N-[(4-carbamimidoylphenyl)methyl]-3-[5-(acetamidomethyl)- 2-methylphenyl]-4- methoxybenzamide,

N-[(4-Carbamimidoylphenyl)methyl]-3-(pyridin-3-yl)benzamide,

N-[(4-Carbamimidoylphenyl)methyl]-3-(lH-pyrazol-5-yl)benzami de,

N-[(4-Carbamimidoylphenyl)methyl]-4-phenylpyridine-2-carboxa mide,

N-[(4-Carbamimidoylphenyl)methyl]-3-(pyridin-4-yl)benzami de,

N-[(4-Carbamimidoylphenyl)methyl]-5-phenylpyridine-3-carboxa mide,

N-[(4-Carbamimidoylphenyl)methyl]-3-(2-methylphenyl)benza mide,

N- [(4-Carbamimidoylphenyl)methyl] -2-hy droxy-5 -phenylbenzamide,

N-[(4-Carbamimidoylphenyl)methyl]-2-fluoro-5-phenylbenzam ide,

2-(4-Bromophenyl)-N-[(4-carbamimidoylphenyl)methyl]benzam ide,

N-[(4-carbamimidoylphenyl)methyl]-2-(pyridin-3-yl)benzami de,

N-[(4-carbamimidoylphenyl)methyl]-2-(3,4-dichlorophenyl)b enzamide,

N-[(4-carbamimidoylphenyl)methyl]-2-(4-chlorophenyl)benza mide,

N-[(4-carbamimidoylphenyl)methyl]-2-(4-methoxyphenyl)benz amide,

N-[(4-carbamimidoylphenyl)methyl]-4-fluoro-2-(4-methoxyph enyl)benzamide, N-[(4-carbamimidoylphenyl)methyl]-4-fluoro-2-(pyridin-3-yl)b enzamide,

N-[(4-carbamimidoylphenyl)methyl]-2-(3,4-dichlorophenyl)- 4-fluorobenzamide, N-[(4-carbamimidoylphenyl)methyl]-2-(4-chlorophenyl)-4-fluor obenzamide,

1 -N- [(4-Carbamimidoylphenyl)methyl] -3 -N-(cyclopropylmethyl)-5 -phenylbenzene- 1,3- dicarboxamide,

N-[(4-carbamimidoylphenyl)methyl]-2-[4-(trifluoromethyl)phen yl]benzamide, N-[(4-Carbamimidoylphenyl)methyl]-2-(4-sulfamoylphenyl)benza mide,

2-ammo-N-[(4-carbamimidoylphenyl)methyl]-5-phenylbenzamid e,

N-[(4-carbamimidoylphenyl)methyl]-2-methyl-3-phenylbenzam ide,

N-[(4-carbamimidoylphenyl)methyl]-4-methyl-3-phenylbenzam ide,

APC00482WO N-[(4-carbamimidoylphenyl)methyl]-4-chloro-3-phenylbenzamide ,

N-[(4-Carbamimidoylphenyl)methyl]-2-chloro-3-phenylbenzam ide,

N-[(4-Carbamimidoylphenyl)methyl]-4-(4-methoxyphenyl)pyri dine-3-carboxamide, N-[(4-carbamimidoylphenyl)methyl]-2-(3-methanesulfonylphenyl )benzamide,

N-[(4-Carbamimidoylphenyl)methyl]-4-(4-sulfamoylphenyl)py ridine-3-carboxamide, N-[(4-Carbamimidoylphenyl)methyl]-3-(4-sulfamoylphenyl)pyrid ine-4-carboxamide, N-[(4-Carbamimidoylphenyl)methyl]-3-(4-sulfamoylphenyl)benza mide,

N-[(4-Carbamimidoylphenyl)methyl]-3-(3-sulfamoylphenyl)be nzamide,

N-[(4-Carbamimidoylphenyl)methyl]-3-[2-(dimethylsulfamoyl )phenyl]benzamide, N-[(4-Carbamimidoylphenyl)methyl]-2-[4-(sulfamoylamino)pheny l]benzamide, N-[(4-Carbamimidoylphenyl)methyl]-4-fluoro-2-(4-sulfamoylphe nyl)benzamide, 4-[(3-Phenylbenzene)sulfonamidomethyl] benzene- 1 -carboximidamide,

N-[(4-Carbamimidoylphenyl)methyl] -3 -(1 -methyl- lH-imidazol-2-yl)benzamide, N-[(4-Carbamimidoylphenyl)methyl]-2-[4-(methylsulfamoyl)phen yl]benzamide, N-[(4-Carbamimidoylphenyl)methyl]-2- {4-[(propan-2-yl)sulfamoyl]phenyl}benzamide, N-[(4-Carbamimidoylphenyl)methyl]-2-[4-(dimethylsulfamoyl)ph enyl]benzamide, N-[(4-Carbamimidoylphenyl)methyl]-2-(3-methyl-4-sulfamoylphe nyl)benzamide, N-[(4-Carbamimidoylphenyl)methyl]-2-(3-fluoro-4-sulfamoylphe nyl)benzamide, N-[(4-Carbamimidoylphenyl)methyl]-3-(2-methyl-4-sulfamoylphe nyl)benzamide, N-[(4-Carbamimidoylphenyl)methyl]-2-(4-carbamoylphenyl)benza mide,

N-[(4-carbamimidoylphenyl)methyl]-2-(2-methyl-4-sulfamoylphe nyl)benzamide, N-[(4-Carbamimidoylphenyl)methyl]-2-[4-(moφholine-4-sulfony l)phenyl]benzamide, N-[(4-Carbamimidoyl-2-chlorophenyl)methyl]-3-phenylbenzamide ,

N-[(4-Carbamimidoyl-2-methylphenyl)methyl]-3-phenylbenzam ide,

N-[(4-carbamimidoylphenyl)methyl]-2-(4-methanesulfonamido phenyl)benzamide, N-[(4-Carbamimidoylphenyl)methyl]-3-(3-cyanophenyl)benzamide ,

N-[(4-Carbamimidoylphenyl)methyl]-3 -phenyl- l,2-thiazole-5-carboxamide,

N-[(4-Carbamimidoylphenyl)methyl]-5-phenyl-lH-pyrazole-3- carboxamide,

N-[(4-Carbamimidoylphenyl)methyl]-3-phenyl-l,2-thiazole-4 -carboxamide,and a pharmaceutically acceptable salt, solvate or hydrate thereof. it is contemplated

that the intermediates described herein can also be used as therapeutic agents to treat or more of the diseases or conditions described throughout

APC00482WO Synthesis

The compounds of formula (I) above may be prepared by, or in analogy with, conventional methods. The preparation of intermediates and compounds according to the Examples of the present invention may in particular be illustrated by, but not limited to, the following Schemes. Definitions of variables in the structures in Schemes herein are commensurate with those of corresponding positions in the formulas delineated herein.

Scheme 1. General synthetic route for preparation of compounds of formula (I)

wherein P, Q, X, Y, R ¹ , R ² and R ³ are as defined in formula (I);

APC00482WO Compounds of general formula (I) can easily be prepared from compounds of general formula II by aminolysis (for example by treatment with HC1 then ammonia, or by treatment with hydroxylamine then acetic anhydride and subsequent hydrogenation). Compounds of general formula (II) can be easily prepared from compounds of general formula (III) by amide coupling with compounds of general formula (IV) and subsequent Suzuki coupling, or by reversing these two steps. Alternatively, compounds of general formula (I) can be prepared from compounds of general formula (V) by amide coupling with compounds of general formula (VI). Compounds of general formula (I) containing a sulfonamide group (Υ=8(¾) can be easily prepared by coupling of sulfonyl chlorides of general formula (VII) with amines of general formula (IV) and subsequent ammolysis.

Optionally, a compound of formula (I) can also be transformed into another compound of formula (I) in one or more synthetic steps. All of these alternatives are exemplified in the experimental section below.

Scheme 2. General s nthetic route for preparation of compounds of formula (IV)

wherein R is as defined in formula (I);

Compounds of general formula (IV) can be easily prepared from benzoic acids of general formula (IVa) by reduction of the carboxylic acid and cyanide displacement of the bromo group to give alcohols of general formula (IVb). Compounds of general formula (IVb) can be easily converted into compounds of general formula (IV), for example by bromo-dehydroxylation and aminolysis (for example with B0C ₂ NH and subsequent Boc deprotection).

APC00482WO The following abbreviations have been used:

APC00482WO PyBop Benzotnazol- 1 -yl-oxytripyrrolidinophosphonium hexafluorophospliate

Rt Retention time

sat saturated

TFA Trifluoroacetic acid

THF Tetrahydrofuran

TLC Thin Layer Chromatography

EXAMPLES AND INTERMEDIATE COMPOUNDS

Experimental Methods

All reagents were commercial grade and were used as received without further purification, unless otherwise specified. Reagent grade solvents were used in all cases. Analytical LCMS was performed on either an Agilent 1100 system equipped with a Phenomenex Synergi, RP-Hydro, 150 x 4.6mm, 4um column (MeCN in water (+0.085% TFA), 200-300nm, 30°C) or Agilent 1 100 system equipped with a Phenomenex Gemini, CI 8, 100 x 4.6mm, 4um column (MeCN in water (lOmM ammonium bicarbonate), 200- 300nm, 40°C). Accurate masses were measured using a Waters QTOF electrospray ion source and corrected using Leucine Enkephalin lockmass. Spectra were acquired in positive electrospray mode. The acquired mass range was m/z 100-1000. Samples were dissolved in DMSO to give lmg/mL solutions which were then further diluted with Acetonitnle (50%) / Water (50%) to ^g/mL solutions prior to analysis. The values reported correspond to the protonated molecular ions [MH]+. Analytical FIPLC was performed on an Agilent 1100 system using a Phenomenex Synergi, RP-Hydro, 150 x 4.6mm, 4um column with a flow rate of 1.5mL per min at 30°C (200-3 OOnm) and a gradient of 5-100% MeCN (+0.085% TFA) in water (+0.1% TFA) over 7mm. Mass directed HPLC was performed on a Waters system using a Phenomenex Gemini, CI 8, 100 x 21mm, 5um column with a flow rate of 20mL per min over 14.5min. Flash chromatography was performed on either a CombiFlash Companion system equipped with RediSep silica columns or a Flash Master Personal system equipped with Strata SI- 1 silica gigatubes or in a glass column under gravity. Reverse Phase HPLC was performed on a Gilson system (Gilson 322 pump with Gilson 321 equilibration pump and Gilson 21 autosampler) equipped with Phenomenex Synergi Hydro RP 150 x 10mm, or YMC ODS-A 100/1 0 x 20mm columns. Microwave irradiations were carried out using a Biotage microwave. Hydrogenations were performed using a Thales H-cube Nano. Reactions were performed at room temperature unless otherwise stated. The

APC00482WO compounds were automatically named using ACD 6.0. All compounds were dried in a vacuum oven overnight. Where yields are not included, the intermediates were used crude. Reactions were monitored by TLC, LCMS or HPLC. INTERMEDIATE 1

-Dimethyl 5-bromobenzene-l,3-dicarboxylate

5-Bromobenzene-l,3-dicarboxylic acid (l.OOg, 4.08mmol) was dissolved in DCM (20mL) and oxalyl chloride (876uL, 10.2mmol) and DMF (200uL) were added. The reaction mixture was stirred for 2h and concentrated in vacuo. The residue was dissolved in MeOH (20mL), stirred for 18h and partitioned between DCM (lOOmL) and sat aq Na2CC>3 (50mL). The aqueous fraction was extracted with DCM (50mL) and the combined organic fractions were dried (MgS04) and concentrated in vacuo. The residue was purified by column chromatography to give the title compound (1.04g, 93%) as a white solid. LCMS: ES ⁺ 272.9, 274.9 [MH] ⁺ .

INTERMEDIATE 2

-Dimethyl 5-phenylbenzene-l,3-dicarboxylate

Intermediate 1 (1.04g, 3.81mmol), phenylboronic acid (510mg, 4.19mmol), Pd(PPh ₃ ) ₄ (220mg, 0.19mmol) and CS2CO3 (1 36g, 4.19mmol) were dissolved in water (2mL),

PhMe (21mL) and EtOH (4mL). The reaction mixture was heated under reflux at 100°C for 6h, filtered through celite and concentrated in vacuo. The residue was purified by column chromatography to give the title compound (843 mg, 82%) as a white solid. LCMS: ES ⁺ 271.2 [MH] ⁺ .

INTERMEDIATE 3

3-(Ethoxycarbonyl)-5-phenylbenzoic acid

APC00482WO

Intermediate 2 (843mg, 3.12mmol) was dissolved in dioxane (lOmL) and EtOH (30mL), Lithium hydroxide monohydrate (260mg, 6.21mmol) was added and the reaction mixture was stirred 3d. The reaction mixture was acidified to pH 2 with 1M aq HC1, diluted with water (lOmL) and extracted with EtOAc (3x20mL). The combined organic fractions were washed with brine (l OmL), dried (MgSC>4) and concentrated in vacuo to give the title compound (843mg, 100%) as a white solid. LCMS: ES ⁺ 271.3 [MH] ⁺ .

INTERMEDIATE 4

4'-Meth lsulfamoyl-biphenyl-2-carboxylic acid

2-Carboxybenzeneboronic acid (0.20g, 1.21mmol), 4-bromo-N- methylbenzenesulphonamide (0.36g, 1.45mmol), Na ₂ C ⁽ ¾ (0.26g, 2.42mmol) and Pd(PPh ₃ ) ₄ (140mg, 0.12mmol) were suspended in dioxane/water (4mL, 1 : 1) and heated using a microwave reactor at 150°C for 20min. The reaction mixture was concentrated in vacuo, dissolved in EtOAc (30mL) and washed with water (2x3 OmL). The combined aqueous fractions were acidified to pH 1 with 1M aq HC1 and extracted with EtOAc (2x30mL). The combined organic fractions were washed with brine (30mL), dried (MgS0 ₄ ) and concentrated in vacuo to give the title compound (259mg, 74%) as a white solid. LCMS: ES ⁺ 292.0 [MH] ⁺ .

INTERMEDIATE 5

- 4- Propan-2-yl)sulfamoyl]phenyl}benzoic acid

APC00482WO 4-Chlorobenzenesulfonyl chloride (150mg, 0.71mmol), isopropylamine (0.18mL, 2.13mmol) and DIPEA (0.13mL, 0.78mmol) were dissolved in THF and stirred for lh. The reaction mixture was concentrated in vacuo and purified by column chromatography. The residue, 2-carboxybenzeneboronic acid (210mg, 1.42mmol), Pd(PPh ₃ ) ₄ (70.0mg, 0.06mmol) and Na ₂ C0 ₃ (130mg, 1.24mmol) were suspended in dioxane / water (4mL, 1 : 1) and heated using a microwave reactor at 150°C for 20min. The reaction mixture was concentrated in vacuo and purified by column chromatography. The residue was suspended in Et ₂ 0, sonicated, filtered and concentrated in vacuo to give the title compound (91.0mg, 40%) as a pale yellow solid. LCMS: ES ⁺ 320.0 [MH] ⁺ .

INTERMEDIATE 6

4'-Dimeth lsulfamoyl-biphenyl-2-carboxylic acid

Intermediate 6 was prepared similarly to Intermediate 5, using dimethylamine instead of isopropylamine, to give the title compound (238mg, 82%) as a white solid. LCMS: ES ⁺ 306.0 [MH] ⁺ .

INTERMEDIATE 7

2- 4-(Morpholine-4-sulfonyl)phenyl] benzoic acid

ntermediate 7 was prepared similarly to Intermediate 5, using morpholine instead of isopropylamine, to give the crude title compound (432mg) as a white solid. LCMS: ES ⁺ 348.0 [MH] ⁺ .

INTERMEDIATE 8

APC00482WO 3'-Meth l-4'-sulfamoyl-biphenyl-2-carboxylic acid

2-Carboxybenzeneboronic acid (200mg, 1.21mmol), 4-bromo-2- methylbenzenesulphonamide (360mg, 1.45mmol), NaiCC (0.26g, 2.42mmol) and Pd(PPh ₃ ) ₄ (140mg, 0.12mmol) were suspended in dioxane / water (4mL, 1 : 1) and heated using a microwave reactor at 150°C for 20min. The reaction mixture was concentrated in vacuo, dissolved in EtOAc (30mL) and washed with 1M aq Na2CC>3 (2x3 OmL). The combined aqueous fractions were acidified to pH 1 with 1M aq HCl and extracted with EtOAc (2x40mL). The combined organic fractions were washed with brine (50mL), dried (MgSC^) and concentrated in vacuo to give the title compound (273 mg, 78%) as a yellow solid. LCMS: ES ⁺ 292.0 [MH] ⁺ .

INTERMEDIATE 9

3'-Fluoro-4'-sulfamoyl-biphenyl-2-carboxylic

Intermediate 9 was prepared similarly to Intermediate 8, using 4-bromo-2- fluorobenzenesulphonamide instead of 4-bromo-2-methylbenzenesulphonamide, to give the crude title compound (360mg) as a white solid. LCMS: ES ⁺ 296.0 [MH] ⁺ .

INTERMEDIATE 10

2- 3-(4-sulfamoylphenyl)phenyl] acetic acid

Intermediate 10 was prepared similarly to Intermediate 8 using 2-(3-bromophenyl)acetic APC00482WO acid instead of 4-bromo-2-methylbenzenesulphonamide and (4- sulfamoylphenyl)boronic acid instead of 2-carboxybenzeneboronic acid, to give the crude title compound (1.09g) as a black solid. LCMS: ES ⁺ 291.9 [MH] ⁺ .

INTERMEDIATE 11

3- 2-Methyl-4-sulfamoylphenyl)benzoic

Intermediate 11 was prepared similarly to Intermediate 8, using 4-bromo-3- methylbenzenesulphonamide instead of 4-bromo-2-methylbenzenesulphonamide and 3- methoxycarbonylphenylboronic acid instead of 2-carboxybenzeneboronic acid, to give the title compound (218mg, 27%) as a white solid. LCMS: ES ⁺ 292.0 [MH] ⁺ .

INTERMEDIATE 12

2- 4-Carbamoylphenyl)benzoic

Intermediate 12 was prepared similarly to Intermediate 8, using 4-bromobenzamide instead of 4-bromo-2-methylbenzenesulphonamide and 2- methoxycarbonylphenylboronic acid instead of 2-carboxybenzeneboronic acid, to give the title compound (216mg, 54%) as a grey solid. LCMS: ES ⁺ 242.0 [MH] ⁺ .

INTERMEDIATE 13

2'-Meth l-4'-sulfamoyl-biphenyl-2-carboxylic acid

Intermediate 13 was prepared similarly to Intermediate 8, using 4-bromo-3- methylbenzenesulphonamide instead of 4-bromo-2-methylbenzenesulphonamide, to give

APC00482WO the crude title compound (161mg) as a white solid. LCMS: ES ⁺ 292.0 [MH] ⁺ .

INTERMEDIATE 14

-Bromo-2-chlorophenyl)methanol

4-Bromo-2-chlorobenzoic acid (4.70g, 20.0mmol) was dissolved in THF (50mL), cooled and 1M BH ₃ in THF (22mL, 22.0mmol) was added drop-wise. The reaction mixture was stirred at room temperature overnight, cooled to -10°C, quenched with water (15mL), poured onto sat aq NaHCOs (lOOmL) and extracted with EtOAc. The combined organic fractions were washed with water, brine, dried (MgSO^ and concentrated in vacuo to give the crude title compound (4.45g) as a white solid. LCMS: ES ⁺ 220 [MH] ⁺ .

INTERMEDIATE 15

-Bromo-2-methylphenyl)methanol

Intermediate 15 was prepared similarly to Intermediate 14, using 4-bromo-2- methylbenzoic acid instead of 4-bromo-2-chlorobenzoic acid, to give the title compound (8.80g, 88%) as a white solid. LCMS: ES ^" 199 [M-H] ^" .

INTERMEDIATE 16

-Chloro-4-(hydroxymethyl)benzonitrile

Intermediate 14 (1.77g, 8.00mmol), ZnCN ₂ (564mg, 4.80mmol) and Pd(PPh ₃ ) ₄ (370mg, 0.32mmol) were dissolved in DMF (20mL) and the reaction mixture was heated at 100°C overnight. The reaction mixture was cooled to room temperature, diluted with water, brine and EtOAc and filtered through celite. The aqueous fraction was extracted with EtOAc and the combined organic fractions were washed with 2M aq NH ₄ OH, brine, dned (MgSO/t) and concentrated in vacuo. The residue was triturated from hexanes to give the crude title compound (1.15 g) as a pink solid. LCMS: ES ⁺ 167.2 [MH] ⁺ .

APC00482WO INTERMEDIATE 17

-(Hydroxymethyl)-3-methylbenzonitrile

Intermediate 17 was prepared similarly to Intermediate 16, using Intermediate 15 instead Intermediate 14, to give the title compound (4.70g, 41%) as a white solid. LCMS: ES ^" 146 [M-H] ^" .

INTERMEDIATE 18

-(Bromomethyl)-3-chlorobenzonitrile

Intermediate 16 (5.03g, 30.0mmol) and CBr ₄ (10.9g, 33.0mmol) were suspended in DCM (90mL), cooled to 5°C and PPh ₃ (8.66g, 33.0mmol) was added. The reaction mixture was stirred at room temperature for 2.5h. Hexane / EtOAc (200mL, 9: 1) was added and the reaction mixture was filtered. The filtrate was concentrated in vacuo and purified by normal phase chromatography to give the title compound (6.20g, 90%) as a white solid. HPLC: 98% purity.

INTERMEDIATE 19

-(Bromomethyl)-3-methylbenzonitrile

Intermediate 19 was prepared similarly to Intermediate 18, using Intermediate 17 instead Intermediate 16, to give the title compound (5.50g, 90%) as a white solid.

INTERMEDIATE 20

-(Aminomethyl)-3-chlorobenzonitrile hydrochloride

Intermediate 18 (4.61g, 20.0mmol) was dissolved in THF (20mL) and added drop-wise to a suspension of NaH (960mg, 60% suspension in mineral oil, 24.0mmol) in THF

APC00482WO (20mL) at 5°C. A solution of (Boc) ₂ NH (4.80g, 22.0mmol) in THF (20mL) was added drop-wise over lOmin and the reaction mixture was stirred at 5°C for 15min and warmed to room temperature overnight. The reaction mixture was quenched with 15% aq NH ₄ C1 (50mL) and the THF was removed in vacuo. The aqueous solution was diluted with brme and extracted with DCM (1L). The organic fraction was washed with brine, dried (MgS04) and concentrated in vacuo. The residue was dissolved in EtOAc (150mL), cooled and HC1 (g) was bubbled through the solution for 15min. The reaction mixture was stirred at room temperature for 6h and allowed to stand at 5°C overnight. The resulting precipitate was collected by filtration and washed with hexanes to give the crude title compound (3.47g, 85%) as a white solid.

INTERMEDIATE 21

-(Aminomethyl)-3-methylbenzonitrile hydrochloride

Intermediate 21 was prepared similarly to Intermediate 20, using Intermediate 19 instead Intermediate 18, to give the title compound (3.25g, 89%) as a white solid.

INTERMEDIATE 22

-[(4-Cyanophenyl)methyl]-3-phenylbenzamide

Biphenyl-3-carboxylic acid (500mg, 2.52mmol) and 4-aminomethyl-benzonitrile (425mg, 2.52mmol) were suspended in DCM (25mL) and DIPEA (873uL, 5.04mmol), EDC (484mg, 2.52mmol) and HOBt (386mg, 2.52mmol) were added. The reaction mixture was stirred overnight, concentrated in vacuo and partitioned between EtOAc (lOOmL) and water (lOOmL). The organic fraction was washed with water (l OOmL), sat aq NaHCC>3 (50mL), brine (50mL), dried (MgSC ) and concentrated in vacuo to give the title compound (738mg, 94%) as a brown solid. LCMS: ES ⁺ 313.0 [MH] ⁺ .

INTERMEDIATE 23

N- [(2-Chloro-4-cyanophenyl)methyl] -3-phenylbenzamide

APC00482WO

Intermediate 23 was prepared similarly to Intermediate 22, using Intermediate 20 instead of 4-aminomethyl-benzonitnle, to give the title compound (1.44g, 69%) as a white solid. LCMS: ES ⁺ 347.1 [MH] ⁺ .

INTERMEDIATE 24

- [(4-Cyano-2-methylphenyl)methyl] -3-phenylbenzamide

Intermediate 24 was prepared similarly to Intermediate 22, using Intermediate 21 instead 0 of 4-aminomethyl-benzonitnle, to give the title compound (1.20g, 61%) as a white solid.

LCMS: ES ⁺ 327 [MH] ⁺ .

INTERMEDIATES 25-39

Intermediates 25-39 were prepared similarly to Intermediate 22 using 4-aminomethyl- 5 benzonitrile and the appropriate carboxylic acid; see Table 1 below.

Table 1: Amide couplings.

APC00482WO 2-(4-Bromophenyl)-N-[(4- Used ES ⁺ 391.1 cyanophenyl)methyl]benzamide crude [MH] ⁺

Br

4'-Amino-biphenyl-2-carboxylic Used ES ⁺ 328.0 acid 4-cyano-benzylamide crude [MH] ⁺

NH ₂

N-[(4-Cyanophenyl)methyl]-2- Used ES ⁺ 314.3 (pyridin-3 -yl)benzamide crude [MH] ⁺

Used

N-[(4-Cyanophenyl)methyl]-2-[4- crude ES ⁺ 405.9

(methylsulfamoyl)phenyl]

from [MH] ⁺ benzamide

o=s=o Int 4

^NH

Used

N-[(4-Cyanophenyl)methyl]-2-{4- crude ES ⁺ 434.0

[(propan-2-yl)sulfamoyl]phenyl}

from [MH] ⁺ o=s=o benzamide

Int 5

Used

N-[(4-Cyanophenyl)methyl]-2-[4- crude

(dimethylsulfamoyl)phenyl] - from

benzamide

o=s=o Int 6

APC00482WO

APC00482WO

INTERMEDIATE 40

3-{[(4-Cyanophenyl)methyl]carbamoyl}-5-phenylbenzoic acid

Intermediate 38 (711mg, 1.85mmol) was dissolved in THF/water (40mL, 1 :1), lithium hydroxide monohydrate (171mg, 4.07mmol) was added and the reaction mixture was stirred for 18h. The THF was removed in vacuo and the aqueous solution was acidified to pH 1 with 1M aq HCl and extracted with EtOAc (3x50mL). The combined organic fractions were dried (MgSC^) and concentrated in vacuo to give the title compound (415mg, 63%) as a white solid. LCMS: ES ⁺ 357.2 [MH] ⁺ .

INTERMEDIATE 41

l-N-[(4-Cyanophenyl)methyl]-3-N-(cyclopropylmethyl)-5-phenyl benzene-l,3- dicarboxamide

Intermediate 40 (200mg, 0.56mmol) was dissolved in DCM (5mL), EDC.HC1 (131mg, 0.84mmol), HONB (151mg, 0.84mmol), cyclopropanemethylamine (49uL, 0.56mmol) and DIPEA (440uL, 2.53mmol) were added and the reaction mixture was stirred for 18h The reaction mixture was concentrated in vacuo and partitioned between DCM (lOOmL)

APC00482WO and sat aq Na2CC>3 solution (50mL). The aqueous fraction was extracted with DCM

(50mL) and the combined organic fractions were dried (MgSC^) concentrated in vacuo.

The residue was purified by column chromatography to give the title compound (121mg, 53%) as a white solid. LCMS: ES ⁺ 410.2 [MH] ⁺ .

INTERMEDIATE 42

-Bromo-N- [(4-cyanophenyl)methyl] benzamide

3-Bromobenzoic acid (2.51g, 12.5mmol) was dissolved in DCM (40mL), oxalyl chloride 10 (1.95mL, 22.7mmol) and DMF (lOOuL) were added and the reaction mixture was stirred for 2h. The reaction mixture was concentrated in vacuo and the residue was dissolved in DCM (40mL) and DIPEA (9.88mL, 56.8mmol), and 4-(aminomethyl)benzonitrile (1.50g, 11.4mmol) was added. The reaction mixture was stirred for 3d and partitioned between DCM (200mL) and sat aq Na2C03 (lOOmL). The aqueous fraction was

15 extracted with DCM (lOOmL) and the combined organic fractions were dried (MgSC^) and concentrated in vacuo to give the crude title compound (3.58g) as a brown gum. LCMS: ES ⁺ 314.9, 316.8 [MH] ⁺ .

INTERMEDIATES 43-51

20 Intermediates 43-51 were prepared similarly to Intermediate 42, using 4- (aminomethyl)benzonitrile and the appropriate carboxylic acid; see Table 2 below.

Table 2: Amide couplings.

APC00482WO 2- Amino-5 -bromo-N- [(4- LCMS: ES ⁺ 330.9,

20%

cyanophenyl)methyl]benzamide 331.9 [MH] ⁺ o

3-Bromo-4-chloro-N-[(4- LCMS: ES ⁺ 348.8,

78%

cyanophenyl)methyl]benzamide 350.9 [MH] ⁺ o

3-Bromo-2-chloro-N-[(4- LCMS: ES ⁺ 348.8,

58%

cyanophenyl)methyl]benzamide 350.9 [MH] ⁺

CI o

2-(3-Bromophenyl)-N-[(4- Used LCMS: ES ⁺ 328.9, cyanophenyl)methyl]acetamide crude 330.9 [MH] ⁺

Br

4-Chloro-N-[(4-

LCMS: ES ⁺ 271.9 cyanophenyl)methyl]pyridine-3- 69%

[MH] ⁺

CI o carboxamide

2-Chloro-N-[(4- LCMS: ES ⁺ 271.2

54%

cyanophenyl)methyl]benzamide [MH] ⁺

CI o

3 -Bromo-N- [(4-

LCMS: ES ⁺ 315.9, cyanophenyl)methyl]pyridine-4- 72%

317.9 [MH] ⁺

Br O carboxamide

2-Chloro-N-[(4-

LCMS: ES ⁺ 289.1 cyanophenyl)methyl]-4- 57%

[MH] ⁺

CI 0 fluorobenzamide

3-(3-Bromophenyl)-N-[(4-

LCMS: ES ⁺ 342.9, cyanophenyl)methyl] 75%

344.8 [MH] ⁺ o propanamide

INTERMEDIATE 52

N-[(4-Cyanophenyl)methyl]-3-[3-(acetamidomethyl)phenyl]benza mide

APC00482WO

Intermediate 42 (575mg, 1.82mmol), N-{[3-

(dihydroxyboranyl)phenyl]methyl}acetamide (352mg, 1.82mmol), Pd(PPh ₃ )4 (169mg, 0.146mmol) and Na2CC>3 (483mg, 4.56mmol) were dissolved in water (2mL) and

5 dioxane (2mL) and heated using a microwave reactor at 150°C for 20min. The reaction mixture was concentrated in vacuo and the residue was partitioned between DCM (50mL) and water (30mL). The aqueous fraction was extracted with DCM (30mL) and the combined organic fractions were dried (MgSOzi) and concentrated in vacuo. The residue was purified by column chromatography to give the title compound (401mg, 10 57%) as a yellow solid. LCMS: ES ⁺ 384.0 [MH] ⁺ .

INTERMEDIATES 53-84

Intermediates 53-84 were prepared similarly to Intermediate 52 using Intermediate 42 (unless otherwise stated) and the appropriate boronic acid / ester; see Table 3 below.

15

Table 3: Suzuki couplings.

APC00482WO N-[(4-Cyanophenyl)methyl]-4- ES ⁺ 314.0

91%

Χ phenylpyridine-2-carboxamide [MH] ⁺ o

N-[(4-Cyanophenyl)methyl]-3- ES ⁺ 314.0

69%

(pyridin-4-yl)benzamide [MH] ⁺

N-[(4-Cyanophenyl)methyl]-5- ES ⁺ 314.0

97%

X phenylpyridine-3 -carboxamide [MH] ⁺ o

N-[(4-Cyanophenyl)methyl]-3-(2- ES ⁺ 327.2

42%

methylphenyl)benzamide [MH] ⁺

N-[(4-Cyanophenyl)methyl]-2- ES ⁺ 329.3

9%

hydroxy-5-phenylbenzamide [MH] ⁺

N-[(4-Cyanophenyl)methyl]-2- ES ⁺ 331.2

30%

fluoro-5-phenylbenzamide [MH] ⁺ o

N-[(4-Cyanophenyl)methyl]-2- Used ES ⁺ 381.1 (3,4-dichlorophenyl)benzamide crude [MH] ⁺

CI

2-(4-Chlorophenyl)-N-[(4- Used ES ⁺ 347.2 cyanophenyl)methyl]benzamide crude [MH] ⁺

CI

APC00482WO

APC00482WO

APC00482WO

APC00482WO

INTERMEDIATE 85

tert-Butyl N-{[4-(2-{[(4- }phenyl)phenyl]sulfamoyl}carbamate

Chlorosulfonyl isocyanate (27.0uL, 0.31mmol) was dissolved in DCM (lmL), cooled to 0°C and i-butanol (29.0uL, 0.31mmol) was added. The reaction mixture was stirred for 90min and added drop-wise with EtsN (0.14mL, l.Olmmol) to a solution of Intermediate 27 (lOOmg, 0.31mmol) and Et ₃ N (0.08mL, 0.58mmol) in DCM at 0°C. The reaction mixture was warmed to room temperature, stirred for 3h, diluted with DCM (20mL) and washed with sat aq NH ₄ C1 (20mL), brine (20mL), dried (MgS0 ₄ ) and concentrated in vacuo. The residue was dissolved in EtOAc (5mL), filtered and purified by column chromatography to give the title compound (120mg, 82%) as a yellow oil. INTERMEDIATE 86

-[(4-Cyanophenyl)methyl]-3-phenylbenzene-l-sulfonamide

3- Phenylbenzene-l-sulfonyl chloride (250mg, 0.99mmol), Et ₃ N (412uL, 2.97mmol) and

4- (aminomethyl)benzonitrile (261mg, 1.98mmol) were dissolved in DCM (lOmL) and stirred for 3h. The reaction mixture was quenched with water and diluted with DCM (50mL). The organic fraction was washed with brine (30mL), dried (MgSO/i) and concentrated in vacuo to give the crude title compound (345mg) as a yellow liquid.

APC00482WO LCMS: ES ⁺ 349.0 [MH] ⁺ .

INTERMEDIATE 87

Eth l 3-(3-cyanophenyl)benzoate

Ethyl 3-iodobenzoate (939mg, 3.40mmol) was dissolved in DMF (2mL) and a solution of 3-cyanophenylboronic acid (500mg, 3.40mmol) in DMF (5mL) was added. A solution of Pd(OAc) ₂ (15mg, 0.068mmol) and Na ₂ C0 ₃ (858mg, 10.2mmol) in water (2mL) was added and the reaction mixture was heated at 110°C for 2d. The reaction mixture was concentrated in vacuo and partitioned between DCM (150mL) and water (50mL). The aqueous fraction was extracted with DCM (50mL) and the combined organic fractions were dried (MgSC^) and concentrated in vacuo. The residue was purified by column chromatography to give the title compound (767mg, 90%) as a pale yellow liquid. LCMS: ES ⁺ 252.1 [MH] ⁺ .

INTERMEDIATE 88

3- 3-Cyanophenyl)benzoic

Intermediate 87 (767mg, 3.05mmol) was dissolved in THF/water (20mL, 1 :1), Lithium hydroxide monohydrate (282mg, 6.72mmol) was added and the reaction mixture was stirred for 18h. The THF was removed in vacuo and the aqueous fraction was acidified to pH 1 with 1M aq HCl and extracted with EtOAc (3x30mL). The combined organic fractions were dried (MgSC^) and concentrated in vacuo to give the title compound

(600mg, 88%) as a white solid. LCMS: ES ⁺ 224.0 [MH] ⁺ .

INTERMEDIATE 89

Eth l 3-(l-methyl-lH-imidazol-2-yl)benzoate

1-Methyl-lH-imidazole (446mg, 5.43mmol) was dissolved in THF (40mL) and the

APC00482WO reaction mixture was cooled to -78°C. n-Butyllithium (3.5mL, 1.6M in hexanes, 5.61mmol) was added drop-wise over 30min and the reaction mixture was stirred at - 78°C for 30min. The reaction mixture was warmed to 0°C and a solution of zinc (II) chloride (2.22g, 16.3mmol) in ether (16.3mL) was added drop-wise over lOmin. The reaction mixture was stirred at 0°C for lh and at room temperature for 30min. Ethyl 3- lodobenzoate (l.OOg, 3.62mmol) and Pd(PPl¾)4 (210mg, 0.18mmol) were added and the reaction mixture was heated under reflux at 65°C for lh, cooled to room temperature and concentrated in vacuo. The residue was purified by column chromatography to give the title compound (145mg, 17%) as a yellow gum LCMS: ES ⁺ 231.2 [MH] ⁺ .

INTERMEDIATE 90

3- l-Methyl-lH-imidazol-2-yl)benzoic acid

Intermediate 89 (145mg, 0.63mmol) was dissolved in THF/water (lOmL, 1 :1), lithium hydroxide monohydrate (58.0mg, 1.39mmol) was added and the reaction mixture was stirred for 18h. The THF was removed in vacuo and the aqueous solution was acidified to pH 1 with 1M aq HC1 and concentrated in vacuo to give the crude title compound (127mg) as a brown gum. LCMS: ES ⁺ 203.2 [MH] ⁺ . INTERMEDIATE 91

-[(4-Cyanophenyl)methyl]-3-(l-methyl-lH-imidazol-2-yl)benzam ide

Intermediate 90 (127mg, 0.63mmol) and 4-(aminomethyl)benzomtrile.HCl (106mg, 0.63mmol) were dissolved in DCM (5mL) and DIPEA (493uL, 2.83mmol). EDC (147mg, 0.94mmol) and HONB (169mg, 0.94mmol) were added. The reaction mixture was stirred for 18h and partitioned between DCM (20mL) and sat aq Na2CC>3 (lOmL).

The aqueous fraction was extracted with DCM (2x20mL) and the combined organic fractions were dried (MgSC>4) and concentrated in vacuo. The residue was purified by column chromatography to give the title compound (40.0mg, 20%) as a colourless gum. LCMS: ES ⁺ 317.3 [MH] ⁺ .

APC00482WO INTERMEDIATES 92 and 93

Methyl 3-phenyl-l,2-thiazole-5-carboxylate and methyl 3-phenyl-l,2-thiazole-4- carboxylate

Benzamide (l.OOg, 8.25mmol) was dissolved in dioxane (20mL), chlorocarbonylsulfenyl chloride (0.70mL, 8.25mmol) was added and the reaction mixture was heated under reflux at 101°C for 6h. The reaction mixture was concentrated in vacuo and the residue was dissolved in m-xylene (30mL). Methyl propiolate (0.84mL, 14.0mmol) was added and the reaction mixture was heated under reflux at 139° C for 24h. The reaction mixture was concentrated in vacuo and the residue was partitioned between DCM (50mL) and water (30mL). The aqueous fraction was extracted with DCM (2x20mL) and the combined organic fractions were dried (MgSC^) and concentrated in vacuo. The residue was purified by column chromatography to give methyl 3-phenyl-l,2-thiazole-5- carboxylate (Intermediate 92, 348mg, 19%) as a yellow gum (LCMS: ES ⁺ 220.0 [MH] ⁺ ) and methyl 3-phenyl-l,2-thiazole-4-carboxylate (Intermediate H93, 285mg, 16%) as a yellow gum (LCMS: ES ⁺ 220.0 [MH] ⁺ ).

INTERMEDIATE 94

-Phenyl-l,2-thiazole-5-carboxylic

Intermediate 92 (348mg, 1.59mmol) was dissolved in THF/water (lOmL, 1 :1), lithium hydroxide monohydrate (146mg, 3.49mmol) was added and the reaction mixture was stirred for 4h. The THF was removed in vacuo and the aqueous solution was acidified to pH 1 with 1M aq HCl and extracted with DCM (2x50mL). The combined organic fractions were dried (MgSC^) and concentrated in vacuo to give the title compound

(303mg, 93%) as a brown gum. LCMS: ES ⁺ 205.9 [MH] ⁺ .

INTERMEDIATE 95

3-Phenyl-l,2-thiazole-4-carboxylic acid

APC00482WO

Intermediate 95 was prepared similarly to Intermediate 94, using Intermediate 93 instead of Intermediate 92, to give the title compound (207mg, 78%) as a brown gum. LCMS: ES ⁺ 205.9 [MH] ⁺ .

INTERMEDIATE 96

Methyl 5-phenyl-lH-pyrazole-3-carboxylate

Methyl 2,4-dioxo-4-phenylbutanoate (470mg, 2.28mmol) was dissolved in AcOH (5mL), hydrazine hydrate (122uL, 2.51mmol) was added and the reaction mixture was heated under reflux at 118°C for 2h. The reaction mixture was concentrated in vacuo to give the title compound (320mg, 69%) as a yellow solid. LCMS: ES ⁺ 203.0 [MH] ⁺ .

INTERMEDIATE 97

5-Phenyl- lH-pyrazole-3-carboxylic acid

Intermediate 96 (320mg, 1.58mmol) was dissolved in THF/water (15mL, 1 :1), lithium hydroxide monohydrate (146mg, 3.48mmol) was added and the reaction mixture was stirred for 2h. The THF was removed in vacuo and the aqueous solution was acidified to pH 1 with 1M aq HCl. The precipitate was collected by filtration to give the title compound (232mg, 78%) as a yellow solid. LCMS: ES ⁺ 189.0 [MH] ⁺ .

INTERMEDIATES 98-100

Intermediates 98-100 were prepared similarly to Intermediate 42, using 4- (aminomethyl)benzonitrile and the appropriate carboxylic acid; see Table 4 below.

APC00482WO Table 4: Amide couplings.

5

INTERMEDIATE 101

N- 4-Cyanophenyl)methyl]-2- [3-(3-sulfamoylphenyl)phenyl] acetamide

Intermediate 46 (293mg, 0.89mmol), 3-(dihydroxyboranyl)benzene-l -sulfonamide (215mg, 1.07mmol), Pd(PPh ₃ ) ₄ (99.0mg, 0.09mmol) and K ₂ C0 ₃ (246mg, 1.78mmol) were suspended in water (5mL) and dioxane (5mL) and heated at 90°C for 90min. The reaction mixture was filtered through Celite, concentrated in vacuo and the residue was partitioned between DCM (50mL) and water (50mL). The aqueous fraction was extracted with DCM (50mL) and the combined organic fractions were dried (MgSC^) and concentrated in vacuo.

The residue was purified by column chromatography to give the title compound (180mg, 50%) as a white solid. LCMS: ES ⁺ 406 [MH] ⁺ . INTERMEDIATE 102

N-[(4-Cyanophenyl)methyl]-2-(3-{4-[(2hydroxyethyl)sulfamoyl] phenyl}phenyl) acetamide

Intermediate 46 (500mg, 1.52mmol), {4-[(2-hydroxyethyl)sulfamoyl]phenyl}boronic acid (450mg, 1.82mmol), Pd(PPh ₃ ) ₄ (170mg, 0.15mmol) and K ₂ C0 ₃ (420mg, 3.04mmol) were suspended in water (5mL) and dioxane (5mL) and heated in a microwave reactor at 150°C for 30min. Further Pd(PPh ₃ ) ₄ (lOOmg, 0.09mmol) was added ad the reaction mixture was heated in a microwave reactor at 150°C for 30min. The reaction mixture was filtered through Celite, concentrated in vacuo and the residue was partitioned between DCM (50mL) and water (50mL). The aqueous fraction was extracted with DCM (50mL) and the combined organic fractions were dried (NaiSOzi) and concentrated in vacuo. The residue was purified by column chromatography to give the title compound (264mg, 39%) as a colourless oil. LCMS: ES ⁺ 449.9 [MH] ⁺ . INTERMEDIATE 103

2- [2-(2-Methyl-4-sulfamoylphenyl)phenyl] acetic acid

4-Bromo-3-methylbenzene-l -sulfonamide (322mg, 1.29mmol), [3-(2-methoxy-2-oxo- ethyl)phenyl]boronic acid (250mg, 1.29mmol), K ₂ C0 ₃ (356 mg, 2.56mmol) and Pd(PPh ₃ ) ₄

(149mg, 0.13mmol) were suspended in dioxane / water (4mL, 1:1) and heated in a microwave reactor at 150°C for 30min. The mixture was filtered through Celite and concentrated in vacuo to give the title compound (198mg, 50%) as a white solid. LCMS: ES ⁺ 306.0 [MH] ⁺ .

REFERENCE EXAMPLE 1

N- [(4-Carbamimidoylphenyl)methyl]-3-phenylbenzamide hydrochloride

Intermediate 22 (738mg, 2.37mmol) was dissolved in a solution of 14M HCl in EtOH (20mL) and stirred overnight in a sealed flask. The reaction mixture was concentrated in vacuo and a solution of 7M NH ₃ in MeOH (20mL) was added. The reaction mixture was stirred for 44h and concentrated in vacuo to give the crude title compound (738mg). 300mg of this material was purified by mass directed HPLC. The residue was dissolved in a solution of 1.25M HCl in EtOH (5mL), stirred for 5min and concentrated in vacuo to give the title compound (113mg, 42%) as a white solid. LCMS: ES ⁺ 330.0 [MH] ⁺ , HPLC: Rt 4.87min, 98.8%.

REFERENCE EXAMPLE 2

N-[(4-Carbamimidoylphenyl)methyl]-3-[3-(acetamidomethyl)phen yl]benzamide hydrochloride

hydrochloride

Intermediate 52 (200mg, 0.52mmol) was dissolved in MeOH (15mL) and hydroxylamine hydrochloride (72.5mg, 1.04mmol) and DIPEA (182uL, 1.04mmol) were added. The reaction mixture was heated under reflux at 65°C for 18h. The reaction mixture was concentrated in vacuo and the residue was partitioned between water (20mL) and DCM (30mL). The aqueous fraction was extracted with DCM (3x20mL) and the combined organic fractions were concentrated in vacuo. The residue was dissolved in glacial AcOH (5mL), Ac ₂ 0 (73.8uL, 0.78mmol) was added and the reaction mixture was stirred for 30min. The reaction mixture was concentrated in vacuo and the residue was dissolved in AcOH (lOmL) and hydrogenated using an H-Cube over 10% Pd/C. The reaction mixture was concentrated in vacuo and purified by reverse phase HPLC. The residue was dissolved in a solution of 4M HCl in dioxane and concentrated in vacuo to give the title compound (10.2mg, 4%) as an off-white solid. LCMS: ES ⁺ 401.0 [MH] ⁺ , HPLC: Rt 4.19min, 98.2%. EXAMPLES 30 and 53; and REFERENCE EXAMPLES 3-29 and 31 -52

Examples 30 and 53, and Reference Examples 3-29 and 31-53 were prepared similarly to Reference Example 1; see Table 5 below.

Table 5: Benzamidine formations

REFERENCE EXAMPLE 54

2,2,2-Trifluoroacetic acid; N- [(4-carbamimidoylphenyl)methyl] -2-(4- methanesulfonamido phenyl)benzamide

Intermediate 27 (lOOmg, 0.31mmol) and DIPEA (O.lOmL, 0.62mmol) were dissolved in THF (2mL), methanesulfonyl chloride (26.0uL, 0.34mmol) was added and the reaction mixture was stirred for 2h. Further methanesulfonyl chloride (12.0uL, 0.16mmol) was added and the reaction mixture was stirred for lh, quenched with MeOH and concentrated in vacuo. The residue was purified by column chromatography, dissolved in sat HCl in EtOH (5mL) and stirred for 2d. The reaction mixture was concentrated in vacuo and the residue dissolved in 7M NH ₃ in MeOH (5mL) and stirred for 48h. The reaction mixture was concentrated in vacuo and purified by column chromatography and reverse phase HPLC. The residue was dissolved in MeOH (5mL) and TFA (ImL), stirred for lh and concentrated in vacuo to give the title compound (3.00mg, 2%) as a white solid. LCMS: ES ⁺ 422.9 [MHf, HPLC: Rt 4.14min, 99.1%.

REFERENCE EXAMPLE 55

N-[(4-Carbamimidoylphenyl)methyl]-3-(3-cyanophenyl)benzamide hydrochloride

Intermediate 88 (300mg, 1.34mmol), 4-(aminomethyl)benzene-l-carboximidamide dihydrochloride (328mg, 1.48mmol) and DIPEA (585uL, 3.36mmol) were dissolved in

DMF (15mL), cooled to 0°C and PyBOP (699mg, 1.34mmol) was added. The reaction mixture was stirred at 0°C for 15min and at room temperature for 3d. The reaction mixture was concentrated in vacuo and purified by column chromatography and reverse phase HPLC. The residue was dissolved in 4M HCl in dioxane and concentrated in vacuo to give the title compound (30.1mg, 6%) as a white solid. LCMS: ES ⁺ 355.3 [MH] , HPLC: 4.85min, 95.8%.

EXAMPLE 56

N-[(4-Carbamimidoylphenyl)methyl]-2-[3-(4-sulfamoylphenyl)ph enyl]acetamide hydrochloride

Example 56 was prepared similarly to Reference Example 55, using Intermediate 10 instead of Intermediate 88, to give the title compound (65.7mg, 8%) as a white solid. LCMS: ES ⁺ 422.9 [MHf, HRMS (ESI+) calcd for C22H22N403S 423.149 found 423.148, HPLC: Rt 4.01min, 100%.

REFERENCE EXAMPLES 57-59

Reference Examples 57-59 were prepared similarly to reference Example 1; see Table 6 below.

Table 6: Benzamidine formations

EXAMPLE 60

N-[(4-Carbamimidoylphenyl)methyl]-2-[3-(3-sulfamoylphenyl)ph enyl]acetamide hydrochloride

Intermediate 101 (180mg, 0.44mmol) was dissolved in a solution of sat HCl in EtOH (5mL) and stirred overnight. The reaction mixture was concentrated in vacuo and a solution of 7M NH ₃ in MeOH (5mL) was added. The reaction mixture was stirred overnight and concentrated in vacuo. The residue was purified by column chromatography. The residue was dissolved in a solution of 1.25M HCl in EtOH (5mL), stirred for 20min and concentrated in vacuo to give the title compound (115mg, 19%) as a white solid. LCMS: ES ⁺ 422.9 [MH] ⁺ , HRMS (ESI+) calcd for C22H22N403S 423.149 found 423.148, HPLC: Rt 4.03min, 99.1%.

EXAMPLE 61

A^-[(4-Carbamimidoylphenyl)methyl]-2-(3-{4-[(2-hydroxyeth yl)sulfamoyl]phenyl} phenyl)acetamide hydrochloride

Intermediate 102 (264mg, 0.59mmol) was dissolved in a solution of sat HCl in EtOH (6mL) and stirred overnight. The reaction mixture was concentrated in vacuo and a solution of 7M NH ₃ in MeOH (5mL) was added. The reaction mixture was stirred for 3 days and concentrated in vacuo. The residue was purified by column chromatography. The residue was dissolved in a solution of 1.25M HCl in EtOH (lOmL), stirred for lOmin and concentrated in vacuo to give the title compound (217mg, 73%) as a white solid. LCMS: ES ⁺ 466.9 [MH] ⁺ , HPLC: Rt 4.35min, 100%. EXAMPLE 62

N-[(4-Carbamimidoylphenyl)methyl]-2-[3-(2-methyl-4-sulfamoyl phenyl)phenyl] acetamide h drochloride

Intermediate 103 (190mg, 0.64mmol), HBTU (240mg, 0.64mmol) and DIPEA (0.16mL, 0.95mmol) were dissolved in DMF (4mL) and stirred for 15min. 4-Cyanobenzylamine (0.08mL, 0.64mmol) was added and the reaction mixture was stirred overnight and concentrated in vacuo. The residue was dissolved in EtOAc (30mL) and washed with, sat aq NaHC03 (2 x 30mL), water (30mL), brine (30mL), dried (MgSC^) and concentrated in vacuo. The residue was purified by column chromatography, dissolved in a solution of sat HCl in EtOH (4mL) and stirred overnight. The reaction mixture was concentrated in vacuo and a solution of 7M NH ₃ in MeOH (4mL) was added. The reaction mixture was stirred overnight and concentrated in vacuo. The residue was purified by column chromatography. The residue was dissolved in a solution of 1.25M HCl in EtOH (4mL), stirred for 30min and concentrated in vacuo to give the title compound (65mg, 22%) as a pale yellow solid. LCMS: ES ⁺ 437.0 [MH] ⁺ , HRMS (ESI+) calcd for C23H24N403S 437.165 found 437.164, HPLC: Rt 4.48min, 99.6%.

BIOLOGICAL TESTS PAR2 studies

The PAR2 receptor couples through the Gq signaling pathway and results in activation of calcium mobilization. The functional activity of test compounds was routinely tested by measuring the ability of compounds to antagonize PAR2 (trypsin challenge) activity in a dose dependent manner, in Wild type HEK-293 cells, 1321N1 cells transfected with the rat PAR2 receptor and 1321N1 cells transfected with the mouse PAR2 receptor, using a calcium flux Fluorescent Imaging Plate Reader FLIPR assay.

The selectivity of compounds for PAR2 versus the PARI and PAR4 receptors was evaluated using the native 1321N1 cell line. In order to confirm that activity at the PAR2 receptor was due to direct inhibition of the PAR2 receptor as opposed to inhibition of trypsin, a series of serine protease assays was developed to measure the activity of in-house compounds on enzyme activity.

Functional calcium mobilisation studies

Briefly, test compounds were dissolved in DMSO to a concentration of 50mM and stored in matrix screenmate racks. The required amount of compound was transferred to 96-well compound plates on the day of assay and dose-response measurements were assayed by making 1 :3.16 serial dilutions to produce 10 point curves. Compounds were then diluted in assay buffer to the required final concentration before being transferred to 384-well assay plates ready for use. Top concentrations were adjusted depending on the potency of the compounds with a typical concentration range of 200 uM to 6.3 nM being used. The assay buffer used was HBSS buffer supplemented with 20 mM HEPES, 2.5 mM Probenicid, adjusted to pH7.4. The loading/wash buffers were the same as the assay buffer.

Wild type HEK-293 cells were cultured in MEM AQmedia containing Glutamine, supplemented with 10% FBS, 1% (5 ml) Penicillin/Streptomycin and 1% Non Essential Amino Acids. Rat or Mouse PAR2 transfected 1321N1 cells were cultured in Dulbecco's modified Eagles medium (DMEM) supplemented with 10% dialyzed FBS, 1% Penicillin/Streptomycin, 378.5 ug/ml Geneticin G418 sulphate and maintained at 37 °C in a humidified, 5% C02 controlled atmosphere. Sub-cultivations were performed every 2-3 d. At confluence, the cells were lifted using 0.25% Trypsin (IX), spun at 1000 rpm for 3 min and re-suspended in medium at 3.3 x 10 ⁵ cells/mL, transferred (30 μΐ/well) to 384-well black/clear BD plates (SLS # 56663BD) and incubated at 37 °C in a 5% C02/95% air humidified incubator for 16 h. The cells were washed with assay buffer at 37 °C using the Biotek ELx 405, washing 3 times, leaving 20 ul buffer in the well. After washing, the cells were loaded with Fluo-4 AM dye (Molecular probes) at 2 uM containing 0.48 ug/mL pluronic acid for 60 min at 37 °C under 5% C02. Following the incubation, cells were washed in assay buffer at 37 °C using the Biotek ELx 405, washing 3 times, leaving 40 μΐ in each well and incubated for 10 min at 37 °C before use.

A combined agonist/antagonist protocol was used to measure changes in intracellular calcium concentration. Compound (antagonist) was added to the cell plate using a Fluorometric Imaging Plate Reader (FLIPR) (Molecular Devices, Sunnyvale, CA, USA). Basal fluorescence was recorded every second for 10 sec prior to compound addition (10 uL) and fluorescence recorded every second for 1 min then every 6 sec for a further 1 min. Trypsin (EC ₅₀ concentration) was then added using the FLIPR and fluorescense recorded as described above. Curve-fitting and parameter estimation were carried out using GraphPad Prism 4.0 (GraphPad Software Inc., San Diego, CA). Trypsin enzyme inhibition

The commercially available protease assay kit from Calbiochem (Cat # 539125) was used to determine inhibition of trypsin activity. The kit quantifies trypsin activity by measuring the cleaved product of FTC-casein. To measure enzyme inhibition activity, compounds were pre-incubated with trypsin before the addition of substrate. Compound IC50 was determined as percentage inhibition of trypsin.

Surprisingly, the Examples have significantly higher antagonist activity at the PAR2 receptor than the corresponding Reference Examples. Without being bound by theory, the surprisingly improved activity arises by virtue of the Examples having n>0. (See Tables 7A, 7B, and 7C).

Table 7A: PAR2 antagonist activity

(A: O. luM, B: 0.1-0.5uM, C: 0.5-luM, D: 1-lOuM, E: 10-25uM)

Table 7B: PAR2 antagonist activity of the Reference Example

(A: O. luM, B: 0.1-0.5uM, C: 0.5-luM, D: 1-lOuM, E: 10-25uM)

*Rat 1321N1 cell line Table 7C: PAR2 antagonist activity of the Examples v corresponding Reference Examples

(A: O. luM, B: 0.1-0.5uM, C: 0.5-luM, D: 1-lOuM, E: 10-25uM)

PAR2 antagonist activity is assessed in rat 1321N1 cell line