WO2009074518A1

WO2009074518A1 - Combinations of prolinamide p2x7 modulators with further therapeutic agents

Info

Publication number: WO2009074518A1
Application number: PCT/EP2008/066890
Authority: WO
Inventors: Anton Duval Michel; Daryl Simon Walter
Original assignee: Glaxo Group Limited
Priority date: 2007-12-12
Filing date: 2008-12-05
Publication date: 2009-06-18

Abstract

The invention provides a combination comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof: formula (I), together with a further therapeutic agent or agents; and the use of the combination in the treatment or prevention of pain, inflammation or a neurodegenerative disease. The compounds of formula (I) are thought to be P2X7 receptor antagonists. The compound of formula (I) is preferably N-{[2-chloro-3-(trifluoromethyl)phenyl]methyl}- 1 -methyl-5-oxo-L-prolinamide. The further therapeutic agent can e.g. be methotrexate, an NSAID, a TNFα inhibitor, sulfasalazine, a statin, a glucocorticoid agent, an anti-IL-1 monoclonal antibody, or an anti-CD20 monoclonal antibody; e.g. for the treatment of rheumatoid arthritis. The further therapeutic agent can be capable of treating inflammatory or neuropathic pain, e.g. paracetamol, an opioid (e.g. morphine, fentanyl, oxycodone, or tramadol), pregabalin, gabapentin, gabapentin enacarbil, or carbamazepine.

Description

Combinations of prolinamide P2X7 modulators with further therapeutic agents

The present invention relates to combinations of heterocyclic (prolinamide) derivatives which modulate P2X7 receptor function and are capable of antagonizing the effects of ATP at the P2X7 receptor (P2X7 receptor antagonists) together with a further therapeutic agent or agents; to pharmaceutical compositions containing the combinations; and to the use of such combinations in therapy.

The P2X7 receptor is a ligand-gated ion-channel which is expressed in cells of the hematopoietic lineage, e.g. macrophages, microglia, mast cells, and lymphocytes (T and B) (see, for example, CoIIo, et al., Neuropharmacology, Vol.36, pp1277-1283 (1997)), and is activated by extracellular nucleotides, particularly adenosine triphosphate (ATP). Activation of P2X7 receptors has been implicated in giant cell formation, degranulation, cytolytic cell death, CD62L shedding, regulation of cell proliferation, and release of proinflammatory cytokines such as interleukin 1 beta (IL- 1 β) (e.g. Ferrari, et al., J. Immunol., Vol.176, pp3877-3883 (2006)) and tumour necrosis factor alpha (TNFα) (e.g. Hide, et al., Journal of Neurochemistry, Vol.75, pp965-972 (2000)). P2X7 receptors are also located on antigen presenting cells, keratinocytes, parotid cells, hepatocytes, erythrocytes, erythroleukaemic cells, monocytes, fibroblasts, bone marrow cells, neurones, and renal mesangial cells. Furthermore, the P2X7 receptor is expressed by presynaptic terminals in the central and peripheral nervous systems and has been shown to mediate glutamate release in glial cells (Anderson, C. et al., Drug. Dev. Res., Vol.50, page 92 (2000)).

The localisation of the P2X7 receptor to key cells of the immune system, coupled with its ability to release important inflammatory mediators from these cells suggests a potential role of P2X7 receptor antagonists in the treatment of a wide range of diseases including pain and neurodegenerative disorders. Recent preclinical in vivo studies have directly implicated the P2X7 receptor in both inflammatory and neuropathic pain (DelϊAntonio et al., Neurosci. Lett, Vol.327, pp87-90 (2002),. Chessell, I. P., et al., Pain, Vol.1 14, pp386-396 (2005), Honore et al., J. Pharmacol. Exp. Ther., Vol.319, p1376-1385 (2006)) while there is in vitro evidence that P2X7 receptors mediate microglial cell induced death of cortical neurons (Skaper, S. D., et al., GHa, Vol.54, p234-242 (2006)). In addition, up-regulation of the P2X7 receptor has been observed around β-amyloid plaques in a transgenic mouse model of Alzheimer's disease (Parvathenani, L. et al., J. Biol. Chem., Vol.278(15), pp13309- 13317 (2003)). The present invention provides combinations which modulate P2X7 receptor function and are capable of antagonizing the effects of ATP at the P2X7 receptor (P2X7 receptor antagonists).

In a first aspect of the invention there is provided a combination comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof:

(I) wherein:

R¹ represents C_1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3_6 cycloalkyl, C3_6 cycloalkylmethyl- or pyridinylmethyl-, any of which is optionally substituted with 1 , 2 or 3 halogen atoms; or unsubstituted phenyl or benzyl;

R² and R³ independently represent hydrogen, halogen, Ci_-5 alkyl, arylmethyl-, C_2-6 alkenyl, C₂_₆ alkynyl or C_3-6 cycloalkylmethyl-; and any of said C_1^3 alkyl, arylmethyl-, C_2-6 alkenyl, C_2-6 alkynyl or C₃.₆ cycloalkylmethyl- is optionally substituted with 1 , 2 or 3 halogen atoms;

R⁴, R⁵ and R⁶ independently represent hydrogen, fluorine or methyl; and R⁷, R⁸, R⁹, R¹⁰ and R¹¹ independently represent hydrogen, halogen, cyano, C_1-5 alkyl, C_2-6 alkenyl, C₂.₆ alkynyl, C_3-6 cycloalkyl or phenyl, and any of said C_1-6 alkyl, C_2-6 alkenyl, C₂.₆ alkynyl, C₃.₆ cycloalkyl or phenyl is optionally substituted with 1 , 2 or 3 halogen atoms; or R¹⁰ and R¹¹ together with the carbon atoms to which they are attached form a benzene ring which is optionally substituted with 1 , 2 or 3 halogen atoms; with the proviso that when R⁷ and R¹¹ are both selected from hydrogen or fluorine, at least one of R⁸, R⁹ and R¹⁰ is a halogen atom, or R⁸, R⁹ and R¹⁰ are selected from the group consisting of hydrogen and CF₃ and one, but not more than one, of R⁸, R⁹ and R¹⁰ is CF₃; together with a further therapeutic agent or agents.

These compounds of formula (I) or pharmaceutically acceptable salts thereof, as defined herein, and their uses as P2X7 receptor antagonists, are disclosed in copending PCT application PCT/EP2007/056675, filed on 3 July 2007, and published on 10 January 2008 as WO 2008/003697 A1.

In one embodiment of the present invention, the further therapeutic agent or agents may be medicaments claimed to be useful in the treatment of a condition which is mediated by P2X7 receptors as described herein.

Suitable examples of other such therapeutic agents may include a β2-agonist (also known as β2 adrenoceptor agonists; e.g. formoterol) and/or a corticosteroid (e.g. budesonide, fluticasone (e.g. as propionate or furoate esters), mometasone (e.g. as furoate), beclomethasone (e.g. as 17-propionate or 17,21 -dipropionate esters), ciclesonide, triamcinolone (e.g. as acetonide), flunisolide, rofleponide or butixocort (e.g. as propionate ester)), for the treatment of a respiratory disorder (such as asthma or chronic obstructive pulmonary disease (COPD)), e.g. as described in WO 2007/008155 and/or WO 2007/008157.

A further therapeutic agent may include a 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitor (e.g. atorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatin, or simvastatin), for the treatment of a cardiovascular disorder (such as atherosclerosis), e.g. as described in WO 2006/083214.

A further therapeutic agent may include a non-steroid anti-inflammatory drug (NSAID; e.g. ibuprofen, naproxen, aspirin, celecoxib, diclofenac, etodolac, fenoprofen, indomethacin, ketoprofen, ketoralac, oxaprozin, nabumetone, sulindac, tolmetin, rofecoxib, valdecoxib, lumaricoxib, meloxicam, etoricoxib or parecoxib; or e.g. paracetamol, loxoprofen or aceclofenac; in particular celecoxib, paracetamol, ibuprofen or diclofenac); for the treatment of an inflammatory disease or disorder (such as rheumatoid arthritis or osteoarthritis, and/or inflammatory pain), e.g. as described in WO 2005/025571. Celecoxib (a COX-2 inhibitor) can for example be administered orally at a dosage regimen of 100 mg or 200 mg (measured as the free base) once or twice daily.

A further therapeutic agent may in particular include a tumour necrosis factor α (TNFα) inhibitor (e.g. etanercept or an anti- TNFα antibody such as infliximab and adalimumab) (e.g. for parenteral administration such as subcutaneous or intravenous administration), for the treatment of an inflammatory disease or disorder (such as rheumatoid arthritis or osteoarthritis, and/or inflammatory pain), e.g. as described in A further therapeutic agent may in particular include an anti-CD20 monoclonal antibody (e.g. for parenteral such as intravenous administration), such as ofatumumab (HuMax-CD20 ™, developed in part by Genmab AS) (e.g. ofatumumab for intravenous administration), rituximab, PRO70769, AME-133 (Applied Molecular

Evolution), or hA20 (Immunomedics, Inc.); in particular ofatumumab or rituximab.

This further therapeutic agent can e.g. be for the treatment of an inflammatory disease or disorder (such as rheumatoid arthritis or osteoarthritis, and/or inflammatory pain).

A further therapeutic agent may include 2-hydroxy-5- [ [4- [ (2- pyridinylamino) sulfonyl] phenyl] azo] benzoic acid (sulfasalazine) for the treatment of an inflammatory disease or disorder (such as rheumatoid arthritis or osteoarthritis; in particular rheumatoid arthritis), e.g. as described in WO 2004/105797.

A further therapeutic agent may in particular include N-[4-[[(2, 4-diamino-6-pteridinyl) methyl] methylamino] benzoyl]- L-glutamic acid (methotrexate) for the treatment of an inflammatory disease or disorder (such as rheumatoid arthritis or osteoarthritis; in particular rheumatoid arthritis), e.g. as described in WO 2004/105796.

A further therapeutic agent may include an inhibitor of pro TNFα convertase enzyme (TACE), for the treatment of an inflammatory disease or disorder (such as rheumatoid arthritis or osteoarthritis; in particular rheumatoid arthritis), e.g. as described in WO 2004/073704.

A further therapeutic agent may in particular include:

a) sulfasalazine; b) a statin (e.g. for oral administration), such as atorvastatin, lovastatin, pravastatin, simvastatin, fluvastatin, cerivastatin, crilvastatin, dalvastatin, rosuvastatin, tenivastatin, fluindostatin, velostatin, dalvastatin, nisvastatin, bervastatin, pitavastatin, rivastatin, glenvastatin, eptastatin, tenivastatin, flurastatin, rosuvastatin or itavastatin; c) a glucocorticoid agent (e.g. for oral or skin-topical administration), such as dexamethasone, methylprednisolone, prednisolone, prednisone and hydrocortisone; d) an inhibitor of p38 kinase (e.g. for oral administration); e) an anti-IL-6-receptor antibody, e.g. an anti-IL-6-receptor monoclonal antibody (e.g. for parenteral such as intravenous administration); f) anakinra; g) an anti-IL-1 (e.g. I L-1 β) monoclonal antibody (e.g. for parenteral such as intravenous administration); h) an inhibitor of JAK3 protein tyrosine kinase; i) an anti-macrophage colony stimulation factor (M-CSF) monoclonal antibody; or j) an anti-CD20 monoclonal antibody (e.g. for parenteral such as intravenous administration), such as rituximab, ofatumumab (HuMax-CD20 ™, developed in part by Genmab AS) (e.g. ofatumumab for intravenous administration), PRO70769, AME- 133 (Applied Molecular Evolution), or hA20 (Immunomedics, Inc.); in particular rituximab or ofatumumab;

for the treatment of an IL-1 (e.g. IL-1 β) mediated disease (such as rheumatoid arthritis or osteoarthritis, and/or inflammatory or neuropathic pain; in particular rheumatoid arthritis), e.g. as described in WO 2006/003517.

In a particular embodiment of the invention, when the further therapeutic agent includes a non-steroid anti-inflammatory drug (NSAI D) (e.g. ibuprofen, naproxen, aspirin, celecoxib, diclofenac, etodolac, fenoprofen, indomethacin, ketoprofen, ketoralac, oxaprozin, nabumetone, sulindac, tolmetin, rofecoxib, valdecoxib, lumaricoxib, meloxicam, etoricoxib or parecoxib; or e.g. paracetamol, loxoprofen or aceclofenac; in particular celecoxib, paracetamol, ibuprofen or diclofenac), then the non-steroid anti-inflammatory drug and/or the combination comprising the non-steroid anti-inflammatory drug is for the treatment of an inflammatory disease or disorder.

Alternatively, in one further embodiment of the invention, the further therapeutic agent does not include a non-steroid anti-inflammatory drug (NSAID) (e.g. ibuprofen, naproxen, aspirin, celecoxib, diclofenac, etodolac, fenoprofen, indomethacin, ketoprofen, ketoralac, oxaprozin, nabumetone, sulindac, tolmetin, rofecoxib, valdecoxib, lumaricoxib, meloxicam, etoricoxib or parecoxib; or e.g. paracetamol, loxoprofen or aceclofenac).

Additionally or alternatively, in one further embodiment of the invention, the further therapeutic agent does not include ethanol, cocaine, nicotine, or an opioid (typically a dependence-inducing opioid, e.g. morphine). In a more particular embodiment, the further therapeutic agent does not include an opioid (typically a dependence-inducing opioid, e.g. morphine), or nicotine, or a dependence-inducing CNS depressant (e.g. ethanol), or a dependence-inducing psychostimulant (e.g. cocaine). In a still more particular embodiment, the further therapeutic agent does not include a dependence- inducing agent (e.g. an opioid such as morphine, or nicotine, or a dependence- inducing CNS depressant such as ethanol, or a dependence-inducing psychostimulant such as cocaine). CNS means central nervous system.

Additionally or alternatively, in one further embodiment of the invention, the further therapeutic agent does not include a cancer chemotherapeutic agent.

In a particular embodiment of the invention, the further therapeutic agent or agents can be a therapeutic agent or agents capable of treating inflammatory pain, such as paracetamol and/or an opioid (such as morphine, fentanyl, oxycodone, tramadol, hydrocodone, hydromorphone, oxymorphone, methadone or buprenorphine; in particular morphine, fentanyl, oxycodone, or tramadol). This/these therapeutic agent(s), and/or the combination comprising this/these therapeutic agent(s), can be for the treatment of inflammatory pain, e.g. in a mammal such as a human.

For example, in the present combination, paracetamol can be administered at a human oral dosage regimen of 500 mg to 1000 mg (e.g. 500 mg, 650 mg or 1000 mg, in particular 650 mg) of paracetamol (measured as the free base / free compound), administered two, three or four times daily.

In a particular embodiment of the invention, the further therapeutic agent or agents can be a therapeutic agent or agents capable of treating neuropathic pain, such as:

- an opioid (such as morphine, fentanyl, oxycodone, tramadol, hydrocodone, hydromorphone, oxymorphone, methadone or buprenorphine; in particular morphine, fentanyl, oxycodone, or tramadol),

- a monoamine reuptake inhibitor (such as duloxetine or amytriptyline),

- pregabalin,

- gabapentin,

- gabapentin enacarbil (XP13512), and/or - carbamazepine.

This/these therapeutic agent(s), and/or the combination comprising this/these therapeutic agent(s), can be for the treatment of neuropathic pain, e.g. in a mammal such as a human.

For example, in the present combination, pregabalin can be administered orally e.g. for neuropathic pain; e.g. at a human oral dosage regimen of 150 mg to 600 mg total pregabalin per day (measured as the free base), split between two to three doses per day. For example, for postherpetic neuralgia (a neuropathic pain condition), pregabalin can be administered at a starting oral dosage regimen of 150 mg total pregabalin per day (split between 2 or 3 doses per day), escalating (e.g. in about one week) to an oral dosage regimen of 300 mg pregabalin total per day, and optionally escalating up to a maximum oral dosage regimen of 600 mg total pregabalin per day. For painful diabetic neuropathy (another neuropathic pain condition), an oral dosage regimen of 150 mg to 300 mg total pregabalin per day can be administered. For fibromyalgia, an oral dosage regimen of 150 mg to 450 mg (e.g. 300 or 450 mg) total pregabalin per day can be administered. Pregabalin can e.g. be administered separately from the compound of formula (I) or the salt thereof.

For example, in the present combination, gabapentin can be administered orally, e.g. for neuropathic pain. Oral dosage units can e.g. contain 100 mg, 300 mg, 400 mg, 600 mg or 800 mg of gabapentin (measured as the free base/acid). The gabapentin dosage regimen for neuropathic pain can e.g. be from 300 mg once, twice or three times per day up to a total dose of 3600 mg / day. Some gradual up-titration of the dosage regimen is usually performed. For example, for peripheral neuropathic pain in adults, gabapentin therapy can be initiated by titrating the dose thus: day 1 = 300 mg of gabapentin (measured as the free base/acid) once a day, day 2 = 300 mg two times a day, and day 3 = 300 mg three times a day; alternatively the starting dose can be 900 mg / day of gabapentin (measured as the free base/acid), administered as three equally divided doses. Thereafter, e.g. based on individual patient response and tolerability, the dose can be further increased, typically in 300 mg / day increments every 2-3 days, up to a maximum total dose of 3600 mg / day of gabapentin (measured as the free base/acid). Slower titration of gabapentin dosage may be appropriate for individual patients. The minimum time to reach a total dose of 1800 mg / day is typically one week, to reach 2400 mg / day is typically a total of 2 weeks, and to reach 3600 mg / day is typically a total of 3 weeks. Gabapentin can e.g. be administered separately from the compound of formula (I) or the salt thereof.

For example, in the present combination, gabapentin enacarbil (XP13512, (±)-1-([(α- isobutanoyloxyethoxy)carbonyl]-aminomethyl)-1-cyclohexane acetic acid, which is a prodrug of gabapentin) can be administered orally, e.g. to a human, e.g. separately from the compound of formula (I) or the salt thereof. In one embodiment, gabapentin enacarbil (XP13512) is for example administered orally, e.g. to a human such as a human adult, e.g. at a total daily dose having an equivalent molar quantity of gabapentin enacarbil as the molar quantity present in 900 mg / day to 3600 mg / day of gabapentin (see e.g. page 81 lines 24-32 of WO 02/100347). A 600 mg dose of gabapentin enacarbil (measured as the free acid) contains the molar equivalent of 312 mg of gabapentin. See also K.C. Cundy et al., "Clinical Pharmacokinetics of XP13512, a Novel Transported Prodrug of Gabapentin", J. CHn. Pharmacol., 2008, e-publication 30 September 2008, incorporated herein by reference, and the Materials and Methods - Formulation and Study Designs sections therein, for examples of some oral doses, dosage regimens and formulations of XP13512 used in human pharmacokinetic studies.

In a particular embodiment of the invention, when the further therapeutic agent includes an opioid (such as morphine, fentanyl, oxycodone, tramadol, hydrocodone, hydromorphone, oxymorphone, methadone or buprenorphine; in particular morphine, fentanyl, oxycodone, or tramadol), then the opioid and/or the combination comprising the opioid is for the treatment of pain, in particular inflammatory or neuropathic pain, e.g. in a mammal such as a human.

When the compounds are used in combination with other therapeutic agents, the compounds may be administered either sequentially or simultaneously by any convenient route.

The individual components of the combination of the invention (i.e. the compound of formula (I) or the salt thereof, and the further therapeutic agent or agents) may be present as separate pharmaceutical formulations / compositions, or may be present as a combined pharmaceutical formulation / composition (e.g. may be together in a single combined oral dosage form, e.g. a single combined tablet or capsule). The individual components of this combination can for example be administered either sequentially in separate pharmaceutical formulations / compositions (e.g. oral), or simultaneously in separate or combined pharmaceutical formulation(s) / composition(s) (e.g. oral); in a particular embodiment they are administered sequentially in separate pharmaceutical formulations / compositions (e.g. oral). The combinations referred to herein may optionally be presented for use in the form of a pharmaceutical formulation and thus pharmaceutical formulations comprising a combination as defined herein together with a pharmaceutically acceptable carrier or excipient comprise a further aspect of the invention. The individual components of such combinations may be administered either sequentially or simultaneously in separate or combined pharmaceutical formulations.

When a compound of formula (I) or a pharmaceutically acceptable salt thereof is used in combination with a second (further) therapeutic agent active against the same disease state the dose of each compound may differ from that when the compound is used alone.

In one embodiment, a combination comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof:

(I) wherein:

R¹ represents C_1-6 alkyl, C_2-6 alkenyl, C₂_₆ alkynyl, C_3-5 cycloalkyl or C_3-6 cycloalkylmethyh any of which may be optionally substituted with 1 , 2 or 3 halogen atoms; or unsubstituted phenyl or benzyl;

R² and R³ independently represent hydrogen, halogen, C_1-6 alkyl, arylmethyl-, C_2-6 alkenyl, C₂.₆ alkynyl or C_3-6 cycloalkylmethyl-; and any of said C₁^₃ alkyl, arylmethyl-,

C_2-6 alkenyl, C₂.₆ alkynyl or C_3-6 cycloalkylmethyl- may be optionally substituted with 1 ,

2 or 3 halogen atoms;

R⁴, R⁵ and R⁶ independently represent hydrogen or fluorine; and

R⁷, R⁸, R⁹, R¹⁰ and R¹¹ independently represent hydrogen, halogen, cyano, C_1-6 alkyl,

C_2-6 alkenyl, C_2-6 alkynyl, C₃.₆ cycloalkyl, or phenyl; and any of said C_1-6 alkyl, C_2-6 alkenyl, C₂.₆ alkynyl, C_3-6 cycloalkyl or phenyl may be optionally substituted with 1 , 2 or 3 halogen atoms; with the proviso that when R⁷ and R¹¹ independently represent hydrogen or fluorine, at least one of R⁸, R⁹ and R¹⁰ is a halogen atom; together with a further therapeutic agent or agents.

(I) wherein:

R¹ represents Ci_-6 alkyl, C₂.₆ alkenyl, C_2-6 alkynyl, C_3-6 cycloalkyl, C_3-6 cycloalkylmethyl- or pyridinylmethyl-, any of which is optionally substituted with 1 , 2 or 3 halogen atoms; or unsubstituted phenyl or benzyl;

R² and R³ independently represent hydrogen, halogen, C-ι-6 alkyl, arylmethyl-, C2-6 alkenyl, C₂.₆ alkynyl or C3-6 cycloalkylmethyl-; and any of said C1-3 alkyl, arylmethyl-, C2-6 alkenyl, C_2-6 alkynyl or C3.6 cycloalkylmethyl- is optionally substituted with 1 , 2 or 3 halogen atoms; R⁴, R⁵ and R⁶ independently represent hydrogen, fluorine or methyl; and

R⁷, R⁸, R⁹, R¹⁰ and R¹¹ independently represent hydrogen, halogen, cyano, C_1-6 alkyl, C_2-6 alkenyl, C₂.₆ alkynyl, C_3-6 cycloalkyl or phenyl, and any of said C_1-6 alkyl, C_2-6 alkenyl, C₂.₆ alkynyl, C₃.₆ cycloalkyl or phenyl is optionally substituted with 1 , 2 or 3 halogen atoms; or R¹⁰ and R¹¹ together with the carbon atoms to which they are attached form a benzene ring which is optionally substituted with 1 , 2 or 3 halogen atoms; with the proviso that when R⁷ and R¹¹ are both selected from hydrogen or fluorine, at least one of R⁸, R⁹ and R¹⁰ is a halogen atom, or not more than one of R⁸, R⁹ and R¹⁰ is a CF₃ group; together with a further therapeutic agent or agents.

As used herein, the term "alkyl" (when used as a group or as part of a group) refers to a straight or branched hydrocarbon chain containing the specified number of carbon atoms. For example, C₁^ alkyl means a straight or branched hydrocarbon chain containing at least 1 and at most 6 carbon atoms. Examples of alkyl include, but are not limited to; methyl (Me), ethyl (Et), n-propyl, i-propyl, n-hexyl and i-hexyl. As used herein, the term "alkenyl" refers to a straight or branched hydrocarbon chain containing the specified number of carbon atoms wherein at least one carbon-carbon bond is a double bond. Examples of alkenyl include, but are not limited to ethenyl, propenyl, n-butenyl, i-butenyl, n-pentenyl and i-pentenyl.

As used herein, the term "alkynyl" refers to a straight or branched hydrocarbon chain containing the specified number of carbon atoms wherein at least one carbon-carbon bond is a triple bond. Examples of alkynyl include, but are not limited to ethynyl, propynyl, butynyl, i-pentynyl, n-pentynyl, i-hexynyl and n-hexynyl.

The term 'cycloalkyl' unless otherwise stated means a closed 3 to 6 membered non- aromatic ring, for example cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.

The term 'aryl' as used herein refers to a Cβ-io monocyclic or bicyclic hydrocarbon ring wherein at least one ring is aromatic. Examples of such groups include phenyl and naphthyl.

The term 'halogen' is used herein to describe, unless otherwise stated, a group selected from fluorine, chlorine, bromine or iodine.

In certain embodiments of the invention, R¹ represents C₁^ alkyl, C₂_₆ alkenyl, C_2-6 alkynyl, C₃.₆ cycloalkyl or pyridinylmethyl-, any of which is optionally substituted with 1 , 2 or 3 halogen atoms; or unsubstituted phenyl or benzyl. In one embodiment, R¹ represents unsubstituted C_1-6 alkyl, C₂.₆ alkenyl, C₂.₆ alkynyl, C₃.₆ cycloalkyl, pyridinylmethyl-, phenyl or benzyl. In another embodiment, R¹ represents unsubstituted Ci_-4 alkyl, C_3-5 cycloalkyl, pyridinylmethyl-, phenyl or benzyl. In yet another embodiment, R¹ represents methyl or ethyl.

In certain embodiments of the invention, R² and R³ independently represent hydrogen, halogen, Ci_-6 alkyl, benzyl, C_2-6 alkenyl, C_2-6 alkynyl or C_3-6 cycloalkylmethyl-; and any of said Ci_-6 alkyl, benzyl, C₂.₆ alkenyl, C₂.₆ alkynyl or C_3-6 cycloalkylmethyl- may be optionally substituted with 1 , 2 or 3 halogen atoms.

In one embodiment, R² and R³ independently represent hydrogen or halogen; unsubstituted Ci_₆ alkyl, benzyl, C₂_₆ alkenyl, C₂_₆ alkynyl or C₃^ cycloalkylmethyl-.

In another embodiment, R² and R³ independently represent hydrogen, fluorine or methyl. In a further embodiment, R² and R³ both represent hydrogen.

In one embodiment of the invention, R⁴ and R⁵ independently represent hydrogen or methyl. In another embodiment, R⁶ represents hydrogen or methyl. In a further embodiment, R⁴, R⁵ and R⁶ all represent hydrogen.

In another embodiment of the invention, R⁷, R⁸, R⁹, R¹⁰ and R¹¹ independently represent hydrogen, halogen, cyano, trifluoromethyl or unsubstituted Ci_-6 alkyl; or R¹⁰ and R¹¹ together with the carbon atoms to which they are attached form an unsubstituted benzene ring. In a further embodiment, R⁷, R⁸, R⁹, R¹⁰ and R¹¹ independently represent hydrogen, halogen, cyano, methyl or trifluoromethyl; or R¹⁰ and R¹¹ together with the carbon atoms to which they are attached form an unsubstituted benzene ring. In yet another embodiment, R⁷, R⁸, R⁹, R¹⁰ and R¹¹ independently represent hydrogen, chlorine, fluorine, bromine, methyl or trifluoromethyl.

In one embodiment of the invention, there is provided a compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein:

R¹ represents unsubstituted C_1-4 alkyl, C₂-₄ alkenyl, C_3-5 cycloalkyl, pyridinylmethyl-, phenyl or benzyl;

R² and R³ both represent hydrogen;

R⁴, R⁵ and R⁶ independently represent hydrogen or methyl; and

R⁷, R⁸, R⁹, R¹⁰ and R¹¹ independently represent hydrogen, chlorine, fluorine, bromine, methyl or trifluoromethyl; with the proviso that when R⁷ and R¹¹ are both selected from hydrogen or fluorine, at least one of R⁸, R⁹ and R¹⁰ is a halogen atom, or R⁸, R⁹ and R¹⁰ are selected from the group consisting of hydrogen and CF₃ and one, but not more than one, of R⁸, R⁹ and

R¹⁰ is CF₃.

Particular compounds of formula (I) include the compounds of Examples 1-136 as shown below, or a pharmaceutically acceptable salt thereof.

Preferably, the compound of formula (I) or the pharmaceutically acceptable salt thereof is Λ/-{[2-chloro-3-(trifluoromethyl)phenyl]methyl}-1-methyl-5-oxoprolinamide:

Most preferably, the compound of formula (I) or the pharmaceutically acceptable salt thereof is /V-^-chloro-S-^rifluoromethylJphenylJmethylJ-i-methyl-S-oxo-/.- prolinamide:

In one particular embodiment of the combination of the invention, the compound of formula (I) or the pharmaceutically acceptable salt thereof is Λ/-{[2-chloro-3- (trifluoromethyl)phenyl]methyl}-1 -methyl-5-oxo-L-prolinamide:

and the further therapeutic agent or agents is or are:

- a β2-agonist (also known as β2 adrenoceptor agonists; e.g. formoterol) and/or a corticosteroid (e.g. budesonide, fluticasone (e.g. as propionate or furoate esters), mometasone (e.g. as furoate), beclomethasone (e.g. as 17-propionate or 17,21- dipropionate esters), ciclesonide, triamcinolone (e.g. as acetonide), flunisolide, rofleponide or butixocort (e.g. as propionate ester)), e.g. for the treatment of a respiratory disorder (such as asthma or chronic obstructive pulmonary disease (COPD)); or

- a 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitor (e.g. atorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatin, or simvastatin), e.g. for the treatment of a cardiovascular disorder (such as atherosclerosis); or

- a non-steroid anti-inflammatory drug (NSAID; e.g. ibuprofen, naproxen, aspirin, celecoxib, diclofenac, etodolac, fenoprofen, indomethacin, ketoprofen, ketoralac, oxaprozin, nabumetone, sulindac, tolmetin, rofecoxib, valdecoxib, lumaricoxib, meloxicam, etoricoxib or parecoxib; or e.g. paracetamol, loxoprofen or aceclofenac); in particular celecoxib, paracetamol, ibuprofen or diclofenac; more particularly celecoxib administered orally at a dosage regimen of 100 mg or 200 mg (measured as the free base) once or twice daily; e.g. for the treatment of an inflammatory disease or disorder (such as rheumatoid arthritis or osteoarthritis, and/or inflammatory pain); and/or

- a tumour necrosis factor α (TNFα) inhibitor (e.g. etanercept or an anti- TNFα antibody such as infliximab and adalimumab) (e.g. for parenteral administration such as subcutaneous or intravenous administration), e.g. for the treatment of an inflammatory disease or disorder (such as rheumatoid arthritis or osteoarthritis, and/or inflammatory pain); and/or

- 2-hydroxy-5- [ [4- [ (2- pyridinylamino) sulfonyl] phenyl] azo] benzoic acid (sulfasalazine); e.g. for the treatment of an inflammatory disease or disorder (such as rheumatoid arthritis or osteoarthritis; in particular rheumatoid arthritis); and/or e.g. for the treatment of an IL-1 (e.g. IL-1 β) mediated disease (such as rheumatoid arthritis or osteoarthritis, and/or inflammatory or neuropathic pain; in particular rheumatoid arthritis); and/or

- N-[4-[[(2, 4-diamino-6-pteridinyl) methyl] methylamino] benzoyl]- L-glutamic acid (methotrexate), e.g. for the treatment of an inflammatory disease or disorder (such as rheumatoid arthritis or osteoarthritis; in particular rheumatoid arthritis); and/or

- pro TNFα convertase enzyme (TACE), e.g. for the treatment of an inflammatory disease or disorder (such as rheumatoid arthritis or osteoarthritis; in particular rheumatoid arthritis); and/or

- a statin (e.g. for oral administration), such as atorvastatin, lovastatin, pravastatin, simvastatin, fluvastatin, cerivastatin, crilvastatin, dalvastatin, rosuvastatin, tenivastatin, fluindostatin, velostatin, dalvastatin, nisvastatin, bervastatin, pitavastatin, rivastatin, glenvastatin, eptastatin, tenivastatin, flurastatin, rosuvastatin or itavastatin; e.g. for the treatment of an IL-1 (e.g. IL-1 β) mediated disease (such as rheumatoid arthritis); and/or

- a glucocorticoid agent (e.g. for oral or skin-topical administration), such as dexamethasone, methylprednisolone, prednisolone, prednisone and hydrocortisone; e.g. for the treatment of an I L- 1 (e.g. IL-1 β) mediated disease (such as rheumatoid arthritis); and/or

- an inhibitor of p38 kinase (e.g. for oral administration); e.g. for the treatment of an IL-1 (e.g. IL-1 β) mediated disease (such as rheumatoid arthritis); and/or

- an anti-IL-6-receptor antibody, e.g. an anti-IL-6-receptor monoclonal antibody (e.g. for parenteral such as intravenous administration); e.g. for the treatment of an IL-1 (e.g. IL-1 β) mediated disease (such as rheumatoid arthritis); and/or

- anakinra; e.g. for the treatment of an IL-1 (e.g. I L- 1 β ) mediated disease (such as rheumatoid arthritis); and/or

- an anti-IL-1 (e.g. I L-1 β) monoclonal antibody (e.g. for parenteral such as intravenous administration); e.g. for the treatment of an IL-1 (e.g. I L-1 β) mediated disease (such as rheumatoid arthritis); and/or

- an inhibitor of JAK3 protein tyrosine kinase; e.g. for the treatment of an IL-1 (e.g. IL- 1 β) mediated disease (such as rheumatoid arthritis); and/or

- an anti-macrophage colony stimulation factor (M-CSF) monoclonal antibody; e.g. for the treatment of an IL-1 (e.g. I L- 1 β ) mediated disease (such as rheumatoid arthritis); and/or

- an anti-CD20 monoclonal antibody (e.g. for parenteral such as intravenous administration), such as ofatumumab (HuMax-CD20 ™, developed in part by Genmab AS) (e.g. ofatumumab for intravenous administration), rituximab, PRO70769, AME-133 (Applied Molecular Evolution), or hA20 (Immunomedics, Inc.); in particular ofatumumab or rituximab; e.g. for the treatment of an IL-1 (e.g. IL-1 β) mediated disease (such as rheumatoid arthritis or osteoarthritis, and/or inflammatory or neuropathic pain; in particular rheumatoid arthritis); and/or e.g. for the treatment of an inflammatory disease or disorder (such as rheumatoid arthritis or osteoarthritis, and/or inflammatory pain); and/or

- a therapeutic agent or agents capable of treating inflammatory pain; such as paracetamol (e.g. oral paracetamol, e.g. 500 mg to 1000 mg for oral administration two, three or four times daily) and/or an opioid (such as morphine, fentanyl, oxycodone, tramadol, hydrocodone, hydromorphone, oxymorphone, methadone or buprenorphine; in particular morphine, fentanyl, oxycodone, or tramadol); and e.g. this/these therapeutic agent(s), and/or the combination comprising this/these therapeutic agent(s), can be for the treatment of inflammatory pain, e.g. in a mammal such as a human; and/or

- a therapeutic agent or agents capable of treating neuropathic pain; such as an opioid (such as morphine, fentanyl, oxycodone, tramadol, hydrocodone, hydromorphone, oxymorphone, methadone or buprenorphine; in particular morphine, fentanyl, oxycodone, or tramadol); a monoamine reuptake inhibitor (such as duloxetine or amytriptyline); pregabalin (e.g. for oral administration e.g. as described herein), gabapentin (e.g. for oral administration e.g. as described herein), gabapentin enacarbil (XP13512) (e.g. for oral administration e.g. as described herein), and/or carbamazepine; and e.g. this/these therapeutic agent(s), and/or the combination comprising this/these therapeutic agent(s), can be for the treatment of neuropathic pain, e.g. in a mammal such as a human.

In the above-defined particular combination of the invention, the Λ/-{[2-chloro-3- (trifluoromethyl)phenyl]methyl}-1 -methyl-5-oxo-L-prolinamide and the further therapeutic agent or agents may be present as separate pharmaceutical formulations

/ compositions, or may be present as a combined pharmaceutical formulation / composition (e.g. may be together in a single combined oral dosage form, e.g. a single combined tablet or capsule). These components of this combination can be administered either sequentially in separate pharmaceutical formulations / compositions (e.g. oral), or simultaneously in separate or combined pharmaceutical formulation(s) / composition(s) (e.g. oral); in a particular embodiment they are administered sequentially in separate pharmaceutical formulations / compositions

(e.g. oral).

Antagonists of P2X7 may be useful in preventing, treating, or ameliorating a variety of pain states (e.g. neuropathic pain, inflammatory pain (e.g. chronic), and visceral pain), inflammation and neurodegeneration, in particular Alzheimer's disease. P2X7 antagonists may also constitute useful therapeutic agents in the management of rheumatoid arthritis and inflammatory bowel disease. Compounds of formula (I) or salts thereof, within the combinations of the present invention, modulate P2X7 receptor function and are capable of antagonizing the effects of ATP at the P2X7 receptor ("P2X7 receptor antagonists"), and may be competitive antagonists, inverse agonists, or negative allosteric modulators of P2X7 receptor function.

Certain compounds of formula (I) may in some circumstances form acid addition salts thereof. It will be appreciated that for use in medicine compounds of formula (I) may be used as salts, in which case the salts should be pharmaceutically acceptable. Pharmaceutically acceptable salts include those described by Berge, Bighley and Monkhouse , J. Pharm. ScL, 1977, 66, 1-19.

When a compound of formula (I) is basic, in one embodiment a pharmaceutically acceptable salt is formed from a pharmaceutically acceptable acid such as an inorganic or organic acid. Such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid, and the like. In a particular embodiment, the pharmaceutically acceptable acid is benzenesulfonic, camphorsulfonic, ethanesulfonic, hydrobromic, hydrochloric, methanesulfonic, nitric, phosphoric, sulfuric, or p-toluenesulfonic acid.

Examples of pharmaceutically acceptable salts include those formed from maleic, fumaric, benzoic, ascorbic, pamoic, succinic, hydrochloric, sulfuric, bismethylenesalicylic, methanesulfonic, ethanedisulfonic, propionic, tartaric, salicylic, citric, gluconic, aspartic, stearic, palmitic, itaconic, glycolic, p-aminobenzoic, glutamic, benzenesulfonic, cyclohexylsulfamic, phosphoric and nitric acids.

The compounds of formula (I) or pharmaceutically acceptable salts thereof may be prepared in crystalline or non-crystalline form (e.g. in crystalline or amorphous solid form), and, in particular if crystalline, may optionally be solvated, e.g. as the hydrate. This invention includes within its scope solvates (e.g. hydrates) of compounds of formula (I) or pharmaceutically acceptable salts thereof, for example stoichiometric solvates (e.g. hydrates); as well as compounds or salts containing variable amounts of solvent (e.g. water). Compounds of formula (I) or salts thereof may be capable of existing in stereoisomeric forms (e.g. diastereomers and enantiomers) and the invention extends to each of these stereoisomeric forms and to mixtures thereof including racemates. The different stereoisomeric forms may be separated one from the other by the usual methods, or any given isomer may be obtained by stereospecific or asymmetric synthesis. In examples where the stereochemical composition of the final product has been determined by chiral HPLC (more specifically by methods (A), (B), (C) or (D) as set out in the Examples), the corresponding stereospecific name and structure have been assigned to the final product where the enantiomeric excess of said product is greater than 70%. Assignment of absolute stereochemistry is based on the known chirality of the starting material. In examples where the composition of the final product has not been characterised by chiral HPLC, the stereochemistry of the final product has not been indicated. However, the chirality of the main component of the product mixture will be expected to reflect that of the starting material and the enatiomeric excess will depend on the synthetic method used and is likely to be similar to that measured for an analogous example (where such an example exists). Thus compounds shown in one chiral form are expected to be able to be prepared in the alternative chiral form using the appropriate starting material. Alternatively, if racemic starting materials are used, it would be expected that a racemic product would be produced and the single enatiomers could be separated by the usual methods. The invention also extends to any tautomeric forms and mixtures thereof.

The combinations of the subject invention may also include isotopically-labeled compounds, which are identical to those recited in formula (I) or salts thereof, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number most commonly found in nature. Examples of isotopes that can be incorporated into compounds or salts within the combinations of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, iodine, and chlorine, such as 3H, 11 C, 14C,

18F, 1231 and 1251.

Compounds within the combinations of the present invention and pharmaceutically acceptable salts of said compounds that contain the aforementioned isotopes and/or other isotopes of other atoms are within the scope of the present invention. Isotopically-labeled compounds or salts within the combinations of the present invention, for example those into which radioactive isotopes such as 3H, 14C are incorporated, are potentially useful in drug and/or substrate tissue distribution assays. Tritiated, i.e., 3H, and carbon-14, i.e., 14C, isotopes are for example optionally chosen for their (in some cases) ease of preparation and detectability. 1 1 C and 8F isotopes can be useful in PET (positron emission tomography), and 1251 isotopes can be useful in SPECT (single photon emission computerized tomography). PET and SPECT can be useful in brain imaging. Further, substitution with heavier isotopes such as deuterium, i.e., 2H, can sometimes afford certain effects resulting from greater metabolic stability, for example increased in vivo half- life or reduced dosage requirements and, hence, may be chosen in some circumstances, lsotopically labeled compounds of formula (I) or salts thereof within this invention are in one embodiment and in some cases prepared by carrying out the procedures disclosed in the Schemes and/or in the Examples below, by substituting an available isotopically labeled reagent for a non-isotopically labeled reagent.

In a further particular embodiment of the invention, the compound of formula (I) or the pharmaceutically acceptable salt thereof is not a radioactive isotopically-labelled compound or salt. In a particular embodiment, the compound or salt is not an isotopically-labelled compound or salt.

Preparation of compounds

(I)

Compounds of formula (I), wherein the variables are as defined above, and pharmaceutically acceptable salts thereof may be prepared by the methodology described hereinafter.

Compounds of formula (I) or a pharmaceutically acceptable salt thereof may be prepared according to the following process comprising process (a), (b), (c) or (d) (described below), and optionally preparing a pharmaceutically acceptable salt of the compound. The process comprises: (a) Coupling of a carboxylic acid of formula (2) (or an activated derivative thereof) with an amine of formula (3) (see Scheme 1 ), wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰ and R¹¹ are as defined above. Compounds (2) and (3) are optionally protected;

(b) The reaction of a dicarbonyl compound of formula (4), an isocyanide of formula (5) and an amine of formula (6) in a suitable solvent such as methanol and at a suitable temperature such as 100⁰C (see Scheme 2), wherein R¹, R², R³, R⁴, R⁵, R⁷, R⁸, R⁹, R¹⁰ and R¹¹ are as defined above and R⁵ = H or methyl. Compounds (4), (5) and (6) are optionally protected. Processes of this type have been described previously in the chemical literature (e.g. H.Tye, and M.Whittaker, Org.Biomol.Chem., 2004, 2, 813-815; G. C. B. Harriman WO 9900362 A1 );

(c) Deprotecting a compound of formula (I) which is protected. Examples of protecting groups and the means for their removal can be found in T.W. Greene and P. G. M. Wuts 'Protective Groups in Organic Synthesis' (J.Wiley and Sons, 3^rd Ed. 1999); or

(d) I ntercon version of compounds of formula (I) to other compounds of formula (I). Examples of conventional interconversion procedures include epimerisation, oxidation, reduction, alkylation, aromatic substitution, nucleophilic substitution, amide coupling and ester hydrolysis.

Scheme 1.

(2) (D

The coupling of an acid of formula (2) and an amine of formula (3) typically comprises the use of activating agents, such as N-(3-dimethylaminopropyl)-N'- ethylcarbodiimide hydrochloride or polymer-supported carbodiimide, 1- hydroxybenzotriazole (HOBT) or 1 -hydroxy-7-azabenzotriazole (HOAt), and optionally a suitable base such as a tertiary alkylamine (e.g. diisopropylethylamine, N-ethyl morpholine, triethylamine) or pyridine, in a suitable solvent such as DMF and/or dichloromethane and at a suitable temperature e.g. between O⁰C and room temperature. Alternatively the coupling of (2) and (3) may be accomplished by treatment with O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate and a suitable tertiary alkylamine such as diisopropylethylamine in a suitable solvent such as dimethylformamide at a suitable temperature such as room temperature. Alternatively, the compound of formula (2) may be employed as an activated derivative (e.g. acid chloride, mixed anhydride, active ester (e.g. O-acyl-isourea)), and under such circumstances process (a) typically comprises treatment of said activated derivative with an amine (Ogliaruso, M.A.; Wolfe, J. F. in The Chemistry of Functional Groups (Ed. Patai, S.) Suppl.B: The Chemistry of Acid Derivatives, Pt. 1 (John Wiley and Sons, 1979), pp442-8; Beckwith, A.L.J, in The Chemistry of Functional Groups (Ed. Patai, S.) Suppl.B: The Chemistry of Amides (Ed. Zabricky, J.j(John Wiley and Sons, 1970), pp 73 ff).

Scheme 2.

Representative methods for the preparation of compounds of formula (2) are shown in Schemes 3-9 below: Scheme 3

wherein R¹, R², R³, R⁴ and R⁵ are as defined above, R⁶ = H or F, and P¹ and P² represent suitable protecting groups such as Ci_-6 alkyl or P¹ and P² = H.

Analogous processes to those described below for the transformations outlined in scheme 3 have been described previously in the chemical literature (e.g. G. Verardo, P. Geatti, E. Pol, and A.G. Giumanini, Can.J.Chem., 80: 779-788 (2002); T. Godet, et.al., Organic Letters, (2004), 6(19), 3281 -3284)

Step (i) typically comprises initial treatment of (7) with a base such as sodium hydroxide in a suitable solvent such as methanol at a suitable temperature such as 0⁰C followed by reductive alkylation which typically comprises subsequent treatment with an aldehyde or ketone and an acid, such as acetic acid, and then addition of a reducing agent such as sodium borohydride at a suitable temperature such as between 0°C and room temperature.

Step (ii) may occur spontaneously, in which case (9) is isolated directly from the reaction of (7) as described in step (i) above, but more typically compound (8) is heated at a suitable temperature, such as 1 10⁰C, in a suitable solvent, such as toluene, to afford compound (9) .

Deprotection step (iii) typically comprises a standard procedure for conversion of a carboxylic ester to an acid, such as use of an appropriate hydroxide salt (e.g. sodium hydroxide) in an appropriate solvent such as methanol at a suitable temperature such as between O⁰C and room temperature. Scheme 4

(10) (11 ) (2)

wherein R¹, R², R³, R⁴ and R⁵ are as defined above, R⁶ = H or F, L¹ is a suitable group such as halogen (e.g. chlorine or bromine) or a boronic acid or boronic ester and P³ represents a suitable protecting groups such as C_1-6 alkyl.

Analogous processes to those described below for the transformations outlined in scheme 4 have been described previously in the chemical literature (e.g. T. Itoh, et.al., Tetrahedron., 59 (2003), 3527-3536; T. Simandan and M. B. Smith, Synthetic Communications, 26(9), 1827-1838 (1996)).

Step (i) typically comprises treatment of (10) with a base such as sodium hydride and an alkylating agent such as an alkyl halide in a suitable solvent such as tetrahydrofuran at a suitable temperature such as between 0⁰C and room temperature or alternatively it may comprise treatment of (10) with an aryl halide or aryl or alkenyl boronic acid (or ester) in a suitable solvent such as toluene in the presence of a suitable catalyst such as a mixture of tris(dibenzylideneacetone)dipalladium(0) and Xantphos™ (9,9-dimethyl-4,5- bis(diphenylphosphino)xanthene) and a suitable base such as cesium carbonate at a suitable temperature such as 120⁰C.

Deprotection (ii) typically comprises a standard procedure for conversion of a carboxylic ester to an acid, such as use of an appropriate hydroxide salt (e.g. sodium hydroxide) in an appropriate solvent such as methanol at a suitable temperature such as between 0⁰C and room temperature; or use of an appropriate acid (e.g. trifluoroacetic acid) in an appropriate solvent such as dichloromethane at a suitable temperature such as between O⁰C and room temperature. Scheme 5

(12) (2)

wherein R¹, R², R³, R⁴ and R⁵ are as defined above and R⁶ = H or F.

Analogous processes to those described below for the transformation outlined in scheme 5 have been described previously in the chemical literature (e.g. S. Aoki, et.al., Tetrahedron, 60 (2004) 7053-7059)

Step (i) typically comprises heating (12) in an autoclave or sealed tube in a suitable solvent, such as water, and at a suitable temperature such as from 100-140⁰C with or without microwave irradiation.

Scheme 6

(16) (2) wherein R¹, R⁴ and R⁵ are as defined above, R² represents a group as defined above other than hydrogen or halogen, R⁶ = H or F, L¹ is a suitable leaving group such as halogen (e.g. chlorine or bromine), and P⁴ and P⁵ represent suitable protecting groups such as Ci_-S alkyl and C_1-6 alkoxycarbonyl respectively.

Analogous processes to those described below for the transformations outlined in scheme 6 have been described previously in the chemical literature (e.g. A. Bassoli, et.al., Eur. J. Org. Chem., 2005, 2518-2525).

Step (i) typically comprises protection of (13) by standard protocols such as treatment with an alkoxycarbonyl anhydride, such as di-tertbutyl dicarbonate, and a base such as triethylamine and a catalyst such as 4-dimethylaminopyridine in a suitable solvent such as dichloromethane at a suitable temperature such as room temperature.

Step (ii) typically comprises treatment of (14) with a base such as lithium bis(trimethylsilyl)amide and an alkylating agent such as an alkyl halide in a suitable solvent such as tetrahydrofuran at a suitable temperature such as between -78⁰C and room temperature.

Step (iii) typically comprises deprotection of (15) by standard protocols such as, for the case when P⁵ is a tertbutoxy carbonyl group, treatment with hydrogen chloride in a suitable solvent such as dioxane and at a suitable temperature such as room temperature.

Step (iv) typically comprises the process described above for the steps shown in Scheme 4.

Scheme 7

(17)

wherein R¹, R², R³, R⁴ and R⁵ are as defined above and R⁶ = H or F. P⁵, P⁶ and P⁷ represent suitable protecting groups, for example P⁵ can be a C_1-6 alkoxycarbonyl and P⁶ and P⁷ can be C₁^ alkyl (P⁶ and P⁷ need not be the same). L¹ is a suitable leaving group such as halogen (e.g. chlorine or bromine).

Step (i) typically comprises treatment of (17) with a suitable base, such as potassium hexamethyldisilazide, and an alkylating agent such as an alkyl halide in a suitable solvent such as tetrahydrofuran at a suitable temperature such as between -78°C and room temperature.

Step (N) typically comprises a standard procedure for conversion of a carboxylic ester to an acid, such as treatment with a suitable acid (e.g. trifluoroacetic acid) in an appropriate solvent such as dichloromethane at a suitable temperature such as room temperature.

Scheme 8

wherein R¹, R², R³, R⁴ and R⁵ are as defined above and R⁶ = H or F. P⁸, P⁹ and P¹⁰ represent suitable protecting groups such as Ci_-6 alkyl in the cases of P⁸ and P⁹ (P⁸ and P⁹ need not be the same) and a group derived from a suitable acyclic or cyclic ketone in the case of P¹⁰.

Analogous processes to those described below for the transformations outlined in steps (i)-(iϋ) of scheme 8 have been described previously in the chemical literature (e.g. J. Wehbe, et.al., Tetrahedron: Asymmetry., 14 ( 2003), 1123-1126).

Step (i) typically comprises treatment of (19) with a suitable ketone, such as (1 R,2R,5f?)-2-hydroxypinan-3-one, and a lewis acid such as boron trifluoride etherate in a suitable solvent such as toluene at a suitable temperature such as 1 10⁰C.

Step (ii) typically comprises treatment of (20) with a Grignard reagent, such as methyl magnesium bromide, and a base, such as 1 ,8-diazabicyclo[5.4.0]undec-7-ene, followed by treatment with an unsaturated ester (21 ), such as ethyl crotonate in a suitable solvent such as tetrahydrofuran at a suitable temperature such as -3O⁰C.

Step (iii) typically comprises a standard procedure for conversion of an imine to an amine, such as treatment with a suitable acid (e.g. 15% aqueous citric acid) in an appropriate solvent such as tetrahydrofuran at a suitable temperature such as room temperature.

Step (iv) typically comprises heating (23) in a suitable solvent, such as toluene, at a suitable temperature such as between room temperature and 12O⁰C.

Step (v) typically comprises the process described above for the steps shown in Scheme 4.

Scheme 9

wherein R¹, R⁴ , R⁵ and R⁶ are as defined above, R² and R³ each represent a group as defined above other than halogen, L¹ and L² are suitable leaving groups such as halogen (e.g. chlorine or bromine), and P¹¹ represents a suitable protecting group such as trityl. Step (i) typically comprises treatment of (25) with a base such as sodium hydride and an alkylating agent such as an alkyl halide in a suitable solvent such as dimethylformamide at a suitable temperature such as between 0⁰C and room temperature.

Step (ii) typically comprises treatment of (26) with a base such as lithium diisopropylamide and an alkylating agent such as an alkyl halide in a suitable solvent such as tetrahydrofuran at a suitable temperature such as between -78°C and room temperature.

Step (iii) typically comprises treatment of (27) with a base such as lithium diisopropylamide and an alkylating agent such as an alkyl halide in a suitable solvent such as tetrahydrofuran at a suitable temperature such as between -78°C and room temperature.

Step (iv) typically comprises a standard procedure for deprotecting an alcohol. For example, when P¹¹ is a trityl group, treatment of (28) with a suitable acid such as Amberlyst 15® in a suitable solvent such as methanol and at a suitable temperature such as room temperature.

Step (v) typically comprises a standard protocol for oxidation of an alcohol to the corresponding carboxylic acid such as treatment of the alcohol (29) with an oxidising agent such as a combination of sodium chlorite, TEMPO (2,2,6,6-tetramethyl-1- piperidinyloxy free radical) and bleach (sodium hypochlorite solution) in a suitable solvent such as a mixture of aqueous sodium phosphate monobasic buffer solution and acetonitrile at a suitable temperature such as 40⁰C.

Step (ii) or step (iii) can be omitted as required to prepare compounds where R² = H or R³ = H respectively.

Compounds of the general formulae (3), (4), (5), (6), (7), (10), (12), (13), (17), (19),

(21 ) and (25) are typically either available from commercial sources or can be prepared by a person skilled in the art using methods described in the chemical literature (or using analogous methods).

Where relevant, pharmaceutically acceptable salts may be prepared conventionally by reaction with the appropriate acid or acid derivative. Clinical Indications

It is believed that, as the compounds of formula (I) or the pharmaceutically acceptable salts thereof within the combinations of the present invention modulate P2X7 receptor function and are capable of antagonizing the effects of ATP at the P2X7 receptor, they may be useful in the treatment of pain, including acute pain, chronic pain, chronic articular pain, musculoskeletal pain, neuropathic pain, inflammatory pain, visceral pain, pain associated with cancer, pain associated with migraine, tension headache and cluster headaches, pain associated with functional bowel disorders, lower back and neck pain, pain associated with sprains and strains, sympathetically maintained pain; myositis, pain associated with influenza or other viral infections such as the common cold, pain associated with rheumatic fever, pain associated with myocardial ischemia, post operative pain, cancer chemotherapy, headache, toothache and dysmenorrhea.

The chronic articular pain condition can be rheumatoid arthritis, osteoarthritis, rheumatoid spondylitis (ankylosing spondylitis), gouty arthritis or juvenile arthritis.

The inflammatory pain condition can be rheumatoid arthritis, osteoarthritis, rheumatoid spondylitis (ankylosing spondylitis) or fibromyalgia.

In particular, the compounds of formula (I) or pharmaceutically acceptable salts thereof, and/or the combinations of the present invention, may be useful in the treatment or prevention of pain (e.g. inflammatory pain) in arthritis, such as pain (e.g. inflammatory pain) in rheumatoid arthritis or osteoarthritis.

Pain associated with functional bowel disorders includes non-ulcer dyspepsia, non- cardiac chest pain and irritable bowel syndrome.

The neuropathic pain condition can be: diabetic neuropathy (e.g. painful diabetic neuropathy), sciatica, non-specific lower back pain, trigeminal neuralgia, multiple sclerosis pain, fibromyalgia, HIV-related neuropathy, post-herpetic neuralgia, trigeminal neuralgia, or lumbar radiculopathy; or pain resulting from physical trauma, amputation, phantom limb syndrome, spinal surgery, cancer, toxins or chronic inflammatory conditions. Alternatively, the neuropathic pain can be: pain associated with normally non-painful sensations such as "pins and needles" (paraesthesias and dysesthesias), increased sensitivity to touch (hyperesthesia), painful sensation following innocuous stimulation (dynamic, static, thermal or cold allodynia), increased sensitivity to noxious stimuli (thermal, cold, mechanical hyperalgesia), continuing pain sensation after removal of the stimulation (hyperpathia), or an absence of or deficit in selective sensory pathways (hypoalgesia).

The acute pain condition can be post-surgical pain or dysmenorrhea (e.g. primary dysmenorrhea).

Other conditions which could potentially be treated by the combinations of the present invention include fever, inflammation, immunological diseases, abnormal platelet function diseases (e.g. occlusive vascular diseases), impotence or erectile dysfunction; bone disease characterised by abnormal bone metabolism or resorbtion; hemodynamic side effects of non-steroidal anti-inflammatory drugs (NSAI D's) such as cyclooxygenase-2 (COX-2) inhibitors, cardiovascular diseases; neurodegenerative diseases and neurodegeneration, neurodegeneration following trauma, tinnitus, dependence on a dependence-inducing agent such as opioids (e.g. morphine), CNS (central nervous system) depressants (e.g. ethanol), psychostimulants (e.g. cocaine) or nicotine; complications of Type I diabetes, kidney dysfunction, liver dysfunction (e.g. hepatitis, cirrhosis), gastrointestinal dysfunction (e.g. diarrhoea), colon cancer, overactive bladder and urge incontinence. Depression and alcoholism could potentially also be treated by the combinations of the present invention.

Inflammation and the inflammatory conditions associated with said inflammation include arthritis (in particular rheumatoid arthritis or osteoarthritis), skin conditions

(e.g. sunburn, burns, eczema, dermatitis, allergic dermatitis, psoriasis), meningitis, ophthalmic diseases such as glaucoma, retinitis, retinopathies, uveitis and of acute injury to the eye tissue (e.g. conjunctivitis), inflammatory lung disorders (e.g. asthma, allergic rhinitis, respiratory distress syndrome, pigeon fancier's disease, farmer's lung, chronic obstructive pulmonary disease (COPD, which includes bronchitis and/or emphysema), or airways hyperresponsiveness); gastrointestinal tract disorders (e.g. aphthous ulcer, Crohn's disease, atopic gastritis, gastritis varialoforme, ulcerative colitis, coeliac disease, regional ileitis, irritable bowel syndrome, inflammatory bowel disease, or gastrointestinal reflux disease); organ transplantation and other conditions with an inflammatory component such as vascular disease, migraine, periarteritis nodosa, thyroiditis, aplastic anaemia, Hodgkin's disease, sclerodoma, myaesthenia gravis, multiple sclerosis, sorcoidosis, nephrotic syndrome, Bechet's syndrome, gingivitis, myocardial ischemia, pyrexia, systemic lupus erythematosus, polymyositis, tendinitis, bursitis, and Sjogren's syndrome. Inflammation or an inflammatory condition associated with said inflammation can in particular be arthritis (e.g. rheumatoid arthritis or osteoarthritis).

Immunological diseases include autoimmune diseases, immunological deficiency diseases or organ transplantation.

Bone diseases characterised by abnormal bone metabolism or resorbtion include osteoporosis (especially postmenopausal osteoporosis), hyper-calcemia, hyperparathyroidism, Paget's bone diseases, osteolysis, hypercalcemia of malignancy with or without bone metastases, rheumatoid arthritis, periodontitis, osteoarthritis, ostealgia, osteopenia, cancer cacchexia, calculosis, lithiasis (especially urolithiasis), solid carcinoma, gout and ankylosing spondylitis, tendinitis and bursitis.

Cardiovascular diseases include hypertension or myocardiac ischemia; atherosclerosis; functional or organic venous insufficiency; varicose therapy; haemorrhoids; and shock states associated with a marked drop in arterial pressure (e.g. septic shock).

Neurodegenerative diseases include dementia, particularly degenerative dementia (such as senile dementia, dementia with Lewy bodies, Alzheimer's disease, Pick's disease, Huntingdon's chorea, Parkinson's disease and Creutzfeldt-Jakob disease, ALS, or motor neuron disease; in particular Alzheimer's disease); vascular dementia (including multi-infarct dementia); as well as dementia associated with intracranial space occupying lesions; trauma; infections and related conditions (including HIV infection, meningitis and shingles); metabolism; toxins; anoxia and vitamin deficiency; and mild cognitive impairment e.g. associated with ageing, particularly age associated memory impairment.

The neurodegenerative disease, e.g. to be treated by the compound of formula (I) or salt thereof or by the combination, can for example be degenerative dementia (in particular Alzheimer's disease), vascular dementia (in particular multi-infarct dementia), or mild cognitive impairment (MCI) e.g. MCI associated with ageing such as age associated memory impairment.

The combinations of the present invention may also be useful as neuroprotectants and in the treatment of neurodegeneration following trauma such as stroke, cardiac arrest, pulmonary bypass, traumatic brain injury, spinal cord injury or the like.

The combinations of the present invention may also be useful in the treatment of malignant cell growth and/or metastasis, and myoblastic leukaemia.

Complications of Type 1 diabetes include diabetic microangiopathy, diabetic retinopathy, diabetic nephropathy, macular degeneration, glaucoma, nephrotic syndrome, aplastic anaemia, uveitis, Kawasaki disease and sarcoidosis.

Kidney dysfunction includes nephritis, glomerulonephritis, particularly mesangial proliferative glomerulonephritis and nephritic syndrome.

It is to be understood that reference to treatment includes both treatment of established symptoms and prophylactic treatment, unless explicitly stated otherwise.

According to a further aspect of the invention, we therefore provide a combination as defined herein, for use in human or veterinary medicine and/or for use in therapy.

According to another aspect of the invention, we provide a combination as defined herein for use in the treatment or prevention (e.g. treatment) of a condition which is mediated by P2X7 receptors. The combination can be for use in the treatment or prevention (e.g. treatment) of pain, inflammation (e.g. rheumatoid arthritis or osteoarthritis) or a neurodegenerative disease, in particular for use in the treatment of inflammatory pain, neuropathic pain, visceral pain, rheumatoid arthritis or osteoarthritis; e.g. in a mammal such as a human.

According to a further aspect of the invention, we provide a method of treating a human or animal (e.g. rodent e.g. rat) subject, for example a human subject, suffering from a condition which is mediated by P2X7 receptors, for example a condition or disease disclosed herein (in particular pain, inflammation, rheumatoid arthritis, osteoarthritis or a neurodegenerative disease, more particularly pain such as inflammatory pain, neuropathic pain or visceral pain), which comprises administering to said subject an effective amount of a combination as defined herein.

According to a further aspect of the invention we provide a method of treating a human or animal subject suffering from pain, inflammation (e.g. rheumatoid arthritis or osteoarthritis), or a neurodegenerative disease (more particularly rheumatoid arthritis or osteoarthritis, and/or pain such as inflammatory pain, neuropathic pain or visceral pain), which method comprises administering to said subject an effective amount of a combination as defined herein.

According to a yet further aspect of the invention we provide a method of treating a human or animal (e.g. rodent e.g. rat) subject, for example a human subject, suffering from inflammatory pain, neuropathic pain or visceral pain (e.g. pain, such as inflammatory pain, in arthritis (e.g. rheumatoid arthritis or osteoarthritis)), which method comprises administering to said subject an effective amount of a combination as defined herein.

According to a further aspect of the invention we provide a method of treating a subject, for example a human subject, suffering from Alzheimer's disease which method comprises administering to said subject an effective amount of a combination as defined herein.

According to another aspect of the invention, we provide the use of a combination as defined herein for the manufacture of a medicament for the treatment or prevention (e.g. treatment) of a condition which is mediated by the action of P2X7 receptors, for example a condition or disease disclosed herein, e.g. in a mammal such as a human or rodent e.g. human or rat e.g. human.

According to another aspect of the invention we provide the use of a combination as defined herein for the manufacture of a medicament for the treatment or prevention (e.g. treatment) of pain (e.g. inflammatory pain, neuropathic pain or visceral pain), inflammation (e.g. rheumatoid arthritis or osteoarthritis), or a neurodegenerative disease; e.g. in a mammal such as a human or rodent e.g. human or rat e.g. human.

According to another aspect of the invention we provide the use of a combination as defined herein for the manufacture of a medicament for the treatment or prevention (e.g. treatment) of inflammatory pain, neuropathic pain or visceral pain (in particular inflammatory pain or neuropathic pain; such as inflammatory pain in arthritis such as rheumatoid arthritis or osteoarthritis); e.g. in a mammal such as a human or rodent e.g. human or rat e.g. human.

In one aspect of the invention we provide the use of a combination as defined herein for the manufacture of a medicament for the treatment or prevention (e.g. treatment) of Alzheimer's disease; e.g. in a mammal such as a human or rodent e.g. human or rat e.g. human.

In order to use a combination as defined herein for the treatment of humans and/or other mammals it can optionally be formulated in accordance with pharmaceutical practice as a pharmaceutical composition. Therefore in another aspect of the invention there is provided a pharmaceutical composition comprising a combination as defined herein, adapted for use in human or veterinary medicine.

In order to use a combination as defined herein in therapy, it can optionally be formulated into a pharmaceutical composition in accordance with pharmaceutical practice. The present invention also provides a pharmaceutical composition, which comprises a combination as defined herein, or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier.

A pharmaceutical composition comprising the combination of the invention, and/or a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier (but not comprising the further therapeutic agent(s)), which may be prepared by admixture, e.g. at ambient temperature and/or at atmospheric pressure, is usually adapted for oral, parenteral or rectal administration and, as such, may be in the form of a tablet, a capsule, an oral liquid preparation, a powder, a granule, a lozenge, a reconstitutable powder, an injectable or infusable solution or suspension, or a suppository.

An orally administrable pharmaceutical composition comprising the combination of the invention, and/or an orally administrable pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier (but not comprising the further therapeutic agent(s)), are generally preferred.

Tablets and capsules for oral administration may be in unit dose form, and may contain one or more excipients, such as a binding agent (e.g. hydroxypropylmethylcellulose or povidone), a filler(s) (e.g. lactose monohydrate, anhydrous lactose, and/or microcrystalline cellulose), a lubricant e.g. tabletting lubricant (e.g. magnesium stearate or calcium stearate), a disintegrant (e.g. croscarmellose sodium), and/or an acceptable wetting agent. The tablets may be coated according to methods well known in normal pharmaceutical practice.

Oral liquid preparations may be in the form of, for example, aqueous or oily suspension, solutions, emulsions, syrups or elixirs, or may be in the form of a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain additive(s) such as suspending agent(s), emulsifying agent(s), non-aqueous vehicle(s) such as edible oil(s), preservative(s), and/or, if desired, flavouring(s) or colourant(s).

For parenteral administration, fluid unit dosage forms are typically prepared utilising a compound or salt within the combination of the invention or pharmaceutically acceptable salt thereof and a sterile vehicle. The compound or salt within the combination, depending on the vehicle and concentration used, can e.g. be either suspended or dissolved in the vehicle. In preparing solutions, the compound or salt within the combination can e.g. be dissolved for injection and filter sterilised before filling into a suitable vial or ampoule and sealing. In one embodiment, an adjuvant(s) such as a local anaesthetic, a preservative(s) and/or a buffering agent(s) are dissolved in the vehicle. To enhance the stability, the composition can e.g. be frozen after filling into the vial and the water removed under vacuum. Parenteral suspensions are typically prepared in substantially the same manner, except that the compound or salt within the combination is suspended in the vehicle instead of being dissolved, and sterilization is not usually accomplished by filtration. The compound or salt within the combination can be sterilised, e.g. by exposure to ethylene oxide, before suspension in a sterile vehicle. In one embodiment, a surfactant or wetting agent is included in the composition, e.g. to facilitate uniform distribution of the compound or salt.

In one embodiment, the composition contains from 0.1% to 99% (by weight of the composition), in particular from 0.1 to 60% or from 1 to 60% or from 10 to 60% by weight, of the active material (e.g. the compound of formula (I) or pharmaceutically acceptable salt of the invention), e.g. depending on the method of administration. The carrier(s) and/or excipient(s) contained in the composition can for example be present in from 1% to 99.9%, e.g. from 10% to 99%, by weight of the composition.

The dose of the compound of formula (I) or the pharmaceutically acceptable salt thereof (e.g. of /V-{[2-chloro-3-(trifluoromethyl)phenyl]methyl}-1-methyl-5-oxo-/.- prolinamide) within the combination, e.g. as used in the treatment of the aforementioned disorders, may vary in the usual way with the seriousness of the disorders, the weight of the sufferer, and other similar factors. However, as a general guide suitable unit doses (e.g. orally-administrable unit doses) may be 0.05 to 2000 mg or 0.05 to 1000 mg, for example 0.05 to 200 mg, or may be for example 0.3 to 2000 mg or 10 to 2000 mg or 10 to 1000 mg or 20 to 1000 mg such as 20 to 40 mg, of the compound of formula (I) or the pharmaceutically acceptable salt thereof (e.g. of /V-{[2-chloro-3-(trifluoromethyl)phenyl]methyl}-1 -methyl-5-oxo-/--prolinamide). In one embodiment, such unit doses are typically administered once a day, or more than once a day (e.g. twice a day); e.g. orally and/or e.g. to a human. Such therapy may for example extend for a number of days, weeks, months or years.

In particular, Λ/-{[2-Chloro-3-(trifluoromethyl)phenyl]methyl}-1-methyl-5-oxo-L- prolinamide (e.g. which can be prepared as described in Example 51 herein) can for example be administered to a human at an oral dosage regimen of 0.3 mg to 2000 mg once or twice per day, e.g. 10 mg to 2000 mg once or twice per day, e.g. 10 mg to 1000 mg once or twice per day or 20 mg to 1000 mg once or twice per day. The treatment may for example extend for a number of days, weeks, months or years.

The following Descriptions and Examples illustrate the preparation of compounds of formula (I) but are not intended to be limiting.

EXAMPLES

The general methods (a)-(d), along with the synthetic methods outlined in Schemes 1 -9 above, for the preparation of compounds of formula (I) are further illustrated by the following examples.

Example 1 Λ/-[(2-chloro-4-fluorophenyl)methyl]-5-oxo-1-(phenylmethyl)-prolinamide (E1)

5-Oxo-i -(phenylmethyl)-proline (0.176 g, 0.80 mmol, prepared as described below) was dissolved in dichloromethane (3 ml) and to this was added 1- hydroxybenzotriazole (0.119 g, 0.88 mmol), triethylamine (0.1 13 ml, 0.81 mmol), [(2- chloro-4-fluorophenyl)methyl]amine (0.134 g, 0.84 mmol) and N-(3- dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (0.169 g, 0.88 mmol) under an atmosphere of argon. The mixture was stirred at room temperature overnight. The mixture was diluted with dichloromethane and washed sequentially with 2M aqueous hydrogen chloride and saturated aqueous sodium hydrogen carbonate. The organic layer was filtered through a phase separator and then evaporated to give the crude product. The crude material was purified by mass-directed automated HPLC to give Λ/-[(2-chloro-4-fluorophenyl)methyl]-5-oxo-1 -(phenylmethyl)- prolinamide (0.1 12 g) as a white solid. LC/MS [M+H]⁺ = 361.2, retention time = 2.55 minutes.

The 5-OXO-1 -(phenylmethyl)-proline used in the above procedure was prepared as follows:

(i) Dimethyl L-glutamate hydrochloride (0.500 g, 2.37 mmol) was dissolved in methanol (10 ml) and cooled to 0⁰C. The mixture was then treated with sodium hydroxide (0.099 g, 2.49 mmol) followed by acetic acid (0.136 ml, 2.37 mmol) and benzaldehyde (0.361 ml, 3.55 mmol). After stirring for 10 minutes at 0°C, sodium borohydride (0.088 g, 2.37 mmol) was added and the mixture was left to warm to room temperature and stirred overnight. The mixture was again cooled to 0⁰C and treated with a further quantity of sodium borohydride (0.044 g, 1.18 mmol). The mixture was again left to warm to room temperature and stirred overnight. Evaporation of the methanol gave a residue which was taken up in ethyl acetate and filtered. The filtrate was then washed with saturated aqueous sodium hydrogen carbonate, filtered through a phase separator (with stirring) and evaporated to give a clear oil (0.56 g). The oil was dissolved in methanol and heated in a sealed tube in a microwave reactor at 12O⁰C for 10 minutes and then for 15 minutes at 14O⁰C (LC/MS indicated that this heating phase had not altered the composition of the mixture significantly). The solvent was evaporated and the residue purified by flash-silica column chromatography, eluting with a 15-20% gradient of ethyl acetate in hexane, to give methyl 5-oxo-1-(phenylmethyl)-prolinate (0.212 g) as a clear oil. LC/MS [M+H]⁺ = 234, retention time = 2.15 minutes.

(ii) Methyl 5-oxo-1-(phenylmethyl)-prolinate (0.212 g, 0.91 mmol) was dissolved in water (3 ml) and methanol (0.5 ml) and treated with 2M aqueous sodium hydroxide (0.682 ml, 1.36 mmol). The mixture was stirred overnight at room temperature and then washed with dichloromethane. The aqueous layer was evaporated and the residue treated with an excess of 1 M hydrogen chloride in ether (~5 ml). The mixture was evaporated once more and the residue was triturated with dichloromethane. The solid material was discarded and the combined dichloromethane fractions were evaporated to give 5-oxo-1-(phenylmethyl)-proline (0.182 g) as a yellow oil which was used without further purification. LC/MS [M+H]⁺ = 220, retention time = 1.72 minutes.

Example 2 /V-[(2-chloro-4-fluorophenyl)methyl]-1-(1-methylethyl)-5-oxo-prolinamide (E2)

1-(1-Methylethyl)-5-oxo-proline (0.060 g, 0.35 mmol, prepared as described below) was dissolved in dichloromethane (3 ml) and dimethylformamide (1 ml) and to this was added 1-hydroxybenzotriazole (0.052 g, 0.39 mmol), [(2-chloro-4- fluorophenyl)methyl]amine (0.061 g, 0.39 mmol) and N-(3-dimethylaminopropyl)-N'- ethylcarbodiimide hydrochloride (0.074 g, 0.39 mmol) under an atmosphere of argon. The mixture was stirred at room temperature overnight. The mixture was diluted with dichloromethane and washed sequentially with 2M aqueous hydrogen chloride and saturated aqueous sodium hydrogen carbonate. The organic layer was filtered through a phase separator and then evaporated to give the crude product. The crude material was purified by mass-directed automated HPLC to give Λ/-[(2-chloro- 4-fluorophenyl)methyl]-1 -(1-methylethyl)-5-oxo-prolinamide (0.032 g) as a white solid. LC/MS [M+H]⁺ = 313.1 , retention time = 2.26 minutes.

The 1-(1-methylethyl)-5-oxo-proline used in the above procedure was prepared as follows: (i) Dimethyl L-glutamate hydrochloride (0.500 g, 2.37 mmol) was dissolved in methanol (4 ml) and tetrahydrofuran (8 ml)and the mixture was then treated with crushed sodium hydroxide (0.099 g, 2.49 mmol) for 10 minutes. At this stage acetic acid (0.136 ml, 2.37 mmol) and acetone (0.261 ml, 3.55 mmol) were added together to the mixture as a solution in tetrahydrofuran (1 ml). After stirring for 10 minutes the mixture was cooled to 0⁰C and treated with sodium borohydride pellets (0.088 g, 2.37 mmol). The mixture was then left to warm to room temperature and stirred overnight. Evaporation of the methanol gave a residue which was taken up in ethyl acetate and filtered. The filtrate was then washed with saturated aqueous sodium hydrogen carbonate, filtered through a phase separator (with stirring) and evaporated to give a clear oil (0.217 g). The oil was purified by flash-silica column chromatography to give dimethyl Λ/-(1-methylethyl)-glutamate (0.200 g).

(ii) Dimethyl Λ/-(1-methylethyl)-glutamate (0.200 g) was dissolved in methanol and heated in a sealed tube in a microwave reactor at 140⁰C for 20 minutes. Thin-layer chromatography indicated that the starting material remained intact so the solvent was evaporated and replaced with toluene. The mixture was heated at reflux temperature for ~3 hrs and then evaporated to give methyl 1-(1-methylethyl)-5-oxo- prolinate (0.152 g) as a light yellow oil which was used in the subsequent step without further purification, (iii) Methyl 1 -(1-methylethyl)-5-oxo-prolinate (0.152 g, 0.82 mmol) was dissolved in water (3 ml) and methanol (0.5 ml) and treated with 2M aqueous sodium hydroxide (0.682 ml, 1.36 mmol). The mixture was stirred for ~4 hrs at room temperature and then washed with dichloromethane. The aqueous layer was evaporated and the residue treated with an excess of 1 M hydrogen chloride in ether (~5 ml). The mixture was evaporated once more and the residue was triturated with dichloromethane. The solid material was discarded and the combined dichloromethane fractions were evaporated to give 1-(1-methylethyl)-5-oxo-proline (0.060 g) as a yellow oil which crystallized on standing. Example 3 Λ/-[(2-chloro-4-fluorophenyl)methyl]-1-ethyl-5-oxo-prolinamide (E3)

Methyl 1-ethyl-5-oxo-prolinate (0.135 g, 0.79 mmol, prepared as described below) was dissolved in methanol (4 ml) and treated with 2M aqueous sodium hydroxide (0.592 ml, 1.18 mmol). The mixture was stirred for ~4 hrs at room temperature and then evaporated to give a residue which was then treated with an excess of 1 M hydrogen chloride in ether (~5 ml) for 10 minutes. The mixture was evaporated once more and the residue was dissolved in dichloromethane (4 ml) and dimethylformamide (2 ml) and filtered to remove solids. The resulting solution was transferred to a reaction tube and 1-hydroxybenzotriazole (0.117 g, 0.87 mmol), [(2- chloro-4-fluorophenyl)methyl]amine (0.138 g, 0.87 mmol) and N-(3- dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (0.167 g, 0.87 mmol) were then added. The mixture was flushed with argon and then stirred at room temperature over the weekend. The mixture was then diluted with dichloromethane and washed sequentially with 2M aqueous hydrogen chloride and saturated aqueous sodium hydrogen carbonate. The organic layer was filtered through a phase separator and then evaporated to give the crude product. The crude material was purified by mass-directed automated HPLC to give Λ/-[(2-chloro-4- fluorophenyl)methyl]-1-ethyl-5-oxo-prolinamide (0.086 g) as a white solid. LC/MS [M+H]⁺ = 299.1 , retention time = 2.13 minutes.

Enantiomeric excess = 100.0%, as determined by chiral chromatography method B, indicative of Λ/-[(2-chloro-4-fluorophenyl)methyl]-1-ethyl-5-oxo-L-prolinamide retention time = 8.05 minutes

The methyl 1 -ethyl-5-oxo-prolinate used in the above procedure was prepared as follows:

(i) Dimethyl L-glutamate hydrochloride (0.500 g, 2.37 mmol) was dissolved in methanol (4 ml) and tetrahydrofuran (8 ml)and the mixture was then treated with crushed sodium hydroxide (0.099 g, 2.49 mmol) for 10 minutes. At this stage acetic acid (0.136 ml, 2.37 mmol) and acetaldehyde (0.199 ml, 3.55 mmol) were added together to the mixture as a solution in tetrahydrofuran (1 ml). After stirring for 10 minutes the mixture was cooled to 0⁰C and treated with sodium borohydride pellets (0.088 g, 2.37 mmol). The mixture was then left to warm to room temperature. Once the mixture had reached room temperature it was diluted with ethyl acetate (30 ml) and washed with saturated aqueous sodium hydrogen carbonate, filtered through a phase separator (with stirring) and evaporated to give an oily residue. The oil was dissolved in toluene and heated at reflux for 4 hrs. To ensure complete reaction the mixture was then heated overnight at reflux. The solvent was then evaporated and the resulting residue was purified by flash-silica column chromatography, eluting with a gradient of 30-50% ethyl acetate in hexane, to give crude methyl 1 -ethyl-5-oxo- prolinate (0.135 g) as a clear oil which was used without further purification.

Examples 4-8

In a manner analogous to that described for Example 3 above the compounds tabulated below (Table 1 ) were prepared by substituting the appropriate aldehyde (or ketone) for the acetaldehyde used in the above procedure. All of the aldehydes and ketones used to make the compounds shown in Table 1 are available from commercial sources or can be prepared using routes described previously in the chemical literature.

Table 1

Example 9 Λ/-[(2-chloro-4-f luorophenyl)methyl]-5-oxo-1 -phenyl-prolinamide (E9)

5-0X0-1 -phenyl-proline (0.047 g, 0.23 mmol, prepared as described below) was dissolved in dichloromethane (~2 ml) and dimethylformamide (1 ml) and to this was added 1-hydroxybenzotriazole (0.034 g, 0.25 mmol), [(2-chloro-4- fluorophenyl)methyl]amine (0.040 g, 0.25 mmol), N-ethyl morpholine (0.032 ml, 0.25 mmol) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (0.048 g, 0.25 mmol). The mixture was stirred at room temperature for 4.5 hrs. The mixture was diluted with more dichloromethane and washed sequentially with 2M aqueous hydrogen chloride and saturated aqueous sodium hydrogen carbonate. The organic layer was filtered through a phase separator and then evaporated to give the crude product. The crude material was purified by mass-directed automated HPLC to give Λ/-[(2-chloro-4-fluorophenyl)methyl]-5-oxo-1 -phenyl-prolinamide (0.032 g) as a white solid. LC/MS [M+H]⁺ = 347.1 , retention time = 2.51 minutes.

The 5-OXO-1 -phenyl-proline used in the above procedure was prepared as follows: (i) Methyl 5-oxo-L-prolinate (0.204 ml, 1.75 mmol) was dissolved in toluene (5 ml) and treated with tris(dibenzylideneacetone)dipalladium (0) (0.024 g, 0.03 mmol), bromobenzene (0.184 ml, 1.75 mmol), cesium carbonate (0.795 g, 2.45 mmol) and Xantphos™ (0.040 g, 0.07 mmol). The resulting mixture was heated at 120⁰C for -18 hrs and then allowed to cool to room temperature. The mixture was diluted with ethyl acetate and washed sequentially with 2M aqueous hydrogen chloride, saturated aqueous sodium hydrogen carbonate, and brine. Filtration through a phase separator followed by evaporation gave a yellow/brown oil (~ 0.200 g). The crude material was purified by mass-directed automated HPLC to give methyl 5-oxo-1- phenylprolinate (0.054 g) as an oil which crystallised on standing. LC/MS [M+H]⁺ = 220, retention time = 2.03 minutes.

(ii) Methyl 5-oxo-1 -phenylprolinate (0.054 g, 0.25 mmol) was combined with 2M aqueous sodium hydroxide (0.160 ml, 0.32 mmol) in methanol (1 ml) and stirred overnight at room temperature. The solvent was then evaporated and the residue taken up in ethyl acetate and washed with 2M aqueous hydrogen chloride. The aqueous layer was separated and washed twice more with ethyl acetate and then the combined ethyl acetate layers were dried using a phase separator and evaporated to give 5-OXO-1 -phenyl-proline (0.047 g) as a clear oil.

Example 10 Λ/-[(2,4-dichlorophenyl)methyl]-1 -methyl-5-oxo-prolinamide (E10)

1-Methyl-5-oxo-proline (0.057 g, 0.4 mmol, prepared as described below) was dissolved in anhydrous dichloromethane (6 ml) and to this was added 1- hydroxybenzotriazole (0.060 g, 0.4 mmol), [(2,4-dichloro-phenyl)methyl]amine (0.055 ml, 0.4 mmol), diisopropylamine (0.140 ml, 0.8 mmol) and O-(7-azabenzotriazol-1-yl)- N,N,N',N'-tetramethyluronium hexafluorophosphate (0.152 g, 0.4 mmol). The mixture was stirred at room temperature (20⁰C) under argon for 3 hrs and then overnight. The mixture was diluted with more dichloromethane (25 ml) and washed sequentially with 2M aqueous hydrogen chloride (20 ml), saturated aqueous sodium hydrogen carbonate (20 ml), 10% aqueous sodium carbonate (20 ml) and brine (20 ml). The organic layer was filtered through a hydrophobic frit and then evaporated to give the crude product. The crude material was dissolved in a mixture of dimethylsulphoxide (0.9 ml) and acetonitrile (0.9 ml) and then purified by mass- directed automated HPLC to give Λ/-[(2,4-dichlorophenyl)methyl]-1-methyl-5-oxo- prolinamide (0.085 g) as a white solid. LC/MS [M+H]⁺ = 301 , retention time = 2.16 minutes.

The 1 -methyl-5-oxo-proline used in the above procedure was prepared as follows: (i) /V-methyl-L-glutamic acid (0.500 g, 3.1 mmol) was dissolved in water (1 ml) and heated in a sealed tube at 14O⁰C for 30 minutes in a microwave reactor. The water was then evaporated and the residue triturated with ether to give, after drying, 1 - methyl-5-oxo-proline (0.298 g) as a white solid.

Λ/-[(2,4-dichlorophenyl)methyl]-1-methyl-5-oxo-prolinamide can also be prepared as described below:

1 -Methyl-5-oxo-proline (36.79g, 0.257moles, prepared as described above) was suspended in DCM (dichloromethane) (500ml). EEDQ (2-Ethoxy-1-ethoxycarbonyl- 1 ,2-dihydroquinoline, 66.7g, 0.27moles, 1.05eq) was added in one portion. All material seemed to dissolve to give an opaque mixture and the temperature dropped from 21⁰C to 10⁰C. This was stirred for 20 minutes under argon and then a solution of 2,4-dichlorobenzylamine (36ml, 0.27moles, 1.05eq) in DCM (100ml) was added dropwise over a period of 40 minutes. During addition, a white precipitate formed in the dropping funnel. The mixture bubbled gently and an ice/water bath was used to maintain the temperature between 15-20⁰C. Upon complete addition of amine, the dropping funnel was rinsed with further DCM (50ml) to rinse all the precipitate into the reaction mixture. The mixture was then allowed to warm to room temperature and stirred for approx. 18 hours. Saturated aqueous sodium hydrogen carbonate (200ml) was added to the mixture and stirred for 5 minutes. The organic layer was then separated and washed with 2N HCI (3 x 250ml). During the acid washes, crystals started to form in the organic layer, so this was diluted with further DCM (200ml). The organic layer was dried by passing through a hydrophobic frit and then concentrated under vacuum to give 65g of pink solids. The solids had formed large lumps so the crude material was ground up in a pestle and mortar. These were then triturated with diethyl ether (400ml) and the solids filtered off and washed with further Et₂O (2 x 200ml). Drying then gave 52.96g of pale pink solids. This material was combined with 2 further batches, prepared in the same way, (141.42g total) and then suspended in ethanol (430ml) and water (715ml) and gradually warmed to 65°C (temperature of solution). The mixture gave an almost clear solution (deep pink), except for a very fine solid suspension. After heating at 65⁰C for 20minutes, the flask was removed from the heat and allowed to warm to room temperature overnight. After this time, white needles had precipitated from solution. The mixture was cooled in an ice bath for 20 minutes to ensure all solids had precipitated. The white solids were then filtered off from the pink solution and washed with portions of 3:5 EtOH/H₂O (2 x 400ml), which had been cooled in an ice bath. The solids were dried in a vacuum oven (4O⁰C) for a total of 5 days to give Λ/-[(2,4-dichlorophenyl)methyl]- 1-methyl-5-oxo-prolinamide (125.37 g) as colourless crystals. LC/MS [M+H]⁺ = 301 , retention time = 2.34 minutes. 1H NMR (CDCI₃, 500MHz) δ 2.01 (m, 1 H), 2.34 (m, 1 H), 2.37 (m, 1 H), 2.46 (m, 1 H), 2.80 (s, 3H), 3.99 (dd, 1 H, J = 9.1 , 4.2 Hz), 4.49 (dd, 1 H, J = 14.9, 5.9 Hz), 4.55 (dd, 1 H, J = 14.8, 6.1 Hz), 6.56 (broad t, 1 H, J = 5.7 Hz), 7.24 (dd, 1 H, J = 8.2, 2.1 Hz), 7.33 (d, 1 H, J = 8.2 Hz), 7.40 (d, 1 H, J = 2.1 Hz); ¹³C NMR δ 175.9, 171 .3, 134.5, 134.3, 133.7, 131.5, 129.6, 127.5, 63.8, 41.2, 29.4, 29.2, 23.4.

Enantiomeric excess = 99.5%, as determined by chiral chromatography method A, indicative of Λ/-[(2,4-dichlorophenyl)methyl]-1-methyl-5-oxo-L-prolinamide

retention time = 9.89 minutes

[α]_D = -2.1 ° (c= 1 , MeOH), Temperature = 29.3⁰C, wavelength = 589nm melting point = 144.0-144.8⁰C

Example 1 1 Λ/-[(2-chloro-4-fluorophenyl)methyl]-1-methyl-5-oxo-prolinamide (E11 )

1-Methyl-5-oxo-proline (0.050 g, 0.35 mmol, prepared as described below) was dissolved in anhydrous dichloromethane (-7 ml) and to this was added 1- hydroxybenzotriazole (0.047 g, 0.42 mmol), [(2-chloro-4-fluorophenyl)methyl]amine (0.056 ml, 0.42 mmol), N-ethyl morpholine (0.166 ml, 1.04 mmol) and N-(3- dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (0.067 g, 0.42 mmol). The mixture was stirred at room temperature overnight. A further aliquot of [(2-chloro-4- fluorophenyl)methyl]amine (0.100 ml, 0.8 mmol) was added to the mixture and stirring continued for a while longer but HPLC indicated that no further product was forming. The mixture was washed sequentially with 2M aqueous hydrogen chloride (5 ml) and saturated aqueous sodium hydrogen carbonate (5 ml). The organic layer was collected and evaporated to give the crude product. The crude material was purified by mass-directed automated HPLC to give Λ/-[(2-chloro-4- fluorophenyl)methyl]-1-methyl-5-oxo-prolinamide (0.015 g) as a white solid. LC/MS [M+H]⁺ = 285, retention time = 2.04 minutes.

The 1 -methyl-5-oxo-proline used in the above procedure was prepared as follows: (i) (7-J-pyroglutamic acid methyl ester (1 g, 6.99 mmol) was dissolved/mixed with tetrahydrofuran (10 ml) and cooled to 0⁰C using an ice-bath. Sodium hydride (0.201 g of a 60% suspension in oil, 8.38 mmol) was added to the mixture. After bubbling stopped, methyl iodide (0.522 ml, 8.38 mmol) was added and the mixture was allowed to warm to room temperature and then stirred for 1 hr. The solvent was evaporated and water was added (1 ml). The aqueous layer was then extracted with dichloromethane. Evaporation of the dichloromethane gave crude methyl 1-methyl- 5-oxo-prolinate (0.308 g) which was used in the next step without further purification. (H) Methyl 1-methyl-5-oxo-prolinate (0.308 g, 1.96 mmol) was dissolved in methanol (-10 ml) and to this was added a solution of sodium hydroxide (0.157 g, 3.92 mmol) in water (-10 ml). The mixture was heated at reflux for 3 hrs, then cooled and evaporated to leave a minimal amount of water. This was acidified to pH 1 using 2M aqueous hydrogen chloride. The aqueous layer was washed with dichloromethane and then separated and evaporated to give 1-methyl-5-oxo-proline as a white solid (0.300 g).

Example 12 1-Ethyl-5-oxo-Λ/-[(2,3,4-trifluorophenyl)methyl]-prolinamide (E12)

1-Ethyl-5-oxo-proline (0.050 g, 0.32 mmol) was dissolved in anhydrous dichloromethane (~7 ml) and dimethylformamide (1 ml) and to this was added 1- hydroxybenzotriazole (0.052 g, 0.38 mmol), [(2,3,4-trifluorophenyl)methyl]amine (0.103 g, 0.64 mmol), N-ethyl morpholine (0.151 ml, 0.95 mmol) and N-(3- dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (0.073 g, 0.38 mmol). The mixture was shaken at room temperature over the weekend. A further aliquot of [(2,3,4-trifluorophenyl)methyl]amine (0.051 g, 0.32 mmol) was added to the mixture and stirring continued for a while longer until HPLC indicated that no further product was forming. The mixture was diluted with 2M aqueous hydrogen chloride (5 ml) and then filtered through a phase seperator. The organic layer was then washed with saturated aqueous sodium hydrogen carbonate and again filtered through a phase separator. The organic layer was then evaporated to give the crude product. The crude material was purified by mass-directed automated HPLC to give 1-ethyl-5-oxo- Λ/-[(2,3,4-tήfluorophenyl)methyl]-prolinamide (0.032 g). LC/MS [M+H]⁺ = 301 , retention time = 2.03 minutes.

The 1-ethyl-5-oxo-proline used in the above procedure was prepared as follows (Method A):

(i) Dimethyl L-glutamate hydrochloride (5.0 g, 23.7 mmol) was dissolved in methanol (100 ml) and the mixture was then treated with ground sodium hydroxide (1.0 g, 24.9 mmol) under argon at room temperature. After 5 minutes, acetaldehyde (1.99 ml, 35.5 mmol) was added and stirring continued for 10 minutes. The mixture was cooled to 0⁰C and treated with sodium borohydride granules (0.701 g, 18.95 mmol). Stirring was continued for 1 hr at 0⁰C and then the methanol was evaporated off and the residue was taken up in ethyl acetate and filtered. The filtrate was then washed with brine and the brine washing was extracted with ethyl acetate. The combined ethyl acetate fractions were filtered through a hydrophobic frit and evaporated to give a clear oil (3.2 g). The oil was dissolved in toluene (30 ml) and heated at reflux overnight. The toluene was then evaporated to give a light orange residue which was purified by flash-silica column chromatography, eluting with a gradient of 20-60% ethyl acetate in hexane, to give partially pure methyl 1-ethyl-5-oxo-prolinate (1.9 g) as a clear oil. This was used in the next step without further purification, (ii) Methyl 1-ethyl-5-oxo-prolinate (1.91 g, 1 1.17 mmol) was dissolved in methanol (25 ml) and treated with 2M aqueous sodium hydroxide (7.3 ml, 14.52 mmol). The mixture was stirred for 4 hrs at room temperature and then washed with dichloromethane. The aqueous layer was evaporated and the residue treated with an excess of 1 M hydrogen chloride in ether (~5 ml). The mixture was evaporated once more and the residue was triturated with dichloromethane. The solid material was discarded and the combined dichloromethane fractions were evaporated to give a clear oil which crystallized on standing. Trituration with hexane and ether and drying gave 1 -ethyl-5-oxo-proline (0.271 g) as a white solid.

Alternatively, 1 -ethyl-5-oxo-proline may be prepared as follows (Method B):

(i) 1 ,1-dimethylethyl 5-oxo-L-prolinate (2.7 g, 12 mmol, prepared as described in Synth. Comm. , 2005, 35(8), 1 129) was added to a suspension of sodium hydride (0.428 g (60% suspension in oil), 10.7 mmol) in tetrahydrofuran (6 ml) and the mixture was stirred at room temperature for 5 minutes. Ethyl iodide (1.67 g, 10.7 mmol) was then added and the mixture was heated at 4O⁰C for 2 hrs. A further quantity of sodium hydride (0.24 g) was added and stirring continued overnight at room temperature. An additional quantity of ethyl iodide (0.86 ml) was added to the mixture at this stage and the mixture was left to stand at room temperature over the weekend. Water (-10 ml) was added to the mixture and this was stirred for 15 minutes. The tetrahydrofuran was evaporated and the remaining aqueous layer was extracted with dichloromethane (2 x 50 ml) and a 3: 1 mixture of chloroform and isopropanol (50 ml). The combined organic layers were filtered through a hydrophobic frit and evaporated to give a yellow oil. Toluene was added to the mixture and then evaporated to a yellow oil once more. This material was purified by automated silica flash-column chromatography (Biotage SP4), eluting with a 15- 100% gradient of ethyl acetate in hexane, to give 1 ,1-dimethylethyl 1 -ethyl-5-oxo- prolinate.

(ii) 1 ,1-Dimethylethyl 1 -ethyl-5-oxo-prolinate (0.965 g) was dissolved in dichloromethane (~5 ml) and treated with trifluoroacetic acid (1 ml). The mixture was stirred at room temperature for 1 .5 hrs and then evaporated. The resulting material was mostly starting material so a further amount of trifluoroacetic acid (1 ml) and dichloromethane (~5 ml) was added and the mixture stirred at room temperature for 36 hrs. The mixture was evaporated and then toluene was added to the residue and this was in turn also evaporated. After repeating this process once more, crude 1- ethyl-5-oxo-proline was obtained as a dark yellow oil which was used without additional purification.

Alternatively, 1 -ethyl-5-oxo-proline may be prepared as follows (Method C): (i) 1 -(1 ,1 -Dimethylethyl) 5-methyl-L-glutamate hydrochloride (5.0 g, 19.71 mmol) was dissolved in a mixture of methanol (30 ml) and tetrahydrofuran (60 ml) and the mixture was then treated with crushed, powdered sodium hydroxide (0.828 g, 20.69 mmol) under argon at room temperature. After stirring for 10 minutes, acetaldehyde (1.1 1 ml, 19.71 mmol) and acetic acid (1.13 ml, 19.71 mmol) were added and stirring continued for 10-15 minutes. The mixture was cooled to 0⁰C in an ice-bath and treated with sodium borohydride pellets (0.746 g, 19.71 mmol). Stirring was continued for -1 hr at O⁰C under argon. The mixture was allowed to warm to room temperature to give a thick suspension. Fine white solids were filtered off and then the methanol was evaporated off and the residue was taken up in dichloromethane (-50 ml) and washed with saturated aqueous sodium hydrogen carbonate (-25 ml). The organic layer was separated using a phase separator and then the aqueous layer was back extracted with more dichloromethane (2 x 20 ml). The combined organic layers were evaporated to give a colourless oil (-4 g). The oil (3g, assume 14.8 mmol) was dissolved in toluene (30 ml) and heated at reflux for -16 hrs overnight to give an orange solution. The toluene was then evaporated to give an orange oil (2.6 g). This was combined with a further batch of oil (0.850 g) which was obtained in the same manner and then purified by automated flash-silica column chromatography (Biotage SP4), eluting with a gradient of 20-80% ethyl acetate in hexane, to give 1 ,1-dimethylethyl 1-ethyl-5-oxoprolinate (2.14 g). (ii) 1 ,1-Dimethylethyl 1-ethyl-5-oxoprolinate (0.933 g) was dissolved in dichloromethane (-5 ml) and treated with trifluoroacetic acid (1 ml). The mixture was stirred for 3 hrs at room temperature and then evaporated. The residue was taken up in toluene and evaporated once more. This gave partially pure (>95%) 1-ethyl-5- oxo-proline as an orange/yellow oil (0.914 g) which was used without further purification.

Examples 13-36

In a manner analogous to that described for Example 12 above the compounds tabulated below (Table 2) were prepared by substituting the appropriate amine (or salt thereof) for the [(2,3,4-trifluorophenyl)methyl]amine used in the above procedure. All of the amines used to make the compounds shown in Table 2 are available from commercial sources or can be prepared using routes described previously in the chemical literature or by methods analogous to those. The 1-ethyl-5-oxo-proline used in the reaction was prepared, in each case, by the method indicated. Where determined (by chiral HPLC), the enantiomeric excess (e.e.) of the isomer shown is also listed along with its stereospecific name, the chiral separation method used in parantheses and the corresponding retention time (r.t.) in that method.

Table 2

The [(2-chloro-3,4-difluorophenyl)methyl]amine hydrochloride required for the synthesis of Λ/-[(2-chloro-3,4-difluorophenyl)methyl]-1-ethyl-5-oxoprolinamide (Example 36) was prepared in the following manner : (i) A solution of N,N,N',N'-tetramethylethylenediamine (39.6 ml, 264 mmol) in tetrahydrofuran (170 ml) was cooled under argon to -70⁰C before the addition of sec- butyl lithium (205 ml, 288 mmol). To the mixture 3,4-difluorobenzoic acid (19 g, 120 mmol) was then added as a solution in tetrahydrofuran (80 ml) over a period of 40 minutes ensuring that the temperature of the mixture did not rise above -6O⁰C. The mixture was then stirred at a temperature of -68°C to -7O⁰C for 1 hr before adding a solution of hexachloroethane (100 g, 422 mmol) in tetrahydrofuran (170 ml) over a period of 35 minutes whilst keeping the temperature of the mixture below -60⁰C. The mixture was stirred at a temperature of -65⁰C to -7O⁰C for 2 hrs. The mixture was allowed to warm to -10⁰C and then water (500 ml) was added to quench the reaction. The mixture was diluted with diethyl ether (250 ml) and the two resulting layers were separated. The aqueous layer was acidified to pH1 using concentrated aqueous hydrogen chloride and then extracted with 2 x 500 ml aliquots of diethyl ether. The combined organic extracts were passed through a hydrophobic frit and reduced in vacuo to give a yellow solid. This was recrystallised from ethyl acetate to give two crops (8.35 g and 4.47 g) of 2-chloro-3,4-difluorobenzoic acid, (ii) 2-Chloro-3,4-difluorobenzoic acid (2 g, 10.4 mmol) was treated with thionyl chloride (3.04 ml) and the mixture was heated to 80°C for 90 minutes. The mixture was then cooled and reduced in vacuo. The residue was dissolved in anhydrous 1 ,4- dioxane (10 ml) and the mixture was then cooled in an ice-water bath. 0.88 Ammonia (aqueous, 25 ml) was added dropwise to the mixture which was subsequently allowed to warm to 22°C over a period of 2 hrs. This process was repeated using 10.8 g of 2-chloro-3,4-difluorobenzoic acid, 8.2 ml of thionyl chloride, and 45 ml of 0.88 ammonia and then both mixtures were combined and partitioned between ethyl acetate (150 ml) and water (100 ml). The aqueous layer was separated and extracted with 2 x 150 ml aliquots of ethyl acetate. The combined organic extracts were then washed with saturated aqueous sodium hydrogen carbonate (100 ml), dried using a hydrophobic frit, and reduced in vacuo to give 2- chloro-3,4-difluorobenzamide (11.86 g) as a white solid. LC/MS [M+H]⁺ = 192/194, retention time = 1.69 minutes.

(iii) 2-Chloro-3,4-difluorobenzamide (1 1.85 g, 62 mmol) was dissolved in tetrahydrofuran (200 ml) and treated with 1 M borane tetrahydrofuran (247 ml, 247 mmol). The mixture was heated to 70⁰C and stirred for 18 hrs. The mixture was then cooled in an ice-water bath and concentrated aqueous hydrogen chloride (150 ml) was added dropwise. Heating, with stirring, at 70⁰C was then resumed for a further 2 hrs. The mixture was then allowed to cool and the solvent was evaporated in vacuo. The residue was partitioned between ethyl acetate (200 ml) and 2N aqueous hydrogen chloride (200 ml). The aqueous layer was separated and the pH was adjusted to 8-9 by dropwise addition of 5N aqueous sodium hydroxide. The resulting cloudy suspension was extracted with ethyl acetate (4 x 200 ml) and the combined organic extracts were then passed through a hydrophobic frit and reduced in volume to -200 ml. The mixture was then acidified by the addition of 1 M ethereal hydrogen chloride (100 ml) resulting in formation of a precipitate. The solvent was evaporated in vacuo to give a white solid. The solid was recrystallised from methylated spirit (60 ml) to give three crops of [(2-chloro-3,4- difluorophenyl)methyl]amine hydrochloride (combined mass = 4.46 g) as a white solid.

Example 37 Λ/-[(2-chloro-4-fluorophenyl)methyl]-1-ethyl-5-oxo-4-(phenylmethyl)- prolinamide (E37)

Crude 1 -ethyl-5-oxo-4-(phenylmethyl)-proline (0.052 g, 0.09 mmol, prepared as described below) was suspended in a mixture of dichloromethane (0.5 ml) and dimethylformamide (0.5 ml) and to this was added N-ethyl morpholine (0.034 ml, 0.27 mmol) causing most of the material to dissolve. 1-Hydroxybenzotriazole (0.016 g, 0.12 mmol) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (0.022 g, 0.12 mmol) were then added and the mixture was stirred for 10 minutes before adding [(2-chloro-4-fluorophenyl)methyl]amine (0.019 g, 0.12 mmol). The mixture was then left to stand at room temperature overnight. Saturated aqueous sodium hydrogen carbonate (~2 ml) was added to the mixture and stirred for 10 minutes. The organic layer was isolated by filtering through a phase separator and then washed with 2M aqueous hydrogen chloride. The organic layer was separated again and evaporated to give a yellow oil which was purified by mass-directed automated HPLC to give Λ/-[(2-chloro-4-fluorophenyl)methyl]-1-ethyl-5-oxo-4- (phenylmethyl)-prolinamide (0.004 g) as a colourless oil. LC/MS [M+H]⁺ = 389, retention time = 2.90 minutes.

The 1 -ethyl-5-oxo-4-(phenylmethyl)-proline used in the above procedure was prepared as follows (Method A): (i) Methyl (S)-(+)-2-pyrrolidinone-5-carboxylate (0.85 g, 5.94 mmol) was dissolved in dichloromethane (5 ml) and treated with triethylamine (0.869 ml, 6.24 mmol) and 4- dimethylaminopyridine (0.010 g). To this was added di-tertbutyl dicarbonate (1 .36 g, 6.24 mmol) and the resulting orange solution was left to stir overnight. The mixture turned blue/grey and evaporation of the solvent gave a grayish oil (1.4 g). This was purified by automated flash-silica column chromatography (Biotage SP4), eluting with a 0-60% gradient of ethyl acetate in hexane, to give 1-(1 ,1-dimethylethyl) 2-methyl-5- oxo-1 ,2-pyrrolidinedicarboxylate (1.37 g) as colourless oil which crystallized on standing. (ii) 1 -(1 ,1-Dimethylethyl) 2-methyl-5-oxo-1 ,2-pyrrolidinedicarboxylate (0.324 g, 1.33 mmol) was dissolved in tetrahydrofuran (3 ml) and the mixture was cooled to -78°C, using an acetone/cardice bath, under an atmosphere of argon. A 1 M solution of lithium bis(trimethylsilyl)amide in tetrahydrofuran (1.4 ml, 1 .40 mmol) was added dropwise and stirred under argon for 1 hour. To this was then added benzyl bromide (0.174 ml, 1.46 mmol) and the mixture was stirred at -78⁰C for a further 2.5 hrs. The mixture was then allowed to warm to room temperature and quenched by the addition of saturated aqueous ammonium chloride (-5 ml) and then left to stand overnight at room temperature. The organic layer was separated and the aqueous layer was diluted with more water (5 ml) and extracted with ethyl acetate (3 x 10 ml). The combined organic layers were dried over anhydrous sodium sulphate and then filtered and concentrated to give a yellow oil (0.700 g). This was purified by automated flash-silica column chromatography (Biotage SP4), eluting with a 0-35% gradient of ethyl acetate in hexane, to give 1 -(1 ,1-dimethylethyl) 2-methyl-5-oxo-4- (phenylmethyl)-1 ,2-pyrrolidinedicarboxylate as a white solid (0.418 g) after evaporation of solvent.

(iii) 1-(1 ,1-Dimethylethyl) 2-methyl-5-oxo-4-(phenylmethyl)-1 ,2- pyrrolidinedicarboxylate (0.415 g, 1.24 mmol) was dissolved in 4M hydrogen chloride in dioxane (2 ml) and stirred at room temperature for 2 hrs. The solvent was evaporated to give a colourless oil which crystallized on standing to give methyl-5- oxo-4-(phenylmethyl)-prolinate as a creamy/white solid. (0.205 g). This was used without further purification in the next step.

(iv) Methyl-5-oxo-4-(phenylmethyl)-prolinate (0.205 g, 0.88 mmol) was dissolved in tetrahydrofuran (2.5 ml) and treated with ethyl iodide (0.077 ml, 0.97 mmol). The mixture was then cooled to 0⁰C and treated with sodium hydride (0.037 g of a 60% suspension in oil, 0.92 mmol). After stirring at O⁰C for 10-15 minutes the solution was warmed to room temperature and stirred for a further 3.5 hrs. The mixture was then treated with saturated aqueous ammonium chloride solution (~2 ml) and subsequently diluted with dichloromethane (5 ml). The organic layer was separated by filtering through a hydrophobic frit (washing the aqueous with further aliquots of dichloromethane (2 x 5 ml)). Evaporation of the combined organic phases gave a brown oil (~ 0.100 g). This was purified by automated flash-silica column chromatography, eluting with a 0-100% gradient of ethyl acetate in hexane, to give partially purified (-90% pure) methyl 1-ethyl-5-oxo-4-(phenylmethyl)-prolinate (0.024 g) as a yellow oil which was used in the subsequent step without further purification, (v) Methyl 1-ethyl-5-oxo-4-(phenylmethyl)-prolinate (0.024 g, 0.09 mmol) was dissolved in methanol (0.5 ml) and cooled to O⁰C in an ice-bath. 2M aqueous sodium hydroxide (0.137 ml, 0.27 mmol) was added to the mixture and stirring continued at 0⁰C for 3 hrs. The solvent was evaporated and the residue was acidified by treatment with 2M aqueous hydrogen chloride (~ 0.2 ml) to give a cloudy solution. Evaporation then gave crude 1-ethyl-5-oxo-4-(phenylmethyl)-proline (0.052 g) as a mixture of white solids and yellow oily residues. This was used without further purification.

Alternatively 1-ethyl-5-oxo-4-(phenylmethyl)-proline could also be prepared in the following manner (Method B):

(i) (S)-(+)-L-5-trityloxymethyl-2-pyrrolidinone (1 .88 g, 20 mmol) was dissolved in dimethylformamide (9 ml) at 0⁰C and treated with sodium hydride (60% suspension in oil, 0.220 g, 5.5 mmol). The mixture was stirred at 0⁰C for 30 mins and then treated with ethyl iodide (0.444 ml, 5.5 mmol). The mixture was allowed to warm to room temperature and then stirred overnight. The mixture was then partitioned between ethyl acetate and saturated aqueous ammonium chloride and extracted with ethyl acetate (x3). The combined organic extracts were washed sequentially with water, 50% aqueous sodium chloride solution (x2), and saturated aqueous sodium chloride solution, and then dried over sodium sulphate. Concentration gave a beige solid which was purified by automated flash silica-gel column chromatography (Biotage SP4), eluting with a 0-100% gradient of ethyl acetate in hexane, to give 1 - ethyl-5-{[(triphenylmethyl)oxy]methyl}-2-pyrrolidinone (1.78 g).

(ii) A 2M solution of lithium diisopropylamine in tetrahydrofuran (1.050 ml, 2.1 mmol) was added, at -78°C, to a solution of 1-ethyl-5-{[(triphenylmethyl)oxy]methyl}-2- pyrrolidinone (0.771 g, 2 mmol) in tetrahydrofuran (10 ml) and the resulting mixture was stirred for 1 hr at -78°C. Benzyl bromide (0.262 ml, 2.2 mmol) was then added and after stirring for a further 1 hr at -78⁰C the mixture was allowed to warm to room temperature overnight. The mixture was quenched with saturated aqueous ammonium chloride and then extracted with ethyl acetate (x3). The combined organic extracts were then washed with water and then with saturated aqueous sodium chloride solution (x2), dried over anhydrous magnesium sulphate, and concentrated to a crude oil (1 .27 g). The crude solid was purified by automated flash silica-gel column chromatography (Biotage SP4), eluting with a 0-100% gradient of ethyl acetate in hexane, to give the desired product (i.e. 1-ethyl-3-(phenylmethyl)-5- {[(triphenylmethyl)oxy]methyl}-2-pyrrolidinone (0.561 g)) which was used in the next step, as well as unreacted starting material and the dialkylation product, 1-ethyl-3,3- bis(phenylmethyl)-5-{[(triphenylmethyl)oxy]methyl}-2-pyrrolidinone (0.053 g). (iii) 1-Ethyl-3-(phenylmethyl)-5-{[(triphenylmethyl)oxy]methyl}-2-pyrrolidinone (0.561 g, 1.1 mmol) was stirred for 24 hrs at room temperature in a mixture of acetonitrile (21 ml) and formic acid (3 ml). The reaction was not complete at this stage so the solvent was evaporated and replaced with formic acid (10 ml) and stirring was continued for 3 hrs. Reaction was still not complete so the mixture was concentrated in vacuo (azeotroping with methanol to remove all of the formic acid) and then dissolved in methanol (20 ml). Amberlyst 15® was then added to the mixture and stirring was continued at room temperature overnight. The resin was filtered off, washing with more methanol, and the filtrate was concentrated to a gum (0.625 g). The gum was purified by automated flash silica-gel column chromatography (Biotage SP4), eluting with a 0-100% gradient of ethyl acetate in hexane, to give 1-ethyl-5- (hydroxymethyl)-3-(phenylmethyl)-2-pyrrolidinone (0.170 g) which was used in the next step.

(iv) 1-Ethyl-5-(hydroxymethyl)-3-(phenylmethyl)-2-pyrrolidinone (0.748 g, 3.21 mmol) was dissolved in acetonitrile (5 ml) and a 1 M aqueous sodium phosphate monobasic buffer solution (3.69 ml, 3.69 mmol), a few crystals of TEMPO (2,2,6,6-tetramethyl-1- piperidinyloxy free radical), and sodium chlorite (0.580 g, 6.41 mmol) were added and the mixture was warmed to 40⁰C. Approximately 1 drop of bleach (sodium hypochlorite solution, available chlorine >12%) was then added to the mixture and stirring continued at 40⁰C for 3 hrs. The mixture was then poured onto ice-water containing 1 % w/w sodium sulphite and the resulting mixture was adjusted to pH2 using 5N aqueous hydrogen chloride and then extracted with ethyl acetate (x3). The combined organic extracts were washed with saturated aqueous sodium chloride and then dried over magnesium sulphate and concentrated to give 1 -ethyl-5-oxo-4- (phenylmethyl)proline (0.807 g) as a solid which was used without additional purification.

Example 38 Λ/-[(2-chloro-4-fluorophenyl)methyl]-1-(2-methyl-2-propen-1-yl)-5- oxoprolinamide (E38)

Crude 1-(2-methyl-2-propen-1-yl)-5-oxoproline (-0.075 g, -0.41 mmol, prepared as described below) was dissolved in dichloromethane (5 ml) and to this was added 1 - Hydroxybenzotriazole (0.061 g, 0.45 mmol), [(2-chloro-4-fluorophenyl)methyl]arnine (0.068 g, 0.43 mmol), and N-(3-dimethylaminopropyl)-N'-ethylcarbodiirnide hydrochloride (0.087 g, 0.45 mmol). The mixture was then stirred at room temperature for 24 hrs. The mixture was diluted with more dichloromethane then washed sequentially with 2M aqueous hydrogen chloride and saturated aqueous sodium hydrogen carbonate. The organic layer was filtered through a phase seperator and evaporated to give a brown residue which was purified by mass- directed automated HPLC to give Λ/-[(2-chloro-4-fluorophenyl)methyl]-1-(2-methyl-2- propen-1-yl)-5-oxoprolinamide (0.018 g) as a white solid. LC/MS [M+H]⁺ = 325.1 , retention time = 2.40 minutes.

The 1-(2-methyl-2-propen-1-yl)-5-oxoproline used in the above procedure was prepared as follows:

(i) Methanol (55 ml) was cooled to -1 O⁰C (using a cardice/carbon tetrachloride bath) with stirring and then thionyl chloride was added dropwise over 45 minutes. (D)- glutamic acid (10 g, 67.96 mmol)was then added in three portions over -5 minutes and then the reaction was stirred for 3 hrs whilst warming to 21⁰C. The solvents were evaporated in vacuo to give a clear oil (15 g) which was dissolved in a mixture of water (150 ml) and dioxane (150 ml). To this sodium carbonate (46 g, 340 mmol) was then slowly added with stirring. Benzyl chloroformate (9.64 ml, 68 mmol) was then added and stirring continued overnight. The mixture was cautiously treated with 2N aqueous hydrogen chloride (250 ml) and then extracted with ethyl acetate (2 x 250 ml). The combined organic fractions were washed with brine and then dried and evaporated to give a clear oil (18.7 g). This was dissolved in dichloromethane (400ml) and treated with concentrated sulphuric acid (1 ml). A large excess of isobutylene was then condensed into the mixture and then stirred overnight at 21⁰C. Saturated aqueous sodium hydrogen carbonate (~ 400 ml) was then added carefully to the mixture and then the organic phase was separated, washed with brine, dried and evaporated in vacuo to give a clear oil (21 .9 g). This was purified by silica gel column chromatography, eluting with a 3:1 mixture of cyclohexane and ethyl acetate, to give 1-(1 ,1-dimethylethyl) 5-methyl Λ/-{[(phenylmethyl)oxy]carbonyl}glutamate (4.87 g).

(ii) To a solution of potassium hexamethyldisilazide (10 ml of a 0.6M solution in toluene, 6 mmol) in tetrahydrofuran (25 ml) at -7O⁰C, was added dropwise a solution of 1-(1 ,1-dimethylethyl) 5-methyl Λ/-{[(phenylmethyl)oxy]carbonyl}glutamate (1.05 g, 3 mmol) in tetrahydrofuran (10 ml) over -5 minutes. The mixture was stirred at -7O⁰C for 1 hr and then treated with methallyl iodide (2.18 g, 12 mmol) in tetrahydrofuran (10 ml). Stirring was continued at at -78⁰C for 2 hrs and then warmed to 21⁰C. After stirring for a further 1 hr the mixture was poured into 1 N aqueous hydrogen chloride and extracted with ethyl acetate (2 x 50 ml). The combined organics were washed with brine, dried, and evaporated in vacuo, to give a yellow oil (1.03 g). This was purified by silica column chromatography, eluting with a 4:1 mixture of cyclohexane and ethyl acetate, to give 1 ,1 -dimethylethyl 1 -(2-methyl-2-propen-1-yl)-5-oxoprolinate as a clear oil (0.322 g). (iii) 1 ,1-dimethylethyl 1-(2-methyl-2-propen-1-yl)-5-oxoprolinate (0.099 g, 0.41 mmol) was dissolved in a mixture of dichloromethane (2 ml) and trifluoroacetic acid (2 ml) and stirred overnight at room temperature. The solvent was evaporated (azeotroping with toluene to remove traces of trifluoroacetic acid) to give crude 1-(2-methyl-2- propen-1-yl)-5-oxoproline as a brown oil which was used without further purification.

Example 39 1 -Cyclopropyl-N-[(2,4-dichlorophenyl)methyl]-5-oxoprolinamide (E39)

To a solution of (2,4-dichlorophenyl)methyl isocyanide (0.047 g, 0.25 mmol) and A- oxobutanoic acid (15% in water, 0.26 ml, 0.4 mmol) in methanol (1.75 ml) was added cyclopropylamine (0.042 ml, 0.6 mmol). The mixture was heated to 100°C for 30 minutes in a microwave reactor. The solvent was removed in vacuo and the residue was purified by mass-directed automated HPLC to give 1-Cyclopropyl-Λ/-[(2,4- dichlorophenyl)methyl]-5-oxoprolinamide (0.072 g) as a white solid. LC/MS [M+H]⁺ = 326/328, retention time = 2.29 minutes.

Example 40 Λ/-{[2-Chloro-3-(trifluoromethyl)phenyl]methyl}-1-cyclopropyl-5- oxoprolinamide (E40)

To a solution of [2-chloro-3-(trifluoromethyl)phenyl]methyl isocyanide (0.088 g, 0.4 mmol) and succinic semialdehyde (15% in water, 0.26 ml, 0.4 mmol) in methanol (1.75 ml) was added cyclopropylamine (0.042 ml, 0.6 mmol). The mixture was heated to 100⁰C for 30 minutes in a microwave reactor. The solvent was removed in vacuo and the residue was purified by mass-directed automated HPLC to give Λ/^^-Chloro-S-^rifluoromethyOphenyllmethy^-i -cyclopropyl-S-oxoprolinamide (0.076 g) as a white solid. LC/MS [M+H]⁺ = 361/363, retention time = 2.39 minutes.

The [2-chloro-3-(trifluoromethyl)phenyl]methyl isocyanide used in the above procedure was prepared as follows:

(i) A solution of {[2-chloro-3-(trifluoromethyl)phenyl]methyl}arnine (1.05 g, 5 mmol) in anhydrous tetrahydrofuran (10 ml) was added dropwise to a solution of N-formyl benzotriazole (0.772 g, 5.25 mmol) in anhydrous tetrahydrofuran (10 ml). The reaction was stirred at 22⁰C for 18 hrs then reduced in vacuo and the residue partitioned between dichloromethane (75 ml) and 2N aqueous sodium hydroxide (40 ml). The organic layer was separated and extracted with 2N aqueous sodium hydroxide (40 ml). The organic layer was passed through a hydrophobic frit and reduced in vacuo to give a white solid. The crude product was purified by automated flash silica column chromatography (Biotage SP4), eluting with a solvent gradient of 0-10% ethyl acetate in dichloromethane, to give {[2-chloro-3- (trifluoromethyl)phenyl]methyl}forrnamide as a white solid, (ii) A solution of {[2-chloro-3-(trifluoromethyl)phenyl]methyl}formamide (0.67 g, 2.82 mmol) in anhydrous dichloromethane (20 ml) was cooled under argon in an ice-water bath before the addition of diisopropylamine (1 .78 ml, 12.7 mmol) followed by phosphorus oxychloride (0.393 ml, 4.23 mmol). The reaction was stirred at between 2-5°C for 2 hrs. The mixture was then reduced in vacuo and the residue treated with saturated aqueous sodium hydrogen carbonate (20 ml) and extracted with dichloromethane (20 ml). The organic layer was passed through a hydrophobic frit and the reduced in vacuo to give a yellow solid. Further drying in vacuo gave [2- chloro-3-(trifluoromethyl)phenyl]methyl isocyanide as an orange gum (0.66 g) which was used without further purification. Example 41 Λ/-[(2-chloro-4-fluorophenyl)methyl]-1 -cyclopropyl-5-oxoprolinamide (E41)

To a solution of [2-chloro-4-fluoro-phenyl]methyl isocyanide

(0.068 g, 0.4 mmol) and succinic semialdehyde (15% in water, 0.26 ml, 0.4 mmol) in methanol (1.75 ml) was added cyclopropylamine (0.042 ml, 0.6 mmol). The mixture was heated to 100⁰C for 30 minutes in a microwave reactor. The solvent was removed in vacuo and the residue was purified by mass-directed automated HPLC to give a colourless gum which was triturated with diethyl ether to give Λ/-[(2-chloro-4- fluorophenyl)methyl]-1-cyclopropyl-5-oxoprolinamide as a pale cream solid (0.058 g). LC/MS [M+H]⁺ = 310, retention time = 2.16 minutes.

The [2-chloro-4-fluoro-phenyl]methyl isocyanide used as the starting material was prepared in an analogous manner to that described for the preparation of [2-chloro-3- (trifluoromethyl)phenyl]methyl isocyanide in example 40 but using 2-chloro-4- fluorophenyl]methyl}amine in the place of 2-chloro-3-

(trifluoromethyl)phenyl]methyl}amine.

Example 42 Λ/-[(2,4-dichlorophenyl)methyl]-1-ethyl-4,4-dimethyl-5-oxoprolinamide (E42)

(2,4-Dichlorophenyl)methyl isocyanide (0.075 g, 0.4 mmol) and crude 2,2-dimethyl-4- oxobutanoic acid (0.1 15 g, 0.6 mmol) were dissolved in methanol (2 ml). Ethylamine solution (2M in water, 0.3 ml, 0.6 mmol) was added and the mixture was heated in a sealed vessel at 100⁰C for 30 minutes in a microwave reactor. The mixture was left to stand over the weekend and then the solvent was removed in vacuo and the resulting orange oil was purified by mass-directed automated HPLC to give a clear oily gum which was triturated with diethyl ether to give Λ/-[(2,4- dichlorophenyl)methyl]-1 -ethyl-4,4-dimethyl-5-oxoprolinamide as a white solid (0.024 g). LC/MS [M+H]⁺ = 343, retention time = 2.57 minutes. The 2,2-dimethyl-4-oxobutanoic acid used as the starting material in the above procedure was prepared as follows:

(i) 2,2-dimethyl-4-pentenoic acid was dissolved in dichloromethane (25 ml) and cooled to -78°C in a CO₂/acetone bath and oxygen was bubbled through the mixture for 5 minutes. The ozone generator was switched on and ozone was bubbled through the mixture for 15 minutes. The flow of ozone was then stopped and the mixture was flushed with oxygen for 5 minutes and then with argon for 2 minutes. TLC indicated that the reaction had not progressed significantly so ozone was bubbled throught the mixture for a further 15 minutes after which time a pale blue colour persisted and a suspension had formed. The ozone flow was switched off and the mixture was flushed with oxygen for 5 minutes and then with argon for 10 minutes (until the exhaust gas gave a negative response to wetted starch/iodine paper). Dimethyl sulphide (1.72 ml, 23.41 mmol) was then added to the mixture and the mixture was allowed to warm to room temperature. After stirring at room temperature for 2 hrs the mixture was concentrated to give a colourless oil (1.5 g). 1.4 g of this material was purified by flash silica column chromatography , eluting with a gradient of 0-50% ethyl acetate in dichloromethane, to give 2,2-dimethyl-4- oxobutanoic acid as a colourless oil (0.649 g).

Example 43 Λ/-{[2-chloro-3-(trifluoromethyl)phenyl]methyl}-1 -(1 -methylethyl)-5- oxoprolinamide (E43)

1-(1-Methylethyl)-5-oxoproline (0.100 g, 0.58 mmol) was dissolved in dichloromethane (20 ml) and to this was added N-(3-dimethylaminopropyl)-N'- ethylcarbodiimide hydrochloride (0.1 11 g, 0.58 mmol), 1-Hydroxybenzotriazole (0.078 g, 0.58 mmol), and N-ethyl morpholine (0.223 ml, 1.75 mmol). Finally {[2-chloro-3- (trifluoromethyl)phenyl]methyl}amine was added to the mixture and stirring continued for -48 hrs. The mixture was then treated with saturated aqueous sodium hydrogen carbonate (20 ml) and stirred vigorously. The aqueous layer was removed using a phase separator and then the solvent was removed from the organic layer using an argon blow-down unit. The resulting residue was treated with a mixture of water and ethylacetate (25 ml, 1 :1 ) and the aqueous layer was subsequently discarded. The organic layer was filtered through a phase separator and evaporated to give an oil. This was triturated with diethyl ether to give a solid and this was subsequently purified by mass-directed automated HPLC to give Λ/-{[2-chloro-3- (trifluoromethyl)phenyl]methyl}-1 -(1-methylethyl)-5-oxoprolinamide (0.097 g) as a white solid. LC/MS [M+H]⁺ = 363, retention time = 2.48 minutes.

The 1-(1-Methylethyl)-5-oxoproline used in the above procedure was prepared in an analogous manner to that described previously for the synthesis of methyl 1-ethyl-5- oxo-prolinate (see example 3) but using acetone in the place of acetaldehyde and with the addition of a subsequent ester deprotection step (using standard conditions, i.e. sodium hydroxide in methanol) being carried out (as opposed to the combined deprotection and amide coupling described in example 3).

Examples 44-49

In a manner analogous to that described for Example 43 above the compounds tabulated below (Table 3) were prepared by substituting the appropriate amine (or salt thereof) for the {[2-chloro-3-(trifluoromethyl)phenyl]methyl}amine used in the above procedure. All of the amines used to make the compounds shown in Table 3 are available from commercial sources or can be prepared using routes described previously in the chemical literature unless stated otherwise.

Table 3

The [(2,3-dichloro-4-fluorophenyl)methyl]amine hydrochloride required for the synthesis of Λ/-[(2,3-dichloro-4-fluorophenyl)methyl]-1-(1-methylethyl)-5-oxo-L- prolinamide (example 48) was prepared in the following manner: (i) Sodium nitrite (0.172 g, 2.5 mmol) was added to a stirred solution of 2-chloro-6- fluoro-3-methyl-phenylamine (0.400 g, 2.5 mmol) in water (20 ml) and 37% aqueous hydrogen chloride (5 ml) at -5°C. The mixture was stirred at -5°C for 5 minutes and then added in one pot to a solution of copper (I) chloride (0.742 g, 7.5 mmol) in 37% aqueous hydrogen chloride (5 ml) whilst maintaining the temperature at -5 to 0⁰C. The reaction mixture was heated to 38°C and stirred for 1 hr then the mixture was cooled and diethyl ether (20 ml) was added. The organic phase was separated and washed with 1 N aqueous hydrogen chloride and then with water. The organic layer was then dried over sodium sulphate and concentrated in vacuo. The crude residue was purified by flash silica column chromatography, eluting with petroleum ether, to give 2,3-dichloro-1 -fluoro-4-methylbenzene (0.090 g, 0.5 mmol) as a white solid, (ii) 2,3-dichloro-1-fluoro-4-methylbenzene (0.090 g, 0.5 mmol) was added to a stirred mixture of potassium dichromate (0.284 g, 1 mmol) in acetic acid (1 ml). 97% Sulphuric acid (0.5 ml) was then added slowly to the mixture which was subsequently heated at 100⁰C for 2 hrs. After cooling to room temperature, water and ice were added and the green solid thus obtained was filtered off and washed with cold water to afford 2,3-dichloro-4-fluorobenzoic acid (0.056 g, 0.27 mmol) as a white solid, (iii) A solution of 2,3-dichloro-4-fluorobenzoic acid (0.200 g, 0.92 mmol) in dichloromethane (~4 ml) was treated with 1 -hydroxybenzotriazole (0.162 g, 1.2 mmol), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (0.230 g, 1.2 mmol), and triethylamine (0.56 ml, 4.0 mmol) under argon at room temperature. The mixture was stirred at room temperature for 40 minutes then treated with ) 32% aqueous ammonium hydroxide (0.088 ml) and stirred overnight at room temperature. The mixture was diluted with dichloromethane and washed sequentially with water and then with saturated aqueous sodium hydrogen carbonate. The organic layer was separated and dried over sodium sulphate then concentrated to give 2,3- dichloro-4-fluorobenzamide (0.156 g) as a white solid that was used without further purification. (iv) A solution of 2,3-dichloro-4-fluorobenzamide (0.750 g, 3.62 mmol) in dry tetrahydrofuran (2 ml) was heated to 90⁰C under nitrogen. A 10M solution of boron hydride dimethyl sulphide complex in tetrahydrofuran (1.05 ml, 5.43 mmol) was added to the hot solution and stirring was continued for 4 hrs. The mixture was then treated with 6N aqueous hydrogen chloride and heating continued for 30 minutes. The solvents were evaporated and the crude residue was purified by SCX cartridge and subsequent flash silica column chromatography eluting with 5% methanol in dichloromethane. The amine obtained was treated with ethereal hydrogen chloride to give [(2,3-dichloro-4-fluorophenyl)methyl]amine hydrogen chloride (0.360 g) as a white solid.

Example 50 Λ/-[(2,3-dimethylphenyl)methyl]-1-ethyl-5-oxoprolinamide (E50)

1-ethyl-5-oxoproline (0.080 g, 0.51 mmol, prepared in an analogous manner to that described for example 12, method A) was dissolved in dichloromethane (5 ml) and to this was added N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (0.1 17 g, 0.61 mmol), N-ethyl morpholine (0.195 ml, 1 .53 mmol), and 2,3-dimethyl benzylamine (0.082 g, 0.61 mmol). The mixture was stirred for -17 hrs and then left to stand over the weekend. The mixture was then treated with saturated aqueous sodium hydrogen carbonate (~3 ml) and stirred vigorously for -10 minutes. The organic layer was separated using a hydrophobic frit and the aqueous layer was extracted with more dichloromethane (-2 ml). The combined organic layers were concentrated to give a yellow oil (-0.2 g). This was purified further by mass-directed automated HPLC to give Λ/-[(2,3-dimethylphenyl)methyl]-1-ethyl-5-oxoprolinamide (0.072 g) as white solid. LC/MS [M+H]⁺ = 275, retention time = 2.12 minutes.

Example 51 Λ/-{[2-chloro-3-(trifluoromethyl)phenyl]methyl}-1-methyl-5- oxoprolinamide (E51 )

1-methyl-5-oxoproline (2.27 g, 15.88 mmol, prepared as described below) was dissolved in dichloromethane (150 ml) and to this was added N-(3- dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (3.35 g, 17.47 mmol), and 1-hydroxybenzotriazole (2.36 g, 17.47 mmol). The mixture was stirred for -10 minutes and then triethylamine (2.21 ml, 15.88 mmol) and {[2-chloro-3- (trifluoromethyl)phenyl]methyl}amine (3.66 ml, 17.47 mmol) were added and the mixture was left stirring at room temperature overnight (-17 hrs). A white precipitate formed during this time. The mixture was then treated with saturated aqueous sodium hydrogen carbonate (-100 ml) and stirred for 10 minutes. The organic layer was separated using a hydrophobic frit and then 2N aqueous hydrogen chloride was added and mixed and separated again. The organic layer was concentrated to give white solids (-2.5 g). The solid was dissolved in ethyl acetate (-200 ml) and washed with water (4 x 50 ml) followed by brine (50 ml). The organic layer was then dried by passing through a phase separator and concentrated to give Λ/-{[2-chloro-3- (trifluoromethyl)phenyl]methyl}-1 -methyl-5-oxo-L-prolinamide

as a fine white solid (2.48 g).

LC/MS [M+H]⁺ = 335, retention time = 2.24 minutes.

¹H NMR (CDCI₃, 500MHz) δ 2.02 (m, 1 H), 2.35 (m, 1 H), 2.39 (m, 1 H), 2.47 (m, 1 H), 2.81 (s, 3H), 4.00 (dd, 1 H, J = 8.9, 4.2 Hz), 4.60 (dd, 1 H, J = 15.1 , 6.2 Hz), 4.65 (dd, 1 H, J = 15.1 , 6.2 Hz), 6.56 (broad t, 1 H, J = 5.8 Hz), 7.38 (t, 1 H, J = 7.7 Hz), 7.60 (dd, 1 H, J = 7.6, 1.0 Hz), 7.68 (dd, 1 H, J = 7.9, 1.2 Hz); ¹³C NMR δ 176.0, 171.5, 137.5, 133.9, 131.7, 129.3, 127.4, 127.0, 122.8, 63.8, 41.8, 29.4, 29.2, 23.4.

The 1-methyl-5-oxoproline used as the starting material was prepared in the following manner:

(i) /V-methyl-L-glutamic acid (9.81 g, 60.87 mmol) was split into two equal batches and each was suspended in water (15 ml) and heated in a sealed tube at 14O⁰C for 30 minutes in a microwave reactor to give a clear solution. The two batches were then combined and the water was evaporated and dried under vacuum to give a white solid. The solid was triturated with ether then filtered and washed with more ether to give, after drying, 1-methyl-5-oxo-proline (7.47 g) as a white solid.

/V-{[2-chloro-3-(thfluoromethyl)phenyl]methyl}-1 -methyl-5-oxoprolinamide may also be prepared as described below: 1-Methyl-5-oxoproline (49.0 g, 0.342 mol, prepared as described above) was suspended in DCM (600ml) (internal temperature drops from 2O⁰C to 13.7⁰C). EEDQ (2-ethoxy-1-ethoxycarbonyl-1 ,2-dihydroquinoline, 75.26 g, 0.359 mol, 1.05eq) was added in one portion and the mixture was stirred at room temperature for 15 minutes. A solution of 1-[2-chloro-3-(trifluoromethyl)phenyl]methanamine (88.77 g, 0.359 mol, 1.05eq) in DCM (250ml) was then added dropwise to the mixture over 20 minutes (slight exotherm to 19⁰C) and any remaining solids were then washed into the mixture using additional DCM (150 ml). The mixture was then stirred at room temperature overnight. Saturated aqueous sodium hydrogen carbonate (300 ml) was added and the mixture was stirred for 5 minutes at room temperature. The organic layer was separated, and washed sequentially with water (300 ml), 2N aqueous hydrogen chloride (3 x 300 ml), water (300 ml) and saturated aqueous sodium chloride solution (300 ml). The organic solution was dried over anhydrous sodium sulphate, filtered and evaporated in vacuo. The resulting solid was then triturated with ether (-500 ml) and the solid was collected, washed with ether and dried (3O⁰C, vac oven over the weekend) to give a colourless solid (91 .1 g, 80%). This material was combined with a similar batch, prepared in an analagous manner, and the combined material (total of 178 g) was dissolved in ethyl acetate (2.75 I) with heating (gentle reflux, overhead stirring). The resulting hot clear solution was gently stirred and cooled to room temperature overnight. The solid was collected, washed with cold ethyl acetate (500 ml) and dried (5O⁰C in vacuum oven, ~3 days) to give Λ/-{[2-chloro-3- (trifluoromethyl)phenyl]methyl}-1 -methyl-5-oxoprolinamide, as colourless needles (148.4 g).

LC/MS [M+H]⁺ = 335/337, retention time = 2.26 minutes.

¹H NMR (DMSO, 500MHz) δ 1.86 (m, 1 H), 2.21 (m, 1 H), 2.24 (m, 1 H), 2.28 (m, 1 H), 2.64 (s, 3H), 4.12 (dd, 1 H, J = 8.3, 3.5 Hz), 4.47 (d, 2H, J = 5.8 Hz), , 7.58 (t, 1 H, J = 7.8 Hz), 7.65 (dd, 1 H, J = 7.8, 1.0 Hz), 7.80 (dd, 1 H, J = 7.8, 1.2 Hz), 8.81 (broad t, 1 H, J = 5.7 Hz); ¹³C NMR δ 174.4, 171.4, 138.8, 133.1 , 129.8, 127.5, 127.1 , 126.6, 122.9, 61.6, 40.2, 29.1 , 28.0, 22.5.

Enantiomeric excess = 99.1 %, as determined by chiral chromatography method A, indicative of Λ/-{[2-chloro-3-(trifluoromethyl)phenyl]methyl}-1-methyl-5-oxo-L- prolinamide retention time = 6.99 minutes

[α]_D = -0.8° (c= 1 , MeOH), Temperature = 29.3⁰C, wavelength = 589nm melting point = 173°C

Example 52 Λ/-[(2,3-dichloro-4-fluorophenyl)methyl]-1-methyl-5-oxoprolinamide

(E52)

1-methyl-5-oxoproline (0.060 g, 0.42 mmol, prepared as described above for example 51 ) was dissolved in dichloromethane (5 ml) and to this was added N-(3- dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (0.096 g, 0.5 mmol), 1- hydroxybenzotriazole (0.068 g, 0.5 mmol), and N-ethyl morpholine (0.160 ml, 1.26 mmol). The mixture was stirred for -10 minutes and then [(2,3-dichloro-4- fluorophenyl)methyl]amine hydrochloride (0.081 g, 0.42 mmol, prepared as described previously for example 48) was added and the mixture was left to stir overnight (-17 hrs) and then over the weekend. The mixture was then treated with saturated aqueous sodium hydrogen carbonate (-3 ml) and stirred vigorously for 10 minutes.

The organic layer was separated using a hydrophobic frit , washing the aqueous with additional dichloromethane (-2 ml). The combined organic fractions were concentrated to give a cream coloured solid. The solid was partitioned between ethyl acetate (-20 ml) and water (-10 ml) and the organic layer was then seperated by passing through a phase separator and concentrated to give Λ/-[(2,3-dichloro-4- fluorophenyl)methyl]-1-methyl-5-oxoprolinamide as an off-white solid. LC/MS [M+H]⁺ = 319, retention time = 2.2 minutes.

Examples 53-64

In a manner analogous to that described for Example 52 above the compounds tabulated below (Table 4) were prepared by substituting the appropriate amine (or salt thereof) for the [(2,3-dichloro-4-fluorophenyl)methyl]amine hydrochloride used in the above procedure. All of the amines used to make the compounds shown in Table 4 are available from commercial sources or can be prepared using routes described previously in the chemical literature or using analogous methods.

Table 4

The amines required for the synthesis of example 62-64 were prepared according to the procedures described, respectively, below:

1 ) {[2-Methyl-3-(trifluoromethyl)phenyl]methyl}amine hydrochloride (Amine used to prepare Example 62)

Borane tetrahydrofuran (1 M, 39.4 ml, 39.4 mmol) was added to a solution of 2- methyl-3-trifluoromethyl benzamide (2 g, 9.85 mmol) in tetrahydrofuran (75 ml) and stirred at 7O⁰C for 5 hrs. LCMS showed incomplete reaction so heating at 70⁰C under argon was continued overnight and then for a further 5 hrs following this. The reaction mixture was treated with 2N aqueous hydrogen chloride and stirred at 100⁰C for 4 hrs and then left to cool over the weekend. The mixture was reduced to dryness undervacuum and then partitioned between dichloromethane and 2N aqueous sodium hydroxide. The organic layer was separated using a hydrophobic frit and reduced to give a residue which was purified by flash silica column chromatography (eluting with 0-5% 2N ammonia/methanol in dichloromethane). The solvent was evaporated and the residue taken up in diethyl ether and treated with 1 M ethereal hydrogen chloride. The solid that precipitated was collected by filtration and this was then triturated with dichloromethane and after filtration 2-Methyl-3- (trifluoromethyl)phenyl]methyl}amine hydrochloride (1.4 g) was obtained as a white solid. LC/MS [M+H]⁺ = 173, retention time = 1.30 minutes.

2) [(2-bromo-4-fluorophenyl)methyl]amine hydrochloride (Amine used to prepare Example 63)

(i) 2-Bromo-4-fluorobenzyl bromide (5g 18.8 mmol) and potassium phthalimide (4g, 21.6 mmol) were combined in dimethylformamide (200 ml) and stirred at 80⁰C for 18 hrs overnight. The mixture was reduced under vacuum and the residue was partitioned between diethyl ether and water. Solids were filtered off and the aqueous layer was washed with more ether (2 x 50 ml). The ether layers were combined and dried over sodium sulphate then filtered and evaporated to give an off-white solid (3.36 g). The solid was triturated with methanol and filtration gave 2-[(2-bromo-4- fluorophenyl)methyl]-1 H-isoindole-1 ,3(2/-/)-dione as a solid (2.06 g) which was used without further purification in the next step.

LC/MS [M+H]⁺ = 334, retention time = 3.30 minutes.

(ii) Hydrazine hydrate (0.655 ml, 21 mmol) was added to a suspension of 2-[(2- bromo-4-fluorophenyl)methyl]-1 H-isoindole-1 ,3(2/-/)-dione (2 g, 6 mmol) in ethanol (60 ml) and stirred at room temperature overnight. The reaction had not gone to completion at this stage so the mixture was heated at 100⁰C for a total of 2 hrs (the mixture turned white and cloudy during this time). The mixture was filtered to remove solids then cooled and filtered again. The solids were washed with cold ethanol and then the combined ethanol fractions were evaporated to dryness under vacuum. The resulting residue was partitioned between 2N aqueous hydrogen chloride and dichloromethane. The organic phase was separated using a hydrophobic frit. The aqueous layer was washed with more dichloromethane and separated again. The aqueous layer was then reduced under vacuum to leave a pale yellow solid (0.876 g). The solid was taken up in saturated aqueous sodium hydrogen carbonate solution and extracted with dichloromethane. Seperation by hydrophobic frit and evaporation gave a residue which was dissolved in diethyl ether and treated with ethereal hydrogen chloride. A pale yellow solid precipitated from the mixture. Evaporation and drying gave [(2-bromo-4-fluorophenyl)methyl]amine hydrochloride (0.789 g).

LC/MS [M+H]⁺ = 203, retention time = 1.08 minutes.

3) {[3-fluoro-2-(trifluoromethyl)phenyl]methyl}amine hydrochloride (Amine used to prepare Example 64)

Borane tetrahydrofuran (1 M, 19.2 ml, 19.2 rmmol) was added dropwise to a solution of 3-fluoro-2-(trifluoromethyl)benzamide (1 g, 4.8 mmol) in tetrahydrofuran (40 ml) under argon at room temperature. The mixture was heated at 70⁰C and then a further aliquot of borane tetrahydrofuran (10 ml, 10 mmol) was added and heating at 7O⁰C was continued over the weekend. The reaction mixture was cooled to room temperature and then treated with 2M aqueous hydrogen chloride (15 ml) and stirred at room tempertaure for 15 minutes. Aqueous sodium hydroxide solution was added until the pH of the mixture was between 8-9 and then the mixture was extracted with ethyl acetate (3 x 30 ml). The combined organic layers were filtered through a hydrophobic frit and then evaporated under vacuum. The residue was redissolved in dichloromethane, filtered through a hydrophobic frit and evaporated to give a yellow oil. The oil was dissolved in 2M aqueous hydrogen chloride. A white precipitate formed and this was collected by vacuum filtration and then loaded equally onto 4 x 10 g SCX columns. The columns were flushed with methanol and water and then aqueous ammonia was used to wash off the product. These latter fractions were reduced under vacuum to give a yellow oil (0.4 g). The oil was dissolved in diethyl ether and treated with 1 M ethereal hydrogen chloride until no more precipitate formed. The mixture was reduced under vacuum to give {[3-fluoro-2- (trifluoromethyl)phenyl]methyl}amine hydrochloride as a white solid. LC/MS [M+H]⁺ = 193, retention time = 1.15 minutes.

Examples 65-69

The examples tabulated below (Table 5) were prepared in a manner analogous to that described for Example 12 by substituting the appropriate amine (or salt thereof) for the [(2,3,4-trifluorophenyl)methyl]amine used in the procedure described for example 12. All of the amines used ito make the compounds shown in Table 5 are available from commercial sources or can be prepared using routes described previously in the chemical literature unless stated otherwise. The 1 -ethyl-5-oxo- proline used to prepare these examples was in turn prepared using method C as described for example 12 apart from in the case of example 65 where method A was used.

Table 5

Example 70 /V-[(2-chloro-4-fluorophenyl)methyl]-1-ethyl-5-oxoprolinamide (E70)

1-ethyl-5-oxoproline (0.100 g, 0.64 mmol) was dissolved in a mixture of dichloromethane (3 ml) and dimethylformamide (0.5 ml) and to this was added N-(3- dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (0.147 g, 0.77 mmol), 1- hydroxybenzotriazole (0.104 g, 0.77 mmol), and N-ethyl morpholine (0.244 ml, 1.92 mmol). The mixture was stirred for 10 minutes and then 2-chloro-4- fluorobenzylamine was added to the mixture and stirring continued overnight (-16 hrs) at room temperature. The mixture was then treated with saturated aqueous sodium hydrogen carbonate (~2 ml) and stirred vigorously for -10 minutes. The aqueous layer was removed using a phase separator and extracted with more dichloromethane (2 x 1 ml). The combined organic layers were concentrated to give a yellow oil and this was subsequently purified by mass-directed automated HPLC to give Λ/-[(2-chloro-4-fluorophenyl)methyl]-1 -ethyl-5-oxo-D-prolinamide (0.065 g) as a white solid. LC/MS [M+H]⁺ = 299, retention time = 2.16 minutes.

Enantiomeric excess = 80.9%, as determined by chiral chromatography method B, indicative of Λ/-[(2-chloro-4-fluorophenyl)methyl]-1-ethyl-5-oxo-D-prolinamide retention time = 5.91 minutes

The 1-ethyl-5-oxoproline used in the above procedure was prepared as described below:

(i) D-pyroglutamic acid ethyl ester (4.17 g, 26.53 mmol) was dissolved in tetrahydrofuran (30 ml) and ethyl iodide (2.23 ml, 27.86 mmol) was added to give a pale yellow solution. This was cooled to 0°C and sodium hydride (60% in oil, 1.1 1 g, 27.86 mmol) was added portionwise. After addition of all the sodium hydride the mixture was stirred at 0⁰C for a further 20 minutes until most of the bubbling had stopped. The mixture was then warmed to room temperature and stirred overnight under argon. The mixture was then treated with saturated aqueous ammonium chloride solution (-5 ml). The organic layer was separated and the aqueous layer was extracted with more dichloromethane (3 x 20 ml). The combined organic layers were dried by passing through a phase separator and then concentrated to a green/brown oil (3.2 g). This was purified by automated flash silica column chromatography (Biotage SP4), eluting with a 0-100% gradient of ethyl acetate in hexane, to give ethyl 1-ethyl-5-oxoprolinate as a yellow oil (1.33 g) which was used in the next step without further purification.

(ii) Ethyl 1 -ethyl-5-oxoprolinate (1.33 g, 7.18 mmol) was dissolved in ethanol (10 ml) and cooled to 0⁰C in an ice bath. To this was added 12.5M aqueous sodium hydroxide solution (1.72 ml, 21.53 mmol) and the mixture was stirred for ~4 hours at 0⁰C. The ethanol was evaporated under vacuum and the aqueous residue was acidified with 2N aqueous hydrogen chloride to pH1. The volume of the aqueous phase was reduced to ~3 ml under vacuum and then extracted with a 3:1 mixture of chloroform and isopropanol using a phase separator. The combined organic layers were concentrated to a pale yellow oil which on drying in vacuo crystallized to give 1 - ethyl-5-oxoproline as a white solid (1.12 g).

Examples 71-82

In a manner analogous to that described for Example 70 above the compounds tabulated below (Table 6) were prepared by substituting the appropriate amine (or salt thereof) for the 2-chloro-4-fluorobenzylamine used in the above procedure. All of the amines used to make the compounds shown in Table 6 are available from commercial sources or can be prepared using routes described previously in the chemical literature unless stated otherwise. Where determined (by chiral HPLC), the enantiomeric excess (e.e.) of the isomer shown is also listed along with its stereospecific name, the chiral separation method used in parantheses and the corresponding retention time (r.t.) in that method.

Table 6

Example 83 Λ/-{[4-fluoro-2-(tπfluoromethyl)phenyl]methyl}-1-methyl-5-oxoprolinamide (E83)

Λ/-{[4-fluoro-2-(trifluoromethyl)pheπyl]methyl}-1-methyl-5-oxoprolinamide was prepared in a manner analogous to that described above for the synthesis of Λ/-[(2- chloro-4-fluorophenyl)methyl]-1 -ethyl-5-oxoprolinamide (Example 70) but 1-methyl-5- oxoproline (prepared as describe below) was substituted for 1-ethyl-5-oxoproline and {[4-fluoro-2-(trifluoromethyl)phenyl]methyl}amine was substituted for 2-chloro-4- fluorobenzylamine. LC/MS [M+H]⁺ = 319, retention time = 2.14 minutes.

The 1 -methyl-5-oxoproline used in the above procedure was prepared as described below:

(i) D-pyroglutamic acid ethyl ester (4.0 g, 25.5 mmol) was dissolved in tetrahydrofuran (25 ml) and cooled to 0⁰C . Methyl iodide (1.66 ml, 26.7 mmol) was added and stirring continued for 10 minutes under argon at 0⁰C. Sodium hydride (60% in oil, 1.6 g, 26.7 mmol) was then added portionwise (allowing each portion to react). After addition of all the sodium hydride the mixture was allowed to warm to room temperature and stirred overnight under argon. The mixture was then treated with saturated aqueous ammonium chloride solution (-15 ml) and stirred for 4 hrs. The organic layer was separated and the aqueous layer was extracted with more dichloromethane. The combined organic layers were dried over magnesium sulphate and then concentrated to a dark oil. This was purified by flash silica column chromatography, eluting with a 0-75% gradient of ethyl acetate in hexane, to give ethyl 1-methyl-5-oxoprolinate as a colourless oil (0.27 g) which was used in the next step without further purification.

(ii) Ethyl 1 -methyl-5-oxoprolinate (0.27 g, 1.58 mmol) was dissolved in ethanol (5 ml) and cooled to 0⁰C in an ice bath. To this was added 2M aqueous sodium hydroxide solution (3 ml) and the mixture was stirred for ~4 hours at 0⁰C. The ethanol was evaporated under vacuum and the aqueous residue was acidified with 2N aqueous hydrogen chloride to pH1. The volume of the aqueous phase was reduced to ~3 ml under vacuum and then extracted with a 3:1 mixture of chloroform and isopropanol using a phase separator. The combined organic layers were concentrated to give 1 - methyl-5-oxoproline which was used without further purification. Examples 84-90

Furthermore, and also in a manner analogous to that described for Example 70 above the compounds tabulated below (Table 7) were prepared by substituting the appropriate amine (or salt thereof) for the 2-chloro-4-fluorobenzylamine used in Example 70. All of the amines used to make the compounds shown in Table 7 are available from commercial sources or can be prepared using routes described previously in the chemical literature unless stated otherwise. 1-Methyl-5-oxoproline (prepared as describe above for example 81 ) was substituted for the 1-ethyl-5- oxoproline used in Example 70. Where determined (by chiral HPLC), the enantiomeric excess (e.e.) of the isomer shown is also listed along with its stereospecific name, the chiral separation method used in parantheses and the corresponding retention time (r.t.) in that method.

Table 7

Example 91 Λ/-[(2-chloro-4-fluorophenyl)methyl]-5-oxo-1-phenyl-prolinamide (E91 )

5-0X0-1 -phenyl-proline (0.072 g, 0.35 mmol, prepared as described below) was dissolved in dichloromethane (~2 ml) and dimethylformamide (0.5 ml) and to this was added N-(3-dimethylaminopropyl)-N'-ethylcarbodiirnide hydrochloride (0.081 g, 0.42 mmol), 1-hydroxybenzotriazole (0.057 g, 0.42 mmol), and N-ethyl morpholine (0.134 ml, 1.05 mmol). The mixture was stirred at room temperature for 30 minutes and then [(2-chloro-4-fluorophenyl)methyl]amine (0.067 g, 0.42 mmol) was added. Stirring was continued overnight at room temperature and then the mixture was diluted with more dichloromethane and saturated aqueous sodium hydrogen carbonate. The aqueous layer was separated and extracted with more dichloromethane (3 aliquots). The combined organic layers were then washed with brine before drying over magnesium sulphate. Evaporation of the solvent then gave a yellow oil which was purified by mass-directed automated HPLC. Finally trituration of the material thus obtained with a 1 :1 mixture of dichloromethane and diethyl ether gave, after filtration and drying, Λ/-[(2-chloro-4-fluorophenyl)methyl]-5-oxo-1-phenyl- prolinamide (0.031 g) as a white solid. LC/MS [M+H]⁺ = 347, retention time = 2.46 minutes.

The 5-OXO-1 -phenyl-proline used in the above procedure was prepared as follows: (i) D-pyroglutamic acid ethyl ester (0.200 g, 1.27 mmol) was dissolved in dioxane (5 ml) and treated with tris(dibenzylideneacetone)dipalladium (0) (0.058 g, 0.06 mmol), bromobenzene (0.351 ml, 1.53 mmol), cesium carbonate (0.621 g, 1.91 mmol) and Xantphos™ (0.1 10 g, 0.19 mmol). The resulting mixture was heated at reflux overnight and then allowed to cool to room temperature. The mixture was diluted with methanol and filtered. The filtrate was evaporated in vacuo and then partitioned between dichloromethane and saturated aqueous sodium hydrogen carbonate. The aqueous layer was extracted with more dichloromethane (3 aliquots) and then the combined organic layers were washed with brine and dried over magnesium sulphate. Evaporation of the solvent gave a bright yellow residue which was purified by flash silica column chromatography, eluting with a gradient of 0-50% ethyl acetate in hexane, to give methyl 5-oxo-1 -phenylprolinate (0.078 g) as a yellow oil. This was used in the next step without further purification.

(ii) Methyl 5-oxo-1-phenylprolinate (0.078 g, 0.36 mmol) was combined with 2N aqueous sodium hydroxide (2 ml) in ethanol (2 ml) at 0⁰C. The mixture was stirred at between -10⁰C and 0°C for 5 hours. The solvent was then evaporated in vacuo and the residue was acidified to pH1 by the addition of 2M aqueous hydrogen chloride. To this was added dichloromethane and the mixture was passed through a phase separator. The aqueous layer was washed with more dichloromethane and then the combined dichloromethane layers were evaporated to give 5-oxo-1 -phenyl-proline (0.072 g) as a yellow gum which was used without further purification in the next step.

Example 92 Λ/-{[2-chloro-3-(trifluoromethyl)phenyl]methyl}-5-oxo-1 -(phenylmethyl)- prolinamide (E92)

5-0X0-1 -(phenylmethyl)proline (0.100 g, 0.46 mmol, prepared as described below) was dissolved in a mixture of dichloromethane (2.5 ml) and dimethylformamide (0.5 ml) and to this were added N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (0.105 g, 0.55 mmol), 1-hydroxybenzotriazole (0.074 g, 0.55 mmol), and N-ethyl morpholine (0.143 ml, 1.37 mmol). The mixture was stirred for 10 minutes and then {[2-chloro-3-(trifluoromethyl)phenyl]methyl}amine (0.115 g, 0.55 mmol) was added and the mixture was stirred for 1 hr. Saturated aqueous sodium hydrogen carbonate (10 ml) was added and the mixture stirred vigorously for 15 minutes. The organic phase was separated with a phase separator and the aqueous phase was washed with further aliquots of dichloromethane (3 x 10 ml). The organic fractions were combined and dried over magnesium sulphate. The solvent was then evaporated and the residue was purified by mass-directed automated HPLC to give Λ/-{[2-chloro-3-(trifluoromethyl)phenyl]methyl}-5-oxo-1-(phenylmethyl)-D-prolinamide LC/MS [M+H]⁺ = 41 1 , retention time = 2.77 minutes.

Enantiomeric excess = 100.0%, as determined by chiral chromatography method D, indicative of Λ/-{[2-chloro-3-(trifluoromethyl)phenyl]methyl}-5-oxo-1 -(phenylmethyl)-D- prolinamide retention time = 10.58 minutes

The 5-OXO-1 -(phenylmethyl)proline used in the method described above was prepared as follows:

D-glutamic acid (1 .47 g, 10 mmol) was dissolved in 2N aqueous sodium hydroxide (10 ml, 20 mmol) and stirred for 15 minutes. The mixture was then treated with a solution of benzaldehyde (1.1 ml, 10 mmol) in ethanol (3 ml) and stirred at room temperature for 30 minutes. The mixture was cooled to O⁰C and treated with sodium borohydride (0.030 g). The mixture was allowed to warm to room temperature with stirring over 4 hrs and then washed with diethyl ether (three times) before acidifying with concentrated hydrochloric acid to pH2. The resulting precipitate was filtered off and washed with diethyl ether before slurrying in ethanol and azeotroping three times with more ethanol. Finally the remaining material was slurried in ethanol (50 ml) and heated at reflux for 16 hrs. The mixture was then cooled to room temperature and evaporated in vacuo. Drying afforded 5-oxo-1-(phenylmethyl)proline. Example 93 /V-[(2-chloro-4-fluorophenyl)methyl]-5-oxo-1 -(phenylmethyl)prolinamide (E93)

/V-[(2-chloro-4-fluorophenyl)methyl]-5-oxo-1 -(phenylmethyl)prolinamide was prepared in an analogous manner to that described for the synthesis of Λ/-{[2-chloro-3- (trifluoromethyl)phenyl]methyl}-5-oxo-1 -(phenylmethyl)prolinamide (Example 92) above but using [(2-chloro-4-fluorophenyl)methyl]amine in the place of {[2-chloro-3- (trifluoromethyl)phenyl]methyl}amine.

LC/MS [M+H]⁺ = 361 , retention time = 2.54 minutes.

Example 94 Λ/-[(2-chloro-4-fluorophenyl)methyl]-1-cyclopentyl-5-oxoprolinamide (E94)

i-Cyclopentyl-5-oxoproline (0.100 g, 0.51 mmol, prepared as described below) was dissolved in a mixture of dichloromethane (2.5 ml) and dimethylformamide (0.5 ml) and to this were added N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (0.1 17 g, 0.61 mmol), 1-hydroxybenzotriazole (0.082 g, 0.61 mmol), and N-ethyl morpholine (0.2 ml, 1.52 mmol). The mixture was stirred for 10 minutes and then [(2-chloro-4-fluorophenyl)methyl]amine (0.097 g, 0.61 mmol) was added and the mixture was stirred overnight. Saturated aqueous sodium hydrogen carbonate (10 ml) was added and the mixture stirred vigorously for 15 minutes. The organic phase was separated with a phase separator and the aqueous phase was washed with further aliquots of dichloromethane (3 x 10 ml). The organic fractions were combined and dried over magnesium sulphate. The solvent was then evaporated and the residue was purified by mass-directed automated HPLC to give /V-[(2-chloro-4-fluorophenyl)methyl]-1-cyclopentyl-5-oxoprolinamide. LC/MS [M+H]⁺ = 339, retention time = 2.4 minutes.

The i-Cyclopentyl-5-oxoproline used in the above procedure was prepared as follows:

(i) Dimethyl D-glutamate hydrochloride (2.1 g, 10.00 mmol) was dissolved in methanol (7.5 ml) and tetrahydrofuran (15 ml)and the mixture was then treated with crushed sodium hydroxide (0.402 g, 10.05 mmol) for 20 minutes under argon. At this stage acetic acid (0.575 ml, 10.05 mmol) and cyclopentanone (0.889 ml, 10.05 mmol) were added to the mixture. After stirring for 10-15 minutes the mixture was cooled to O⁰C in an ice-bath and treated with sodium borohydride pellets (0.380 g, 10.05 mmol). The mixture was stirred for 3 hrs under argon and allowed to warm to room temperature. Once the mixture had reached room temperature the methanol was evaporated off and the residue was diluted with dichloromethane (20 ml) and washed with saturated aqueous sodium hydrogen carbonate (-25 ml). The organic layer was separated and the aqueous layer was back-extracted with more dichloromethane (2 x 20 ml). The combined organic layers were concentrated in vacuo to give an oil. The oil was dissolved in toluene (10 ml) and heated at reflux overnight. The solvent was then evaporated and the resulting residue was purified by flash-silica column chromatography, eluting with a gradient of 0-10% methanol in dichloromethane, to give crude methyl 1 -cyclopentyl-5-oxoprolinate which was used without further purification in the next step, (ii) Methyl 1-cyclopentyl-5-oxoprolinate (0.560 g, 2.65 mmol) was dissolved in ethanol (10 ml) and cooled to 0°C in an ice-bath. 2M aqueous sodium hydroxide (5 ml) was added and the mixture was stirred at ice temperature for 4 hrs. The ethanol was then evaporated under vacuum and the aqueous residue was acidified to pH1 by the addition of 2N aqueous hydrogen chloride. The volume of resulting aqueous mixture was reduced under vacuum to ~3 ml and this was then extracted with a 3:1 mixture of chloroform and isopropanol respectively using a phase separator. The aqueous layer was washed with more dichloromethane and then the combined organic fractions were evaporated to give crude 1 -cyclopentyl-5-oxoproline which was used in subsequent reactions without further purification.

Examples 95-99

In a manner analogous to that described for Example 94 above the compounds tabulated below (Table 8) were prepared by substituting the appropriate amine (or salt thereof) for the [(2-chloro-4-fluorophenyl)methyl]amine used in the above procedure and/or substituting the appropriate aldehyde or ketone for the cyclopentanone used in the above procedure. All of the amines used to make the compounds shown in Table 8 are available from commercial sources or can be prepared using routes described previously in the chemical literature unless stated otherwise,

Table 8

Example 100 Λ/-{[2-chloro-3-(trifluoromethyl)phenyl]methyl}-1 -(2,2-dimethylpropyl)-5- oxoprolinamide (E100)

1-(2,2-dimethylpropyl)-5-oxoproline (0.100 g, 0.5 mmol, prepared as described below) was dissolved in dichloromethane (5 ml) and to this was added N-(3- dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (0.191 g, 1 mmol), and 1- hydroxybenzotriazole (0.135 g, 1 mmol). The mixture was stirred for 30 minutes at room temperature and then {[2-chloro-3-(trifluoromethyl)phenyl]methyl}amine (0.209 g, 1 mmol) was added and the mixture was stirred overnight at room temperature. The mixture was then washed sequentially with water, 3N aqueous citric acid, and three more times with water then dried by filtering through a hydromatrix cartridge (Varian 5 g). The solvent was then evaporated and the residue was purified by mass-directed automated HPLC to give Λ/-{[2-chloro-3-

(trifluoromethyl)phenyl]methyl}-1 -(2,2-dimethylpropyl)-5-oxoprolinamide. LC/MS [M+H]⁺ = 391/393, retention time = 2.78 minutes.

The 1-(2,2-dimethylpropyl)-5-oxoproline used in the method described above was prepared as follows:

L-glutamic acid (1.47 g, 10 mmol) was dissolved in 2N aqueous sodium hydroxide (10 ml, 20 mmol) and treated with a solution of trimethylacetaldehyde (1 .09 ml, 10 mmol) in ethanol (5 ml) and then stirred at room temperature for 30 minutes. The mixture was cooled to O⁰C and treated with sodium borohydride (0.130 g). The mixture was allowed to warm to room temperature with stirring over 4 hrs and then acidified to neutral pH. Concentration in vacuo was followed by slurrying in ethanol and azeotroping three times with more ethanol. Finally the remaining material was suspended in ethanol (50 ml) and heated at reflux for 48 hrs. The mixture was then cooled to room temperature, salts were filtered off and the solvent was evaporated in vacuo to give a gum. Trituration with diethyl ether followed by drying afforded solid 1-(2,2-dimethylpropyl)-5-oxoproline (1.1 g).

Example 101 Λ/-{[2-chloro-3-(trifluoromethyl)phenyl]methyl}-5-oxo-1 -(phenylmethyl)- prolinamide (E101)

Λ/-{[2-chloro-3-(trifluoromethyl)phenyl]methyl}-5-oxo-1-(phenylmethyl)-D-prolinamide was prepared in an analogous manner to that described for the synthesis of Λ/-{[2- chloro-3-(trifluoromethyl)phenyl]methyl}-1-(2,2-dimethylpropyl)-5-oxoprolinamide (example 100) above but using 5-oxo-1 -(phenylmethyl)proline in the place of 1-(2,2- dimethylpropyl)-5-oxoproline.

LC/MS [M+H]⁺ = 41 1/413, retention time = 2.77 minutes.

Enantiomeric excess = 100.0%, as determined by chiral chromatography method D, indicative of Λ/-{[2-chloro-3-(trifluoromethyl)phenyl]methyl}-5-oxo-1 -(phenylmethyl)-D- prolinamide retention time = 8.09 minutes

5-OXO-1 -(phenylmethyl)proline was prepared in an analogous manner to that described above for the synthesis of 1-(2,2-dimethylpropyl)-5-oxoproline (example 100) but using benzaldehyde in the place of trimethylacetaldehyde.

Example 102 Λ/-[(2,4-dichlorophenyl)methyl]-1-methyl-5-oxoprolinamide (E102)

N-[(2,4-dichlorophenyl)methyl]-1-methyl-5-oxoprolinamide was prepared in a manner analogous to that described above for the synthesis of Λ/-{[4-fluoro-2- (trifluoromethyl)phenyl]methyl}-1 -methyl-5-oxoprolinamide (Example 83) but [(2,4- dichlorophenyl)methyl]amine was substituted for {[4-fluoro-2-

(trifluoromethyl)phenyl]methyl}amine. LC/MS [M+H]⁺ = 300.9, retention time = 2.13 minutes.

Enantiomeric excess = 97.8%, as determined by chiral chromatography method A, indicative of Λ/-[(2,4-dichlorophenyl)methyl]-1-methyl-5-oxo-D-prolinamide retention time = 6.25 minutes

Example 103 1-ethyl-Λ/-{[2-fluoro-3-(trifluoromethyl)phenyl]methyl}-5-oxoprolinamide (E103)

1-θthyl-Λ/-{[2-fluoro-3-(trifluoromethyl)phenyl]methyl}-5-oxoprolinamidθ was prepared in a manner analagous to that decribed above (see example 70) for Λ/-[(2-chloro-4- fluorophenyl)methyl]-1-ethyl-5-oxo-D-prolinamide but using 2-fluoro-3- trifluoromethylbenzylamine in the place of 2-chloro-4-fluorobenzylamine. LC/MS [M+H]⁺ = 333, retention time = 2.24 minutes.

Examples 104-109

The examples tabulated below (Table 9) were prepared in a manner analogous to that described for Example 12 by substituting the appropriate amine (or salt thereof) for the [(2,3,4-trifluorophenyl)methyl]amine used in the procedure described for Example 12. All of the amines used to make the compounds shown in Table 9 are available from commercial sources or can be prepared using routes described previously in the chemical literature unless stated otherwise. The 1 -ethyl-5-oxo- proline used to prepare these examples was in turn prepared using method C as described for Example 12.

Table 9

The 2-(aminomethyl)-6-(trifluoromethyl)benzonitrile trifluoroacetate required for the synthesis of example 105 was prepared as follows:

(i) {[2-Fluoro-3-(trifluoromethyl)phenyl]methyl}amine (1.93 g, 10 mmol) was dissolved in dichloromethane (40 ml) and treated with a solution of bis(1 ,1- dimethylethyl) dicarbonate (2.18 g, 10 mmol) in dichloromethane (10 ml). After stirring at room temperature for 2 hrs the solvent was evaporated to give a pale yellow solid which was purified by silica-gel column chromatography, eluting with a 1 :10-1 :5 gradient of ethyl acetate in hexanes, to give 1 ,1-dimethylethyl {[2-fluoro-3- (trifluoromethyl)phenyl]methyl}carbamate (2 g).

(ii) 1 ,1-Dimethylethyl {[2-fluoro-3-(trifluoromethyl)phenyl]methyl}carbamate (1.17 g, 4 mmol) was dissolved in dimethylsulphoxide (5 ml) and treated with potassium cyanide (0.260 g, 4 mmol). The mixture was then heated at 80⁰C under argon for 1.5 hrs and then at 120⁰C overnight (16 hrs). Additional potassium cyanide (0.260 g, 4 mmol) was then added and heating continued at 120⁰C for a further 24 hrs. The mixture was then cooled to room temperature, quenched with water, and diluted with ethyl acetate. The organic extracts were separated and washed three times with water and then with saturated aqueous sodium chloride solution. Drying and evaporation gave a brown gum which was purified by silica-gel column chromatography, eluting with a 1 :10-1 :5 gradient of ethyl acetate in hexanes, to give partially pure 1 ,1-dimethylethyl {[2-cyano-3-(trifluoromethyl)phenyl]methyl}carbamate as a dark solid/semi-solid which was used in the next step without further purification. LC/MS [M-BOC+H]⁺ = 201 , retention time = 1.19 minutes. (iii) 1 ,1-Dimethylethyl {[2-cyano-3-(trifluoromethyl)phenyl]methyl}carbamate (0.190 g, 0.63 mmol) was dissolved in dichloromethane (4 ml) and treated with trifluoroacetic acid (4 ml). The mixture was stirred at room temperature for 1 hr and then evaporated. The residue was twice taken up in dichloromethane and evaporated again to give crude 2-(aminomethyl)-6-(trifluoromethyl)benzonitrile trifluoroacetate which was used without further purification.

Example 1 10 1 -methyl-Λ/-(1-naphthalenylmethyl)-5-oxoprolinamide (E110)

1-methyl-5-oxoproline (0.050 g, 0.35 mmol, prepared in a manner analogous to that described above for example 51 ), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (0.081 g, 0.42 mmol), 1-hydroxybenzotriazole (0.057 g, 0.42 mmol), N- ethyl morpholine (0.166 ml, 1 .05 mmol) and (i-naphthalenylmethyl)amine were combined in dichloromethane (~8 ml) and the mixture was stirred for -20 hrs at room temperature. The mixture was then washed with 2M aqueous hydrogen chloride (5 ml) and the organic layer was separated using a phase separator. The organic layer was washed with saturated aqueous sodium hydrogen carbonate, separated as before, and then evaporated. The residue was purified by mass-directed automated HPLC to give 1-methyl-Λ/-(1-naphthalenylmethyl)-5-oxoprolinamide as a white solid (0.062 g). LC/MS [M+H]⁺ = 283, retention time = 2.1 minutes.

Example 1 1 1 Λ/-{[2-chloro-4-fluoro-3-(trifluoromethyl)phenyl]methyl}-1-methyl-5- oxoprolinamide (E111)

1-methyl-5-oxoproline (0.057 g, 0.4 mmol, prepared in a manner analogous to that described above for example 51 ) dissolved in dichloromethane (4 ml) and treated with 2-ethoxy-1-ethoxycarbonyl-1 ,2-dihydroquinoline (0.104 g, 0.42 mmol). {[2- Chloro-4-fluoro-3-(trifluoromethyl)phenyl]methyl}amine hydrochloride (0.105 g, 0.4 mmol, prepared as described below) was then added and the mixture was stirred at room temperature for 4 hrs. The mixture was treated with saturated aqueous sodium hydrogen carbonate (10 ml) and stirred for 5 minutes. The organic phase was separated using a hydrophobic frit and then washed with 2N aqueous hydrogen chloride (2 x 10 ml). Evapotation of the organic phase then gave Λ/-{[2-chloro-4- fluoro-3-(trifluoromethyl)phenyl]methyl}-1-methyl-5-oxoprolinamide (0.106 g). LC/MS [M+H]⁺ = 353, retention time = 2.49 minutes.

The {[2-Chloro-4-fluoro-3-(trifluoromethyl)phenyl]methyl}amine hydrochloride used in the method described above was prepared as follows:

(i) 1-Chloro-3-fluoro-2-(trifluoromethyl)benzene (10 g , 50 mmol) was dissolved in tetrahydrofuran (100 ml), cooled to -70⁰C under argon, and treated with a 1 .4M solution of sec-butyl lithium in cyclohexane (37.5 ml, 52.5 mmol). Stirring was continued for 2 hrs and then trimethylsilyl chloride (6.7 ml, 52.5 mmol) was added and stirring continued, still at -70⁰C, for a further 1 hr. The mixture was allowed to warm to room temperature and the tetrahydrofuran was then removed in vacuo. The residue was partitioned between diethyl ether and water and then the organic layer was separated and washed with 2N aqueous hydrogen chloride. The organic phase was separated and concentrated to give the crude product which was purified by flash silica-gel column chromatography, eluting with hexane, to give [4-chloro-2- fluoro-3-(trifluoromethyl)phenyl](trimethyl)silane as a clear oil (10.35 g). (ii) 2,2,6,6-tetramethylpiperidine (3.3 ml, 19.44 mmol) was added slowly to a solution of n-butyl lithium (2.5M in toluene, 7.7 ml, 19.44 mmol) in tetrahydrofuran (75 ml) at - 75⁰C under argon and stirred for 15 minutes. A solution of [4-chloro-2-fluoro-3- (trifluoromethyl)phenyl](trimethyl)silane (5 g, 18.5 mmol) in tetrahydrofuran (10 ml) was then added dropwise to the mixture, ensuring that the temperature of the mixture was kept below -65°C, and stirring was continued for 2 hrs. Excess solid carbon dioxide, which had previously been washed with tetrahydrofuran at -65°C, was added in lumps and the mixture was allowed to warm to room temperature over 2 hrs. The mixture was reduced under vacuum to give a pale yellow solid. This material was partitioned between water which had been acidified to pH1 (200 ml) and diethyl ether (200 ml). The organic layer was separated and dried over anhydrous sodium sulphate. Evaporation gave a pale brown solid which was recrystallised from toluene to give 2-chloro-4-fluoro-3-(trifluoromethyl)-5-(trimethylsilyl)benzoic acid (3.85 g, in 3 batches) as white needles. LC/MS [M-H]^" = 312, retention time = 3.29 minutes.

(iii) A solution of potassium fluoride (0.367 g, 9.55 mmol) in water (15 ml) was added to a solution of 2-chloro-4-fluoro-3-(trifluoromethyl)-5-(trimethylsilyl)benzoic acid (1 g, 3.18 mmol) in tetrahydrofuran (50 ml) and the mixture was stirred at 100⁰C overnight. An additional aliquot of water (15 ml) and potassium fluoride (0.370 g, 9.62 mmol) was added and heating at 100⁰C was continued for a further 4 hrs. The tetrahydrofuran was evaporated in vacuo and replaced with enough dimethylformamide to dissolve all solids. The mixture was heated overnight at 100⁰C but starting material still remained so more potassium fluoride (0.367 g, 9.55 mmol) was added and heating at 100⁰C continued for 7 days. At this stage almost all of the starting material had disappeared so the reaction was evaporated to dryness under vacuum and taken up in 2N aqueous hydrogen chloride (75 ml) and diethyl ether (50 ml). The aqueous layer was separated and extracted with more diethyl ether (2 x 50 ml) and then the combined organic fractions were dried over sodium sulphate and evaporated to give crude product as a white solid. This was purified by recrystallisation from toluene to give 2-chloro-4-fluoro-3-(trifluoromethyl)benzoic acid (0.566 g) as a white solid.

(iv) 2-Chloro-4-fluoro-3-(trifluoromethyl)benzoic acid (0.560 g, 2.31 mmol), ammonium 1 H-1 ,2,3-benzotriazol-1-olate (0.534 g, 3.47 mmol, prepared as described below), N-(3-dimethylaminopropyl)-N'-ethylcarbodiirnide hydrochloride (0.643 g, 3.47 mmol), and N-ethyl morpholine (0.594 ml, 4.62 mmol) were stirred together in dichloromethane (30 ml) for a total of 3 hrs. Saturated aqueous sodium hydrogen carbonate (30 ml) was added and the mixture was stirred for 15 minutes. The organic layer was separated using a hydrophobic frit and then washed with 2N aqueous hydrogen chloride (2 x 50 ml). Seperation of the organic layer, again using a hydrophobic frit, and evaporation in vacuo gave 2-chloro-4-fluoro-3- (trifluoromethyl)benzamide (0.493 g) as an off-white solid which was used without further purification in the subsequent step. The ammonium 1 /-/-1 ,2,3-benzotriazol-1-olate used in the step described above was prepared as follows:

Ammonium hydroxide (4.15 ml, 75 mmol) was added slowly to a solution of 1- hydroxybenzotriazole (10 g, 74 mmol) in tetrahydrofuran (100 ml) at 0⁰C (ice-bath) and stirred for 2 hrs. Filtration and washing with tetrahydrofuran gave ammonium 1/-/-1 ,2,3-benzotriazol-1-olate (10.57 g) as a white solid.

(v) 2-Chloro-4-fluoro-3-(trifluoromethyl)benzamide (0.490 g, 2.03 mmol) was treated with 1 M borane in tetrahydrofuran (20.33 ml, 20.33 mmol) and stirred at 60⁰C overnight. The mixture was then treated with 2N aqueous hydrogen chloride until gas evolution ceased and then stirred at 100⁰C for 2 hrs. The mixture was reduced in vacuo and the residue was taken up in a minimum of water and washed with dichloromethane (30 ml). The pH of the aqueous layer was adjusted to pH1 1 by the addition of 2N aqueous sodium hydroxide solution and then extracted with dichloromethane (2 x 25 ml). The dichloromethane layers were separated using a hydrophobic frit, combined and evaporated in vacuo to leave a pale yellow oil. A 1 M solution of hydrogen chloride in diethyl ether (3 ml, 3 mmol) was added and the resulting white solid was filtered off to give {[2-Chloro-4-fluoro-3- (trifluoromethyl)phenyl]methyl}amine hydrochloride (0.210 g) which was used without further purification.

Example 1 12 Λ/-{[2-chloro-3-(trifluoromethyl)phenyl]methyl}-1 -cyclobutyl-5- oxoprolinamide (E112)

i-Cyclobutyl-5-oxoproline (0.238 g, 0.82 mmol) was suspended in dichloromethane (3 ml) and to this was added N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (0.188 g, 0.98 mmol), 1 -Hydroxybenzotriazole (0.132 g, 0.98 mmol), and N-ethyl morpholine (0.313 ml, 2.46 mmol). The mixture was stirred at room temperature for 30 minutes and then {[2-chloro-3-

(trifluoromethyl)phenyl]methyl}amine (0.205 g, 0.98 mmol) was added to the mixture and stirring continued for -20 hrs at room temperature. The mixture was then diluted with more dichloromethane and treated with saturated aqueous sodium hydrogen carbonate. The dichloromethane layer was separated and the aqueous layer was extracted with 3 further aliquots of dichloromethane. The combined organic extracts were washed with water and then with brine, dried over anhydrous magnesium sulphate and evaporated in vacuo to give the crude product. This was further purified by mass-directed automated HPLC to give Λ/-{[2-chloro-3- (trifluoromethyl)phenyl]methyl}-1 -cyclobutyl-5-oxoprolinamide (0.105 g) as a white solid. LC/MS [M+H]⁺ = 375, retention time = 2.53 minutes.

The i-Cyclobutyl-5-oxoproline used in the above procedure was prepared in an analogous manner to that described previously for the synthesis of methyl 1-ethyl-5- oxo-prolinate (see example 3) but using cyclobutanone in the place of acetaldehyde and with the addition of a subsequent ester deprotection step (using standard conditions, i.e. sodium hydroxide in methanol) being carried out (as opposed to the combined deprotection and amide coupling described in example 3).

Examples 1 13-1 17

In a manner analogous to that described for Example 112 above the compounds tabulated below (Table 10) were prepared by substituting the appropriate amine (or salt thereof) for the {[2-chloro-3-(trifluoromethyl)phenyl]methyl}amine used in the above procedure. All of the amines used to make the compounds shown in Table 10 are available from commercial sources or can be prepared using routes described previously in the chemical literature unless stated otherwise,.

Table 10

The [(2-chloro-3,4-difluorophenyl)methyl]amine hydrochloride required for the synthesis of /V-[(2-chloro-3,4-difluorophenyl)methyl]-1 -cyclobutyl-5-oxoprolinamide (Example 115) was prepared as described above for example 36.

Example 1 18 /V-{[2-chloro-3-(trifluoromethyl)phenyl]methyl}-1 -(2,2-dimethylpropyl)-5- oxoprolinamide (E118)

N-{[2-chloro-3-(trifluoromethyl)phenyl]methyl}-1 -(2,2-dimethylpropyl)-5- oxoprolinamide was prepared in an analogous manner to that described for example 100 but using 1-(2,2-dimethylpropyl)-5-oxoproline prepared as described below. LC/MS [M+H]⁺ = 391/393, retention time = 2.76 minutes.

The 1-(2,2-dimethylpropyl)-5-oxoproline used in the method described above was prepared as follows: D-glutamic acid (2.21 g, 15 mmol) was dissolved in 2N aqueous sodium hydroxide (15 ml, 30 mmol), cooled to 0⁰C, and treated with a solution of trimethylacetaldehyde (1.63 ml, 15 mmol) in ethanol (3 ml) and then stirred at room temperature for 45 minutes. The mixture was again cooled to 0⁰C and treated portion-wise with sodium borohydride (0.189 g, 5 mmol). The mixture was allowed to warm to room temperature with stirring over 4 hrs then after washing with diethyl ether it was acidified to ~pH4 using concentrated hydrochloric acid. The resulting precipitate was collected by filtration, washed with diethyl ether and then dried in a vacuum oven overnight. The solid was then suspended in ethanol (50 ml) and the mixture was heated at reflux for 24 hrs. Concentration and trituration with hexane then afforded 1-(2,2-dimethylpropyl)-5-oxoproline (1.51 g) as a solid which was used without further purification.

Example 1 19 Λ/-{[2-chloro-3-(trifluoromethyl)phenyl]methyl}-5-oxo-1-(2- pyridinylmethyl)prolinamide (E119)

5-OXO-1 -(2-pyridinylmethyl)proline (0.220 g, 1 mmol, prepared as described below), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (0.384 g, 2 mmol), and 1-hydroxybenzotriazole (0.308 g, 2 mmol) were stirred together in dichloromethane (10 ml) at room temperature for 30 minutes. The mixture was then treated with {[2-chloro-3-(trifluoromethyl)phenyl]methyl}amine (0.314 g, 1.5 mmol) and the mixture was stirred overnight at room temperature. The mixture was concentrated and partitioned between ethyl acetate and saturated aqueous sodium hydrogen carbonate solution. The aqueous layer was separated and extracted with ethyl acetate and then the combined ethyl acetate fractions were washed with 3 portions of water and then with saturated aqueous sodium chloride solution. Drying over sodium sulphate and concentration gave a solid residue which was purified by mass-directed automated HPLC to give Λ/-{[2-chloro-3- (trifluoromethyl)phenyl]methyl}-5-oxo-1-(2-pyridinylmethyl)prolinamide (0.263 g) as a buff-coloured solid. LC/MS [M+H]⁺ = 412/414, retention time = 2.15 minutes.

The 5-OXO-1 -(2-pyridinylmethyl)proline used in the method described above was prepared as follows:

D-glutamic acid (2.21 g, 15 mmol) was dissolved in 2N aqueous sodium hydroxide (15 ml, 30 mmol) at O⁰C and then treated with pyridine-2-carboxaldehyde (1.43 ml, 15 mmol). The mixture was stirred at room temperature for 45 minutes and then cooled to 0°C and treated with sodium borohydride (0.189 g, 5 mmol). The mixture was allowed to warm to room temperature with stirring over 4 hrs then after washing twice with diethyl ether it was acidified to pH5-6. The aqueous layer was concentrated then azeotroped three times with toluene and then with a 1 :1 ethanol:toluene mixture and finally with ethanol. The residue was then taken up in ethanol (50 ml) and refluxed for 8 hrs. Concentration gave an oil which when dried in vacuo gave 5-oxo-1-(2-pyridinylmethyl)proline (2.60 g) as a foam which was used without any further purification.

Example 120 Λ/-{[2-chloro-3-(trifluoromethyl)phenyl]methyl}-5-oxo-1-(3- pyridinylmethyl)prolinamide (E120)

5-OXO-1 -(3-pyridinylmethyl)proline (0.210 g, 1 mmol, prepared as described below), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (0.383 g, 2 mmol), and 1-hydroxybenzotriazole (0.306 g, 2 mmol) were stirred together in dichloromethane (10 ml) at room temperature for 30 minutes. The mixture was then treated with {[2-chloro-3-(trifluoromethyl)phenyl]methyl}amine (0.314 g, 1.5 mmol) and the mixture was stirred overnight at room temperature. The mixture was concentrated and partitioned between ethyl acetate and saturated aqueous sodium hydrogen carbonate. The aqueous layer was separated and extracted with more ethyl acetate and then the combined ethyl acetate fractions were washed sequentially with 3 portions of water and then with saturated aqueous sodium chloride solution. Drying over magnesium sulphate and concentration gave a solid residue which was purified by mass-directed automated HPLC to give Λ/-{[2-chloro-3- (trifluoromethyl)phenyl]methyl}-5-oxo-1-(3-pyridinylmethyl)prolinamide (0.031 g). LC/MS [M+H]⁺ = 412/414, retention time = 1.83 minutes.

The 5-OXO-1 -(3-pyridinylmethyl)proline used in the method described above was prepared as follows:

D-glutamic acid (2.21 g, 15 mmol) was dissolved in 2N aqueous sodium hydroxide (15 ml, 30 mmol) at 0°C and then treated with pyridine-3-carboxaldehyde (1.41 ml, 15 mmol) in ethanol (3 ml). The mixture was stirred at room temperature for 30 minutes and then cooled to 0⁰C and treated portion-wise with sodium borohydride (0.189 g, 5 mmol). The mixture was allowed to warm to room temperature with stirring over 4 hrs then after washing with diethyl ether it was acidified to pH5-6 using concentrated hydrochloric acid. The resulting precipitate was collected by filtration, washed with diethyl ether, and dried in vacuo. This material was then taken up in ethanol (50 ml) and refluxed overnight. Fine solids were removed by filtration and then concentration gave 5-oxo-1 -(3-pyridinylmethyl)proline (2.04 g) as a white solid which was used without any further purification.

Example 121 /V-[(2,4-dichlorophenyl)methyl]-5-oxo-1-(3-pyridinylmethyl)prolinamide (E121 )

Λ/-[(2,4-dichlorophenyl)methyl]-5-oxo-1-(3-pyridinylmethyl)prolinamide was prepared in a manner analogous to that described above for the synthesis of Λ/-{[2-chloro-3-

(trifluoromethyl)phenyl]methyl}-5-oxo-1-(3-pyridinylmethyl)prolinamide (E120) but using [(2,4-dichlorophenyl)methyl]amine in the place of {[2-chloro-3-

(trifluoromethyl)phenyl]methyl}amine.

LC/MS [M+H]⁺ = 378/380/382, retention time = 1.70 minutes.

Example 122 1-cyclopropyl-Λ/-[(2,4-dichlorophenyl)methyl]-2-methyl-5- oxoprolinamide (E122)

To a solution of (2,4-dichlorophenyl)methyl isocyanide (0.047 g, 0.25 mmol) and levulinic acid (0.041 ml, 0.4 mmol) in methanol (2 ml) was added cyclopropylamine (0.042 ml, 0.6 mmol). The mixture was heated to 100⁰C for 30 minutes in a microwave reactor. The solvent was removed in vacuo and the residue was purified by mass-directed automated HPLC to give 1-cyclopropyl-Λ/-[(2,4- dichlorophenyl)methyl]-2-methyl-5-oxoprolinamide (0.054 g) as a white solid. LC/MS [M+H]⁺ = 341/343, retention time = 2.57 minutes.

Examples 123-126

In a manner analogous to that described for Example 122 above the compounds tabulated below (Table 1 1 ) were prepared by substituting the appropriate amine for the cyclopropylamine used in the above procedure. All of the amines used to make the compounds shown in Table 11 are available from commercial sources or can be prepared using routes described previously in the chemical literature,.

Table 1 1

Example 127 Λ/-[(2,4-dichlorophenyl)methyl]-1 ,3,3-trimethyl-5-oxoprolinamide (E127)

To a solution of (2,4-dichlorophenyl)methyl isocyanide (0.094 g, 0.5 mmol) and 3,3- dimethyl-4-oxobutanoic acid (0.065 mg, 0.5 mmol, prepared as described below) in methanol (2 ml) was added methylamine (0.080 ml, 33% solution in ethanol). The mixture was heated to 100⁰C for 30 minutes in a microwave reactor. The solvent was removed in vacuo and the residue was purified by mass-directed automated HPLC to give a colourless gum which was triturated with diethyl ether to give Λ/-[(2,4- dichlorophenyl)methyl]-1 ,3,3-trimethyl-5-oxoprolinamide (0.043 g) as a sticky white solid. LC/MS [M+H]⁺ = 329/331 , retention time = 2.42 minutes. The 3,3-dimethyl-4-oxobutanoic acid used in the procedure described above was prepared as follows:

3,3-dimethyl-4-pentenoic acid (1 .3 g, 10.14 mmol) was dissolved in dichloromethane

(25 ml) and cooled to -78⁰C in a CO₂/acetone bath. Oxygen was bubbled through the mixture for 5 minutes followed by ozone for 25 minutes (giving a blue solution).

Oxygen was bubbled through the mixture for a further 5 minutes followed by argon for 10 minutes. Dimethylsulphide (2.23 ml, 30.4 mmol) was then added to the mixture and the mixture was removed from the cooling bath and stirred for 2.5 hrs.

The resulting colourless solution was reduced in vacuo to give 3,3-dimethyl-4- oxobutanoic acid as a colourless oil which was used without further purification.

Example 128 Λ/-{[2-chloro-3-(trifluoromethyl)phenyl]methyl}-1 ,3,3-trimethyl-5- oxoprolinamide (E128)

/V-{[2-chloro-3-(trifluoromethyl)phenyl]methyl}-1 ,3,3-trimethyl-5-oxoprolinamide was prepared in a manner analogous to that described above for the synthesis of Λ/-[(2,4- dichlorophenyl)methyl]-1 ,3,3-trimethyl-5-oxoprolinamide (E127) but using [2-chloro-3- (trifluoromethyl)phenyl]methyl isocyanide (prepared as described in example 40) in the place of (2,4-dichlorophenyl)methyl isocyanide. LC/MS [M+H]⁺ = 363/365, retention time = 2.49 minutes.

Example 129 Λ/-{[2-chloro-3-(trifluoromethyl)phenyl]methyl}-1 ,3-dimethyl-5- oxoprolinamide (E129)

1 ,3-Dimethyl-5-oxoproline (0.620 g, 3.6 mmol, prepared as described below) ,N-(3- dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (0.822 g, 4.3 mmol), 1-

Hydroxybenzotriazole (0.581 g, 4.3 mmol), N-ethyl morpholine (1.4 ml, 10.8 mmol), and {[2-chloro-3-(tπfluoromethyl)phenyl]methyl}amine (0.828 g, 3.96 mmol) were combined in a mixture of dichloromethane (10 ml) and dimethylformamide (5 ml) and stirred under argon overnight. The mixture was then washed sequentially with water (50 ml), 0.5 N aqueous hydrogen chloride (50 ml), water (50 ml), saturated aqueous sodium hydrogen carbonate (50 ml) and water (50 ml). The dichloromethane layer was passed through a hydrophobic frit and evaporated in vacuo to give the crude product. This was further purified by mass-directed automated HPLC (10 x 0.100 g injections) to give Λ/-{[2-chloro-3-(trifluoromethyl)phenyl]methyl}-1 ,3-dimethyl-5- oxoprolinamide (0.613 g). LC/MS [M+H]⁺ = 349, retention time = 2.31 , 2.38 minutes (two diastereoisomers).

The 1 ,3-dimethyl-5-oxoproline used in the above procedure was prepared as follows: (i) (f?,f?,f?)-2-hydroxypinen-3-one (10.9 g, 64.8 rmmol) and glycine-t-butyl ester (13 g, 97.2 mmol) in anhydrous toluene (200 ml) was treated with boron trifluoride-diethyl etherate (0.460 g, 3.24 mmol) and then heated, under argon, for 6 hrs at reflux. The mixture was then cooled to room temperature and stirred overnight. Filtration through a sinter followed by evaporation gave a yellow gum which was purified by automated flash silica-gel column chromatography (using a Biotage SP4), eluting with a mixture of 25% ethyl acetate in hexane, to give some 1 ,1-dimethylethyl N- [(I R^R.δR^-hydroxy^.e.e-trimethylbicyclofS.i .i lhept-S-ylidenejglycinate (3.68 g) and some mixed fractions. The impure material was further purified, again using automated flash silica-gel column chromatography (Biotage SP4), but eluting with a gradient of 0-25% ethyl acetate in hexane (0-15% over 10 column volumes and 15- 25% over 5 column volumes), to give a further crop of 1 ,1-dimethylethyl N- [(I R^R.δR^-hydroxy^e.e-trimethylbicycloβ.i .i lhept-S-ylidenetølycinate (1.73 g). The two batches of 1 ,1-dimethylethyl Λ/-[(1 R,2R,5R)-2-hydroxy-2,6,6- trimethylbicyclo[3.1.1]hept-3-ylidene]glycinate were combined (5.41 g) and this material was used in the next step.

(Note: The glycine-t-butyl ester used above could also be replaced with glycine-t- butyl ester hydrochloride and a molar equivalent of potassium carbonate) (ii) A solution of 1 ,1-dimethylethyl Λ/-[(1 R,2R,5R)-2-hydroxy-2,6,6- trimethylbicycloβ.i .iJhept-S-ylideneJglycinate (11 .05 g, 39.3 mmol) in anhydrous tetrahydrofuran (100 ml) was cooled to -30⁰C and treated with a 3M solution of methylmagnesium bromide in diethyl ether (17.1 ml, 51.1 mmol). 1 ,8-

Diazabicyclo[5.4.0]undec-7-ene (7.78 g, 51 .1 mmol) was then added and the mixture was stirred for an additional 20 minutes at -30⁰C. The mixture was then treated with ethyl crotonate and stirring continued for 1 hr. The mixture was quenched by addition of saturated aqueous ammonium chloride solution (35 ml) and then extracted with ethyl acetate (3 x 100 ml). The combined organic extracts were dried over sodium sulphate, filtered and evaporated to give a yellow oil. This material was purified by automated flash silica-gel column chromatography (using a Biotage SP4), eluting with a gradient of 0-20% (over 5 column volumes) then 20-35% (over 14 column volumes) ethyl acetate in hexane, to give 1-(1 ,1-dimethylethyl) 5-ethyl N- [(1 R,2f?,5R)-2-hydroxy-2,6,6-trimethylbicyclo[3.1.1 ]hept-3-ylidene]-3-methylglutamate (4.2 g) which was used in the next step.

(iii) A 10% aqueous solution of citric acid (1 1 ml, 5.6 mmol) was added to a solution of 1-(1 ,1-dimethylethyl) 5-ethyl Λ/-[(1 R2/?,5fi)-2-hydroxy-2,6,6- trimethylbicyclo[3.1.1]hept-3-ylidene]-3-methylglutamate (2g) in tetrahydrofuran (10 ml) and the mixture was stirred at room temperature for 4 days. The mixture was the evaporated and the residue suspended in water (50 ml) and washed with diethyl ether (100 ml). The aqueous phase was then adjusted to pH~7 using aqueous sodium hydrogen carbonate solution and then extracted with diethyl ether (3 x 100 ml). The organic fractions were combined, dried over sodium sulphate, filtered and evaporated to give 1 -(1 , 1 -dimethylethyl) 5-ethyl 3-methylglutamate (1.1 g) as a yellow oil which was used in the next step without further purification, (iv) 1-(1 ,1-Dimethylethyl) 5-ethyl 3-methylglutamate (1.1 g, 4.5 mmol) was left to stand, attached to a high vacuum line, overnight and then over a weekend. Starting material was still evident at this stage so toluene (30 ml) was added and the resulting mixture was heated at 1 10⁰C overnight. Evaporation gave 1 ,1 -dimethylethyl 3- methyl-5-oxoprolinate (0.79 g) which was used in the next step without further purification, (v) 1 ,1 -dimethylethyl 3-methyl-5-oxoprolinate (0.79 g, 3.96 mmol) was dissolved in tetrahydrofuran (8 ml) and treated with methyl iodide (0.27 ml, 4.36 mmol). The mixture was then cooled to 0⁰C and treated portion-wise with sodium hydride (60% in oil, 0.170 g, 4.36 mmol). The mixture ceased bubbling after 30 minutes at 0⁰C and was then allowed to warm to room temperature and stirred overnight. The mixture was quenched by addition of saturated aqueous ammonium chloride solution (10 ml) and the organic layer was separated and put aside. The aqueous layer was extracted with dichloromethane (3 x 20 ml) and the combined extracts were dried using a hydrophobic frit. All of the organic fractions (including that put aside earlier) were combined and evaporated to give crude 1 ,1 -dimethylethyl 1 ,3-dimethyl-5- oxoprolinate (0.770 g) as a yellow gum which was used without further purification. (vi) 1 ,1 -Dimethylethyl 1 ,3-dimethyl-5-oxoprolinate (0.770 g, 3.62 mmol) was suspended in dichloromethane (5 ml) and treated with trifluoroacetic acid (0.4 ml, 5.4 mmol). The mixture was stirred for 5 hrs and then evaporated. Azeotroping the resulting residue with toluene then gave unreacted starting material (0.600 g). This was again taken up in dichloromethane (2 ml) and treated with trifluoroacetic acid (2 ml) once more. After stirring for 2 hrs the mixture was evaporated and the residue again azeotroped with toluene (10 ml) to give crude 1 ,3-dimethyl-5-oxoproline (0.760 g) which was used without any additional purification.

Example 130 Λ/-[(2-chloro-4-fluorophenyl)methyl]-1-ethyl-4,4-dimethyl-5- oxoprolinamide (E130)

1-ethyl-4,4-dimethyl-5-oxoproline (0.130 g, 0.702 mmol, prepared as described below), 1-Hydroxybenzotriazole (0.161 g, 1.053 mmol), and N-(3- dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (0.202 g, 1.053 mmol) were dissolved in dichloromethane (5 ml) and stirred for 15 minutes at room temperature. [(2-chloro-4-fluorophenyl)methyl]amine (0.134 g, 0.842 mmol) and diisopropylethylamine (0.184 ml, 1.053 mmol) were then added to the mixture and stirring continued overnight at room temperature. The mixture was then concentrated in vacuo and the residue partitioned between ethyl acetate and water and extracted with ethyl acetate. The combined organic layers were washed sequentially with 3N citric acid, water, saturated aqueous sodium carbonate, water (x3), and then brine and dried over anhydrous sodium sulfate. Concentration gave a crude solid which was subsequently purified by mass-directed automated HPLC to give Λ/-[(2-chloro-4- fluorophenyl)methyl]-1-ethyl-4,4-dimethyl-5-oxoprolinamide (0.146 g) as a solid. LC/MS [M+H]⁺ = 327/329, retention time = 2.35 minutes.

The 1-ethyl-4,4-dimethyl-5-oxoproline used in the above procedure was prepared as described below:

(i) (S)-(+)-L-5-trityloxymethyl-2-pyrrolidinone (7.51 g, 20 mmol) was dissolved in dimethylformamide (25 ml) at O⁰C and treated portion wise with sodium hydride (60% suspension in oil, 0.880 g, 22 mmol). The mixture was stirred at O⁰C for 1 hr and then treated with ethyl iodide (1.78 ml, 22 mmol). The mixture was allowed to warm to room temperature and then stirred overnight. The mixture was then poured onto ice and extracted with ethyl acetate (x3). The combined organic extracts were washed sequentially with water, 50% aqueous sodium chloride solution (3x), and saturated aqueous sodium chloride solution, and then dried over sodium sulphate. Concentration gave a crude solid which was purified by automated flash silica-gel column chromatography (Biotage SP4), eluting with a 0-100% gradient of ethyl acetate in hexane, to give 1-ethyl-5-{[(triphenylmethyl)oxy]methyl}-2-pyrrolidinone (7.09 g).

(ii) To a 2M solution of lithium diisopropylamide in tetrahydrofuran (1.912 ml, 3.82 mmol) at -78°C was added, drop-wise, a solution of 1-ethyl-5- {[(triphenylmethyl)oxy]methyl}-2-pyrrolidinone (1.34 g, 3.48 mmol) in tetrahydrofuran (10 ml) and the resulting mixture was stirred for 1 hr at -78°C. lodomethane (0.239 ml, 3.82 mmol) was then added and after stirring for a further 1 hr at -78⁰C the mixture was allowed to warm to room temperature over 3 hrs. The mixture was then re-cooled to -78⁰C and treated, drop-wise, with a further aliquot of a 2M solution of lithium diisopropylamide in tetrahydrofuran (1.912 ml, 3.82 mmol). After stirring for an additional 1 hr at -78°C the mixture was again treated with iodomethane (0.239 ml, 3.82 mmol) and then the mixture was allowed to warm to room temperature and stirred overnight. The mixture was quenched with saturated aqueous ammonium chloride and then extracted with ethyl acetate (2x). The combined organic extracts were then washed with water (3x) and then with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulphate, and concentrated to a crude oily solid (1.7 g). The crude solid was purified by automated flash silica-gel column chromatography (Biotage SP4), eluting with a 0-100% gradient of ethyl acetate in hexane, to give product fractions (i.e. 1-ethyl-3,3-dimethyl-5- {[(triphenylmethyl)oxy]methyl}-2-pyrrolidinone (0.468 g)) as well as monoalkyated material (i.e. 1-ethyl-3-methyl-5-{[(triphenylmethyl)oxy]methyl]-2-pyrrolidinone (0.524 g)) and a mixture of these two (0.240 g). The desired product was set aside while the 1-ethyl-3-methyl-5-{[(triphenylmethyl)oxy]methyl}-2-pyrrolidinone and the mixed material were combined and dissolved in tetrahydrofuran (20 ml). This solution was then added drop wise to a 2M solution of lithium diisopropylamide in tetrahydrofuran (1.912 ml, 3.82 mmol) at -78°C and stirring at this temperature was continued for 1 hr. lodomethane (0.239 ml, 3.82 mmol) was then added to the mixture and the mixture was allowed to warm to room temperature over 4 hrs. Workup as described above gave an additional batch of 1-ethyl-3,3-dimethyl-5- {[(triphenylmethyl)oxy]methyl}-2-pyrrolidinone (0.846 g) as an oil which was combined with the material set aside earlier (total mass = 1.08 g) and used in the next step without further purification.

(iii) Amberlyst 15® (5.56 g, 26.1 mmol) was washed three times with methanol and then a solution of 1-ethyl-3,3-dimethyl-5-{[(triphenylmethyl)oxy]methyl}-2- pyrrolidinone (1.08 g, 2.61 mmol) in methanol (50 ml) was added. The mixture was left to stand for 4 days at room temperature and then the resin was removed by filtration (washing with methanol). The combined methanol fractions were concentrated to give a crude oil (1 .62 g) which was purified by automated flash silica- gel column chromatography (Biotage SP4), eluting with a 0-100% gradient of ethyl acetate in hexane, to give 1 -ethyl-5-(hydroxymethyl)-3,3-dimethyl-2-pyrrolidinone (0.376 g) as an oil that solidified on standing.

(iv) 1-Ethyl-5-(hydroxymethyl)-3,3-dimethyl-2-pyrrolidinone (0.366 g, 2.1 mmol), sodium chlorite (0.387 g, 4.3 mmol), and a 1 M aqueous sodium phosphate monobasic buffer solution (2.46 ml, 2.46 mmol) were combined in acetonitrile (3 ml) and heated to 4O⁰C. A few crystals of TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy free radical) and approximately 1 drop of bleach (sodium hypochlorite solution, available chlorine >12%) were then added to the mixture and stirring continued at 4O⁰C for 4 hrs. The mixture was then poured onto ice containing 1 % w/w sodium sulphite and the resulting mixture was extracted with ethyl acetate (x3). The combined organic extracts were washed with saturated aqueous sodium chloride and then dried over magnesium sulphate and concentrated to give 1 -ethyl-4,4-dimethyl-5- oxoproline (0.392 g) as a white solid which was used without additional purification. LC/MS [M+H]⁺ = 186.

Example 131 Λ/-{[2-chloro-3-(trifluoromethyl)phenyl]methyl}-1-ethyl-4,4-dimethyl-5- oxoprolinamide (E131)

/V-^-Chloro-S-^rifluoromethylJphenyllmethylJ-i-ethyl^^-dimethyl-S-oxoprolinamide was prepared in a manner analogous to that described above for the synthesis of N- [(2-chloro-4-fluorophenyl)methyl]-1-ethyl-4,4-dimethyl-5-oxoprolinamide (E130) but using {[2-chloro-3-(trifluoromethyl)phenyl]methyl}amine in the place of [(2-chloro-4- fluorophenyl)methyl]amine. LC/MS [M+H]⁺ = 377/379, retention time = 2.63 minutes.

Example 132 Λ/-[(2-chloro-3,4-difluorophenyl)methyl]-1 -ethyl-4,4-dimethyl-5- oxoprolinamide (E132)

/V-[(2-chloro-3,4-difluorophenyl)methyl]-1-ethyl-4,4-dimethyl-5-oxoprolinamide was prepared in a manner analogous to that described above for the synthesis of Λ/-[(2- chloro-4-fluorophenyl)methyl]-1 -ethyl-4,4-dimethyl-5-oxoprolinamide (E130) but using [(2-chloro-3,4-difluorophenyl)methyl]amine hydrochloride (prepared as described above for Example 36) in the place of [(2-chloro-4-fluorophenyl)methyl]amine. LC/MS [M+H]⁺ = 345/347, retention time = 2.43 minutes.

Example 133 Λ/-[(2-chloro-4-fluorophenyl)methyl]-1-ethyl-5-oxo-4,4- bis(phenylmethyl)prolinamide (E133)

Λ/-[(2-chloro-4-fluorophenyl)methyl]-1-ethyl-5-oxo-4,4-bis(phenylmethyl)prolinamide was prepared in a manner analogous to that described above for the synthesis of N- [(2-chloro-4-fluorophenyl)methyl]-1-ethyl-4,4-dimethyl-5-oxoprolinamide (E130) but using 1-ethyl-5-oxo-4,4-bis(phenylmethyl)proline in the place of 1-ethyl-4,4-dimethyl- 5-oxoproline. 1-Ethyl-5-oxo-4,4-bis(phenylmethyl)proline was prepared in a manner analogous to that described for 1-ethyl-4,4-dimethyl-5-oxoproline in example 130 above but using 1-ethyl-3,3-bis(phenylmethyl)-5-{[(triphenylmethyl)oxy]methyl}-2- pyrrolidinone (isolated as a side-product in method B, Example 37) in place of 1 - ethyl-3,3-dimethyl-5-{[(triphenylmethyl)oxy]methyl}-2-pyrrolidinone. LC/MS [M+H]⁺ = 479/481 , retention time = 3.32 minutes.

Example 134 Λ/-{[2-chloro-3-(trifluoromethyl)phenyl]methyl}-1 -ethyl-5-oxo-4- (phenylmethyl)prolinamide (E134)

Λ/-{[2-chloro-3-(tπfluoromethyl)phenyl]methyl}-1 -ethyl-5-oxo-4- (phenylmethyl)prolinamide was prepared in a manner analogous to that described above for the synthesis of Λ/-[(2-chloro-4-fluorophenyl)methyl]-1-ethyl-5-oxo-4- (phenylmethyl)-prolinamide (E37), but using {[2-chloro-3-

(trifluoromethyl)phenyl]methyl}amine in the place of [(2-chloro-4- fluorophenyl)methyl]amine. Method B, as described in Example 37, was used to prepare the 1-ethyl-5-oxo-4-(phenylmethyl)-proline. LC/MS [M+H]⁺ = 439/441 , retention time = 2.99 minutes.

Example 135 Λ/-{[2-Cyano-3-(trifluoromethyl)phenyl]methyl}-1 -methyl-5- oxoprolinamide (E135)

/V-{[2-Cyano-3-(trifluoromethyl)phenyl]methyl}-1-methyl-5-oxoprolinamide was prepared in a manner analogous to that described above for the synthesis of Λ/-{[2- cyano-3-(trifluoromethyl)phenyl]methyl}-1 -ethyl-5-oxoprolinamide (E105) but using 1 - methyl-5-oxo-proline in place of 1-ethyl-5-oxo-proline. LC/MS [M+H]⁺ = 326, retention time = 2.02 minutes.

Example 136 Λ/-(2-biphenylylmethyl)-1 -ethyl-5-oxoprolinamide (E136)

/V-(2-biphenylylmethyl)-1 -ethyl-5-oxoprolinamide was prepared in a manner analogous to that described above for the synthesis of Λ/-[(2,3- dimethylphenyl)methyl]-1-ethyl-5-oxoprolinamide (E50) but using (2- biphenylylmethyl)amine in place of 2,3-dimethyl benzylamine. LC/MS [M+H]⁺ = 323, retention time = 2.38 minutes.

Microwave Reactor Where indicated in the above examples, the microwave reactor used was a Biotage Initiator™ . Reactions were carried out using normal power output unless specified otherwise.

Mass-directed automated HPLC

Where indicated in the above examples, purification by mass-directed automated

HPLC was carried out using the following apparatus and conditions:

Hardware

Waters 2525 Binary Gradient Module

Waters 515 Makeup Pump

Waters Pump Control Module Waters 2767 Inject Collect

Waters Column Fluidics Manager

Waters 2996 Photodiode Array Detector

Waters ZQ Mass Spectrometer

Gilson 202 fraction collector Gilson Aspec waste collector

Software

Waters MassLynx version 4 SP2

Column

The columns used are Waters Atlantis, the dimensions of which are 19mm x 100mm (small scale) and 30mm x 100mm (large scale). The stationary phase particle size is 5μrm.

Solvents

A : Aqueous solvent = Water + 0.1 % Formic Acid

B : Organic solvent = Acetonitrile + 0.1 % Formic Acid

Make up solvent = Methanol : Water 80:20

Needle rinse solvent = Methanol

Methods

There are five methods used depending on the analytical retention time of the compound of interest. They have a 13.5-minute runtime, which comprises a 10- minute gradient followed by a 3.5 minute column flush and re-equilibration step. Large/Small Scale 1.0-1.5 = 5-30% B Large/Small Scale 1.5-2.2 = 15-55% B Large/Small Scale 2.2-2.9 = 30-85% B Large/Small Scale 2.9-3.6 = 50-99% B

Large/Small Scale 3.6-5.0 = 80-99% B (in 6 minutes followed by 7.5 minutes flush and re-equilibration)

Flow rate

All of the above methods have a flow rate of either 20mls/min (Small Scale) or 40mls/min (Large Scale).

Chiral HPLC

Apparatus and conditions used to characterize enantiomeric purity of selected samples was as follows:

Method (A) Instrument: Agilent 1100 Series Liquid Chromatogram

Column: Chiralpak AD (250mm x 4.6mm; 10um particle size)

Mobile phase: Heptane:absolute ethanol (70:30) v/v pump-mixed

Flow rate: 1 ml/min

Temperature: Ambient U.V. Wavelength: 215nm

Method (B)

Instrument: Agilent 1100 Series Liquid Chromatogram Column: Chiralpak AD (250mm x 4.6mm; 10um particle size) Mobile phase: Heptane:absolute ethanol (50:50) v/v pump-mixed Flow rate: 1 ml/min Temperature: Ambient U.V. Wavelength: 215nm

Method (C)

Instrument: Agilent 1100 Series Liquid Chromatogram Column: Chiralpak AD (250mm x 4.6mm; 10um particle size) Mobile phase: Heptane:absolute ethanol (80:20) v/v pump-mixed Flow rate: 1 ml/min Temperature: Ambient U.V. Wavelength: 215nm

Method (D)

Instrument: Agilent 1100 Series Liquid Chromatogram Column: Chiralpak AS (250mm x 4.6mm; 10um particle size) Mobile phase: Heptane:absolute ethanol (80:20) v/v pump-mixed Flow rate: 1 ml/min

Temperature: Ambient U.V. Wavelength: 215nm

Liquid Chromatography / Mass Spectrometry

Analysis of the above Examples by Liquid Chromatography / Mass Spectrometry (LC/MS) was carried out using the following apparatus and conditions:

Hardware

Agilent 1 100 Gradient Pump

Agilent 1 100 Autosampler

Agilent 1 100 DAD Detector

Agilent 1 100 Degasser Agilent 1 100 Oven

Agilent 1 100 Controller

Waters ZQ Mass Spectrometer

Sedere Sedex 85

Software

Waters MassLynx version 4.0 SP2

Column

The column used is a Waters Atlantis, the dimensions of which are 4.6mm x 50mm. The stationary phase particle size is 3μm.

Solvents A : Aqueous solvent = Water + 0.05% Formic Acid B : Organic solvent = Acetonitrile + 0.05% Formic Acid

Method The generic method used has a 5 minute runtime.

The above method has a flow rate of 3ml/mins. The injection volume for the generic method is 5ul. The column temperature is 30deg. The UV detection range is from 220 to 330nm.

Pharmaceutical Composition Example for one Compound of Formula (I)

/V-{[2-Chloro-3-(trifluoromethyl)phenyl]methyl}-1 -methyl -5-oxo-L-prolinamide immediate release tablets for oral administration

Immediate release tablets for oral administration, containing Λ/-{[2-Chloro-3- (trifluoromethyl)phenyl]methyl}-1-methyl-5-oxo-/.-prolinamide (a compound of formula (I), which e.g. can optionally be prepared as described in Example 51 ) as the active therapeutic agent (P2X7 receptor antagonist) at 0.3 mg, 1 mg, 5 mg, 25 mg and 125 mg doses, are prepared according to the compositions shown in the table below.

Notes:

1. The active agent is Λ/-{[2-chloro-3-(trifluoromethyl)phenyl]methyl}-1-methyl-5-oxo-/-- prolinamide. 2. The purified water is removed during processing, does not appear in final product. 3. Opadry® White OY-S-28876 contains titanium dioxide (E171 ), macrogol 400, and hypromellose (hydroxypropylmethylcellulose).

/V-{[2-Chloro-3-(trifluoromethyl)phenyl]methyl}-1-methyl-5-oxo-L-prolinamide tablets are manufactured using a process of wet granulation and drying, compression and tablet coating. The manufacture process contains the following steps:

1 ) Two platform granules, one with a 1 .484% w/w drug concentration and the other with a 37.1 1 % w/w drug concentration, are prepared by granulating the required amount of drug substance (Λ/-{[2-chloro-3-

(trifluoromethyl)phenyl]methyl}-1-methyl-5-oxo-L-prolinamide), lactose monohydrate, microcrystalline cellulose, hypromellose (hydroxypropylmethylcellulose) and croscarmellose sodium in a high shear mixer using water as the granulation fluid. 2) The wet granules from 1 ) are then dried.

3) The dried granules from 2) are screened.

4) The screened granules from 3) are blended with the required amount of extragranular excipient(s) in a low shear mixer. For the 0.3 mg, 1 mg and 5 mg dose tablets, the platform granules containing 1.484% w/w of drug substance are used to make the blend; whereas for the 25 mg and 125 mg dose tablets, the platform granules containing 37.1 1 % w/w of drug substance are used to make the blend.

5) The blend from 4) is lubricated with magnesium stearate.

6) The lubricated blend from 5) is compressed into tablet cores at 350 mg core weight.

7) The tablet cores are coated with Opadry® White OY-S-28876 to obtain the final coated tablets as the product.

In one embodiment, one or more of one dose of these tablets can optionally be used as one half (the P2X7 receptor antagonist half) of the separate combinations disclosed in the Combination Examples 1 , 2 and 3 given below. COMBINATION EXAMPLES

Combination Example 1

W-{[2-chloro-3-(trifluoromethyl)phenyl]methyl}-1 -methyl-5-oxo-L-prolinamide and methotrexate, as a separate combination for the treatment of rheumatoid arthritis

The following two therapeutic agents are administered to a human suffering from rheumatoid arthritis, e.g. a human adult suffering from severe, active, classical or definite rheumatoid arthritis, as a separate combination and using the following dosage regimens, with the two therapeutic agents being administered either at substantially the same time of the day and/or at different times of the day as appropriate:

Therapeutic agent 1 (a compound of formula (I))

Λ/-{[2-Chloro-3-(trifluoromethyl)phenyl]methyl}-1-methyl-5-oxo-/--prolinamide is administered to the human at an oral dosage regimen of 0.3 mg to 2000 mg once or twice per day, e.g. 10 mg to 2000 mg once or twice per day, e.g. 20 mg to 1000 mg once or twice per day. The Λ/-{[2-chloro-3-(trifluoromethyl)phenyl]methyl}-1-methyl-5-

oxo-L-prolinamide (

) can be prepared as described in

Example 51 herein.

Therapeutic agent 2 - methotrexate

Methotrexate is administered separately to the human at a dosage regimen of 7.5 mg orally once weekly, or using divided oral doses of 2.5 mg at 12 hour intervals for 3 doses (7.5 mg) as a course once weekly. The schedule can be adjusted gradually to achieve an optimal response, but typically should not exceed a total weekly oral dose of 20mg of methotrexate. Once a response has been achieved, the methotrexate dose is typically reduced to the lowest possible effective dose.

The combination treatment is optionally continued for a number of days, weeks, months or years. Combination Example 2

/V-{[2-chloro-3-(trifluoromethyl)phenyl]methyl}-1 -methyl-5-oxo-L-prolinamide and pregabalin, as a separate combination for the treatment of neuropathic pain

The following two therapeutic agents are administered to a human, for the treatment of neuropathic pain in the human, as a separate combination and using the following dosage regimens, with the two therapeutic agents being administered either at substantially the same time of the day and/or at different times of the day as appropriate:

Therapeutic agent 1 (a compound of formula (I))

oxo-L-prolinamide (

) ^{can De} prepared as described in

Example 51 herein.

Therapeutic agent 2 - pregabalin

Pregabalin is administered separately to the human at an oral dosage regimen of 150 mg to 600 mg total pregabalin per day (measured as the free base), split between two to three doses per day. For example, for postherpetic neuralgia (a neuropathic pain condition), pregabalin can be administered at a starting oral dosage regimen of 150 mg total pregabalin per day (split between 2 or 3 doses per day), escalating (e.g. in about one week) to an oral dosage regimen of 300 mg pregabalin total per day, and optionally escalating up to a maximum oral dosage regimen of 600 mg total pregabalin per day. For painful diabetic neuropathy (another neuropathic pain condition), an oral dosage regimen of 150 mg to 300 mg total pregabalin per day can be administered. For fibromyalgia, an oral dosage regimen of 150 mg to 450 mg (e.g. 300 or 450 mg) total pregabalin per day can be administered.

The combination treatment is optionally continued for a number of days, weeks, months or years. Combination Example 3

/V-{[2-chloro-3-(trifluoromethyl)phenyl]methyl}-1 -methyl-5-oxo-L-prolinamide and paracetamol, as a separate combination for the treatment of osteoarthritis and/or inflammatory pain

The following two therapeutic agents are administered to a human, for the treatment of osteoarthritis and/or inflammatory pain in the human, as a separate combination and using the following dosage regimens, with the two therapeutic agents being administered either at substantially the same time of the day and/or at different times of the day as appropriate:

Therapeutic agent 1 (a compound of formula (I))

/V-{[2-Chloro-3-(trifluoromethyl)phenyl]methyl}-1-methyl-5-oxo--.-prolinamide is administered to the human at an oral dosage regimen of 0.3 mg to 2000 mg once or twice per day, e.g. 10 mg to 2000 mg once or twice per day, e.g. 20 mg to 1000 mg once or twice per day. The Λ/-{[2-chloro-3-(trifluoromethyl)phenyl]methyl}-1-methyl-5-

oxo-L-prolinamide (

) ^{can De} prepared as described in

Example 51 herein.

Therapeutic agent 2 - paracetamol

Paracetamol is administered separately to the human at an oral dosage regimen of 500 mg to 1000 mg (e.g. 500 mg, 650 mg or 1000 mg, in particular 650 mg) of paracetamol (measured as the free base / free compound), administered two, three or four times daily.

The combination treatment is optionally continued for a number of days, weeks, months or years. PHARMACOLOGICAL DATA

Compounds of formula (I) may be tested for in vitro biological activity at the P2X7 receptor in accordance with the following studies:

Ethidium Accumulation Assay

Studies were performed using NaCI assay buffer of the following composition (in mM): 14OmM NaCI, HEPES 10, Λ/-methyl-D-glucamine 5, KCI 5.6, D-glucose 10, CaCb 0.5 (pH 7.4). HEK293 cells, expressing human recombinant P2X7 receptors, were grown in poly-L-lysine pretreated 96 well plates for 18-24 h. (The cloning of the human P2X7 receptor is described in US 6,133,434). The cells were washed twice with 350μl of assay buffer before addition of 50μl of test compound. The cells were then incubated at room temperature (19-21⁰C) for 30 min before addition of ATP and ethidium (100μM final assay concentration). The ATP concentration was chosen to be close to the EC₈₀ for the receptor type and was 1 mM for studies on the human P2X7 receptor. Incubations were continued for 8 or 16 min and were terminated by addition of 25μl of 1.3M sucrose containing 5mM of the P2X7 receptor antagonist reactive black 5 (Aldrich). Cellular accumulation of ethidium was determined by measuring fluorescence (excitation wavelength of 530nm and emission wavelength of 620nm) from below the plate with a Canberra Packard Fluorocount (Pangbourne, UK) or a Flexstation.il (Molecular Devices) Antagonist plC₅₀ values for blocking ATP responses were determined using iterative curve fitting techniques.

Fluorescent Imaging Plate Reader (FLIPR) Ca Assay Studies were performed using NaCI assay buffer of the following composition (in mM) for human P2X7: 137 NaCI; 20 HEPES; 5.37 KCI; 4.17 NaHCO₃; 1 CaCI₂; 0.5 MgSO₄; and 1g/L of D-glucose (pH 7.4).

HEK293 cells, expressing human recombinant P2X7 receptors, were grown in poly- L-lysine pretreated 384 well plates for 42-48h. (The cloning of the human P2X7 receptor is described in US 6,133,434). The cells were washed three times with 80μl of assay buffer, loaded for 1 h at 37°C with 2μM Fluo4 (Teflabs), washed three times again, and left with 30μl buffer before the addition of 10 μl of 4x concentrated test compound. The cells were then incubated at room temperature for 30 mins before addition (online, by FLIPR384 or FLIPR3 instrument (Molecular Devices)) of

Benzoylbenzoyl-ATP (BzATP) 60μM final assay concentration. The BzATP concentration was chosen to be close to the EC_8O for the receptor type. Incubations and reading were continued for 90sec, and intracellular calcium increase was determined by measuring fluorescence (excitation wavelength of 488nm and emission wavelength of 516nm) from below the plate, with FLIPR CCD camera. Antagonist plC₅₀ values for blocking BzATP responses were determined using iterative curve fitting techniques.

The compounds of Examples 1-136 were tested in the FLIPR Ca Assay and/or the Ethidium Accumulation Assay for human P2X7 receptor antagonist activity and found to have plC50 values > 4.7 in the FLIPR Ca Assay and/or plC50 values > 5.5 in the Ethidium Accumulation Assay.

In Vivo data

Rat model of neuropathic pain

By placing loosely constrictive ligatures around the common sciatic nerve, a peripheral mononeuropathy can be produced, which thereby provides a rat model of neuropathic pain, Bennet et ai, Pain, Vol.33, pp87-107 (1988). Adult male Random Hooded rats (180-20Og) from Charles River, UK were anaesthetised with isoflurane (3%). The sciatic nerve in the left leg was exposed at mid thigh level and 4 loose ligatures of Chromic 4.0 gut tied around the nerve as described by Bennet et ai, Pain, Vol.33, pp87-107 (1988). The wound was closed and secured with staples. Sham rats underwent the same procedure but loose ligatures were not applied. The presence of mechanical (tactile) allodynia was assessed using manual application of Von Frey hair monofilaments. Monofilaments were applied in ascending order to the plantar region of the hind paw (range: 1.4g-26g). Each hair was applied for approx. 3- 5 seconds until a paw withdrawal response was observed. After confirmation with reapplication of lower and/or higher hairs, the lowest hair to give a paw withdrawal was recorded as the threshold response (g). When stable allodynia was established rats were dosed orally 26-33 days post surgery with compound twice daily for 8 days with allodynia measurements recorded at least three times during the dosing period. Λ/-[(2,4-dichlorophenyl)methyl]-1-methyl-5-oxo-Z--prolinamide (E10) and Λ/-{[2-chloro- 3-(trifluoromethyl)phenyl]methyl}-1 -methyl-5-oxo-/--prolinamide (E51) significantly reversed CCI-induced mechanical allodynia compared to vehicle response.

Rat model of joint pain

By measuring hypersensitivity post intra-articular injection of FCA into the knee, the effectiveness of a potential analgesic in reversing FCA-induced hypersensitivity can be assessed in a joint pain model of chronic inflammatory pain. Adult male Random Hooded rats (150-18Og) from Charles River, UK were briefly anaesthetised with isoflurane (3%). Rats were then injected with 150 μl of Freund's complete adjuvant (FCA) into the left knee joint (intra-articularly, i.art). The ability to bear weight on each hindlimb (weight bearing, g) was measured prior to and following surgery using a Dual Channel Weight Averager (Linton Instruments). When a stable difference in weight bearing was established between the injected and contralateral paws, rats were typically dosed orally (normally 13-17 days post surgery) with compound twice daily for 5 days with weight bearing measurements recorded daily. Λ/-[(2,4- dichlorophenyl)methyl]-1 -methyl-5-oxo-/--prolinamide (E10) and Λ/-{[2-chloro-3- (trifluoromethyl)phenyl]methyl}-1 -methyl-5-oxo-L-prolinamide (E51) significantly reversed FCA (i.art)-induced difference in weight bearing compared to vehicle response and produced ED50's <20mg/kg obtained from Area Under the Curve (AUC) calculations.

Rat model of acute inflammatory pain

A useful animal model for acute inflammatory pain is the Freund's Complete Adjuvant (FCA)-induced inflammation model. A similar model using carrageenan rather than FCA is described by Clayton et al. in Br. J. Pharmacol. 1997; 120, 219P. Adult male Random Hooded rats (180-22Og) from Charles River, UK received an intraplantar (i.pl) injection of 100 μl of FCA into the plantar surface of the left hind paw. The ability to bear weight on each hindlimb (weight bearing g) was measured prior to and 24 hours after the FCA injection using a Dual Channel Weight Averager (Linton Instruments). After the post-FCA reading, rats were typically dosed orally with compound after which weight bearing measurements were recorded. Λ/-[(2,4- dichlorophenyl)methyl]-1 -methyl-5-oxo-L-prolinamide (E10) and Λ/-{[2-chloro-3- (trifluoromethyl)phenyl]methyl}-1 -methyl-5-oxo-L-prolinamide (E51) significantly reversed FCA (i.pl)-induced difference in weight bearing compared to vehicle response and produced ED50's <20mg/kg obtained from dose response curves.

Claims

CLAIMS:

1. A combination comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof:

(I) wherein:

R² and R³ independently represent hydrogen, halogen, C-ι-6 alkyl, arylmethyl-, C2-6 alkenyl, C₂.₆ alkynyl or C3-6 cycloalkylmethyl-; and any of said C1-6 alkyl, arylmethyl-, C2-6 alkenyl, C_2-6 alkynyl or C3.6 cycloalkylmethyl- is optionally substituted with 1 , 2 or 3 halogen atoms;

R⁴, R⁵ and R⁶ independently represent hydrogen, fluorine or methyl; and R⁷, R⁸, R⁹, R¹⁰ and R¹¹ independently represent hydrogen, halogen, cyano, C_1-6 alkyl, C_2-6 alkenyl, C₂.₆ alkynyl, C_3-6 cycloalkyl or phenyl, and any of said C_1-6 alkyl, C_2-6 alkenyl, C₂.₆ alkynyl, C₃.₆ cycloalkyl or phenyl is optionally substituted with 1 , 2 or 3 halogen atoms; or R¹⁰ and R¹¹ together with the carbon atoms to which they are attached form a benzene ring which is optionally substituted with 1 , 2 or 3 halogen atoms; with the proviso that when R⁷ and R¹¹ are both selected from hydrogen or fluorine, at least one of R⁸, R⁹ and R¹⁰ is a halogen atom, or R⁸, R⁹ and R¹⁰ are selected from the group consisting of hydrogen and CF₃ and one, but not more than one, of R⁸, R⁹ and R¹⁰ is CF₃;

together with a further therapeutic agent or agents.

2. A combination as claimed in claim 1 , wherein:

R¹ represents C_1-6 alkyl, C_2-6 alkenyl, C₂.₆ alkynyl, C_3-6 cycloalkyl or C_3-6 cycloalkylmethyl-, any of which may be optionally substituted with 1 , 2 or 3 halogen atoms; or unsubstituted phenyl or benzyl; R² and R³ independently represent hydrogen, halogen, C_1-6 alkyl, arylmethyl-, C_2-6 alkenyl, C₂_₆ alkynyl or C_3-6 cycloalkylmethyl-; and any of said C₁^₃ alkyl, arylmethyl-, C_2-6 alkenyl, C₂_₆ alkynyl or C_3-5 cycloalkylmethyl- may be optionally substituted with 1 , 2 or 3 halogen atoms; R⁴, R⁵ and R⁶ independently represent hydrogen or fluorine; and

R⁷, R⁸, R⁹, R¹⁰ and R¹¹ independently represent hydrogen, halogen, cyano, Ci_₅ alkyl, C_2-6 alkenyl, C_2-6 alkynyl, C₃.₆ cycloalkyl, or phenyl; and any of said C_1-6 alkyl, C_2-6 alkenyl, C₂.₆ alkynyl, C_3-6 cycloalkyl or phenyl may be optionally substituted with 1 , 2 or 3 halogen atoms; with the proviso that when R⁷ and R¹¹ independently represent hydrogen or fluorine, at least one of R⁸, R⁹ and R¹⁰ is a halogen atom.

3. A combination as defined in claim 1 or 2, wherein:

R¹ represents unsubstituted Ci_-4 alkyl, C2-4 alkenyl, C3-5 cycloalkyl, pyridinylmethyl-, phenyl or benzyl;

R² and R³ both represent hydrogen;

R⁴, R⁵ and R⁶ independently represent hydrogen or methyl; and

R¹⁰ is CF₃.

4. A combination as defined in claim 1 , wherein the compound of formula (I) is a compound of Example 1-136, or a pharmaceutically acceptable salt thereof.

5. A combination as defined in claim 1 , wherein the compound of formula (I) or the salt thereof is Λ/-{[2-chloro-3-(trifluoromethyl)phenyl]methyl}-1-methyl-5- oxoprolinamide:

6. A combination as defined in claim 1 , wherein the compound of formula (I) or the salt thereof is /V-{[2-chloro-3-(trifluoromethyl)phenyl]methyl}-1-methyl-5-oxo-/-- prolinamide:

7. A combination as claimed in any one of claims 1 to 6, wherein when the further therapeutic agent includes a non-steroid anti-inflammatory drug (NSAID), the non-steroid anti-inflammatory drug is for the treatment of an inflammatory disease or disorder.

8. A combination as claimed in any one of claims 1 to 7, wherein the further therapeutic agent does not include ethanol, cocaine, nicotine, or an opioid such as morphine.

9. A combination as claimed in claim 8, wherein the further therapeutic agent does not include nicotine, or an opioid such as morphine, or a dependence-inducing central nervous system depressant such as ethanol, or a dependence-inducing psychostimulant such as cocaine.

10. A combination as claimed in claim 9, wherein the further therapeutic agent does not include a dependence-inducing agent.

11. A combination as claimed in any one of claims 1 to 10, wherein the further therapeutic agent does not include a cancer chemotherapeutic agent.

12. A combination as claimed in any one of claims 1 to 1 1 , wherein:

(i) the further therapeutic agent or agents includes a β2 adrenoceptor agonist and/or a corticosteroid, for the treatment of a respiratory disorder; or

(ii) the further therapeutic agent includes a 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitor for the treatment of a cardiovascular disorder; or

(iii) the further therapeutic agent includes a non-steroid anti-inflammatory drug (NSAID), for the treatment of an inflammatory disease or disorder; and/or

(iv) the further therapeutic agent includes a tumour necrosis factor α (TNFα) inhibitor for the treatment of an inflammatory disease or disorder; and/or

(v) the further therapeutic agent includes 2-hydroxy-5- [ [4- [ (2- pyridinylamino) sulfonyl] phenyl] azo] benzoic acid (sulfasalazine) for the treatment of an inflammatory disease or disorder; and/or

(vi) the further therapeutic agent includes N-[4-[[(2, 4-diamino-6-pteridinyl) methyl] methylamino] benzoyl]- L-glutamic acid (methotrexate) for the treatment of an inflammatory disease or disorder; and/or

(vii) the further therapeutic agent includes an inhibitor of pro TNFα convertase enzyme (TACE) for the treatment of an inflammatory disease or disorder; and/or

(viii) the further therapeutic agent includes: a) sulfasalazine; b) a statin; c) a glucocorticoid agent; d) an inhibitor of p38 kinase; e) an anti-IL-6-receptor antibody; f) anakinra; g) an anti-IL-1 monoclonal antibody; h) an inhibitor of JAK3 protein tyrosine kinase; i) an anti-macrophage colony stimulation factor (M-CSF) monoclonal antibody; or j) an anti-CD20 monoclonal antibody; for the treatment of an IL-1 mediated disease.

13. A combination as claimed in claim 7 or 12, wherein, when the further therapeutic agent includes a non-steroid anti-inflammatory drug (NSAID), the nonsteroid anti-inflammatory drug is ibuprofen, naproxen, aspirin, celecoxib, diclofenac, etodolac, fenoprofen, indomethacin, ketoprofen, ketoralac, oxaprozin, nabumetone, sulindac, tolmetin, rofecoxib, valdecoxib, lumaricoxib, meloxicam, etoricoxib or parecoxib.

14. A combination as claimed in claim 12, wherein:

(i) the further therapeutic agent or agents includes a β2 adrenoceptor agonist being formoterol; and/or a corticosteroid being budesonide, fluticasone as the propionate or furoate ester, mometasone furoate, beclomethasone as the 17-propionate or 17,21-dipropionate ester, ciclesonide, triamcinolone acetonide, flunisolide, rofleponide, or butixocort as the propionate ester; for the treatment of a respiratory disorder; or

(ii) the further therapeutic agent includes a 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitor for the treatment of a cardiovascular disorder, wherein the 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitor is atorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatin, or simvastatin; or

(iii) the further therapeutic agent includes a non-steroid anti-inflammatory drug (NSAID) for the treatment of an inflammatory disease or disorder; wherein the nonsteroid anti-inflammatory drug (NSAID) is ibuprofen, naproxen, aspirin, celecoxib, diclofenac, etodolac, fenoprofen, indomethacin, ketoprofen, ketoralac, oxaprozin, nabumetone, sulindac, tolmetin, rofecoxib, valdecoxib, lumaricoxib, meloxicam, etoricoxib or parecoxib; and/or

(iv) the further therapeutic agent includes a tumour necrosis factor α (TNFα) inhibitor for the treatment of an inflammatory disease or disorder, wherein the tumour necrosis factor α (TNFα) inhibitor is etanercept, the anti-TNFα antibody infliximab, or the anti- TNFα antibody adalimumab; and/or

(vii) the further therapeutic agent includes an inhibitor of pro TNFα convertase enzyme (TACE) for the treatment of an inflammatory disease or disorder; and/or (viii) the further therapeutic agent includes: a) sulfasalazine; b) a statin, which is atorvastatin, lovastatin, pravastatin, simvastatin, fluvastatin, cerivastatin, crilvastatin, dalvastatin, rosuvastatin, tenivastatin, fluindostatin, velostatin, dalvastatin, nisvastatin, bervastatin, pravastatin, rivastatin, glenvastatin, eptastatin, tenivastatin, flurastatin, rosuvastatin or itavastatin; c) a glucocorticoid agent which is dexamethasone, methylprednisolone, prednisolone, prednisone or hydrocortisone; d) an inhibitor of p38 kinase; e) an anti-IL-6-receptor antibody; f) anakinra; g) an anti-IL-1 monoclonal antibody; h) an inhibitor of JAK3 protein tyrosine kinase; i) an anti-macrophage colony stimulation factor (M-CSF) monoclonal antibody; or j) an anti-CD20 monoclonal antibody being rituximab or HuMax-CD20 (ofatumumab); for the treatment of an IL-1 mediated disease.

15. A combination as claimed in claim 14, wherein:

(i) the further therapeutic agent or agents includes a β2 adrenoceptor agonist being formoterol; and/or a corticosteroid being budesonide, fluticasone as the propionate or furoate ester, mometasone furoate, beclomethasone as the 17-propionate or 17,21-dipropionate ester, ciclesonide, triamcinolone acetonide, flunisolide, rofleponide, or butixocort as the propionate ester; for the treatment of asthma or chronic obstructive pulmonary disease; or

(ii) the further therapeutic agent includes a 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitor for the treatment of atherosclerosis, wherein the 3- hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitor is atorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatin, or simvastatin; or

(iii) the further therapeutic agent includes a non-steroid anti-inflammatory drug (NSAID) for the treatment of an inflammatory disease or disorder being rheumatoid arthritis or osteoarthritis; wherein the non-steroid anti-inflammatory drug (NSAID) is ibuprofen, naproxen, aspirin, celecoxib, diclofenac, etodolac, fenoprofen, indomethacin, ketoprofen, ketoralac, oxaprozin, nabumetone, sulindac, tolmetin, rofecoxib, valdecoxib, lumaricoxib, meloxicam, etoricoxib or parecoxib; and/or

(iv) the further therapeutic agent includes a tumour necrosis factor α (TNFα) inhibitor for the treatment of an inflammatory disease or disorder being rheumatoid arthritis or osteoarthritis, wherein the tumour necrosis factor α (TNFα) inhibitor is etanercept, the anti-TNFα antibody infliximab, or the anti-TNFα antibody adalimumab; and/or

(v) the further therapeutic agent includes 2-hydroxy-5- [ [4- [ (2- pyridinylamino) sulfonyl] phenyl] azo] benzoic acid (sulfasalazine) for the treatment of an inflammatory disease or disorder being rheumatoid arthritis; and/or

(vi) the further therapeutic agent includes N-[4-[[(2, 4-diamino-6-pteridinyl) methyl] methylamino] benzoyl]- L-glutamic acid (methotrexate) for the treatment of an inflammatory disease or disorder being rheumatoid arthritis; and/or

(vii) the further therapeutic agent includes an inhibitor of pro TNFα convertase enzyme (TACE) for the treatment of an inflammatory disease or disorder being rheumatoid arthritis; and/or

(viii) the further therapeutic agent includes: a) sulfasalazine; b) a statin, which is atorvastatin, lovastatin, pravastatin, simvastatin, fluvastatin, cerivastatin, crilvastatin, dalvastatin, rosuvastatin, tenivastatin, fluindostatin, velostatin, dalvastatin, nisvastatin, bervastatin, pitavastatin, rivastatin, glenvastatin, eptastatin, tenivastatin, flurastatin, rosuvastatin or itavastatin; c) a glucocorticoid agent which is dexamethasone, methylprednisolone, prednisolone, prednisone or hydrocortisone; d) an inhibitor of p38 kinase; e) an anti-IL-6-receptor antibody; f) anakinra; g) an anti-IL-1 monoclonal antibody; h) an inhibitor of JAK3 protein tyrosine kinase; i) an anti-macrophage colony stimulation factor (M-CSF) monoclonal antibody; or j) an anti-CD20 monoclonal antibody being rituximab or HuMax-CD20 (ofatumumab); for the treatment of an IL-1 mediated disease being rheumatoid arthritis.

16. A pharmaceutical composition which comprises a combination as defined in any one of the preceding claims and a pharmaceutically acceptable carrier or excipient.

17. A combination as claimed in any one of claims 1 to 15, for use in therapy.

18. A combination as claimed in claim 17, for use in the treatment of pain, inflammation or a neurodegenerative disease.

19. A combination as claimed in claim 18, for use in the treatment of inflammatory pain, neuropathic pain, visceral pain, rheumatoid arthritis or osteoarthritis.

20. A method of treating a human or animal subject suffering from pain, inflammation or a neurodegenerative disease, which method comprises administering to said subject an effective amount of a combination as defined in any one of claims 1 to 15.

21. Use of a combination as defined in any one of claims 1 to 15 for the manufacture of a medicament for the treatment or prevention of pain, inflammation or a neurodegenerative disease.

22. Use as claimed in any claim 21 , for the manufacture of a medicament for the treatment or prevention of inflammatory pain, neuropathic pain, visceral pain, rheumatoid arthritis or osteoarthritis.