WO2010125103A1

WO2010125103A1 - Diketopiperazine derivatives as p2x7 modulators

Info

Publication number: WO2010125103A1
Application number: PCT/EP2010/055716
Authority: WO
Inventors: Laura Jane Chambers; David Kenneth Dean; Jorge Munoz-Muriedas; Jon Graham Anthony Steadman; Daryl Simon Walter
Original assignee: Glaxo Group Limited
Priority date: 2009-04-29
Filing date: 2010-04-28
Publication date: 2010-11-04
Also published as: GB0907425D0

Abstract

The invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof: (I) wherein: A represents an aryl, heteroaryl or heterocyclyl group; and any ring or ring system of said aryl or heteroaryl is optionally substituted with 1 to 3 substituents, which may be the same or different, selected from the group consisting of halogen, C_1-6 alkyl, -CF₃, - OCF₃, cyano, C_1-6 alkoxy, -NR¹⁰R¹¹, -X-aryl, -X-heteroaryl and -X-heterocyclyl; R¹, R², R³, R⁴ and R⁵ independently represent hydrogen, fluorine, chlorine, -CF₃, cyano or C_1-6 alkyl, such that at least one of R¹, R², R³, R⁴ and R⁵ is other than hydrogen; R⁶, R⁷, R⁸, R⁹, R¹⁰ and R¹¹ independently represent hydrogen or C_1-6 alkyl; X represents a linker selected from a bond, -(CH₂)_n- and -O-(CH₂)_n-; and n represents an integer from 1 to 3. The compounds or salts modulate P2X7 receptor function and are capable of antagonizing the effects of ATP at the P2X7 receptor ("P2X7 receptor antagonists").

Description

Diketopiperazine derivatives as P2X7 modulators

The present invention relates to diketopiperazine derivatives which modulate P2X7 receptor function and are capable of antagonizing the effects of ATP at the P2X7 receptor ("P2X7 receptor antagonists"); to processes for their preparation; to pharmaceutical compositions containing them; and to the use of such compounds 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 (Dell'Antonio et al., Neurosci. Lett., Vol.327, pp87-90 (2002),. Chessell, IP., et al., Pain, Vol.114, 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., GNa, 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)).

WO 97/36889 (Merck & Co Inc) describes a series of 2,3-diketopiperazine farnesyl protein transferase inhibitors for the treatment of cancer. WO 2006/086445, WO 2005/011656, WO 2005/01 1657 and WO 2005/01 1653 (all of Xenon Pharm Inc) describe a series of piperazine derivatives as stearoyl-CoA desaturase inhibitors for the treatment of a range of disorders.

The present invention provides compounds 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 compound of formula (I) or a pharmaceutically acceptable salt thereof:

(I) wherein: A represents an aryl, heteroaryl or heterocyclyl group; and any ring or ring system of said aryl or heteroaryl is optionally substituted with 1 to 3 substituents, which may be the same or different, selected from the group consisting of halogen, C_1-6 alkyl, -CF₃, -OCF₃, cyano, Ci_-6 alkoxy, -NR¹⁰R¹¹, -X-aryl, -X-heteroaryl and -X-heterocyclyl; R¹, R², R³, R⁴ and R⁵ independently represent hydrogen, fluorine, chlorine, -CF₃, cyano or Ci_-6 alkyl, such that at least one of R¹, R², R³, R⁴ and R⁵ is other than hydrogen;

R⁶, R⁷, R⁸, R⁹, R¹⁰ and R¹¹ independently represent hydrogen or Ci_-6 alkyl; X represents a linker selected from a bond, -(CH₂)_n- and -O-(CH₂)_n-; and n represents an integer from 1 to 3. 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, C_1-6 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.

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

The term 'aryl' as used herein refers to a C_6-io monocyclic or bicyclic hydrocarbon ring wherein at least one ring is aromatic. Examples of such groups include phenyl, naphthyl or tetrahydronaphthyl.

The term "heteroaryl" as used herein means a 5 to 6 membered monocyclic aromatic or a fused 8 to 10 membered bicyclic aromatic ring system containing 1 to 3 heteroatoms selected from oxygen, nitrogen and sulfur. Suitable examples of such monocyclic aromatic rings include thienyl, furanyl, pyrrolyl, triazolyl, imidazolyl, oxazolyl, thiazolyl, oxadiazolyl, isothiazolyl, isoxazolyl, thiadiazolyl, pyrazolyl, pyrimidyl, pyridazinyl, pyrazinyl and pyridyl. Suitable examples of such fused bicyclic aromatic ring systems include quinolinyl, isoquinolinyl, quinazolinyl, quinoxalinyl, cinnolinyl, naphthyridinyl, indolyl, indazolyl, pyrrolopyridinyl, benzofuranyl, dihydrobenzodioxinyl, benzodioxinyl, benzothienyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzoxadiazolyl, benzothiadiazolyl and the like. Heteroaryl groups, as described above, may be linked to the remainder of the molecule via a carbon atom or, when present, a suitable nitrogen atom except where otherwise indicated above.

The term 'heterocyclyl' refers to a 4-7 membered monocyclic ring or a fused 8-12 membered bicyclic ring which may be saturated or partially unsaturated containing 1 to 4 heteroatoms selected from oxygen, nitrogen or sulphur. Examples of such monocyclic rings include pyrrolidinyl, azetidinyl, pyrazolidinyl, oxazolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, thiazolidinyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, dioxolanyl, dioxanyl, oxathiolanyl, oxathianyl, dithianyl, dihydrofuranyl, tetrahydrofuranyl, dihydropyranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, diazepanyl, azepanyl and the like. Examples of such bicyclic rings include indolinyl, isoindolinyl, benzopyranyl, quinuclidinyl, 2,3,4,5-tetrahydro-1 /-/-3-benzazepine, tetrahydroisoquinolinyl, dihydrobenzodioxinyl, dihydrobenzooxazinyl and the like.

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

(I) wherein:

A represents an aryl or heteroaryl group; and any ring or ring system of said aryl or heteroaryl is optionally substituted with 1 to 3 substituents, which may be the same or different, selected from the group consisting of halogen, Ci_-6 alkyl, -CF₃, cyano, Ci_-6 alkoxy and -X-morpholinyl;

R¹, R², R³, R⁴ and R⁵ independently represent hydrogen, fluorine, chlorine, -CF₃, cyano or C_1-6 alkyl, such that at least one of R¹, R², R³, R⁴ and R⁵ is other than hydrogen;

R⁶ and R⁷ independently represents hydrogen or C_1-6 alkyl;

X represents a linker selected from a bond, -(CH₂)_n- and -O-(CH₂)_n-; and n represents an integer from 1 to 3.

It is to be understood that the present invention covers and discloses all possible combinations of particular, preferred, suitable, or other embodiments of groups or features (e.g. of A, R¹ , R², R3, R4, RS₁ RΘ, R7 _{; R}8_{; R}9_; RiO₁ RH ₁ X, and/or n), e.g. covers and discloses all possible combinations of embodiments of different groups or features, which embodiments are described herein.

In certain particular embodiments, A represents a group other than pyridyl. In certain particular embodiments, A represents a group other than pyridazinyl.

In one particular embodiment, A represents a C_6-io aryl group such as phenyl, naphthyl or tetrahydronaphthyl optionally substituted by 1 to 3 substituents, which may be the same or different, selected from halogen, C_1-6 alkyl, -CF₃, -OCF₃, cyano, C-_I-6 alkoxy, -NR¹⁰R¹¹, -X-aryl, -X-heteroaryl and -X-heterocyclyl.

In certain particular embodiments, A represents a C_6-io aryl group such as phenyl, naphthyl or tetrahydronaphthyl optionally substituted by 1 to 3 substituents, which may be the same or different, selected from halogen (e.g. chlorine, fluorine, bromine or iodine), Ci_-6 alkyl (e.g. methyl, ethyl, isopropyl or t-butyl), -CF₃, -OCF₃, cyano, Ci_-6 alkoxy (e.g. methoxy), -NR¹⁰R¹¹ (e.g. -NMe₂ Or -NEt₂), -X-aryl (e.g. -phenyl), -X-heteroaryl (e.g. -pyrrolyl or -imidazolyl), and -X-heterocyclyl (e.g. -piperidinyl, -pyrrolidinyl, -morpholinyl, -(CH₂)-morpholinyl, -(CH₂)₂-morpholinyl or -O-(CH₂)₂-morpholinyl).

In certain particular embodiments, A represents a C_6-10 aryl group such as phenyl, naphthyl or tetrahydronaphthyl optionally substituted by 1 to 3 substituents, which may be the same or different, selected from halogen, C_1-6 alkyl, -CF₃, cyano, C_1-6 alkoxy and -X-morpholinyl.

In certain particular embodiments, A represents phenyl optionally substituted by 1 to 3 substituents, which may be the same or different, selected from halogen (e.g. fluorine, chlorine, bromine or iodine), C_1-6 alkyl (e.g. methyl, ethyl or isopropyl), -CF₃, cyano, C_1-6 alkoxy (e.g. methoxy), and -X-morpholinyl (e.g. -morpholinyl, -(CH₂)- morpholinyl or -(CH₂)₂-morpholinyl).

In certain particular embodiments, A represents a 5 to 6 membered heteroaryl (e.g. pyridinyl or pyrimidinyl) optionally substituted by 1 to 3 substituents, which may be the same or different, selected from halogen, C_1-6 alkyl (e.g. methyl), -CF₃, cyano, C_1-6 alkoxy and -X-morpholinyl. In certain embodiments, A represents a 5 to 6 membered heteroaryl (e.g. pyridinyl or pyrimidinyl) optionally substituted by 1 to 3 substituents, which may be the same or different, selected from C_1-6 alkyl (e.g. methyl).

In certain particular embodiments, A represents an 8 to 10 membered heteroaryl (e.g. indolyl, quinolinyl or isoquinolinyl) optionally substituted by 1 to 3 substituents, which may be the same or different, selected from halogen, C_1-6 alkyl, -CF₃, cyano, C_1-6 alkoxy and -X-morpholinyl.

In certain particular embodiments, A represents an 8 to 10 membered heteroaryl (e.g. indolyl, quinolinyl or isoquinolinyl) optionally substituted by 1 to 3 substituents, which may be the same or different, selected from Ci_-6 alkyl (e.g. methyl).

In certain particular embodiments, A represents an 8 to 10 membered heteroaryl (e.g. benzodioxinyl, quinolinyl or isoquinolinyl) optionally substituted by 1 to 3 substituents, which may be the same or different, selected from halogen, C_1-6 alkyl, - CF₃, cyano, Ci_-6 alkoxy and -X-morpholinyl.

In certain particular embodiments, A represents a heterocyclyl group (e.g. dihydrobenzodioxinyl or dihydrobenzooxazinyl) optionally substituted by 1 to 3 substituents, which may be the same or different, and which is or are C_1-6 alkyl (e.g. methyl).

In certain particular embodiments, A represents phenyl, naphthyl, tetrahydronaphthyl (e.g. 5,6,7,8-tetrahydronaphthalenyl), quinolinyl, pyridinyl or dihydrobenzodioxinyl (e.g. 2,3-dihydro-1 ,4-benzodioxinyl) optionally substituted by 1 to 3 substituents, which may be the same or different, selected from fluorine, chlorine, bromine, iodine, methyl, -CF₃, cyano, methoxy, -morpholinyl, -(CH₂)-morpholinyl, -(CH₂)₂-morpholinyl, and -O-(CH₂)₂-morpholinyl.

In certain particular embodiments, A represents phenyl optionally substituted by 1 to 3 substituents, which may be the same or different, selected from fluorine, chlorine, bromine, iodine, methyl, -CF₃, cyano, methoxy, -morpholinyl, -(CH₂)-morpholinyl, - (CH₂)₂-morpholinyl, and -O-(CH₂)₂-morpholinyl.

In one particular embodiment, R¹ represents hydrogen, fluorine, chlorine, cyano, -CF₃ or C_1-3 alkyl (e.g. methyl). In certain particular embodiments, R¹ represents hydrogen, fluorine, chlorine, -CF₃ or methyl.

Preferably, R¹ represents chlorine. In one particular embodiment, R² represents hydrogen, fluorine, chlorine, -CF₃ or C_1-3 alkyl (e.g. methyl). In certain particular embodiments, R² represents hydrogen, fluorine, chlorine, or -CF₃.

In certain particular embodiments, R² represents hydrogen or chlorine.

Preferably, R³ represents hydrogen, fluorine or chlorine.

In certain particular embodiments, R⁴ represents hydrogen, fluorine, chlorine,, -CF₃, or methyl.

Preferably, R⁴ represents hydrogen.

In one particular embodiment, R⁵ represents hydrogen, fluorine, chlorine, -CF₃, cyano or C_1-3 alkyl (e.g. methyl). In certain particular embodiments, R⁵ represents hydrogen, fluorine, chlorine, -CF₃ or C_1-3 alkyl (e.g. methyl).

In certain particular embodiments, R⁵ represents hydrogen.

In one particular embodiment, R², R⁴ and R⁵ represent hydrogen, R¹ represents chlorine, and R³ represents fluorine or chlorine.

In certain particular embodiments, one of R⁶ and R⁷ represents hydrogen and the other represents C_1-6 alkyl (e.g. methyl). In one embodiment, R⁶ and R⁷ both represent hydrogen.

In certain particular embodiments, one of R⁸ and R⁹ represents hydrogen and the ootthheerr rreepprreesseennttss CC_11--66 alkyl (e.g. methyl). In one embodiment, R⁸ and R⁹ both represent hydrogen.

In one particular embodiment of the first and second aspects of the invention, there is provided a compound of formula (I), or a pharmaceutically acceptable salt thereof, which is:

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(1-naphthalenyl)-2,3-piperazinedione (E1 ); 1-[(2-Chloro-6-fluorophenyl)methyl]-4-(1-naphthalenyl)-2,3-piperazinedione (E2);

1-[1-(2,4-Dichlorophenyl)ethyl]-4-(2-methylphenyl)-2,3-piperazinedione (E3); 1-[(2,4-Dichlorophenyl)methyl]-4-(2-methylphenyl)-2,3-piperazinedione (E4);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(2-methylphenyl)-2,3-piperazinedione (E5);

1-[(2-Chlorophenyl)methyl]-4-(2-methylphenyl)-2,3-piperazinedione (E6);

1-(2-Methylphenyl)-4-[(2,3,4-trifluorophenyl)methyl]-2,3-piperazinedione (E7); 1-[(2,3-Dichlorophenyl)methyl]-4-(2-methylphenyl)-2,3-piperazinedione (E8);

1-{[4-Fluoro-2-(trifluoromethyl)phenyl]methyl}-4-(2-methylphenyl)-2,3-piperazinedione

(E9);

2-{[4-(2-Methylphenyl)-2,3-dioxo-1 -piperazinyl]methyl}benzonitrile (E10);

1-{[2-Chloro-3-(trifluoromethyl)phenyl]methyl}-4-(2-methylphenyl)-2,3- piperazinedione (E1 1 );

1-[(2-Fluorophenyl)methyl]-4-(2-methylphenyl)-2,3-piperazinedione (E12);

1-[(2-Chloro-6-fluorophenyl)methyl]-4-(2-methylphenyl)-2,3-piperazinedione (E 13);

1-[(2,4-Dichloro-6-methylphenyl)methyl]-4-(2-methylphenyl)-2,3-piperazinedione

(E14); 1-(2-Methylphenyl)-4-[(2-methylphenyl)methyl]-2,3-piperazinedione (E15);

1-[(4-Chlorophenyl)methyl]-4-(2-methylphenyl)-2,3-piperazinedione (E 16);

1-[(2,5-Dichlorophenyl)methyl]-4-(2-methylphenyl)-2,3-piperazinedione (E17);

1-[(4-Fluoro-2-methylphenyl)methyl]-4-(2-methylphenyl)-2,3-piperazinedione (E18);

1-[(3-Chlorophenyl)methyl]-4-(2-methylphenyl)-2,3-piperazinedione (E19); 1-(2-Methylphenyl)-4-{[3-(trifluoromethyl)phenyl]methyl}-2,3-piperazinedione (E20);

1-[(2,5-Dimethylphenyl)methyl]-4-(2-methylphenyl)-2,3-piperazinedione (E21 );

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(2,6-dimethylphenyl)-2,3-piperazinedione

(E22);

1-{[2-Chloro-3-(trifluoromethyl)phenyl]methyl}-4-(2_!6-dimethylphenyl)-2,3- piperazinedione (E23);

1-[(2,3-Dichlorophenyl)methyl]-4-(2,6-dimethylphenyl)-2,3-piperazinedione (E24);

1-(2,6-Dimethylphenyl)-4-[(2,3,4-trifluorophenyl)methyl]-2,3-piperazinedione (E25);

1-(2,6-Dimethylphenyl)-4-{[4-fluoro-2-(trifluoromethyl)phenyl]methyl}-2,3- piperazinedione (E26); 1-[(2,4-Dichlorophenyl)methyl]-4-(2_!6-dimethylphenyl)-2,3-piperazinedione (E27);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(4-fluoro-2,6-dimethylphenyl)-2,3- piperazinedione (E28);

1-[(2,4-Dichlorophenyl)methyl]-4-(4-fluoro-2,6-dimethylphenyl)-2,3-piperazinedione

(E29); 1-{[2-Chloro-3-(trifluoromethyl)phenyl]methyl}-4-(4-fluoro-2_!6-dimethylphenyl)-2,3- piperazinedione (E30); 1-[(2,3-Dichlorophenyl)methyl]-4-(4-fluoro-2,6-dimethylphenyl)-2,3-piperazinedione

(E31 );

1-(4-Fluoro-2,6-dimethylphenyl)-4-[(2,3,4-trifluorophenyl)methyl]-2,3-piperazinedione

(E32); 1-(4-Fluoro-2,6-dimethylphenyl)-4-{[4-fluoro-2-(trifluoromethyl)phenyl]methyl}-2,3- piperazinedione (E33);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-phenyl-2,3-piperazinedione (E34);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(2-fluorophenyl)-2,3-piperazinedione (E35);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(2,3-dimethylphenyl)-2,3-piperazinedione (E36);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-(methyloxy)phenyl]-2,3-piperazinedione

(E37);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(3-methylphenyl)-2,3-piperazinedione (E38);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[3-(methyloxy)phenyl]-2,3-piperazinedione (E39);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(2,4-dimethylphenyl)-2,3-piperazinedione

(E40);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(2,5-dimethylphenyl)-2,3-piperazinedione

(E41 ); 1-[(2-Chloro-4-fluorophenyl)methyl]-4-(3,5-dimethylphenyl)-2,3-piperazinedione

(E42);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(3-fluoro-2-methylphenyl)-2,3-piperazinedione

(E43);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(3-iodophenyl)-2,3-piperazinedione (E44); 1-[(2-Chloro-4-fluorophenyl)methyl]-4-(3,4-dimethylphenyl)-2,3-piperazinedione

(E45);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[3-fluoro-2-(methyloxy)phenyl]-2,3- piperazinedione (E46);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(5-fluoro-2-methylphenyl)-2,3-piperazinedione (E47);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-methyl-5-(trifluoromethyl)phenyl]-2,3- piperazinedione (E48);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-methyl-5-(methyloxy)phenyl]-2,3- piperazinedione (E49); 1-[(2-Chloro-4-fluorophenyl)methyl]-4-[5-fluoro-2-(methyloxy)phenyl]-2,3- piperazinedione (E50); 1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-(4-morpholinylmethyl)phenyl]-2,3- piperazinedione hydrochloride (E51 );

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-(1-piperidinyl)phenyl]-2,3-piperazinedione hydrochloride (E52); 1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-(1-pyrrolidinyl)phenyl]-2,3-piperazinedione hydrochloride (E53);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[4-(4-morpholinyl)phenyl]-2,3-piperazinedione

(E54);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-(1 /-/-pyrrol-1-yl)phenyl]-2,3-piperazinedione (E55);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(4-fluoro-2_!3-dimethylphenyl)-2,3- piperazinedione (E56);

1-(4-Bromo-2-methylphenyl)-4-[(2-chloro-4-fluorophenyl)methyl]-2,3-piperazinedione

(E57); 1-[(2-Chloro-4-fluorophenyl)methyl]-4-(5_!6_!7_!8-tetrahydro-1-naphthalenyl)-2,3- piperazinedione (E58);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[3-(4-morpholinyl)phenyl]-2,3-piperazinedione

(E59);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-(4-morpholinyl)phenyl]-2,3-piperazinedione (E60);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(2_!3-dihydro-1 _!4-benzodioxin-6-yl)-2,3- piperazinedione (E61 );

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[3-(4-morpholinylmethyl)phenyl]-2,3- piperazinedione (E62); 1-[(2-Chloro-4-fluorophenyl)methyl]-4-{2-[2-(4-morpholinyl)ethyl]phenyl}-2,3- piperazinedione hydrochloride (E63);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-methyl-3-(4-morpholinylmethyl)phenyl]-2,3- piperazinedione hydrochloride (E64);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(2-methyl-3-{[2-(4- morpholinyl)ethyl]oxy}phenyl)-2,3-piperazinedione hydrochloride (E65);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-methyl-3-(methyloxy)phenyl]-2,3- piperazinedione (E66);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-methyl-3-(4-morpholinyl)phenyl]-2,3- piperazinedione (E67); 1-[(2-Chloro-4-fluorophenyl)methyl]-4-(1 /-/-indol-4-yl)-2,3-piperazinedione (E68);

1-[5-Bromo-2-(methyloxy)phenyl]-4-[(2-chloro-4-fluorophenyl)methyl]-2,3- piperazinedione (E69); 1-[(2-Chloro-4-fluorophenyl)methyl]-4-(1 /-/-indol-7-yl)-2,3-piperazinedione (E70);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[3-chloro-2-(methyloxy)phenyl]-2,3- piperazinedione (E71 );

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[3-chloro-2-(4-morpholinyl)phenyl]-2,3- piperazinedione (E72);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[5-chloro-2-(4-morpholinyl)phenyl]-2,3- piperazinedione (E73);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-(4-morpholinyl)-5-(trifluoromethyl)phenyl]-

2,3-piperazinedione (E74); 1 -[3-Bromo-2-(methyloxy)phenyl]-4-[(2-chloro-4-fluorophenyl)methyl]-2,3- piperazinedione (E75);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[3-methyl-2-(4-morpholinyl)phenyl]-2,3- piperazinedione (E76);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-methyl-5-(4-morpholinyl)phenyl]-2,3- piperazinedione (E77);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[5-methyl-2-(4-morpholinyl)phenyl]-2,3- piperazinedione (E78);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[5-fluoro-2-(4-morpholinyl)phenyl]-2,3- piperazinedione (E79); 1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-(methyloxy)-5-(4-morpholinyl)phenyl]-2,3- piperazinedione (E80);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(8-quinolinyl)-2,3-piperazinedione (E81 );

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(3-isoquinolinyl)-2,3-piperazinedione (E82);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(2-pyrimidinyl)-2,3-piperazinedione (E83); 1-[(2-Chloro-4-fluorophenyl)methyl]-4-(2-pyridinyl)-2,3-piperazinedione (E84);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(6-methyl-2-pyridinyl)-2,3-piperazinedione

(E85);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(2-ethylphenyl)-2,3-piperazinedione (E86);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-(1-methylethyl)phenyl]-2,3-piperazinedione (E87);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(2,3-dihydro-1 _!4-benzodioxin-5-yl)-2,3- piperazinedione (E88);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(2,5-difluorophenyl)-2,3-piperazinedione (E89);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(2-fluoro-3-methylphenyl)-2,3-piperazinedione (E90);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(4-fluoro-2-methylphenyl)-2,3-piperazinedione

(E91 ); 1-(2-Biphenylyl)-4-[(2-chloro-4-fluorophenyl)methyl]-2,3-piperazinedione (E92);

1-[2,6-Bis(methyloxy)phenyl]-4-[(2-chloro-4-fluorophenyl)methyl]-2,3-piperazinedione

(E93);

1-(3-Bromo-2-methylphenyl)-4-[(2-chloro-4-fluorophenyl)methyl]-2,3-piperazinedione (E94);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-chloro-5-(4-morpholinyl)phenyl]-2,3- piperazinedione (E95);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(3-chloro-2-methylphenyl)-2,3-piperazinedione

(E96); 1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-(dimethylamino)phenyl]-2,3-piperazinedione

(E97);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-(methyloxy)-3-(4-morpholinyl)phenyl]-2,3- piperazinedione (E98);

1-(2-Chloro-4-fluorophenyl)-4-[(2-chloro-4-fluorophenyl)methyl]-2,3-piperazinedione (E99);

1 -[(2-Chloro-4-fluorophenyl)methyl]-4-(2-iodophenyl)-2,3-piperazinedione (E100);

2-{4-[(2-Chloro-4-fluorophenyl)methyl]-2,3-dioxo-1-piperazinyl}-6-fluorobenzonitrile

(E101 );

2-{4-[(2-Chloro-6-fluorophenyl)methyl]-2,3-dioxo-1-piperazinyl}benzonitrile (E102); 1-[(2-Chloro-4-fluorophenyl)methyl]-4-(2-chlorophenyl)-2,3-piperazinedione (E103);

1-(5-Bromo-2-chlorophenyl)-4-[(2-chloro-4-fluorophenyl)methyl]-2,3-piperazinedione

(E104);

1-(2-Bromophenyl)-4-[(2-chloro-4-fluorophenyl)methyl]-2,3-piperazinedione (E105);

1 -[(2-Chloro-4-fluorophenyl)methyl]-4-(5-quinolinyl)-2,3-piperazinedione (E106); 1 -[(2-Chloro-4-fluorophenyl)methyl]-4-(4-isoquinolinyl)-2,3-piperazinedione (E107);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-(1 _!1-dimethylethyl)phenyl]-2,3- piperazinedione (E108);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-{2-[(trifluoromethyl)oxy]phenyl}-2,3- piperazinedione (E109); 1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-chloro-3-(methyloxy)phenyl]-2,3- piperazinedione (E1 10);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(4-methyl-3,4-dihydro-2/-/-1 ,4-benzoxazin-5-yl)-

2,3-piperazinedione (E111 );

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-(trifluoromethyl)phenyl]-2,3-piperazinedione (E1 12);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[4-(diethylamino)-2-methylphenyl]-2,3- piperazinedione (E1 13); 1-(2,3-Dichlorophenyl)-4-[(2-methylphenyl)methyl]-2,3-piperazinedione (E1 14);

1-[(2-Chlorophenyl)methyl]-4-(2,3-dichlorophenyl)-2,3-piperazinedione (E115);

1-[(3-Chlorophenyl)methyl]-4-(2,3-dichlorophenyl)-2,3-piperazinedione (E116);

1-[(4-Chlorophenyl)methyl]-4-(2,3-dichlorophenyl)-2,3-piperazinedione (E117); 1-[(2-Chloro-4-fluorophenyl)methyl]-4-(2,3-dichlorophenyl)-2,3-piperazinedione

(E1 18);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(2-methyl-3-pyridinyl)-2,3-piperazinedione

(E1 19);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(3-methyl-4-pyridinyl)-2,3-piperazinedione (E120);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(3,6-dimethyl-2-pyridinyl)-2,3-piperazinedione

(E121 );

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(3-methyl-2-pyridinyl)-2,3-piperazinedione

(E122); 1-[(2-Chloro-4-fluorophenyl)methyl]-4-[3-(1 /-/-imidazol-1-yl)-2-methylphenyl]-2,3- piperazinedione (E123);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-methyl-3-(1-pyrrolidinyl)phenyl]-2,3- piperazinedione (E124);

(5S)-1-[(2,4-Dichlorophenyl)methyl]-5-methyl-4-phenyl-2,3-piperazinedione (E125); or

(5R)-1-[(2,4-dichlorophenyl)methyl]-5-methyl-4-phenyl-2,3-piperazinedione (E126);

or a pharmaceutically acceptable salt thereof.

Antagonists of P2X7 may be useful in the treatment (e.g. amelioration) or prophylaxis (in particular treatment) of a variety of pain states (e.g. neuropathic pain, chronic inflammatory pain, or visceral pain), inflammation (e.g. rheumatoid arthritis or osteoarthritis), or neurodegenerative diseases such as Alzheimer's disease. P2X7 antagonists may constitute useful therapeutic agents in the management of rheumatoid arthritis or inflammatory bowel disease.

Compounds or salts of the present invention which modulate P2X7 receptor function and are capable of antagonizing the effects of ATP at the P2X7 receptor ("P2X7 receptor antagonists") 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. Sci., 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, p-aminobenzoic, ascorbic, aspartic, benzenesulfonic, benzoic, bismethylenesalicylic, camphorsulfonic, citric, cyclohexylsulfamic, ethanedisulfonic, ethanesulfonic, fumaric, gluconic, glutamic, glycolic, hydrobromic, hydrochloric, isethionic, itaconic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, palmitic, pamoic, pantothenic, phosphoric, propionic, salicylic, stearic, succinic, sulfuric, tartaric, p-toluenesulfonic acid, and the like.

In one particular embodiment, the pharmaceutically acceptable salt is is formed from a pharmaceutically acceptable strong acid. For example, the pharmaceutically acceptable salt can be a benzenesulfonate, camphorsulfonate, ethanesulfonate, hydrobromide, hydrochloride, methanesulfonate, nitrate, phosphate, sulfate, or p- toluenesulfonate.

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 thereof containing variable amounts of solvent (e.g. water).

Compounds of formula (I) are 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.

Preparation of compounds

(I)

Compounds of formula (I), wherein the variables are as defined above, or salts thereof may be prepared by the methodology described hereinafter, constituting a further aspect of this invention.

The invention therefore also provides a process for preparing a compound of formula (I) or a pharmaceutically acceptable salt thereof, which comprises step (a), (b), (c), (d), (e), (f), (g), or (h) as follows, and optionally converting the compound into a pharmaceutically acceptable salt thereof: (a) Preparation of a compound of formula (I) wherein R⁶, R⁷, R⁸ and R⁹ each represent hydrogen by condensation of a compound of general formula (6) with an aldehyde of general formula (7) (see Scheme 1 ) wherein A, R¹, R², R³, R⁴, and R⁵ are as defined above. Compounds (6) and (7) are optionally protected.

(b) Preparation of a compound of formula (I) wherein R⁶, R⁷, R⁸ and R⁹ each represent hydrogen by coupling of a compound of general formula (20) with a compound of general formula (21 ), (see Scheme 5) wherein A, R¹, R², R³, R⁴, and R⁵ are as defined above. Compounds (20) and (21 ) are optionally protected.

(c) Preparation of a compound of formula (I) wherein R⁶, R⁷, R⁸ and R⁹ each both represent hydrogen by oxidation of a compound of general formula (23), (see Scheme 6) wherein A, R¹, R², R³, R⁴, and R⁵ are as defined above. Compound (23) may be optionally protected.

(d) Preparation of a compound of formula (I) wherein R⁶ and R⁷ do not both represent hydrogen and R⁸ and R⁹ represent hydrogen by condensation of a compound of general formula (11 ) with an amine of general formula (12) (see Scheme 2) wherein A, R¹, R², R³, R⁴, R⁵, R⁶ and R⁷ are as defined above. Compounds (11 ) and (12) are optionally protected.

(e) Preparation of a compound of formula (I) wherein R⁶ and R⁷ do not both represent hydrogen and R⁸ and R⁹ represent hydrogen by condensation of a compound of general formula (15) with an amine of general formula (8) (see Scheme 3) wherein A, R¹, R², R³, R⁴, R⁵, R⁶ and R⁷ are as defined above. Compounds (8) and (15) are optionally protected.

(f) Preparation of a compound of formula (I) by treating a compound of general formula (27) with a compound of general formula (28) (see Scheme 7) wherein A, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸ and R⁹ are as defined above.

(g) 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). (h) 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, metal- catalysed Buchwald coupling, amide coupling and ester hydrolysis.

Scheme 1

Step (i) typically comprises treatment of compound (2) with a suitable reagent such as di-tertbutyldicarbonate, with a base such as triethylamine, in a suitable solvent such as dichloromethane and at a suitable temperature such as between O⁰C and room temperature.

Step (ii) typically comprises treatment of compound (3) with compound (4) with a suitable base such as triethylamine, in a suitable solvent such as dichloromethane and at a suitable temperature such as between O⁰C and room temperature.

Step (iii) typically comprises treatment of compound (5) with a suitable acid such as 4 Molar hydrochloric acid in a suitable solvent such as 1 ,4-dioxane and at a suitable temperature such as room temperature.

Step (iv) typically comprises treatment of compound (6) with compound (7) with a suitable reducing agent such as sodium triacetoxyborohydride and with a suitable dehydrating agent such as 4A molecular sieves, in a suitable solvent such as dichloromethane and at a suitable temperature such as room temperature. Scheme 2

Step (i) typically comprises treatment of compound (8) with compound (4), in a suitable solvent such as dichloromethane and at a suitable temperature such as O⁰C or room temperature.

Step (ii) typically comprises treatment of compound (9) with a suitable alkylating agent such as allyl bromide, with a suitable base such as sodium hydride, in a suitable solvent such as dimethylformamide and at a suitable temperature such as room temperature.

Step (iii) typically comprises treatment of compound (10) with a suitable oxidising agent such as ozone, in a suitable solvent such as dichloromethane, at a suitable temperature such as -78⁰C and worked up with a suitable reagent such as dimethylsulfide.

Step (iv) typically comprises treatment of compound (11 ) with compound (12) with a suitable reducing agent such as sodium triacetoxyborohydride and with a suitable dehydrating agent such as 4A molecular sieves, an acid such as acetic acid, in a suitable solvent such as dichloromethane and at a suitable temperature such as O⁰C or room temperature. Alternatively the reaction may be carried out with polymer- supported cyanoborohydride as the reducing agent, an acid such as acetic acid, in a suitable solvent such as methanol and at a suitable temperature such as room temperature.

Scheme 3

(16)

Step (i) typically comprises treatment of compound (12) with a suitable reagent such as methyl chlorooxoacetate, with a suitable base such as triethylamine, in a suitable solvent such as dichloromethane and at a suitable temperature such as O⁰C.

Step (ii) typically comprises treatment of compound (13) with a suitable alkylating agent such as allyl bromide, with a suitable base such as sodium hydride, in a suitable solvent such as dimethylformamide and at a suitable temperature such as room temperature.

Step (iii) typically comprises treatment of compound (14) with a suitable oxidising agent such as ozone, in a suitable solvent such as dichloromethane, at a suitable temperature such as -78 ⁰C and worked up with a suitable reagent such as dimethylsulfide.

Compounds of formula (I) may be prepared directly from compound (15) using the conditions mentioned in step (iv) or using a two stage process via compound (16) as outlined in steps (v) and (vi) below.

Step (iv) typically comprises treatment of compound (15) with compound (8) with a suitable reducing agent such as sodium triacetoxyborohydride or polymer-supported cyanoborohydride, optionally with a suitable dehydrating agent such as 4A molecular sieves, with the optional use of an acid such as acetic acid in a suitable solvent such as dichloromethane or 1 ,2-dichloroethane and at a suitable temperature such as between 0 ⁰C and 80 ⁰C. Alternatively the reaction may be carried out with sodium borohydride as the reducing agent and acetic acid as the solvent at a suitable temperature, such as between 80 ⁰C and 105 ⁰C. Alternatively the reaction may be carried out with polymer-supported cyanoborohydride as the reducing agent, using an acid such as acetic acid, in a suitable solvent such as methanol and at a suitable temperature such as room temperature. Alternatively the reaction may be carried out with polymer-supported cyanoborohydride as the reducing agent, using an acid such as acetic acid, in a suitable solvent such as 1 ,2-dichloroethane and at a suitable temperature such as between room temperature and 80 ⁰C. The mixture would then be filtered, concentrated and taken up in a suitable solvent such as 1-butanol and heated at a suitable temperature such as 160 ⁰C.

Step (v) comprises treatment of compound (15) with compound (8) with a suitable reducing agent such as polymer-supported cyanoborohydride, with the use of an acid such as acetic acid in a suitable solvent such as methanol and at a suitable temperature such as room temperature.

Step (vi) comprises heating of compound (16) in a suitable solvent such as methanol or 1-butanol at a suitable temperature such as between 130 ⁰C and 200 ⁰C.

Scheme 4

(16) (17)

(I)

Step (i) typically comprises treatment of compound (16) with a suitable base such as lithium hydroxide, in a suitable solvent such as tetrahydrofuran and water and at a suitable temperature such as room temperature.

Step (ii) typically comprises treatment of compound (17) with a suitable reagent such as thionyl chloride at a suitable temperature such as 4O⁰C.

Step (ii) typically comprises treatment of compound (18) with a suitable reagent such as imidazole in a suitable solvent such as acetonitrile at a suitable temperature such as room temperature. The mixture would then be worked up and the residue dissolved in a suitable solvent such as 1-butanol at a suitable temperature such as 200⁰C.

Scheme 5

step (IN)

Step (i) typically comprises treatment of compound (8) with compound (19) with a suitable reducing agent such as sodium triacetoxyborohydride and a suitable dehydrating agent such as 4A molecular sieves in a suitable solvent such as dichloromethane and at a suitable temperature such as O⁰C or room temperature.

Step (ii) typically comprises treatment of compound (3) with compound (4), with a suitable base such as triethylamine, in a suitable solvent such as dichloromethane and at a suitable temperature such as room temperature.

Step (iii) typically comprises treatment of compound (5) with a suitable acid such as 4 Molar hydrochloric acid in a suitable solvent such as 1 ,4-dioxane and at a suitable temperature such as room temperature, followed by neutralisation with a suitable base such as triethylamine in a suitable solvent such as dichloromethane and at a suitable temperature such as room temperature.

Step (iv) typically comprises treatment of compound (20) with compound (21 ) with a suitable base such as sodium hydride, in a suitable solvent such as dimethylformamide and at a suitable temperature such as 5O⁰C.

Scheme 6

(I)

Step (i) typically comprises treatment of compound (22) with a suitable reagent such as ethylene diamine in a suitable solvent such as 1-methyl-2-pyrrolidinone using microwave irradiation at a suitable temperature such as 22O⁰C and for a suitable time such as between 1 and 2 hours.

Step (ii) typically comprises treatment of compound (2) with compound (7) with a suitable reducing agent such as sodium triacetoxyborohydride and a suitable dehydrating agent such as 4A molecular sieves in a suitable solvent such as dichloromethane and at a suitable temperature such as O⁰C or room temperature.

Step (iii) typically comprises treatment of compound (23) with a suitable reagent such as oxalyl chloride, with a suitable base such as triethylamine, in a suitable solvent such as dichloromethane and at a suitable temperature such as O⁰C or room temperature.

Scheme 7

(27)

Step (i) typically comprises treatment of compound (12) with compound (24) with a suitable reducing agent such as sodium triacetoxyborohydride and a suitable dehydrating agent such as 4A molecular sieves, in a suitable solvent such as dichloromethane and at a suitable temperature such as room temperature.

Step (ii) typically comprises treatment of compound (25) with compound (4) with a suitable base such as triethylamine, in a suitable solvent such as dichloromethane and at a suitable temperature such as between O⁰C and room temperature.

Step (iii) typically comprises treatment of compound (26) with a suitable acid such as 4 Molar hydrochloric acid in a suitable solvent such as 1 ,4-dioxane and at a suitable temperature such as room temperature, followed by neutralisation with a suitable base such as triethylamine in a suitable solvent such as dichloromethane and at a suitable temperature such as room temperature.

Step (iv) typically comprises treatment of compound (27) with compound (28) with a suitable catalyst such as copper (II) acetate, a suitable base such as pyridine, a suitable oxidant such as pyridine N-oxide, a suitable dehydrating agent such as 4A molecular sieves in a suitable solvent such as toluene and at a suitable temperature such as 9O⁰C. Compounds of the general formulae (2), (4), (7), (8), (12), (19), (21 ), (22), (24) and (28) 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, pharmaceutical compositions, and dosages

It is believed that, as the compounds or pharmaceutically acceptable salts of the present invention modulate P2X7 receptor function and are capable of antagonizing the effects of ATP at the P2X7 receptor ("P2X7 receptor antagonists"); they may be useful in the treatment or prophylaxis (in particular treatment) of pain; such as 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 or cluster headaches, pain associated with functional bowel disorders, lower back and/or neck pain, pain associated with sprains and/or 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, or 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 may be useful in the treatment or prophylaxis (in particular treatment) 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 condition can be pain associated with normally non-painful sensations such as "pins and needles" (paraesthesias and/or dysesthesias), increased sensitivity to touch (hyperesthesia), painful sensation following innocuous stimulation (dynamic, static, thermal or cold allodynia), increased sensitivity to noxious stimuli (thermal, cold, or 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).

The compounds or pharmaceutically acceptable salts of the present invention may potentially be useful in the treatment or prophylaxis (e.g. prophylaxis, e.g. reduction, delay or prevention) of the development of tolerence to the analgesic action of an opioid analgesic (such as morphine, fentanyl, oxycodone, tramadol, hydrocodone, hydromorphone, oxymorphone, methadone or buprenorphine; in particular morphine, fentanyl, oxycodone, or tramadol).

Other conditions which could potentially be subject to treatment or prophylaxis (in particular treatment) using the compounds or salts of the present invention are: 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 (e.g. atherosclerosis); neurodegenerative diseases and/or neurodegeneration; neurodegeneration following trauma; tinnitus; dependence on (e.g. addiction to) a dependence-inducing agent such as: an opioid analgesic (e.g. morphine), a CNS (central nervous system) depressant (e.g. ethanol), a psychostimulant (e.g. cocaine) or nicotine; diabetes such as Type 1 or Type 2 diabetes, complications of diabetes such as complications of Type I or Type 2 diabetes, kidney dysfunction, liver dysfunction (e.g. hepatitis, cirrhosis), gastrointestinal dysfunction (e.g. diarrhoea), gastric cancer, colon cancer, overactive bladder, or urge incontinence. Depression and alcoholism could potentially also be subject to treatment or prophylaxis by compounds or salts of the present invention.

Inflammation and/or the inflammatory conditions associated with said inflammation can be: arthritis (in particular rheumatoid arthritis or osteoarthritis), skin conditions (e.g. sunburn, burns, eczema, dermatitis, allergic dermatitis, or psoriasis), meningitis, ophthalmic diseases such as glaucoma, retinitis, retinopathies, uveitis or of acute injury to the eye tissue (e.g. conjunctivitis), an inflammatory lung disorder (e.g. asthma, chronic obstructive pulmonary disease (COPD, which includes bronchitis and/or emphysema), allergic rhinitis, respiratory distress syndrome, pigeon fancier's disease, farmer's lung, or airways hyperresponsiveness); a gastrointestinal tract disorder (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; or 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, or Sjogren's syndrome.

The inflammation and/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 can be: 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, gouty and/or ankylosing spondylitis, tendinitis or 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 which could potentially be subject to treatment or prophylaxis (in particular treatment) using the compounds or salts of the present invention are: dementia, particularly degenerative dementia (such as Alzheimer's disease, senile dementia, dementia with Lewy bodies, temporal lobe dementia, Huntingdon's chorea, Parkinson's disease, Pick's disease, Creutzfeldt-Jakob disease, or Amyotrophic Lateral Sclerosis (ALS); in particular Alzheimer's disease); mild cognitive impairment (MCI) e.g. MCI associated with ageing, particularly age associated memory impairment; motor neuron disease; vascular dementia (including multi-infarct dementia and/or dementia associated with cerebral ischaemia); or a neurodegenerative disease (e.g. dementia) associated with: an intracranial space occupying lesion, head trauma, intracranial and/or cerebral infections or related conditions (such as HIV infection, viral or bacterial meningitis, or cerebral herpes virus infections such as shingles or herpes simplex virus), metabolism, toxins, anoxia, hypoxia or vitamin deficiency.

The neurodegenerative disease, e.g. to be subject to treatment or prophylaxis (in particular treatment) by the compound of formula (I) or salt thereof, can in particular be degenerative dementia (in particular Alzheimer's disease), Parkinson's diesase (in particular dementia in Parkinson's diesase), vascular dementia (in particular multi- infarct dementia), dementia with Lewy bodies, Huntingdon's chorea, or mild cognitive impairment (MCI) e.g. MCI associated with ageing such as age associated memory impairment. The neurodegenerative disease, e.g. to be subject to treatment or prophylaxis (in particular treatment) by the compound of formula (I) or salt thereof, can in particular 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.

In one embodiment, the compound of formula (I) or the salt thereof of the invention is used for treatment or prophylaxis (in particular treatment) of a neurodegenerative disease (such as degenerative dementia e.g. Alzheimer's disease, or vascular dementia, or mild cognitive impairment), by disease modification and/or by neuroprotection. Alternatively or additionally, in one embodiment, the compound of formula (I) or the salt thereof of the invention is used for treatment or prophylaxis (in particular treatment) of a neurodegenerative disease (such as degenerative dementia e.g. Alzheimer's disease, or vascular dementia, or mild cognitive impairment) by symptomatic treatment of cognitive impairment associated with the neurodegenerative disease.

The compounds of formula (I) or pharmaceutically acceptable salts thereof may also be useful for neuroprotection and/or in the treatment or prophylaxis (e.g. treatment) of neurodegeneration following trauma such as stroke, cardiac arrest, pulmonary bypass, traumatic brain injury, spinal cord injury or the like.

The compounds or pharmaceutically acceptable salts of the present invention may also be useful in the treatment or prophylaxis (in particular treatment) of malignant cell growth and/or metastasis, or myoblastic leukaemia.

Complications of Type 1 diabetes can be: diabetic microangiopathy, diabetic retinopathy, diabetic nephropathy, macular degeneration, glaucoma, nephrotic syndrome, aplastic anaemia, uveitis, Kawasaki disease or sarcoidosis.

Kidney dysfunction can be: nephritis, glomerulonephritis, particularly mesangial proliferative glomerulonephritis or nephritic syndrome.

The compounds or pharmaceutically acceptable salts of the present invention may potentially be useful in the treatment or prophylaxis (e.g. treatment) of epilepsy and/or seizures (i.e. as anticonvulsants), for example in a mammal such as a human.

The compounds or pharmaceutically acceptable salts of the present invention may potentially be useful in the treatment or prophylaxis (e.g. treatment) of a human epileptic syndrome, such as: partial and/or generalised seizures (e.g. tonic, tonic- clonic, or absence seizures), temporal lobe epilepsy, absence epilepsies (including childhood, juvenile, myoclonic, photo- or pattern-induced), severe epileptic encephalopathies (including hypoxia-related or Rasmussen's syndrome), febrile convulsions, epilepsy partialis continua, progressive myoclonus epilepsies (including Unverricht-Lundborg disease or Lafora's disease), post-traumatic seizures and/or epilepsy such as those related to head injury, simple reflex epilepsies (including photosensive, somatosensory, proprioceptive, audiogenic or vestibular), metabolic disorders commonly associated with epilepsy such as pyridoxine-dependent epilepsy, Menkes' kinky hair disease, Krabbe's disease, epilepsy due to alcohol and/or drug abuse (e.g. cocaine abuse), cortical malformations associated with epilepsy (e.g. double cortex syndrome or subcortical band heterotopia), or chromosomal anomolies associated with seizures or epilepsy such as Partial monosomy (15Q / Angelman syndrome); in a human.

According to a further aspect of the invention, we therefore provide a compound of formula (I) or a pharmaceutically acceptable salt thereof for use in human or veterinary medicine and/or for use in therapy.

According to another aspect of the invention, we provide a compound of formula (I) or a pharmaceutically acceptable salt thereof for use in the treatment or prophylaxis (e.g. treatment) of a condition which is mediated by P2X7 receptors, for example a condition or disease disclosed herein (in particular pain, inflammation such as rheumatoid arthritis or osteoarthritis, or a neurodegenerative disease (e.g.

Alzheimer's disease or mild cognitive impairment), or epilepsy and/or seizures (e.g. a human epileptic syndrome); more particularly pain such as inflammatory pain, neuropathic pain or visceral pain, or rheumatoid arthritis or osteoarthritis); e.g. in a mammal such as a human or rodent e.g. human or rat e.g. human.

According to a further aspect of the invention, we provide a method of treatment or prophylaxis (e.g. treatment) of 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 such as rheumatoid arthritis or osteoarthritis, or a neurodegenerative disease (e.g. Alzheimer's disease or mild cognitive impairment), or epilepsy and/or seizures (e.g. a human epileptic syndrome); more particularly pain such as inflammatory pain, neuropathic pain or visceral pain, or rheumatoid arthritis or osteoarthritis), which comprises administering to said subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

According to a further aspect of the invention we provide a method of treatment or prophylaxis (e.g. treatment) of a human or animal (e.g. rodent e.g. rat) subject, for example a human subject, suffering from or susceptible to pain, inflammation (e.g. rheumatoid arthritis or osteoarthritis), or a neurodegenerative disease (e.g.

Alzheimer's disease or mild cognitive impairment), or epilepsy and/or seizures (e.g. a human epileptic syndrome), (more particularly pain such as inflammatory pain, neuropathic pain or visceral pain, or rheumatoid arthritis or osteoarthritis), which method comprises administering to said subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

According to a yet further aspect of the invention we provide a method of treatment or prophylaxis (e.g. treatment) of a human or animal (e.g. rodent e.g. rat) subject, for example a human subject, suffering from or susceptible to 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 compound of formula (I) or a pharmaceutically acceptable salt thereof.

According to a further aspect of the invention we provide a method of treatment of a subject, for example a human subject, suffering from Alzheimer's disease or mild cognitive impairment, which method comprises administering to said subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

In one aspect of the invention we provide a method of treatment or prophylaxis (e.g. prophylaxis, e.g. reduction, delay or prevention) of the development of tolerence to the analgesic action of an opioid analgesic (such as morphine, fentanyl, oxycodone, tramadol, hydrocodone, hydromorphone, oxymorphone, methadone or buprenorphine; in particular morphine, fentanyl, oxycodone, or tramadol), in a subject suffering from or susceptible to the development of such opioid analgesic tolerance, which method comprises administering to said subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

According to another aspect of the invention, we provide the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment or prophylaxis (e.g. treatment) of a condition which is mediated by the action of P2X7 receptors, for example a condition or disease disclosed herein (in particular pain, inflammation such as rheumatoid arthritis or osteoarthritis, or a neurodegenerative disease (e.g. Alzheimer's disease or mild cognitive impairment), or epilepsy and/or seizures (e.g. a human epileptic syndrome); more particularly pain such as inflammatory pain, neuropathic pain or visceral pain); 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 compound of formula (I) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment or prophylaxis (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. Alzheimer's disease or mild cognitive impairment), or epilepsy and/or seizures (e.g. a human epileptic syndrome), (more particularly: pain such as inflammatory pain, neuropathic pain or visceral pain, or rheumatoid arthritis or osteoarthritis); 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 compound of formula (I) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment or prophylaxis (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 compound of formula (I) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment or prophylaxis (e.g. treatment) of Alzheimer's disease or mild cognitive impairment; 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 compound of formula (I) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment or prophylaxis (e.g. prophylaxis, e.g. reduction, delay or prevention) of the development of tolerence to the analgesic action of an opioid analgesic (such as morphine, fentanyl, oxycodone, tramadol, hydrocodone, hydromorphone, oxymorphone, methadone or buprenorphine; in particular morphine, fentanyl, oxycodone, or tramadol).

In order to use a compound of formula (I) or a pharmaceutically acceptable salt thereof 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 compound of formula (I), or a pharmaceutically acceptable salt thereof, adapted for use in human or veterinary medicine.

In order to use a compounds of formula (I) or a pharmaceutically acceptable salt thereof in therapy, it will normally be formulated into a pharmaceutical composition in accordance with pharmaceutical practice. The present invention also provides a pharmaceutical composition, which comprises a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

The pharmaceutical composition may be for use in a method of treatment or prophylaxis or in a use or in a treatment or prophylaxis, as described herein.

A pharmaceutical composition of the invention, which may be prepared by admixture, for example at ambient temperature and/or atmospheric pressure, is usually adapted for oral, parenteral or rectal administration. As such, the pharmaceutical composition may be in the form of a tablet, a capsule, a 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 is 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 (e.g. lactose and/or microcrystalline cellulose), a lubricant e.g. a tabletting lubricant (e.g. magnesium stearate or calcium stearate), a disintegrant (e.g. a tablet disintegrant such as sodium starch glycolate or croscarmellose sodium), and/or an acceptable wetting agent. The tablets may be coated e.g. according to methods known in 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 a suspending agent(s), an emulsifying agent(s), a non-aqueous vehicle(s) (such as an edible oil), and/or a preservative(s), and/or, if desired, a flavouring(s) or colourant(s). For parenteral administration, fluid unit dosage forms are typically prepared utilising a compound of the invention or pharmaceutically acceptable salt thereof and a sterile vehicle. In one embodiment, the compound or salt, depending on the vehicle and concentration used, is either suspended or dissolved in the vehicle. In preparing solutions, the compound or salt 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) is or are dissolved in the vehicle. To enhance the stability, the composition can for example 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 is typically suspended in the vehicle instead of being dissolved, and sterilization is not usually accomplished by filtration. The compound or salt 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 of the invention.

In one embodiment, the composition contains from 0.1% to 99% (by weight of the composition), in particular from 0.1 to 60% or 1 to 60% or 10 to 60% by weight, of the active material (the compound 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; and/or in an amount of from 20 mg to 2000 mg such as 50 mg to 1000 mg per unit dose of the composition.

The dose of the compound or pharmaceutically acceptable salt thereof, e.g. for use in the treatment or prophylaxis (e.g. treatment) of the hereinmentioned disorders / diseases / conditions, may vary in the usual way with the seriousness of the disorders, the weight of the sufferer, and/or other similar factors. However, as a general guide, in one embodiment a unit dose of 0.05 to 2000 mg or 0.05 to 1000 mg, for example 0.05 to 200 mg, such as 20 to 40 mg, of the compound or pharmaceutically acceptable salt of the invention (measured as the compound), may be used, e.g. in a pharmaceutical composition. In one embodiment, such a unit dose is for administration once a day e.g. to a mammal such as a human; alternatively such a unit dose may be for administration more than once (e.g. twice or three times) a day e.g. to a mammal such as a human. Such therapy may extend for a number of days, weeks, months or years.

Combinations

Compounds of formula (I) or pharmaceutically acceptable salts thereof may be used in combination with other (further) therapeutic agent(s), for example medicaments claimed to be useful in the treatment or prophylaxis (e.g. treatment) of the above mentioned disorders.

Examples of such further therapeutic agent(s) 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)), e.g. 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) (e.g. for oral administration), e.g. for the treatment of a cardiovascular disorder (such as atherosclerosis), e.g. as described in WO 2006/083214.

A further therapeutic agent may in particular 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) (e.g. for oral administration), e.g. 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 or 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), e.g. as described in WO 2004/105798.

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), e.g. 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), e.g. for oral administration and/or e.g. 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. For the treatment of rheumatoid arthritis, methotrexate can be administered 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 total) as a course once weekly; the schedule can optionally be adjusted gradually to achieve an optimal response, but typically does 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.

A further therapeutic agent may include an inhibitor of 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), e.g. as described in WO 2004/073704. A further therapeutic agent may 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. IL-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;

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), e.g. as described in WO 2006/003517.

In particular, 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 analgesic (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, 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 analgesic (such as morphine, fentanyl, oxycodone, tramadol, hydrocodone, hydromorphone, oxymorphone, methadone or buprenorphine; in particular morphine, fentanyl, oxycodone, or tramadol, most particularly morphine),

- 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, 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, 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, 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 analgesic (such as morphine, fentanyl, oxycodone, tramadol, hydrocodone, hydromorphone, oxymorphone, methadone or buprenorphine; in particular morphine, fentanyl, oxycodone, or tramadol), then the opioid analgesic and/or the combination comprising the opioid analgesic is for the treatment of pain, in particular inflammatory or neuropathic pain, e.g. in a mammal such as a human. In a more particular embodiment of this embodiment, the compound or salt of the present invention is administered (e.g. to a human), e.g. either sequentially or simultaneously, in combination with the opioid analgesic, wherein the opioid analgesic is administered at a reduced dosage compared to the dosage (e.g. human dosage) typically used for said opioid analgesic (i.e. the compound or salt of the invention might give an opioid-sparing effect); this might give adequate pain control and/or might result in a reduction of opioid-analgesic-induced adverse events. In a particular embodiment, the further therapeutic agent may be useful in the treatment or prophylaxis (in particular treatment) of a Neurodegenerative disease. For example the further therapeutic agent may be useful in alleviating the sympthoms of a Neurodegenerative disase.

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 invention thus provides, in a further aspect, a combination comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof together with a further therapeutic agent or agents (e.g. as defined herein).

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 therapeutic agent active against the same disease state the dose of each compound may differ from that when the compound is used alone. The following Examples illustrate the compounds of the invention and methods for their preparation, but are not intended to be limiting.

EXAMPLES

The general methods (a)-(g), along with the synthetic methods outlined in Schemes 1-5 above, for the preparation of compounds of the present invention are further illustrated by the following examples.

Example 1 1-[(2-Chloro-4-fluorophenyl)methyl]-4-(1-naphthalenyl)-2,3-piperazinedione (E1)

To a solution of methyl [(2-aminoethyl)(1-naphthalenyl)amino](oxo)acetate hydrochloride (0.555 mmol, prepared as described below) in dichloromethane (3 ml), cooled to O⁰C under argon, was added 4A molecular sieves (0.2g), 2-chloro-4- fluorobenzaldehyde (0.62 mmol) and sodium triacetoxyborohydride (0.18 g, 0.85 mmol). The reaction was stirred at O⁰C for 15 minutes then the cooling bath removed and the reaction stirred for a further 5 hours. The reaction was reduced in vacuo and the residue purified by mass-directed automated HPLC to yield 1-[(2-chloro-4- fluorophenyl)methyl]-4-(1-naphthalenyl)-2,3-piperazinedione (0.081 g) as a white solid. LC/MS [M+H]⁺ = 383, retention time = 2.88 minutes.

The methyl [(2-aminoethyl)(1-naphthalenyl)amino](oxo)acetate hydrochloride used in the method described above was prepared as follows:

(i) To Λ/-(1-naphthyl)ethylenediamine dihydrochloride (5 g, 19.3 mmol) in anhydrous dichloromethane (40 ml), cooled to O⁰C under argon, was added triethylamine (5.38 ml, 38.6 mmol) and then di-tertbutyldicarbonate (4.21 g, 19.3 mmol). The reaction was allowed to warm to room temperature over a period of 3 hours. LC/MS analysis showed that starting material remained so the reaction was stirred at room temperature for a further 18 hours. The reaction was then cooled in an ice/water bath before the addition of triethylamine (4.03 ml) and methyl chlorooxoacetate (1.86 ml) and the reaction stirred for a further 18 hours. The reaction was then diluted with dichloromethane (100 ml) and washed with 1 N hydrochloric acid (100 ml), saturated aqueous sodium bicarbonate (100ml), passed through a hydrophobic frit and reduced in vacuo to yield a pale brown oil. The residue was purified by flash-silica gel column chromatography, eluting with hexane (4 column volumes) followed by a 0-20% gradient of ethyl acetate in hexane (10 column volumes) and finally 20% ethyl acetate in hexane (5 column volumes). The desired product eluted in 20% ethyl acetate and these fractions were reduced in vacuo to yield methyl [[2-({[(1 ,1- dimethylethyl)oxy]carbonyl}amino)ethyl](1-naphthalenyl)amino](oxo)acetate (1.81 g) as a colourless gum.

(ii) Methyl [[2-({[(1 ,1-dimethylethyl)oxy]carbonyl}amino)ethyl](1- naphthalenyl)amino](oxo)acetate (0.42 g, 1.13 mmol) was dissolved in 4 Molar hydrochloric acid in 1 ,4-dioxane (8 ml). The reaction was stirred at 22⁰C for 1 hour. The reaction was then reduced in vacuo to yield methyl [(2-aminoethyl)(1- naphthalenyl)amino](oxo)acetate hydrochloride (0.36 g) as a cream-coloured solid.

Example 2

1 -[(2-Chloro-6-fluorophenyl)methyl]-4-(1 -naphthalenyl)-2,3-piperazinedione (E2)

In a manner analogous to that described for Example 1 above the compound tabulated below (Table 1 ) was prepared by substituting the appropriate aldehydes (or protected aldehyde) for the 2-chloro-4-fluorbenzaldehyde used in the above procedure. The aldehyde or protected aldehydes that may be used to make the compound shown in Table 1 are available from commercial sources or can be prepared using routes described previously in the chemical literature or analogous methods.

Table 1

Example 3

1 -[1 -(2,4-Dichlorophenyl)ethyl]-4-(2-methylphenyl)-2,3-piperazinedione (E3)

To 1-(2,4-dichlorophenyl)ethanamine (0.122 g, 0.64 mmol) in dichloromethane (5 ml) at O⁰C was added acetic acid (0.109 ml, 1.91 mmol), 4A molecular sieves (-0.2 g) and sodium triacetoxyborohydride (0.203 g, 0.96 mmol). To this mixture was added methyl [(2-methylphenyl)(2-oxoethyl)amino](oxo)acetate (0.150 g, 0.64 mmol, prepared as described below) in dichloromethane (5 ml). The reaction was stirred at O⁰C under argon for 20 minutes before being warmed to room temperature and stirred for 40 hours. The molecular sieves were filtered off and to the filtrate was added further dichloromethane and saturated aqueous sodium bicarbonate. The product was extracted with dichloromethane (three washes) and the combined organic layers washed with water. The organic layer was dried over sodium sulfate and evaporated in vacuo to give 0.185 g of a yellow oil. The residue was purified by mass-directed automated HPLC and triturated with diethyl ether to yield 1-[1-(2,4- dichlorophenyl)ethyl]-4-(2-methylphenyl)-2,3-piperazinedione (0.064 g) as a white solid. LC/MS [M+H]⁺ = 377, retention time = 2.84 minutes.

The methyl [(2-methylphenyl)(2-oxoethyl)amino](oxo)acetate used in the method described above was prepared as follows:

(i) o-Toluidine (2 ml, 18.66 mmol) and triethylamine (2.85 ml, 20.53 mmol) were dissolved in dichloromethane (50 ml) with stirring at room temperature. The reaction vessel was then cooled to O⁰C and methyl chlorooxoacetate (1.89 ml, 20.53 mmol) was added dropwise. The reaction was then stirred at room temperature for 2 hours. The mixture was washed with saturated aqueous sodium bicarbonate (30 ml). The organic layer was separated and the aqueous layer extracted with further dichloromethane (2 x 25 ml). The combined organic layers were washed with 2N hydrochloric acid, separated and the aqueous again extracted with dichloromethane (2 x 25ml). Combined organic extracts were dried over sodium sulfate and concentrated in vacuo to yield methyl [(2-methylphenyl)amino](oxo)acetate (3.76 g), which was used without further purification.

(ii) Methyl [(2-methylphenyl)amino](oxo)acetate (3.76 g, 19.45 mmol) was dissolved in dimethylformamide (75 ml) and cooled to O⁰C. AIIyI bromide (1.85 ml, 21.40 mmol) and sodium hydride (60% dispersion in mineral oil, 0.82 g, 20.42 mmol) were added and the mixture stirred at O⁰C for a further 10 minutes. The reaction was then warmed to room temperature and stirred for 3 hours. The reaction mixture was then diluted with dichloromethane (50 ml) and saturated aqueous ammonium chloride (30 ml) was added. The layers were vigorously mixed, the organic layer separated and the aqueous layer extracted with further dichloromethane (2 x 20 ml). The combined organic layers were dried over sodium sulfate and concentrated in vacuo (azeotroping with toluene to remove dimethylformamide). The residue was purified by flash-silica gel chromatography, eluting with a gradient of 0-100% ethyl acetate in hexane (-1.8 L). Product fractions were concentrated in vacuo to yield methyl [(2-methylphenyl)(2-propen-1-yl)amino](oxo)acetate (3.87 g, combined mass of two collected batches). The product had not eluted cleanly but was used without further purification.

(iii) Methyl [(2-methylphenyl)(2-propen-1-yl)amino](oxo)acetate (1.13 g, 4.86 mmol) was dissolved in dichloromethane (50 ml) and cooled to -78⁰C. The vessel was purged with argon for 5 minutes followed by oxygen for 5 minutes. Ozone was then bubbled through the reaction mixture until a blue colour persisted (approx. 30 minutes). The reaction was then purged with oxygen for 5 minutes followed by argon for 40 minutes. The solution was then warmed to room temperature and dimethylsulfide (1.1 ml, 14.58 mmol) was added. The reaction was stirred for 12 hours. The reaction mixture was concentrated in vacuo to yield crude methyl [(2- methylphenyl)(2-oxoethyl)amino](oxo)acetate (1.499 g), which was used without purification. Examples 4-21 (E4-E21)

In a manner analogous to that described for Example 3 above the compounds tabulated below (Table 2) were prepared by substituting the appropriate amines (or amine salts) for the 1-(2,4-dichlorophenyl)ethanamine used in the above procedure. The amines or amine salts that may be 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 analogous methods. (In the case of examples 9-1 1 , 14-18 and 20-21 , flash-silica gel chromatography was used in addition to mass- directed automated HPLC).

Table 2

Example 22

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(2,6-dimethylphenyl)-2,3-piperazinedione

(E22)

Methyl [(2,6-dimethylphenyl)(2-oxoethyl)amino](oxo)acetate (0.27 g, 1.0 mmol) and 2-chloro-4-fluorobenzylamine (0.19 g, 1.2 mmol) were stirred in 2% acetic acid/methanol (10 ml) for 5 minutes, whereupon polymer-supported cyanoborohydride (0.98 g, 4.0 mmol) was added. The mixture was stirred at room temperature for 16 hours. The resin was filtered off using an SCX cartridge (Varian, 5g), washing through with methanol. The filtrate was concentrated in vacuo to afford the crude product as a gum. The crude material was purified twice by flash-silica gel chromatography (first eluting with a 0-25% gradient of methanol in dichloromethane; then re-purified using a 0-100% gradient of ethyl acetate in iso-hexane) to afford 1- [(2-chloro-4-fluorophenyl)methyl]-4-(2,6-dimethylphenyl)-2,3-piperazinedione (0.11 g)-

LC/MS [M+H]⁺ = 361 , retention time = 2.78 minutes.

The methyl [(2,6-dimethylphenyl)(2-oxoethyl)amino](oxo)acetate used in the method described above was prepared as follows:

(i) Methyl chlorooxoacetate (5.06 ml, 55.0 mmol) was dissolved in dichloromethane (50 ml) and then added dropwise to a solution of 2,6-dimethylaniline (6.19 ml, 50 mmol) and triethylamine (8.36 ml, 60.0 mmol) in dichloromethane (100 ml) at O⁰C. The reaction mixture was warmed to room temperature with stirring overnight. The mixture was concentrated in vacuo and the residue partitioned between ethyl acetate and 2 Molar hydrochloric acid. The organic layer was separated and the aqueous extracted with three portions of ethyl acetate. The combined organic extracts were washed with water, saturated aqueous sodium bicarbonate, water (x3) and brine. The solution was dried over anhydrous magnesium sulfate and concentrated in vacuo to afford 12.36g of a solid. The crude product was purified by flash-silica gel chromatography, eluting with a 0-100% gradient of ethyl acetate in hexane) to afford methyl [(2,6-dimethylphenyl)amino](oxo)acetate (7.05 g).

(ii) Sodium hydride (60% dispersion in mineral oil, 1.50 g, 37.4 mmol) was added portionwise to a solution of methyl [(2,6-dimethylphenyl)amino](oxo)acetate (7.05 g, 34.0 mmol) in N,N-dimethylformamide (90 ml) under argon at O⁰C. The reaction mixture was then allowed to stir at room temperature for one hour. The mixture was then cooled to O⁰C and allyl bromide (3.24 ml, 37.4 mmol) was added. The mixture was warmed to room temperature and stirred for 24 hours. The mixture was poured on to ice and extracted in to ethyl acetate (x3). The combined organic extracts were washed with water (x3), brine, dried over anhydrous magnesium sulfate and concentrated in vacuo to give 8.9g of an oil. The crude product was purified by flash- silica gel chromatography, eluting with a 0-100% gradient of ethyl acetate in hexane to afford methyl [(2,6-dimethylphenyl)(2-propen-1-yl)amino](oxo)acetate (7.17 g).

(iii) Methyl [(2,6-dimethylphenyl)(2-propen-1-yl)amino](oxo)acetate (7.17 g, 29.0 mmol) was dissolved in dichloromethane (150 ml) and cooled to -78⁰C. The mixture was degassed with argon then oxygen. Ozone was bubbled through the solution for

1 hour whereupon the solution had turned blue. Argon was passed trough the solution until no ozone was present, whereupon dimethyl sulfide (6.43 ml, 87 mmol) was added. The solution was stirred to room temperature over 5 hours and concentrated in vacuo. The residue was partitioned between diethyl ether and water. The organic phase was washed with water (x1 ), 50% brine (x3), brine, dried over anhydrous magnesium sulfate and concentrated to yield methyl [(2,6- dimethylphenyl)(2-oxoethyl)amino](oxo)acetate (7.70 g) as a viscous oil.

Examples 23-27 (E23-E27)

In a manner analogous to that described for Example 22 above the compounds tabulated below (Table 3) were prepared by substituting the appropriate amines (or amine salts) for the 2-chloro-4-fluorobenzylamine used in the above procedure. The amines or amine salts that may be 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 or analogous methods.

Table 3

Example 28

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(4-fluoro-2,6-dimethylphenyl)-2,3- piperazinedione (E28)

Methyl [(4-fluoro-2,6-dimethylphenyl)(2-oxoethyl)amino](oxo)acetate (0.15 g, 0.49 mmol) was dissolved in dichloromethane (5 ml) and cooled to O⁰C. 4 Angstrom molecular seives (0.3 g, 0.49 mmol), acetic acid (0.09 ml, 1.48 mmol), 2-chloro-4- fluorobenzylamine (0.07 ml, 0.54 mmol) and sodium triacetoxyborohydride (0.16 g, 0.74 mmol) were added. After 5 minutes, the reaction was warmed to room temperature and stirred for 16 hours overnight. Saturated aqueous sodium bicarbonate (5 ml) was added and the mixture stirred for 10 minutes. The organic layer was separated using a hydrophobic frit and concentrated in vacuo. The crude material was purified by mass-directed automated HPLC to yield 1-[(2-chloro-4- fluorophenyl)methyl]-4-(4-fluoro-2,6-dimethylphenyl)-2,3-piperazinedione (0.083 g) as a white solid. LC/MS [M+H]⁺ = 379, retention time = 2.84 minutes.

The methyl [(4-fluoro-2,6-dimethylphenyl)(2-oxoethyl)amino](oxo)acetate used in the method described above was prepared as follows:

(i) 4-Fluoro-2,6-dimethylaniline (1.5 g, 10.8 mmol) and triethylamine (1.58 ml, 1 1.3 mmol) were dissolved in dichloromethane (40 ml) and cooled to O⁰C. Methyl chlorooxoacetate (1.05 ml, 1 1.3 mmol) was added and the reaction stirred at O⁰C for 30 minutes. The mixture was then warmed to room temperature and stirred for 5 hours. Saturated aqueous sodium bicarbonate (15 ml) was added, the organic layer separated and the aqueous extracted with dichloromethane (2 x 10 ml). The combined organics were then washed with 2 Molar hydrochloric acid (20 ml) and the aqueous extracted with dichloromethane (2 x 15 ml). The combined organic layers were dried over sodium sulfate, filtered and concentrated in vacuo to yield methyl [(4- fluoro-2,6-dimethylphenyl)amino](oxo)acetate (2.38 g) as an off-white solid.

(ii) Methyl [(4-fluoro-2,6-dimethylphenyl)amino](oxo)acetate (2.38 g, 10.6 mmol) and allyl bromide (0.96 ml, 11.1 mmol) were dissolved in N,N-dimethylformamide (40 ml) and cooled to O⁰C. Sodium hydride (60% in mineral oil, 0.444 g, 1 1.1 mmol) was added portionwise, allowing each portion to react before the addition was continued.

Upon complete addition, the mixture was stirred at O⁰C for 20 minutes before warming to room temperature and stirring for 4 hours. The reaction was quenched by adding saturated aqueous ammonium chloride (30 ml). The mixture was then extracted with ethyl acetate (3 x 30 ml). The combined organic layers were dried over sodium sulfate and concentrated in vacuo to give ~10g of a golden brown oil.

The crude material was purified by flash-silica gel chromatography, eluting with a 10-

30% gradient of ethyl acetate in hexane. Product fractions were concentrated in vacuo to yield methyl [(4-fluoro-2,6-dimethylphenyl)(2-propen-1- yl)amino](oxo)acetate (2.45 g) as a pale yellow oil, which was used without further purification. (iii) Methyl [(4-fluoro-2,6-dimethylphenyl)(2-propen-1-yl)amino](oxo)acetate (2.45 g, 9.25 mmol) was dissolved in dichloromethane (50 ml) and cooled to -78 ⁰C. The vessel was purged with argon for 5 minutes followed by oxygen for 5 minutes. Ozone was then bubbled through the reaction mixture until a blue colour persisted (approximately 1 hour). The reaction was then purged with oxygen for 5 minutes followed by argon for 20 minutes until the exhaust gases gave a negative response to wetted starch iodide paper. The solution was then warmed to room temperature and dimethyl sulfide (2.05 ml, 27.8 mmol) added. The mixture was stirred at room temperature for 5 hours and then left to stand overnight. The reaction mixture was washed with water, the organic layer separated with a hydrophobic frit and concentrated in vacuo to yield methyl [(4-fluoro-2,6-dimethylphenyl)(2- oxoethyl)amino](oxo)acetate (2.82 g) as a yellow oil, which was used without purification.

Examples 29-33 (E29-E33)

In a manner analogous to that described for Example 28 above the compounds tabulated below (Table 4) were prepared by substituting the appropriate amines (or amine salts) for the 2-chloro-4-fluorobenzylamine used in the above procedure. The amines or amine salts that may be 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 analogous methods. (In the case of example 33, flash-silica gel chromatography was required in addition to mass-directed automated HPLC).

Table 4

Example 34 1-[(2-Chloro-4-fluorophenyl)methyl]-4-phenyl-2,3-piperazinedione (E34)

Methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (0.13 g, 0.34 mmol, prepared as described below) was dissolved in dichloromethane (8 ml) and cooled to O⁰C. 4A molecular sieves (-0.2 g), aniline (0.031 ml, 0.34 mmol), acetic acid (0.058 ml, 1.02 mmol) and sodium triacetoxyborohydride (0.108 g, 0.51 mmol) were added and the mixture stirred at O⁰C for 10 minutes. The reaction was then warmed to room temperature and stirred for 5/4 hours. The reaction was quenched by adding saturated aqueous sodium bicarbonate and stirred vigorously for 10 minutes. The organic layer was separated through a hydrophobic frit and the aqueous layer extracted with further dichloromethane (5 ml). Combined organic layers were concentrated in vacuo and purified by mass-directed automated HPLC to yield 1-[(2-chloro-4-fluorophenyl)methyl]-4-phenyl-2,3-piperazinedione (0.072 g) as a white solid.

LC/MS [M+H]⁺ = 333, retention time = 2.58 minutes.

The methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate used in the method described above was prepared as follows:

(i) 2-chloro-4-fluorobenzylamine (4.50 g, 28.18 mmol) was dissolved in dichloromethane (40 ml). Triethylamine (3.93 ml, 28.18 mmol) was added and the solution cooled to O⁰C. Methyl chlorooxoacetate (2.59 ml, 28.18 mmol) was added dropwise and the resulting suspension stirred at O⁰C for 90 minutes. The reaction was warmed to room temperature and washed with saturated aqueous sodium bicarbonate (30 ml), followed by 2N hydrochloric acid (30 ml). The organic layer was concentrated in vacuo to yield methyl {[(2-chloro-4- fluorophenyl)methyl]amino}(oxo)acetate (6.84 g), which was used without purification.

(ii) Methyl {[(2-chloro-4-fluorophenyl)methyl]amino}(oxo)acetate (5.56 g, 22.63 mmol) was dissolved in dimethylformamide (45 ml) and allyl bromide (2.06 ml, 23.76 mmol) was added. The reaction was cooled to O⁰C and sodium hydride (60% dispersion in mineral oil, 0.95 g, 23.76 mmol) was added portionwise, allowing each portion to react before addition was continued. The mixture became very thick, so a further portion of dimethylformamide (20 ml) was added. Upon complete addition, the mixture was stirred at O⁰C for 30 minutes before warming to room temperature and stirring for a further 5 hours. The reaction was quenched by adding saturated aqueous ammonium chloride (100 ml). The mixture was then extracted with ethyl acetate (3 x 100ml). The combined organic layers were washed with water (3 x 50 ml), dried by passing through a hydrophobic frit and concentrated in vacuo. The residue was purified by flash-silica gel chromatography, eluting with a 10-30% gradient of ethyl acetate in hexane (1.5 L). Clean product fractions concentrated in vacuo to yield methyl [[(2-chloro-4-fluorophenyl)methyl](2-propen-1- yl)amino](oxo)acetate (4.05 g) as a colourless oil.

(iii) Methyl [[(2-chloro-4-fluorophenyl)methyl](2-propen-1-yl)amino](oxo)acetate (4.05 g, 14.18 mmol) was dissolved in dichloromethane (50 ml) and cooled to -78⁰C. The vessel was purged with argon for 5 minutes followed by oxygen for 5 minutes. Ozone was then bubbled through the reaction mixture until a blue colour persisted (approx. 1 hour). The reaction was then purged with oxygen for 5 minutes followed by argon for 30 minutes. The solution was then warmed to room temperature and dimethylsulfide (3.12 ml, 42.53 mmol) was added. The reaction was stirred for 4 hours before being left to stand overnight. The reaction mixture was concentrated in vacuo to yield crude methyl [[(2-chloro-4-fluorophenyl)methyl](2- oxoethyl)amino](oxo)acetate (5.44 g), which was used without purification.

Examples 35-56 (E35-E56)

In a manner analogous to that described for Example 34 above the compounds tabulated below (Table 5) were prepared by substituting the appropriate amines (or amine salts) for the aniline used in the above procedure. The amines or amine salts that may be used to make the compounds shown in Table 5 are available from commercial sources or can be prepared using routes described previously in the chemical literature or analogous methods. (In the case of examples 35-39 and 48- 49, the products were freeze-dried. In the case of examples 44, 47 and 50, flash- silica gel chromatography was used in addition to mass-directed automated HPLC. In the case of examples 40-42 and 45, flash-silica gel chromatography was used in place of mass-directed automated HPLC. In the case of examples 51-53, the purified material was treated with 1 Molar hydrochloric acid in diethyl ether to prepare the salt). Table 5

Example 57

1-(4-Bromo-2-methylphenyl)-4-[(2-chloro-4-fluorophenyl)methyl]-2,3- piperazinedione (E57)

4-Bromo-2-methylaniline (0.82 g, 4.40 mmol) and acetic acid (0.69 ml, 12.0 mmol) were dissolved in dichloromethane (10 ml) and 4 Angstrom molecular sieves added. To this was added a solution of methyl [[(2-chloro-4-fluorophenyl)methyl](2- oxoethyl)amino](oxo)acetate (prepared as described earlier) in dichloromethane (0.1 17M, 34.2 ml, 4.0 mmol). The mixture was stirred for 5 minutes whereupon sodium triacetoxyborohydride (1.27 g, 6.00 mmol) was added and stirring continued overnight. The reaction mixture was stirred with saturated aqueous sodium bicarbonate (10 ml) for 15 minutes and filtered through a hydrophobic frit. The filtrate was concentrated in vacuo and purified twice by flash-silica gel chromatography. Firstly the mixture was eluted with a 0-25% gradient of methanol in dichloromethane; the product was then further purified with a 0-100 gradient of ethyl acetate in hexane to afford 1 -(4-bromo-2-methylphenyl)-4-[(2-chloro-4-fluorophenyl)methyl]-2,3- piperazinedione (0.36 g). LC/MS [M+H]⁺ = 425/427, retention time = 2.95 minutes.

Example 58

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(5,6,7,8-tetrahydro-1 -naphthalenyl)-2,3- piperazinedione (E58)

The 5,6,7,8-tetrahydro-1-naphthalenamine (0.038 g, 0.26 mmol) was added to a mixture of methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (0.10 g, 0.26 mmol, prepared as described earlier), acetic acid (0.044 ml, 0.78 mmol) and 4A molecular sieves in dichloromethane (6 ml) and stirred at room temperature for 5 minutes. Sodium triacetoxyborohydride (0.083 g, 0.39 mmol) was added and the reaction stirred overnight at room temperature. Saturated aqueous sodium bicarbonate (approx. 10 ml) was added and the reaction stirred vigorously for 10 minutes. The organic layer was separated with a hydrophobic frit, reduced in vacuo and purified using mass-directed automated HPLC. The product fractions were freeze-dried to yield 1-[(2-chloro-4-fluorophenyl)methyl]-4-(5,6,7,8-tetrahydro-1- naphthalenyl)-2,3-piperazinedione (0.021 g) as a white solid. LC/MS [M+H]⁺ = 387.0, retention time = 3.00 minutes.

Examples 59-62 (E59-E62)

In a manner analogous to that described for Example 58 above the compounds tabulated below (Table 6) were prepared by substituting the appropriate amines (or amine salts) for the 5,6,7,8-tetrahydro-1-naphthalenamine used in the above procedure. The amines or amine salts that may be 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 or analogous methods. (In the case of examples 59 and 61 , flash-silica gel chromatography was used in addition to mass-directed automated HPLC).

Table 6

Example 63

1-[(2-Chloro-4-fluorophenyl)methyl]-4-{2-[2-(4-morpholinyl)ethyl]phenyl}-2,3- piperazinedione hydrochloride (E63)

Methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (0.17 g, 0.60 mmol, prepared as described earlier) was dissolved in dichloromethane (10 ml) and cooled to O⁰C. 4A molecular sieves (approx. 0.20 g), 2-[2-(4-morpholinyl)ethyl]aniline (0.12 g, 0.60 mmol), acetic acid (0.10 ml, 1.80 mmol) and sodium triacetoxyborohydride (0.19 g, 0.90 mmol) were added. Upon completion, the reaction was allowed to warm to room temperature and partitioned between dichloromethane (100 ml) and saturated aqueous sodium bicarbonate (100 ml); washed with water (two washes), followed by brine and dried over magnesium sulfate. The solution was concentrated in vacuo and purified by mass-directed automated HPLC, followed by salt formation with 1 Molar hydrochloric acid in diethyl ether to yield 1-[(2-chloro-4-fluorophenyl)methyl]-4-{2-[2-(4-morpholinyl)ethyl]phenyl}- 2,3-piperazinedione hydrochloride (0.074 g). LC/MS [M+H]⁺ = 447.7, retention time = 1.75 minutes.

The 2-[2-(4-morpholinyl)ethyl]aniline used in the method described above was prepared as follows:

(i) Triphenylphosphine (2.62 g, 10.0 mmol) and carbon tetrabromide (3.32 g, 10.0 mmol) were added sequentially to a solution of 2-(2-hydroxyethyl)-nitrobenzene

(1.41 ml, 10.0 mmol) in dichloromethane (50 ml) at O⁰C. The reaction was stirred overnight and quenched with saturated aqueous sodium bicarbonate. The organic layer was washed with brine, dried over anhydrous magnesium sulphate and concentrated in vacuo. The residue was treated with ethyl acetate, and the precipitated triphenylphosphine oxide removed by filtration. Purified by flash-silica gel column chromatography, eluting with a 0-100% gradient of ethyl acetate in hexane to yield 2-(2-Bromoethyl)-nitrobenzene (2.30 g).

(ii) To a solution of 1-(2-bromoethyl)-2-nitrobenzene (0.46 g, 2.00 mmol) in acetonitrile (10 ml) was added morpholine (0.26 ml, 3.00 mmol), potassium carbonate (0.83 g, 6.00 mmol) and sodium iodide (catalytic amount). The reaction mixture was refluxed at 85°C under argon overnight. The mixture was then filtered, washed with ethyl acetate and concentrated in vacuo. Purified by flash-silica gel column chromatography, eluting with a 0-25% gradient of ammonia/methanol in dichloromethane. Product fractions were concentrated in vacuo to yield 4-[2-(2- nitrophenyl)ethyl]morpholine (0.47 g) as a pale brown gum.

(iii) 4-[2-(2-nitrophenyl)ethyl]morpholine (0.32 g, 1.35 mmol) was dissolved in ethanol (15 ml) and 10% palladium on charcoal (30mg, 0.28 mmol) was added. The reaction was stirred under an atmosphere of hydrogen overnight. The mixture was filtered and concentrated in vacuo to afford 2-[2-(4-morpholinyl)ethyl]aniline (0.28 mmol) as a pale gum

Example 64 1 -[(2-Chloro-4-fluorophenyl)methyl]-4-[2-methyl-3-(4- morpholinylmethyl)phenyl]-2,3-piperazinedione hydrochloride (E64)

Methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (0.17 g, 0.60 mmol, prepared as described earlier) was dissolved in dichloromethane (10 ml) and cooled to 0⁰C in an ice bath. 4A molecular sieves (-0.2 g) were added, then 2- methyl-3-(4-morpholinylmethyl)aniline (0.12 g, 0.60 mmol) followed by acetic acid (0.10 ml, 1.80 mmol) and finally sodium triacetoxyborohydride (0.19 g, 0.90 mmol). The reaction mixture was allowed to warm to room temperature and stirred overnight. The reaction mixture was partitioned between dichloromethane (50 ml) and saturated aqueous sodium bicarbonate (50 ml) and washed with two portions of water, brine and then dried over anhydrous magnesium sulfate. The solution was concentrated in vacuo and purified by mass-directed automated HPLC. The product was dissolved in methanol (2 ml), treated with 1 Molar hydrochloric acid in diethyl ether (0.5 ml) and concentrated in vacuo to afford 1-[(2-chloro-4-fluorophenyl)methyl]-4-[2-methyl-3-(4- morpholinylmethyl)phenyl]-2,3-piperazinedione hydrochloride (0.094 g). LC/MS [M+H]⁺ = 446, retention time = 1.70 minutes.

The 2-methyl-3-(4-morpholinylmethyl)aniline used in the method described above was prepared as follows:

(i) To a solution of 1-(chloromethyl)-2-methyl-3-nitrobenzene (0.74 g, 4.0 mmol) in 4- methylpentan-2-one (20 ml) was added morpholine (0.52 ml, 6.0 mmol), potassium carbonate (1.66 g, 12.0 mmol) and a catalytic amount of sodium iodide. The reaction mixture was heated to reflux at 125°C under argon overnight. The reaction mixture was filtered, washed with ethyl acetate and concentrated in vacuo. The residue was purified by flash-silica gel chromatography, eluting with a 0-20% gradient of 2 Molar ammonia in methanol solution in dichloromethane. Clean fractions were concentrated in vacuo to yield 4-[(2-methyl-3-nitrophenyl)methyl]morpholine (0.99 g) as a brown gum, which was used without further purification.

(ii) 4-[(2-methyl-3-nitrophenyl)methyl]morpholine (0.95 g, 4.00 mmol) was dissolved in ethanol (15 ml). 10% Palladium on carbon paste (0.10 g, 0.940 mmol) was added and the reaction stirred under hydrogen overnight. The mixture was then filtered, washed with ethanol and concentrated in vacuo. Analysis of the residue indicated that the reaction had not gone to completion. The residue was redissolved in ethanol (15 ml), 10% Palladium on carbon paste (0.10 g, 0.940 mmol) was added and the reaction stirred under hydrogen overnight. The mixture was then filtered, washed with ethanol and concentrated in vacuo to yield 2-methyl-3-(4- morpholinylmethyl)aniline (0.75 g), which was used without further purification.

Example 65

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(2-methyl-3-{[2-(4- morpholinyl)ethyl]oxy}phenyl)-2,3-piperazinedione hydrochloride (E65)

HCI

Methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (0.17 g, 0.60 mmol, prepared as described earlier) was dissolved in dichloromethane (10 ml) and cooled to 0⁰C in an ice bath. 4A molecular sieves (-0.2 g), 2-methyl-3-{[2-(4- morpholinyl)ethyl]oxy}aniline (0.14 g, 0.60 mmol), acetic acid (0.10 ml, 1.80 mmol) and sodium triacetoxyborohydride (0.19 g, 0.90 mmol) were then added. The reaction mixture was allowed to warm to room temperature and stirred overnight. The reaction mixture was then partitioned between dichloromethane (50 ml) and saturated aqueous sodium bicarbonate (50ml), washed with two portions of water, brine and then dried over anhydrous magnesium sulfate. The residue was concentrated in vacuo and purified by mass-directed automated HPLC. The product was dissolved in methanol (2 ml), treated with 1 Molar hydrochloric acid in diethyl ether (0.5 ml) and concentrated in vacuo to afford 1-[(2-chloro-4- fluorophenyl)methyl]-4-(2-methyl-3-{[2-(4-morpholinyl)ethyl]oxy}phenyl)-2,3- piperazinedione hydrochloride (0.12 g) LC/MS [M+H]⁺ = 476.2, retention time = 1.84 minutes.

The 2-methyl-3-{[2-(4-morpholinyl)ethyl]oxy}aniline used in the method described above was prepared as follows:

(i) Sodium hydride (0.44 g, 1 1.00 mmol, 60% dispersion in mineral oil) was added to a solution of 2-methyl-3-nitrophenol (0.77 g, 5.0 mmol) in N,N-dimethylformamide (10 ml) and stirred for 30 minutes. 4-(2-chloroethyl)morpholine (0.82 g, 5.5 mmol) and a catalytic amount of sodium iodide were then added and the reaction mixture stirred overnight at 60⁰C. The reaction mixture was then partitioned between ethyl acetate and water. The aqueous layer was back extracted with three portions of ethyl acetate. The organic phases were combined and washed with three portions of water, brine and then dried over anhydrous magnesium sulphate. The mixture was filtered and concentrated in vacuo. The residue was then partitioned between ethyl acetate (100 ml) and 2 Molar sodium hydroxide (100 ml). The aqueous layer was back extracted with two portions of ethyl acetate and organic phases combined. These were then washed with two portions of water, brine and then dried over anhydrous magnesium sulphate. The mixture was filtered and concentrated in vacuo. The residue was purified by flash-silica gel chromatography, eluting with a 0- 20% gradient of 2 Molar ammonia in methanol solution in dichloromethane. The clean fractions were concentrated in vacuo to afford 4-{2-[(2-methyl-3- nitrophenyl)oxy]ethyl}morpholine (0.93 g) as a yellow gum. (ii) 4-{2-[(2-methyl-3-nitrophenyl)oxy]ethyl}morpholine (0.93 g, 3.5 mmol) was dissolved in ethanol (15 ml) and 10% Palladium on carbon paste (0.10 g, 0.94 mmol) added. The reaction mixture was stirred under hydrogen overnight. The mixture was then filtered, washed with ethanol and concentrated in vacuo to afford 2-methyl-3-{[2- (4-morpholinyl)ethyl]oxy}aniline (0.83 g), which was used without purification.

Example 66

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-methyl-3-(methyloxy)phenyl]-2,3- piperazinedione (E66)

A solution of methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (0.28 g, 0.94 mmol, prepared as described earlier) in 1 ,2-dichloroethane (5 ml) was cooled to O⁰C in an ice bath. 4A molecular sieves were added, followed by acetic acid (0.16 ml, 2.80 mmol), 3-methoxy-2-methylaniline (0.13 g, 0.94 mmol) and sodium triacetoxyborohydride (0.30 g, 1.40 mmol). The reaction was stirred under argon overnight. Saturated aqueous sodium bicarbonate (5 ml) was added and the reaction stirred vigorously for 15 minutes. The organic layer was separated using a hydrophobic frit and concentrated in vacuo. The residue was purified by flash-silica gel chromatography, eluting with a 10-100% gradient of ethyl acetate in isohexane. The material was then triturated with diethyl ether to yield 1-[(2-chloro-4- fluorophenyl)methyl]-4-[2-methyl-3-(methyloxy)phenyl]-2,3-piperazinedione (0.19 g) as a white solid. LC/MS [M+H]⁺ = 377.1 , retention time = 2.80 minutes.

Examples 67-68 (E67-68)

In a manner analogous to that described for Example 66 above the compounds tabulated below (Table 7) were prepared by substituting the appropriate amines (or amine salts) for the 3-methoxy-2-methylaniline used in the above procedure. The amines or amine salts that may be 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 or analogous methods. Table 7

Example 69

1-[5-Bromo-2-(methyloxy)phenyl]-4-[(2-chloro-4-fluorophenyl)methyl]-2,3- piperazinedione (E69)

Methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (prepared as described earlier, 0.98 g, 3.41 mmol), acetic acid (0.78 ml, 13.7 mmol) and 4 Angstrom molecular sieves in 1 ,2-dichloroethane (10 ml) were cooled to 0 ⁰C. 5- Bromo-2-(methyloxy)aniline (0.69 g, 3.41 mmol) was added and the reaction stirred for 10 minutes. Sodium triacetoxyborohydride (1.09 g, 5.12 mmol) was added, the reaction stirred for five minutes at 0 ⁰C, then allowed to warm to room temperature and stirred overnight. Saturated aqueous sodium bicarbonate was added slowly until the evolution of gas had stopped. The organic layer was separated and the aqueous layer extracted with dichloromethane. The combined organic layers were washed with 3N citric acid and separated using a hydrophobic frit. The solution was concentrated in vacuo and purified by flash-silica gel chromatography, eluting with a 10-100% gradient of ethyl acetate in isohexane. The combined product fractions were further purified by mass-directed automated HPLC to yield 1-[5-bromo-2- (methyloxy)phenyl]-4-[(2-chloro-4-fluorophenyl)methyl]-2,3-piperazinedione (0.30 g) as a white solid. LC/MS [M+H]⁺ = 441/443, retention time = 2.87 minutes.

Example 70 1-[(2-Chloro-4-fluorophenyl)methyl]-4-(1H-indol-7-yl)-2,3-piperazinedione (E70)

Methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (prepared as described earlier, 0.55 g, 1.72 mmol), acetic acid (0.394 ml, 6.88 mmol) and 4 Angstrom molecular sieves in 1 ,2-dichloroethane (10 ml) were cooled to 0 ⁰C. 7- Aminoindole (0.23 g, 1.72 mmol) was added and the reaction stirred at 0⁰C for 15 minutes. Sodium triacetoxyborohydride (0.51 g, 2.41 mmol) was added, the reaction stirred for ten minutes at 0 ⁰C, then allowed to warm to room temperature and stirred overnight. The reaction mixture was diluted with dichloromethane (10 ml) and saturated aqueous sodium bicarbonate was added slowly until the evolution of gas had stopped. The mixture was filtered through celite, then the organic layer was separated and washed with brine. The organic layer was separated using a hydrophobic frit, concentrated in vacuo and purified by flash-silica gel chromatography, eluting with a 10-100% gradient of ethyl acetate in isohexane. The combined product fractions were further purified by mass-directed automated HPLC to yield 1-[(2-chloro-4-fluorophenyl)methyl]-4-(1 H-indol-7-yl)-2,3-piperazinedione (0.009 g).

LC/MS [M+H]⁺ = 371.9, retention time = 2.74 minutes. Example 71

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[3-chloro-2-(methyloxy)phenyl]-2,3- piperazinedione (E71)

Methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (0.27 g, 0.92 mmol, prepared as described earlier), acetic acid (0.21 ml, 3.7 mmol) and 4A molecular sieves in 1 ,2-dichloroethane (5 ml) were cooled to 0⁰C. 3-chloro-2- (methyloxy)aniline (0.15 g, 0.92 mmol) and sodium triacetoxyborohydride (0.29 g, 1.38 mmol) were added and the reaction was stirred at 0⁰C for ten minutes. The reaction was then allowed to warm to room temperature and stirred for 6 hours before being heated to 60⁰C overnight. The reaction was cooled to room temperature and saturated aqueous sodium bicarbonate was added until the evolution of gas stopped. The aqueous layer was separated and washed with dichloromethane, the organic layers separated using a hydrophobic frit and concentrated in vacuo. The residue was purified by flash-silica gel chromatography eluting with a 0-100% gradient of ethyl acetate in isohexane to give 1-[(2-chloro-4- fluorophenyl)methyl]-4-[3-chloro-2-(methyloxy)phenyl]-2,3-piperazinedione (0.055 g) as a low melting solid. LC/MS [M+H]⁺ = 396.92, retention time = 2.83 minutes.

Example 72

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[3-chloro-2-(4-morpholinyl)phenyl]-2,3- piperazinedione (E72)

Methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (0.37 g, 0.95 mmol, prepared as described earlier), acetic acid (0.16 ml, 2.9 mmol) and 4A molecular sieves in 1 ,2-Dichloroethane (5 ml) were cooled to 0⁰C in an ice bath. 3- Chloro-2-morpholin-4-yl-phenylamine (0.30 g, 1.43 mmol) and sodium triacetoxyborohydride (0.30 g, 1.43 mmol) were added, the reaction was allowed to warm to room temperature and stirred overnight. The reaction was then heated to 80 ⁰C for 5 hours, followed by stirring at 80⁰C overnight. The reaction was allowed to cool to room temperature and saturated aqueous sodium bicarbonate was slowly added until gas evolution had stopped. The organic layer was separated using a hydrophobic frit and concentrated in vacuo. The residue was purified by flash-silica gel chromatography, eluting with a 10-75% gradient of ethyl acetate in isohexane. The product was then triturated with diethyl ether to give 1-[(2-chloro-4- fluorophenyl)methyl]-4-[3-chloro-2-(4-morpholinyl)phenyl]-2,3-piperazinedione (0.20 g) as a pale yellow solid. LC/MS [M+H]⁺ = 452.0, retention time = 2.80 minutes.

Example 73

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[5-chloro-2-(4-morpholinyl)phenyl]-2,3- piperazinedione (E73)

Methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (0.41 g, 1.41 mmol, prepared as described earlier), acetic acid (0.32 ml, 5.6 mmol) and 4A molecular sieves in 1 ,2-Dichloroethane (10 ml) were cooled to 0⁰C. 5-chloro-2-(4- morpholinyl)aniline (0.30 g, 1.41 mmol) was added and the reaction allowed to stir for 10 minutes. Sodium triacetoxyborohydride (0.49 g, 2.12 mmol) was added; the reaction was allowed to warm to room temperature and stirred for 3 hours. The reaction was then heated to 80 °C with stirring overnight. The reaction mixture was allowed to cool and saturated aqueous sodium bicarbonate was added until gas evolution stopped. The organic layer was separated using a hydrophobic frit. The aqueous layer was extracted with dichloromethane and the organic layer separated. The combined organic layers were concentrated in vacuo and purified by flash-silica gel chromatography, eluting with a 10-100% gradient of ethyl acetate in isohexane to give 1-[(2-chloro-4-fluorophenyl)methyl]-4-[5-chloro-2-(4-morpholinyl)phenyl]-2,3- piperazinedione (0.24 g) as a cream-coloured solid. LC/MS [M+H]⁺ = 452.0, retention time = 2.84 minutes.

Example 74

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-(4-morpholinyl)-5- (trifluoromethyl)phenyl]-2,3-piperazinedione (E 74)

Methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (0.29 g, 0.74 mmol, prepared as described earlier), acetic acid (0.17 ml, 2.97 mmol) and 4A molecular sieves in 1 ,2-dichloroethane (5 ml) was cooled to 0⁰C. 2-(4-Morpholinyl)- 5-(trifluoromethyl)aniline (0.18 g, 0.74 mmol) and sodium triacetoxyborohydride (0.24 g, 1.12 mmol) were added, and the reaction stirred at 0 ⁰C for 10 minutes. The mixture was then allowed to warm to room temperature and stirred overnight. The reaction was then heated to 80⁰C for 24 hours. Saturated aqueous sodium bicarbonate was added until the evolution of gas had stopped. The organic layer was separated using a hydrophobic frit and reduced in vacuo. Purified by flash-silica gel chromatography, eluting with a 10-100% gradient of ethyl acetate in isohexane to yield 1 -[(2-chloro-4-fluorophenyl)methyl]-4-[2-(4-morpholinyl)-5-

(trifluoromethyl)phenyl]-2,3-piperazinedione (0.14 g) as a pale yellow solid. LC/MS [M+H]⁺ = 486.1 , retention time = 2.98 minutes.

Example 75 1-[3-Bromo-2-(methyloxy)phenyl]-4-[(2-chloro-4-fluorophenyl)methyl]-2,3- piperazinedione (E75)

Methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (0.57 g, 1.98 mmol, prepared as described earlier), acetic acid (0.45 ml, 7.92 mmol), and 4A molecular sieves in 1 ,2-dichloroethane (10 ml) were cooled to 0⁰C. 3-Bromo-2- (methyloxy)aniline (0.40 g, 1.98 mmol) was added and the reaction was stirred for 10 minutes. Sodium triacetoxyborohydride (0.63 g, 2.97 mmol) was added, the reaction allowed to warm to room temperature and stirred overnight. The reaction was then heated to 80⁰C for 4 hours. The reaction mixture was allowed to cool and saturated aqueous sodium bicarbonate was added until gas evolution stopped. The organic layer was separated and the aqueous layer extracted with dichloromethane. The organic layers were separated using a hydrophobic frit and concentrated in vacuo. The residue was purified by flash-silica gel chromatography, eluting with a 10-100% gradient if ethyl acetate in isohexane to give 1-[3-bromo-2-(methyloxy)phenyl]-4-[(2- chloro-4-fluorophenyl)methyl]-2,3-piperazinedione (0.28 g) as a light brown solid. LC/MS [M+H]⁺ = 440.97, retention time = 2.86 minutes.

The 3-Bromo-2-(methyloxy)aniline used in the method described above was prepared as follows:

(i) To a stirred solution of 2-bromoaniline (200 g, 1.16 mol) and triethylamine (202.6 ml_, 1.45 mol) in dichloromethane (600 ml.) at 0°C was added dropwise acetyl chloride (99.2 ml_, 1.40 mol). Upon complete addition, the reaction mixture was allowed to warm to room temperature and was stirred for a further 2 h. Water and dichloromethane were then added to the mixture, the organic layer was separated, and the aqueous layer was further extracted with dichloromethane. The combined organic extracts were washed with brine, dried, and the solvent was removed in vacuo to give Λ/-(2-bromophenyl)acetamide (183.2 g) as a solid, mp 97-100⁰C. R_f 0.40 (7:8 ethyl acetate/hexane).

(ii) To a stirred suspension of Λ/-(2-bromophenyl)acetamide (58.Og, 0.27 mol) and acetic anhydride (29 ml.) in acetic acid (23 ml.) at -5°C was added dropwise a mixture of nitric acid (23 ml.) in acetic acid (23 ml_). After being stirred for 2 h at - 5°C, the reaction mixture was allowed to stand at room temperature overnight. The two identical batches, which were run in parallel were then combined for work up. Water was added, the resultant precipitate collected by filtration and washed with water. The crude product was then recrystallised from dichloromethane to give, after drying, Λ/-(2-bromo-6-nitrophenyl)acetamide (44.8 g) as a colourless solid, mp 197— 199°C.

(iii) Λ/-(2-Bromo-6-nitrophenyl)acetamide (28.0 g, 0.108 mol) was suspended in 6 M aqueous hydrochloric acid (300 ml.) and the resultant reaction mixture was heated at reflux with vigorous stirring for 3 h before being allowed to cool to room temperature. The reaction was then poured into a mixture of sodium hydroxide (80 g, 2.0 mol) and ice (300 g) to which was added ethyl acetate. The organic layer was separated and the aqueous phase was further extracted with ethyl acetate. The combined organic extracts were washed with brine, dried (magnesium sulfate), and the solvent was removed in vacuo to give 2-bromo-6-nitroaniline as a solid, mp 70-72⁰C. R_f 0.39 (1 :3 ethyl acetate/hexane).

(iv) Sodium nitrate (8.20 g, 0.12 mol) was added portionwise to concentrated sulfuric acid (85 ml.) and the resultant mixture was heated at 60-70⁰C before being cooled to 25-30°C. A solution of 2-bromo-6-nitroaniline (23.5 g, 0.11 mol) in hot glacial acetic acid (215 ml.) was then added dropwise while the temperature of the reaction mixture was kept below 35°C. Upon complete addition, the solution was warmed at 35°C for 30 min and was then added portionwise to a mixture of copper(l) chloride (23.5 g, 0.24 mol) in concentrated hydrochloric acid (215 ml_). Upon complete addition, the reaction was heated to 70⁰C, an equal volume of water was then added, and the mixture was cooled to 0⁰C. The resultant yellow solid was collected by filtration and washed with water. The material was subsequently dissolved in ethyl acetate and the insoluble material filtered off. The filtrate was washed with 1 M aqueous hydrochloric acid, water and brine, dried (magnesium sulfate), and the solvent was removed in vacuo. The crude product was recrystallised from hexanes/ethyl acetate to give 1-bromo-2-chloro-3-nitrobenzene (18.4 g) as a solid, mp 58-59°C. R_f 0.33 (1 :3 ethyl acetate/hexanes).

(v) To a stirred solution of 1-bromo-2-chloro-3-nitrobenzene (24.5 g, 0.10 mol) in methanol (250 ml.) in a 1 L autoclave was added sodium methoxide (8.43 g, 0.16 mol). The autoclave was then sealed and heated at 100°C for 3 h. Upon being allowed to cool to room temperature the majority of the solvent was removed under vacuum. Water and ethyl acetate were then added, the two layers were separated, and the aqueous layer was further extracted with ethyl acetate. The combined organic fractions were washed with 1 M aqueous sodium hydroxide (3^χ), water and brine, dried (magnesium sulfate), and the solvent was removed in vacuo. The crude product was recrystallised from hexane to give 1-bromo-2-methoxy-3-nitrobenzene (14.1 g, 58%) as a solid, mp 64-65°C. R_f 0.36 (1 :3 ethyl acetate/hexane).

(vi) To a stirred mixture of tin(ιι) chloride (62.11 g, 0.328 mol) in methanol (300 ml.) and concentrated hydrochloric acid (150 ml.) at 0⁰C was added in a single portion 1- bromo-2-methoxy-3-nitrobenzene (15.2 g, 66.0 mmol). The resultant reaction mixture was stirred at room temperature overnight, ethyl acetate (500 ml.) was added, and the mixture was made basic with the addition of 5 M aqueous sodium hydroxide. A further aliquot of ethyl acetate (500 ml.) was added, the layers were allowed to separate, and the organic fraction was separated. The resultant mixture was then further extracted with ethyl acetate (2^χ). The combined organic layers were washed with water and brine, dried (magnesium sulfate), and the solvent was removed in vacuo to give 3-bromo-2-methoxy-aniline (12.8 g) as a pale tan oil. R_f 0.18 (1 :3 ethyl acetate/hexane).

Example 76

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[3-methyl-2-(4-morpholinyl)phenyl]-2,3- piperazinedione (E76)

Methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (0.27 g, 0.70 mmol, prepared as described earlier), acetic acid (0.12 ml, 2.09 mmol) and 4A molecular sieves in 1 ,2-dichloroethane (5 ml) were cooled to 0⁰C in an ice bath. 3- Methyl-2-(4-morpholinyl)aniline (0.13 g, 0.70 mmol) and sodium triacetoxyborohydride (0.22 g, 1.04 mmol) were added, the reaction was allowed to warm to room temperature and stirred overnight. The reaction was then stirred at 80⁰C for 5 hours. The reaction was allowed to cool to room temperature. Saturated aqueous sodium bicarbonate was added slowly with stirring until gas evolution had stopped. The organic layer was separated using a hydrophobic frit and concentrated in vacuo. The residue was purified by flash-silica gel chromatography, eluting with a 20-100% gradient of ethyl acetate in isohexane to give 1-[(2-Chloro-4- fluorophenyl)methyl]-4-[3-methyl-2-(4-morpholinyl)phenyl]-2,3-piperazinedione (0.13 g) as a pale yellow solid.

LC/MS [M+H]⁺ = 432.0, retention time = 2.74 minutes.

The 3-Methyl-2-(4-morpholinyl)aniline used in the method described above was prepared as follows:

(i) 2-Bromo-1-methyl-3-nitrobenzene (0.68 g, 3.15 mmol) in morpholine (5 ml_, 57.4 mmol) was stirred at 120⁰C for 5 hours, followed by further heating at 120 ⁰C overnight. Further 2-bromo-1-methyl-3-nitrobenzene (1.7 g, 7.87 mmol) was added and the reaction stirred at 120 ⁰C for 7 hours then overnight for 2 nights. The total reaction time was approximately 60 hours. The reaction mixture was cooled to room temperature. Solids were filtered off and washed with dichloromethane. The filtrate solution was concentrated in vacuo and purified by flash-silica gel chromatography, eluting with a 2-20% gradient of ethyl acetate in isohexane to yield 4-(2-methyl-6- nitrophenyl)morpholine (1.29 g) as a bright yellow solid.

(ii) 4-(2-methyl-6-nitrophenyl)morpholine (1.29 g, 5.80 mmol) and palladium on carbon (0.62 g, 0.580 mmol) in ethyl acetate (100 ml) were stirred under hydrogen for 6 hours. The reaction mixture was filtered through celite, and washed with ethyl acetate. The solution was concentrate in vacuo to give 3-methyl-2-(4- morpholinyl)aniline (0.98 g) as an off white solid.

Example 77

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-methyl-5-(4-morpholinyl)phenyl]-2,3- piperazinedione (E77)

Methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (0.35 g, 0.912 mmol, prepared as described earlier), acetic acid (0.16 ml, 2.74 mmol) and 4A molecular sieves in 1 ,2-dichloroethane (5 ml) were cooled to 0⁰C in an ice bath. 2- Methyl-5-(4-morpholinyl)aniline (0.29 g, 1.51 mmol) and sodium triacetoxyborohydride (0.29 g, 1.37 mmol) were added, the reaction was allowed to warm to room temp and stirred overnight. Saturated aqueous sodium bicarbonate was added slowly until gas evolution stopped. The organic layer was separated using a hydrophobic frit, washed with 3 N citric acid solution and separated as before. The organic layer was concentrated in vacuo and purified by flash-silica gel chromatography, eluting with a 20-100% gradient of ethyl acetate in isohexane. The product was then triturated with diethyl ether to yield 1-[(2-chloro-4- fluorophenyl)methyl]-4-[2-methyl-5-(4-morpholinyl)phenyl]-2,3-piperazinedione (0.18 g) as a light brown solid.

LC/MS [M+H]⁺ = 432.1 , retention time = 2.63 minutes.

The 2-Methyl-5-(4-morpholinyl)aniline used in the method described above was prepared as follows:

Potassium phosphate (8.50 g, 40.0 mmol), L-proline (0.46 g, 4.00 mmol) and copper(l) iodide (0.38 g, 2.00 mmol) were added to a solution of 5-bromo-2- methylaniline (2.5 ml, 20.0 mmol) and morpholine (2.62 ml, 30.0 mmol) in dimethyl sulfoxide (15 ml). The reaction was heated to 120 ⁰C under argon overnight. The reaction was then stirred at 120 ⁰C for a further 6 hours. The reaction was allowed to cool before being diluted with water (-40 ml). The mixture was extracted with ethyl acetate (x3), the organic layer separated and washed with water (x2). The aqueous phase was then back extracted with ethyl acetate (x2). The combined organic layers were dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by flash-silica gel chromatography to give crude 2-methyl-5-(4- morpholinyl)aniline (0.51 g), which was used without further purification.

Example 78

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[5-methyl-2-(4-morpholinyl)phenyl]-2,3- piperazinedione (E78)

Methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (0.33 g, 0.86 mmol, prepared as described earlier), acetic acid (0.20 ml, 3.45 mmol) and 4A molecular sieves in 1 ,2-dichloroethane (5 ml) was cooled to 0 ⁰C. 5-methyl-2-(4- morpholinyl)aniline (0.17 g, 0.86 mmol) and sodium triacetoxyborohydride (0.27 g, 1.29 mmol) were then added. The reaction was stirred at 0⁰C for 10 minutes, then allowed to warm to room temperature and stirred overnight. The reaction mixture was then heated to 80⁰C for 3 hours. The reaction was allowed to cool and saturated aqueous sodium bicarbonate was added until gas evolution stopped. The organic layer was separated using a hydrophobic frit and concentrated in vacuo. The residue was purified by flash-silica gel chromatography, eluting with a 10-100% gradient of ethyl acetate in isohexane to yield 1-[(2-chloro-4-fluorophenyl)methyl]-4-[5-methyl-2- (4-morpholinyl)phenyl]-2,3-piperazinedione (0.14 g) as a white solid. LC/MS [M+H]⁺ = 432.1 , retention time = 2.81 minutes.

The 5-methyl-2-(4-morpholinyl)aniline used in the method described above was prepared as follows:

(i) 1-Bromo-4-methyl-2-nitrobenzene (4.51 g, 20.88 mmol) in morpholine (7.27 ml, 84 mmol) was stirred at 120 ⁰C for 3 hours. The reaction was allowed to cool, the solids were filtered off and washed with dichloromethane. The filtrate was concentrated in vacuo and purified by flash-silica gel chromatography, eluting with a 10-20% gradient of ethyl acetate in isohexane to give 4-(4-methyl-2-nitrophenyl)morpholine (5.0 g) as an orange oil.

(ii) 4-(4-Methyl-2-nitrophenyl)morpholine (5.0 g, 22.50 mmol) and palladium on carbon (0.48 g, 0.45 mmol) in ethyl acetate (100 ml) were stirred overnight at room temperature under hydrogen. The reaction mixture was filtered through celite and washed with ethyl acetate. The filtrate was concentrated in vacuo to give 5-methyl-2- (4-morpholinyl)aniline (3.49 g) as a white solid.

Example 79

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[5-fluoro-2-(4-morpholinyl)phenyl]-2,3- piperazinedione (E79)

Methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (prepared as described earlier, 0.36 g, 1.26 mmol), acetic acid (0.29 ml, 5.06 mmol) and 4 Angstrom molecular sieves in 1 ,2-dichloroethane (10 ml) were cooled to 0 ⁰C. 5- Fluoro-2-(4-morpholinyl)aniline (0.40 g, 1.26 mmol) was added and the mixture stirred for 10 minutes. Sodium triacetoxyborohydride (0.40 g, 1.90 mmol) was then added, the reaction mixture was allowed to warm to room temperature and stirred for 3 hours. The mixture was then heated to 80 ⁰C overnight. Saturated aqueous sodium bicarbonate was added until the evolution of gas had stopped. The organic layer was separated and the aqueous layer extracted with dichloromethane. The combined organic layers were reduced in vacuo and purified by flash-silica gel chromatography, eluting with a 10-100% gradient of ethyl acetate in isohexane. The crude product was further purified by mass-directed automated HPLC to yield 1-[(2- chloro-4-fluorophenyl)methyl]-4-[5-fluoro-2-(4-morpholinyl)phenyl]-2,3- piperazinedione (26 mg, 0.060 mmol, 4.72 % yield) as an off-white solid. LC/MS [M+H]⁺ = 436.0, retention time = 2.68 minutes.

The 5-Fluoro-2-(4-morpholinyl)aniline used in the method described above was prepared as follows:

(i) 1-bromo-4-fluoro-2-nitrobenzene (1.78 g, 8.09 mmol) and morpholine (2.12 ml, 24.3 mmol) were stirred at 120 ⁰C for 2 hours. The reaction mixture was allowed to cool, the insoluble material was filtered off and washed with dichloromethane. The filtrate was reduced in vacuo and purified by flash-silica gel chromatography, eluting with a 0-20% gradient of ethyl acetate in isohexane to yield 4-(4-fluoro-2- nitrophenyl)morpholine (1.51 g) as a bright orange oil that crystallised upon standing.

(ii) 4-(4-fluoro-2-nitrophenyl)morpholine (1.5 g, 6.63 mmol) and palladium on carbon

(0.07 g, 0.07 mmol) in ethyl acetate (40 ml) was stirred at room temperature under a hydrogen atmosphere overnight. The mixture was then filtered through celite and washed with further ethyl acetate. The solution immediately started to turn a red colour. The mixture was extracted with 3N citric acid solution, and the aqueous layer adjusted to ~pH 12 by the addition of 2N sodium hydroxide. This was then extracted with ethyl acetate (x2) and the combined organic layers dried over sodium sulfate. The solution was reduced in vacuo to yield 5-Fluoro-2-(4-morpholinyl)aniline as a dark red oil, which was used without further purification.

Example 80

1 -[(2-Chloro-4-fluorophenyl)methyl]-4-[2-(methyloxy)-5-(4-morpholinyl)phenyl]- 2,3-piperazinedione (E80)

Methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (0.59 g, 2.06 mmol, prepared as described earlier), acetic acid (0.47 ml, 8.25 mmol) and 4 Angstrom molecular sieves in 1 ,2-dichloroethane (10 ml) were cooled to 0 ⁰C and stirred for 5 minutes. 2-(Methyloxy)-5-(4-morpholinyl)aniline (0.30 g, 1.456 mmol) was added and the mixture stirred for a further 10 mins. Sodium triacetoxyborohydride (0.61 g, 2.89 mmol) was then added, the mixture allowed to warm to room temperature and stirred overnight. The mixture was diluted with dichloromethane (-10 ml.) and filtered through celite. Saturated aqueous sodium bicarbonate (-20 ml.) was added to the filtrate and the mixture was stirred for 10 minutes. The organic layer was separated using a hydrophobic frit and concentrated in vacuo. The residue was purified by flash-silica gel chromatography, eluting with a 10-100% gradient of ethyl acetate in isohexane to yield 1-[(2-chloro-4- fluorophenyl)methyl]-4-[2-(methyloxy)-5-(4-morpholinyl)phenyl]-2,3-piperazinedione (0.15 g) as a cream-coloured solid. LC/MS [M+H]⁺ = 447.9, retention time = 2.49 minutes.

The 2-(methyloxy)-5-(4-morpholinyl)aniline used in the method described above was prepared as follows:

(i) 4-Bromo-1-(methyloxy)-2-nitrobenzene (4.71 g, 20.3 mmol) was dissolved in 1 ,2- dimethoxyethane (50 ml) and the solution degassed under argon. Morpholine (2.12 ml, 24.4 mmol), potassium phosphate dibasic (4.95 g, 28.4 mmol), 2- biphenylyl(dicyclohexyl)phosphane (0.53 g, 1.52 mmol) and palladium (II) acetate (0.23 g, 1.02 mmol) were added sequentially and the reaction mixture stirred at 80⁰C for 36 hours. A 5 mL aliquot of the mixture was removed and heated to 120 ⁰C for 2 hours in the microwave. This mixture was diluted with ethyl acetate (5 mL), filtered through celite and concentrated in vacuo. The residue was purified by flash-silica gel chromatography, eluting with a 50-100% gradient of dichloromethane in isohexane to yield a bright orange gum that crystallised upon standing. The remainder of the reaction mixture was heated to 130 ⁰C in the microwave for 4 hours. The mixture was diluted with ethyl acetate, filtered through celite and concentrated in vacuo. At this point the crude residue was divided into two; one half was not purified and was used no further. The remaining half was purified by flash-silica gel chromatography, eluting with a 0-1% gradient of methanol in dichloromethane. The two batches of purified material were combined to yield 4-[4-(methyloxy)-3-nitrophenyl]morpholine (0.63 g).

(ii) 4-[4-(methyloxy)-3-nitrophenyl]morpholine (0.63 g, 2.63 mmol) and palladium on carbon (0.03 g, 0.03 mmol) in a mixture of ethanol (100 ml) and ethyl acetate (25 ml) was stirred under a hydrogen atmosphere overnight. The reaction mixture was filtered through celite, which was washed with ethyl acetate. The filtrate was concentrated in vacuo to yield 2-(methyloxy)-5-(4-morpholinyl)aniline (0.54 g).

Example 81

1 -[(2-Chloro-4-fluorophenyl)methyl]-4-(8-quinolinyl)-2,3-piperazinedione (E81 )

Methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (0.15 g, 0.40 mmol, prepared as described earlier) was dissolved in 1 ,2-dichloroethane (1 ml). 8- quinolinamine (0.1 15 g, 0.80 mmol), polymer-supported cyanoborohydride (0.50 g, 2.0 mmol), acetic acid (0.023 ml, 0.40 mmol) and further 1 ,2-dichloroethane (4 ml) were added and the reaction heated to 85⁰C for 16 hours. The resin was filtered off and washed with dichloromethane (3 ml). Combined solutions were concentrated in vacuo and purified by mass-directed automated HPLC, followed by trituration with diethyl ether (10 ml) to yield 1-[(2-chloro-4-fluorophenyl)methyl]-4-(8-quinolinyl)-2,3- piperazinedione (0.024 g) as an off-white/pink solid. LC/MS [M+H]⁺ = 384, retention time = 2.44 minutes.

Example 82

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(3-isoquinolinyl)-2,3-piperazinedione

(E82)

In a manner analogous to that described for Example 81 above the compound tabulated below (Table 8) was prepared by substituting the appropriate amine for the 8-quinolinamine used in the above procedure. The amine (or amine salts) that may be used to make the compound shown in Table 8 are available from commercial sources or can be prepared using routes described previously in the chemical literature or analogous methods.

Table 8

Example 83 1-[(2-Chloro-4-fluorophenyl)methyl]-4-(2-pyrimidinyl)-2,3-piperazinedione (E83)

Methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (0.15 g, 0.40 mmol, prepared as described earlier) was dissolved in 1 ,2-dichloroethane (1 ml). 2- Pyrimidinamine (0.08 g, 0.80 mmol), polymer-supported cyanoborohydride (0.50 g, 2.0 mmol), acetic acid (0.023 ml, 0.40 mmol) and further 1 ,2-dichloroethane (4 ml) were added and the reaction heated to 85⁰C for 16 hours. The reaction mixture was then heated to 16O⁰C for 1 hour using microwave irradiation. The resin was filtered off and washed with dichloromethane (3 ml). The combined solutions were concentrated in vacuo and purified by mass-directed automated HPLC to yield 1-[(2- chloro-4-fluorophenyl)methyl]-4-(2-pyrimidinyl)-2,3-piperazinedione (0.002 g) as a white solid. LC/MS [M+H]⁺ = 335, retention time = 2.07 minutes.

Example 84 1-[(2-Chloro-4-fluorophenyl)methyl]-4-(2-pyridinyl)-2,3-piperazinedione (E84)

Methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (0.10 g, 0.26 mmol, prepared as described earlier) was dissolved in dichloromethane (5 ml). 2- Aminopyridine (0.03 g, 0.29 mmol), acetic acid (0.02ml, 0.29 mmol) and polymer- supported cyanoborohydride (0.33 g, 1.3 mmol) were added and the reaction stirred at room temperature for approximately 65 hours. The reaction mixture was then heated to 4O⁰C for 1.5 hours. The solvent was removed in vacuo, the mixture taken up in 1 ,2-dichloroethane (5 ml) and the reaction heated to 85⁰C for 4.5 hours. The resin was filtered off and washed with dichloromethane (3 ml). The combined solutions were concentrated in vacuo and purified by mass-directed automated HPLC to yield 1-[(2-chloro-4-fluorophenyl)methyl]-4-(2-pyridinyl)-2,3-piperazinedione (0.005 g) as an off-white/pink solid. LC/MS [M+H]⁺ = 334, retention time = 2.40 minutes.

Example 85 1-[(2-Chloro-4-fluorophenyl)methyl]-4-(6-methyl-2-pyridinyl)-2,3- piperazinedione (E85)

Methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (0.15 g, 0.40 mmol, prepared as described earlier) was dissolved in 1 ,2-dichloroethane (2 ml). 6- Methyl-2-pyridinamine (0.09 g, 0.80 mmol), polymer-supported cyanoborohydride (0.50 g, 2.0 mmol), acetic acid (0.023 ml, 0.40 mmol) and further 1 ,2-dichloroethane (3 ml) were added and the reaction heated to 85⁰C for 16 hours. The resin was filtered off and washed with dichloromethane (3 ml). The combined solutions were concentrated in vacuo and purified by mass-directed automated HPLC followed by trituration with diethyl ether (-15 ml) to yield 1-[(2-chloro-4-fluorophenyl)methyl]-4-(6- methyl-2-pyridinyl)-2,3-piperazinedione (0.002 g) as a white solid. LC/MS [M+H]⁺ = 348, retention time = 2.59 minutes.

Example 86

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(2-ethylphenyl)-2,3-piperazinedione (E86)

Methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (0.29 g, 1.00 mmol, prepared as described earlier) and 2-ethylaniline (0.24 g, 2.00 mmol) were dissolved in 2% acetic acid/methanol (10 ml) at room temperature. The mixture was stirred for 15 minutes and polymer-supported cyanoborohydride (0.98 g, 4.00 mmol) was added. The mixture was stirred at room temperature for 48 hours and filtered through an SCX cartridge (Varian, 5g). The filtrate was concentrated in vacuo and purified by mass-directed automated HPLC to afford 1-[(2-chloro-4- fluorophenyl)methyl]-4-(2-ethylphenyl)-2,3-piperazinedione (0.21 g). LC/MS [M+H]⁺ = 361.0, retention time = 2.81 minutes.

Example 87-93

In a manner analogous to that described for Example 86 above the compounds tabulated below (Table 9) were prepared by substituting the appropriate amine for the 2-ethylaniline used in the above procedure. The amines (or amine salts) that may be 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 or analogous methods.

Table 9

Example 94

1-(3-Bromo-2-methylphenyl)-4-[(2-chloro-4-fluorophenyl)methyl]-2,3- piperazinedione (E94)

Methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (0.86 g, 2.39 mmol, prepared as described earlier) and 3-bromo-2-methylaniline (0.67 g, 3.59 mmol) were dissolved in 2% acetic acid/methanol (50 ml) at room temperature. The mixture was stirred for 5 minutes and polymer-supported cyanoborohydride (2.34 g, 9.58 mmol) was added. The mixture was stirred at room temperature for 3 days before being filtered and the filtrate concentrated in vacuo. The crude product was purified by flash-silica gel chromatography, eluting with a 0-25% gradient of methanol in dichloromethane. This purification was then repeated. The crude product was then recrystallised from methanol to afford solid product, which still contained some impurities. The mother liquors were concentrated in vacuo, purified by mass-directed automated HPLC and finally recrystallised from methanol to afford 1-(3-bromo-2- methylphenyl)-4-[(2-chloro-4-fluorophenyl)methyl]-2,3-piperazinedione (0.12 g). LC/MS [M+H]⁺ = 425/427, retention time = 2.97 minutes.

Example 95

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-chloro-5-(4-morpholinyl)phenyl]-2,3- piperazinedione (E95)

Methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (0.20 g, 0.66 mmol, prepared as described earlier) and 2-chloro-5-(4-morpholinyl)aniline (0.28 g, 1.32 mmol) were stirred in 2% acetic acid in Methanol (10 ml) for 15 minutes at room temperature. Polymer-supported cyanoborohydride (0.64 g, 2.63 mmol) was added and the reaction stirred for a total of 64 hours. The resin was filtered off and washed thoroughly with methanol. The filtrate was reduced in vacuo, then dissolved in dichloromethane. The mixture was washed with saturated aqueous sodium bicarbonate, the organic layer separated using a hydrophobic frit and reduced in vacuo. The residue was purified by flash-silica gel chromatography, eluting with a 10-100% gradient of ethyl acetate in isohexane to yield 1-[(2-chloro-4- fluorophenyl)methyl]-4-[2-chloro-5-(4-morpholinyl)phenyl]-2,3-piperazinedione (0.1 16 g) as a white solid.

LC/MS [M+H]⁺ = 452, retention time = 2.84 minutes.

Example 96 1-[(2-Chloro-4-fluorophenyl)methyl]-4-(3-chloro-2-methylphenyl)-2,3- piperazinedione (E96)

Methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (0.36 g, 1.0 mmol, prepared as described earlier) and 2-chloro-3-methylaniline (0.17 g, 1.2 mmol) were dissolved in 2% acetic acid/methanol (50 ml) at room temperature. The mixture was stirred for 5 minutes before polymer-supported cyanoborohydride (0.98 g, 4.0 mmol) was added and the mixture stirred at room temperature for 1 week. The resin was filtered off, washed with methanol and concentrated in vacuo. The residue was purified by flash-silica gel chromatography, eluting with a 0-25% gradient of methanol in dichloromethane to afford product that was impure. This material was repurified by flash-silica gel chromatography, eluting with a 0-100% gradient of ethyl acetate in isohexane to afford product that was still impure. This material was then purified by mass-directed automated HPLC to yield 1-[(2-chloro-4-fluorophenyl)methyl]-4-(3- chloro-2-methylphenyl)-2,3-piperazinedione (0.095 g) as a white solid. LC/MS [M+H]⁺ = 381 , retention time = 2.90 minutes.

Example 97

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-(dimethylamino)phenyl]-2,3- piperazinedione (E97)

Methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (0.36 g, 1.0 mmol, prepared as described earlier) and N,N-dimethyl-1 ,2-benzenediamine (0.16 g, 1.2 mmol) were dissolved in 2% acetic acid/methanol (10 ml) at room temperature. The mixture was stirred for 5 minutes and polymer-supported cyanoborohydride (0.98 g, 4.0 mmol) was added. The mixture was stirred at room temperature for 24 hours. The resin was filtered off, washed with methanol and the filtrate concentrated in vacuo. The residue was purified by flash-silica gel chromatography, eluting with a 0-25% gradient of methanol in dichloromethane. The material was then further purified by flash-silica gel chromatography, eluting with a 0-100% gradient of ethyl acetate in isohexane to afford 1-[(2-chloro-4-fluorophenyl)methyl]-4-[2- (dimethylamino)phenyl]-2,3-piperazinedione (0.252 g). LC/MS [M+H]⁺ = 376, retention time = 2.25 minutes.

The N,N-dimethyl-1 ,2-benzenediamine used in the method described above was prepared as follows:

(i) 1-Fluoro-2-nitrobenzene (3.16 ml, 30 mmol) was dissolved in tetrahydrofuran (50 ml) at O⁰C and treated dropwise with dimethylamine (2M solution in tetrahydrofuran) (45.0 ml, 90 mmol). The mixture was stirred at O⁰C for 2 hours and allowed to warm to room temperature overnight. The mixture was concentrated in vacuo and the residue was partitioned between ethyl acetate and 10% 0.880 ammonia solution. The aqueous layer was extracted with ethyl acetate (x3) and the combined organic layers were washed with water (x3) and brine. The solution was dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash-silica gel chromatography, eluting with a 0-100% gradient of ethyl acetate in hexane to afford N,N-dimethyl-2-nitroaniline (5.88 g).

(ii) N,N-dimethyl-2-nitroaniline (4.99 g, 30 mmol) was hydrogenated at 1 atmosphere hydrogen over 10% palladium on carbon paste (0.64 g, 3.0 mmol) in ethanol (100 ml) for 48 hours. The catalyst was filtered off and the filtrate concentrated in vacuo to yield N,N-dimethyl-1 ,2-benzenediamine (3.89 g) as an oil.

Example 98

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-(methyloxy)-3-(4-morpholinyl)phenyl]- 2,3-piperazinedione (E98)

A solution of methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (0.17 g, 0.58 mmol, prepared as described earlier) and 2-(methyloxy)-3-(4- morpholinyl)aniline (0.12 mg, 0.58 mmol) in 2% acetic acid in methanol (7 ml) was stirred at room temperature for 15 minutes. Polymer-supported cyanoborohydride (0.28 g, 1.15 mmol) was added and the reaction mixture stirred at room temperature for 60 hours. The resin was filtered off and washed with methanol. The filtrate was reduced in vacuo and purified by mass-directed automated HPLC to yield 1-[(2- chloro-4-fluorophenyl)methyl]-4-[2-(methyloxy)-3-(4-morpholinyl)phenyl]-2,3- piperazinedione (0.05 g) LC/MS [M+H]⁺ = 448, retention time = 2.74 min.

The 2-(methyloxy)-3-(4-morpholinyl)aniline used in the method described above was prepared as follows:

(i) Palladium (II) acetate (0.10 g, 0.44 mmol), 2,2'-bis(diphenylphosphino)-1 ,1 '- binaphthalene (0.42 g, 0.67 mmol) and cesium carbonate (2.17 g, 6.66 mmol) in tetrahydrofuran (20 ml) were stirred at room temperature for 30 minutes under argon. Morpholine (1.16 ml, 13.3 mmol) was added, followed by 1-bromo-2-(methyloxy)-3- nitrobenzene (1.03 g, 4.44 mmol) and the mixture stirred for 72 hours at reflux. The reaction mixture was allowed to cool, reduced in vacuo and partitioned between ethyl acetate and water. The organic layer was washed with water and separated, then dried over sodium sulfate and reduced in vacuo. The residue was purified by flash- silica gel chromatography, eluting with a 5-15% gradient of ethyl acetate in hexane to yield 4-[2-(methyloxy)-3-nitrophenyl]morpholine (0.64 g) as a bright yellow solid.

(ii) A mixture of 4-[2-(methyloxy)-3-nitrophenyl]morpholine (0.62 g, 2.60 mmol) and palladium on carbon (0.12 g, 0.11 mmol) in ethyl acetate (30 ml) was stirred at room temperature under hydrogen for 8 hours. The catalyst was filtered off and washed with ethyl acetate. The residue was purified by flash-silica gel chromatography, eluting with a 0-2% gradient of methanol in dichloromethane. The product fractions were combined and extracted into 3N citric acid (x3 washes). The combined aqueous layers were adjusted to pH 10 by the addition of 2N sodium hydroxide. The mixture was then extracted with dichloromethane (x2) and separated using a hydrophobic frit. The organic layer was reduced in vacuo to yield 2-(methyloxy)-3-(4- morpholinyl)aniline (0.122 g) as an orange gum.

Example 99 i-^-Chloro^-fluorophenylJ^-^-chloro^-fluorophenylJmethyl^S- piperazinedione (E99)

Methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (0.29 g, 1.0 mmol, prepared as described earlier) and 2-chloro-4-fluoraniline (0.29 g, 2.0 mmol) were dissolved in 2% acetic acid/methanol (10ml) at room temperature. The mixture was stirred for 15 minutes before polymer-supported cyanoborohydride (0.98 g, 4.0 mmol) was added. The mixture was stirred at room temperature overnight, then filtered through an SCX cartridge (Varian, 5g) and the filtrate concentrated in vacuo. The residue was then dissolved in 1-butanol (1.5 ml) and heated to 200 ⁰C by microwave irradiation for 1 hour. The mixture was concentrated in vacuo and purified by mass-directed automated HPLC to yield 1-(2-chloro-4-fluorophenyl)-4-[(2-chloro- 4-fluorophenyl)methyl]-2,3-piperazinedione (0.161 g). LC/MS [M+H]⁺ = 385, retention time = 2.78 min.

Example 100

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(2-iodophenyl)-2,3-piperazinedione

(E100)

Methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (0.29 g, 1.0 mmol, prepared as described earlier) and 2-iodoaniline (0.30 g, 1.38 mmol) were dissolved in 2% acetic acid/methanol (10ml) at room temperature. The mixture was stirred for 15 minutes and polymer-supported cyanoborohydride (0.98 g, 4.0 mmol) was added. The mixture was stirred at room temperature overnight and filtered through an SCX cartridge (Varian, 5g). The filtrate was concentrated in vacuo, dissolved in 1-butanol (1.5 ml) and heated to 200 ⁰C in the microwave for 2 hours. The residue was purified by mass-directed automated HPLC to yield 1-[(2-chloro-4- fluorophenyl)methyl]-4-(2-iodophenyl)-2,3-piperazinedione (0.012 g).

LC/MS [M+H]⁺ = 458.9, retention time = 2.80 min. Example 101

2-{4-[(2-Chloro-4-fluorophenyl)methyl]-2,3-dioxo-1 -piperazinyl}-6- fluorobenzonitrile (E101)

Methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (0.15 g, 0.37 mmol, prepared as described earlier) in acetic acid (1 ml) was added to a solution of 2-amino-6-fluorobenzonitrile (0.050 g, 0.37 mmol) in acetic acid (1 ml) and stirred at room temperature for 30 minutes. The reaction was then cooled to O⁰C and sodium borohydride (0.014 g, 0.38 mmol) was added. The reaction was warmed to room temperature and heated to 8O⁰C with stirring overnight. The reaction was allowed to cool, water (5 ml) was added and the reaction stirred for 10 minutes. The mixture was extracted with dichloromethane (2 x 10 ml) and separated by hydrophobic frit. The solution was concentrated in vacuo and purified by mass-directed automated HPLC, followed by freeze drying to yield 2-{4-[(2-chloro-4-fluorophenyl)methyl]-2,3- dioxo-1-piperazinyl}-6-fluorobenzonitrile (0.0221 g) as a pale yellow solid. LC/MS [M+H]⁺ = 376, retention time = 2.61 minutes.

Examples 102-103 (E102-103)

In a manner analogous to that described for Example 101 above the compounds tabulated below (Table 10) were prepared by substituting the appropriate anilines (or protected anilines) for the 2-amino-6-fluorobenzonitrile used in the above procedure. The anilines or protected anilines that may be 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 or analogous methods.

Table 10

Example 104

1-(5-Bromo-2<:hlorophenyl)-4-[(2-chloro-4-fluorophenyl)methyl]-2,3- piperazinedione (E104)

5-Bromo-2-chloroaniline (1.767 g, 8.55 mmol) was added to a solution of methyl [[(2- chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (2.46 g, 8.55 mmol, prepared as described earlier) in acetic acid (40 ml) and stirred for 5 minutes. The reaction was cooled to 0⁰C and sodium borohydride (0.34 g, 8.89 mmol) was added slowly, then stirred at 80⁰C for 3 hours. The mixture was allowed to cool, then excess acetic acid was removed in vacuo, azeotroping with methanol. The residue was taken up in dichloromethane and washed with saturated aqueous sodium bicarbonate, then 3N citric acid (a small amount of brine was added to aid the separation). The organic layer was separated using a hydrophobic frit and reduced in vacuo. The residue was purified by flash-silica gel chromatography to yield 1-(5- bromo-2-chlorophenyl)-4-[(2-chloro-4-fluorophenyl)methyl]-2,3-piperazinedione

(0.584 g) as a light yellow solid.

LC/MS [M+H]⁺ = 446.9, retention time = 2.95 min.

Example 105

1-(2-Bromophenyl)-4-[(2-chloro-4-fluorophenyl)methyl]-2,3-piperazinedione

(E105)

Methyl {{2-[(2-bromophenyl)amino]ethyl}[(2-chloro-4-fluorophenyl)methyl]amino}(oxo) acetate (0.065 g, 0.132 mmol) was dissolved in acetic acid (2 ml) and heated to reflux at 105⁰C overnight. The reaction mixture was purified by flash-silica gel column chromatography, eluting with a 0-100% gradient of ethyl acetate in hexane. Product fractions were concentrated in vacuo to yield 1-(2-bromophenyl)-4-[(2-chloro- 4-fluorophenyl)methyl]-2,3-piperazinedione (0.013 g) as a gum. LC/MS [M+H]⁺ = 412.9, retention time = 2.74 minutes.

The methyl {{2-[(2-bromophenyl)amino]ethyl}[(2-chloro-4-fluorophenyl)methyl]amino} (oxo)acetate used in the method described above was prepared as follows:

Methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (0.20 g, 0.60 mmol, prepared as described earlier) was dissolved in dichloromethane (10 ml) and cooled to 0⁰C in an ice bath. 4A molecular sieves were added, then 2-bromoaniline (0.10 g, 0.60 mmol) followed by acetic acid (0.069 ml, 1.20 mmol) and finally sodium triacetoxyborohydride (0.19 g, 0.90 mmol). The reaction mixture was allowed to warm to room temperature and stirred overnight. Saturated aqueous sodium bicarbonate (10 ml) was added and reaction mixture stirred vigorously. The aqueous layer was removed by pipette and the remaining reaction mixture eluted through a HydroMatrix cartridge. Fractions combined and concentrated in vacuo to yield methyl {{2-[(2-bromophenyl)amino]ethyl}[(2-chloro-4-fluorophenyl)methyl]amino}(oxo) acetate (0.25 g), which was used without purification.

Example 106 1-[(2-Chloro-4-fluorophenyl)methyl]-4-(5-quinolinyl)-2,3-piperazinedione (E106)

Methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (0.40 g, 1.39 mmol, prepared as described earlier) was dissolved in 1 ,2-dichloroethane (20 ml) and 5-aminoquinoline (0.40 g, 2.78 mmol) and acetic acid (0.08 ml, 1.39 mmol) were added. The mixture was stirred for 10 minutes before polymer-supported cyanoborohydride (1.74 g, 6.95 mmol) was added and the reaction was stirred at room temperature for approximately 64 hours. The mixture was then to reflux at 85⁰C for 7 hours. The reaction mixture was then concentrated in vacuo and redissolved in 1-butanol (3 ml). The solution was heated to 16O⁰C for 1 hour in the microwave. The mixture was concentrated in vacuo and purified by flash-silica gel chromatography. The mixture was eluted with a 0-100% gradient of ethyl acetate in hexane followed by a flush of 5% methanol in DCM, then 10% methanol in DCM. Clean product fractions were combined and concentrated in vacuo to give a brown oil, which was triturated with a mixture of diethyl ether (10 ml) and methanol (2 ml) to yield 1 -[(2-chloro-4-fluorophenyl)methyl]-4-(5-quinolinyl)-2,3-piperazinedione (0.081 g) as a golden-brown solid. LC/MS [M+H]⁺ = 384, retention time = 2.12 minutes.

Example 107

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(4-isoquinolinyl)-2,3-piperazinedione

(E107)

Methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (0.40 g, 1.39 mmol, prepared as described earlier), 4-aminoisoquinoline (0.40 g, 2.78 mmol) and acetic acid (0.08 ml, 1.39 mmol) were dissolved in 1 ,2-dichloroethane (20 ml) and stirred for 10 minutes. Polymer-supported cyanoborohydride (1.74 g, 6.95 mmol) was added and the reaction stirred at room temperature for approximately 64 hours. The reaction was then heated to reflux at 85⁰C for 7 hours. The reaction mixture was then concentrated in vacuo and redissolved in 1-butanol (3 ml). The solution was heated to 16O⁰C for 1 hour in the microwave. The mixture was concentrated in vacuo and purified by flash-silica gel chromatography. The mixture was eluted with ethyl acetates followed by 5% methanol in dichloromethane and finally 10% methanol in dichloromethane. Clean product fractions were combined and concentrated in vacuo to give a brown oil, which was triturated with a mixture of diethyl ether (10 ml) and methanol (2 ml) to yield 1-[(2-chloro-4-fluorophenyl)methyl]-4-(4-isoquinolinyl)-2,3- piperazinedione (0.032 g) as a cream-coloured solid. LC/MS [M+H]⁺ = 384, retention time = 2.29 minutes.

Example 108 1 -[(2-Chloro-4-fluorophenyl)methyl]-4-[2-(1 ,1 -dimethylethyl)phenyl]-2,3- piperazinedione (E108)

[[(2-Chloro-4-fluorophenyl)methyl](2-{[2-(1 ,1- dimethylethyl)phenyl]amino}ethyl)amino](oxo)acetyl chloride (0.126 g, 0.296 mmol) was dissolved in acetonitrile (10 ml) and imidazole (0.040 g, 0.592 mmol) added.

The reaction was stirred overnight and filtered through an SCX cartridge (Varian, 5g).

The filtrate was concentrated in vacuo, dissolved in 1-butanol (1.5 ml) and heated to

200⁰C in the microwave for 1.5 hours. The mixture was concentrated in vacuo and purified by mass-directed automated HPLC to yield 1-[(2-chloro-4- fluorophenyl)methyl]-4-[2-(1 ,1-dimethylethyl)phenyl]-2,3-piperazinedione (0.037 g).

LC/MS [M+H]⁺ = 389, retention time = 3.08 minutes.

The [[(2-chloro-4-fluorophenyl)methyl](2-{[2-(1 ,1- dimethylethyl)phenyl]amino}ethyl)amino](oxo)acetyl chloride used in the method described above was prepared as follows: (i) Methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (0.288 g, 1 mmol, prepared as described earlier) and 2-(1 ,1-dimethylethyl)aniline (0.298 g, 2.0 mmol) were dissolved in 2% acetic acid/methanol (10 ml) at room temperature. The mixture was stirred for 15 minutes and polymer-supported cyanoborohydride (0.976 g, 4.00 mmol) was added. The mixture was stirred at room temperature for 48 hours and filtered through an SCX cartridge (Varian, 5g). The filtrate was concentrated in vacuo to afford methyl [[(2-chloro-4-fluorophenyl)methyl](2-{[2-(1 ,1- dimethylethyl)phenyl]amino}ethyl)amino](oxo)acetate (0.236 g). LC/MS [M+H]⁺ = 421/423, retention time = 3.67 minutes.

(ii) Methyl [[(2-chloro-4-fluorophenyl)methyl](2-{[2-(1 ,1-dimethylethyl)phenyl]amino} ethyl)amino](oxo)acetate (0.1 18 g, 0.280 mmol) was dissolved in tetrahydrofuran (5 ml) and water (5 ml). Lithium hydroxide monohydrate (0.0129 g, 0.308 mmol) was subsequently added and the reaction mixture stirred overnight. The mixture was then concentrated in vacuo and azeotroped with Toluene to yield [[(2-chloro-4- fluorophenyl)methyl](2-{[2-(1 ,1-dimethylethyl)phenyl]amino}ethyl)amino](oxo)acetic acid (0.121 g), which was used without purification.

(iii) [[(2-chloro-4-fluorophenyl)methyl](2-{[2-(1 ,1-dimethylethyl)phenyl]amino}ethyl) amino](oxo)acetic acid (0.12 g, 0.297 mmol) was dissolved in thionyl chloride (2 ml,

27.5 mmol) and heated at 40 ⁰C for 3 hours. The mixture was concentrated in vacuo and azeotroped with toluene to afford [[(2-chloro-4-fluorophenyl)methyl](2-{[2-(1 ,1- dimethylethyl)phenyl]amino}ethyl)amino](oxo)acetyl chloride (0.126 g), which was used without purification.

Example 109

1 -[(2-Chloro-4-fluorophenyl)methyl]-4-{2-[(trifluoromethyl)oxy]phenyl}-2,3- piperazinedione (E109)

Methyl {[(2-chloro-4-fluorophenyl)methyl][2-({2-[(trifluoromethyl)oxy]phenyl}amino) ethyl]amino}(oxo)acetate (0.155 g, 0.345 mmol) was dissolved in 1-butanol (3 ml) and heated to 200 ⁰C in the microwave for 100 minutes and then concentrated in vacuo. The residue was purified by mass-directed automated HPLC to afford 0.070 g of material,

In parallel, methyl {[(2-chloro-4-fluorophenyl)methyl][2-({2-[(trifluoromethyl)oxy] phenyl}amino)ethyl]amino}(oxo)acetate (0.138 g, 0.307 mmol) was dissolved in methanol (3 ml) and heated to 130 ⁰C in the microwave for 2 hours and then concentrated in vacuo. The residue was purified by mass-directed automated HPLC to afford 0.043 g of material.

The two batches outlined above were combined, before being further purified by- directed automated HPLC to yield 1-[(2-chloro-4-fluorophenyl)methyl]-4-{2- [(trifluoromethyl)oxy]phenyl}-2,3-piperazinedione (0.093 g). LC/MS [M+H]⁺ = 417, retention time = 2.90 minutes.

The methyl {[(2-chloro-4-fluorophenyl)methyl][2-({2-[(trifluoromethyl)oxy]phenyl} amino)ethyl]amino}(oxo)acetate used in the method described above was prepared as follows:

Methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (0.288 g, 1 mmol, prepared as described earlier) and 2-[(trifluoromethyl)oxy]aniline (0.354 g, 2.00 mmol) were dissolved in 2% acetic acid/methanol (10 ml) at room temperature. The mixture was stirred for 15 minutes and polymer-supported cyanoborohydride (0.976 g, 4.00 mmol) was added. The mixture was stirred at room temperature for 48 hours and filtered through an SCX cartridge (Varian, 5g). The filtrate was concentrated in vacuo to afford methyl {[(2-chloro-4-fluorophenyl)methyl][2-({2- [(trifluoromethyl)oxy]phenyl}amino)ethyl]amino}(oxo)acetate (0.293 g), which was used without purification.

Example 110

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-chloro-3-(methyloxy)phenyl]-2,3- piperazinedione (E110)

Methyl [[(2-chloro-4-fluorophenyl)methyl](2-{[2-chloro-3-(methyloxy)phenyl]amino} ethyl)amino](oxo)acetate (0.289 g, 0.673 mmol) was dissolved in 1-butanol (2 ml) and heated at 200 ⁰C in a microwave reactor for 2 hours. The mixture was concentrated in vacuo and purified by mass-directed automated HPLC. The material was then further purified by flash-silica gel chromatography, eluting with a 0-25% gradient of methanol in dichloromethane) to afford 1-[(2-chloro-4- fluorophenyl)methyl]-4-[2-chloro-3-(methyloxy)phenyl]-2,3-piperazinedione (0.117 g). LC/MS [M+H]⁺ = 396.9, retention time = 2.77 minutes.

The methyl [[(2-chloro-4-fluorophenyl)methyl](2-{[2-chloro-3-(methyloxy)phenyl] amino}ethyl)amino](oxo)acetate used in the method described above was prepared as follows:

(i) 3-Nitrophenol (5 g, 35.9 mmol) was warmed to 5O⁰C in concentrated hydrochloric acid (50 ml) until dissolved. The solution was rapidly cooled in an ice-salt bath to produce a fine precipitate. Potassium chlorate (4.40 g, 35.9 mmol) dissolved in water (50 ml) was added dropwise at 0 ⁰C and the solution stirred at this temperature for 2 hours. The aqueous solution was extracted with ethyl acetate (x3), combined extracts washed with water, brine, dried over anhydrous sodium sulfate and concentrated to an orange oil that solidified upon standing. The crude product was purified by flash-silica gel chromatography, eluting with a 0-100% gradient of ethyl acetate in hexane to afford 2-chloro-3-nitrophenol (2.61 g).

(ii) 2-Chloro-3-nitrophenol (2.61 g, 15.04 mmol) and potassium carbonate (3.12 g, 22.56 mmol) were suspended in acetone (50 ml). Methyl iodide (1.034 ml, 16.54 mmol) was added and the mixture heated at 70 ⁰C for 4 hours. The solids were filtered, washed with acetone and the filtrate concentrated to a solid. The crude product was purified by flash-silica gel chromatography, eluting with a 0-100% gradient of ethyl acetate in isohexane to afford 2-chloro-3-nitrophenyl methyl ether (2.44 g). (iii) Iron powder (3.63 g, 65.0 mmol) was suspended in acetic acid (50 ml) and warmed to 4O⁰C with mechanical stirring. 2-Chloro-3-nitrophenyl methyl ether (2.44 g, 13.01 mmol) dissolved in acetic acid (25 ml) was added at such a rate as to maintain a temperature of 45-5O⁰C. The mixture was heated at 80 ⁰C for 2 hours after the addition had been completed. The mixture was filtered through celite and concentrated in vacuo. The residue was partitioned between ethyl acetate and 10% 0.880 ammonia solution. Solids were removed by filtration through celite and the aqueous extracted in to ethyl acetate (x3). The combined extracts were washed with water, brine, dried over anhydrous sodium sulfate and concentrated to an oil. The crude product was purified by flash-silica gel chromatography, eluting with a 0-100% gradient of ethyl acetate in isohexane to afford 2-chloro-3-(methyloxy)aniline (1.66 g).

(iv) Methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (0.288 g, 1 mmol, prepared as described earlier) and 2-chloro-3-(methyloxy)aniline (0.315 g,

2.00 mmol) were dissolved in 2% acetic acid/methanol (10ml) at room temperature.

The mixture was stirred for 15 minutes and polymer-supported cyanoborohydride

(0.976 g, 4.00 mmol) was added. The mixture was stirred at room temperature overnight, filtered through an SCX cartridge (Varian, 5g) and concentrated in vacuo to afford methyl [[(2-chloro-4-fluorophenyl)methyl](2-{[2-chloro-3-(methyloxy)phenyl] amino}ethyl)amino](oxo)acetate (0.289 g), which was used without purification.

Example 111

1 -[(2-Chloro-4-fluorophenyl)methyl]-4-(4-methyl-3,4-dihydro-2H-1,4-benzoxazin- 5-yl)-2,3-piperazinedione (E111)

Methyl ([(2-chloro-4-fluorophenyl)methyl]{2-[(4-methyl-3,4-dihydro-2H-1 ,4- benzoxazin-5-yl)amino]ethyl}amino)(oxo)acetate (0.482 g, 1.106 mmol) was dissolved in 1-butanol (2 ml) and heated at 200 ⁰C in a microwave reactor for 2 hours. The mixture was concentrated in vacuo and purified by mass-directed automated HPLC; then by flash-silica gel chromatography, eluting with a 0-25% gradient of methanol in dichloromethane to afford 1-[(2-chloro-4-fluorophenyl)methyl]- 4-(4-methyl-3,4-dihydro-2/-/-1 ,4-benzoxazin-5-yl)-2,3-piperazinedione (0.079 g). LC/MS [M+H]⁺ = 404, retention time = 2.77 minutes.

The methyl ([(2-chloro-4-fluorophenyl)methyl]{2-[(4-methyl-3,4-dihydro-2H-1 ,4- benzoxazin-5-yl)amino]ethyl}amino)(oxo)acetate used in the method described above was prepared as follows:

(i) 2-Amino-3-nitrophenol (5 g, 32.4 mmol) and 1 ,2-dibromoethane (4.47 ml, 51.9 mmol) were heated to 125 ⁰C in N,N-dimethylformamide (20 ml). Solid potassium hydroxide (3.64 g, 64.9 mmol) was added portionwise. The mixture was heated at

125 ⁰C for 48 hours, cooled and poured on to ice. Extracted in to ethyl acetate (x3), combined extracts washed with water (x3), 50% brine, brine, dried over anhydrous sodium sulfate and concentrated to a red solid. The solid was triturated in dichloromethane and filtered. The filtrate was concentrated in vacuo and purified by flash-silica gel chromatography, eluting with a 0-100% gradient of ethyl acetate in isohexane to afford 5-nitro-3,4-dihydro-2H-1 ,4-benzoxazine (1.04 g).

(ii) Sodium hydride (60% in mineral oil, 0.254 g, 6.35 mmol) was suspended in N, N- dimethylformamide (5 ml) at O⁰C. 5-Nitro-3,4-dihydro-2H-1 ,4-benzoxazine (1.04 g,

5.77 mmol) dissolved in N,N-dimethylformamide (20 ml) was added dropwise and the solution allowed to reach room temperature over 15 minutes. The solution was cooled to O⁰C and iodomethane (0.397 ml, 6.35 mmol) was added. The solution was stirred at room temperature for 4 hours and the bulk of the dimethylformamide removed in vacuo. The residue was partitioned between ethyl acetate and water.

The aqueous phase was extracted with ethyl acetate (x3). The combined extracts were washed with water (x3), brine, dried over anhydrous sodium sulfate and concentrated in vacuo to a red oil. The crude product was purified by flash-silica gel chromatography, eluting with a 0-100% gradient of ethyl acetate in isohexane to afford 4-methyl-5-nitro-3,4-dihydro-2H-1 ,4-benzoxazine (1.06 g).

(iii) 4-Methyl-5-nitro-3,4-dihydro-2H-1 ,4-benzoxazine (1.06 g) was dissolved in ethanol (30 ml) and hydrogenated at room temperature over 10% palladium on carbon paste (0.116 g, 0.546 mmol) under an atmosphere of hydrogen at 1 atmosphere for 7 hours. The catalyst was filtered and the filtrate concentrated to yield 4-methyl-3,4-dihydro-2H-1 ,4-benzoxazin-5-amine (0.835 g). (iv) Methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (0.288 g, 1 mmol, prepared as described earlier) and 4-methyl-3,4-dihydro-2H-1 ,4-benzoxazin- 5-amine (0.328 g, 2.00 mmol) were dissolved in 2% acetic acid/methanol (10ml) at room temperature. The mixture was stirred for 15 minutes and polymer-supported cyanoborohydride (0.976 g, 4.00 mmol) was added. The mixture was stirred at room temperature overnight, filtered through an SCX cartridge (Varian, 5g) and concentrated in vacuo to afford methyl ([(2-chloro-4-fluorophenyl)methyl]{2-[(4- methyl-3,4-dihydro-2H-1 ,4-benzoxazin-5-yl)amino]ethyl}amino)(oxo)acetate (0.482 g), which was used without purification.

Example 112

1 -[(2-Chloro-4-fluorophenyl)methyl]-4-[2-(trifluoromethyl)phenyl]-2,3- piperazinedione (E112)

Methyl [[(2-chloro-4-fluorophenyl)methyl](2-{[2-(trifluoromethyl)phenyl]amino}ethyl) amino](oxo)acetate (0.385 g, 0.89 mmol) was dissolved in 1-butanol (2 ml) was heated at 200 ⁰C in a microwave reactor for 8 hours. The mixture was concentrated in vacuo and purified by mass-directed automated HPLC to yield 1-[(2-chloro-4- fluorophenyl)methyl]-4-[2-(trifluoromethyl)phenyl]-2,3-piperazinedione (0.008 g) LC/MS [M+H]⁺ = 400.9, retention time = 2.85 minutes.

The methyl [[(2-chloro-4-fluorophenyl)methyl](2-{[2-(trifluoromethyl)phenyl]amino} ethyl)amino](oxo)acetate used in the method described above was prepared as follows:

Methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (0.288 g, 1 mmol) and 2-(trifluoromethyl)aniline (0.251 ml, 2.00 mmol) were dissolved in 2% acetic acid/methanol (10ml) at room temperature. The mixture was stirred for 15 minutes and polymer-supported cyanoborohydride (0.976 g, 4.00 mmol) was added. The mixture was stirred at room temperature overnight, filtered through an SCX cartridge (Varian, 5g) and concentrated in vacuo to afford methyl [[(2-chloro-4- fluorophenyl)methyl](2-{[2-(trifluoromethyl)phenyl]amino}ethyl)amino](oxo)acetate (0.385 g), which was used without purification.

Example 113 1-[(2-Chloro-4-fluorophenyl)methyl]-4-[4-(diethylamino)-2-methylphenyl]-2,3- piperazinedione (E113)

A solution of methyl [[(2-chloro-4-fluorophenyl)methyl](2-oxoethyl)amino](oxo)acetate (0.117M in dichloromethane, 8.55 ml, 1 mmol, prepared as described earlier) and 4 Angstrom molecular sieves (0.30 g) were added to (4-amino-3- methylphenyl)diethylamine hydrochloride (0.236 g, 1.100 mmol) in dichloromethane (10 ml), containing diisopropylethylamine (0.192 ml, 1.100 mmol). Acetic acid (0.229 ml, 4.00 mmol) was added and the mixture was stirred for 5 minutes. Sodium triacetoxyborohydride (0.318 g, 1.500 mmol) was added and the mixture stirred overnight. The reaction mixture was stirred with sodium bicarbonate solution (10 ml) for 15 minutes and filtered through a hydrophobic frit. The filtrate was concentrated and purified by flash-silica gel chromatography, eluting with a 0-100% gradient of ethyl acetate in hexane to afford a crude oil. The oil was twice purified by mass- directed automated HPLC afford 1-[(2-chloro-4-fluorophenyl)methyl]-4-[4- (diethylamino)-2-methylphenyl]-2,3-piperazinedione (0.095 g). LC/MS [M+H]⁺ = 418, retention time = 1.96 minutes.

Example 114 1-(2,3-Dichlorophenyl)-4-[(2-methylphenyl)methyl]-2,3-piperazinedione (E114)

1-(2,3-dichlorophenyl)-2,3-piperazinedione (0.1 1 g, 0.42 mmol) was dissolved in dimethylformamide (4 ml) with stirring at room temperature. To the resulting solution was added sodium hydride (0.02 g, 0.50 mmol) at O C. The mixture was allowed to stir for approx. 15 minutes at room temperature, before adding 1-(bromomethyl)-2- methylbenzene (0.08 ml, 0.62 mmol). The reaction temperature was raised to 50 C, and left to stir for approx. 15 hours. The reaction was quenched with saturated aqueous ammonium chloride (5 ml). Dichloromethane (10 ml) was added, and the layers mixed thoroughly with vigorous stirring. The organic layer was separated and the solvent removed. The residue was suspended in dichloromethane and a white precipitate filtered off. The filtrate was concentrated in vacuo and partitioned between water (10 ml) and dichloromethane (10 ml). The organic layer was removed and concentrated in vacuo. The residue was purified by mass-directed automated HPLC to yield 1-(2,3-dichlorophenyl)-4-[(2-methylphenyl)methyl]-2,3-piperazinedione (0.066 g) as a white solid. LC/MS [M+H]⁺ = 363, retention time = 2.83 minutes.

The 1-(2,3-dichlorophenyl)-2,3-piperazinedione used in the method described above was prepared as follows:

(i) N-Boc-2-aminoacetaldehyde (2.0 g, 12.6 mmol) was dissolved in dichloromethane (50 ml). 4A molecular sieves (0.3g) were added and the solution cooled to O⁰C. 2,3-dichloroaniline (2.14 g, 13.2 mmol) and sodium triacetoxyborohydride (3.99 g, 18.9 mmol) were added and the suspension warmed to room temperature. The reaction was stirred for 16 hours. The reaction was quenched with saturated aqueous sodium bicarbonate (30 ml) and stirred for 20 minutes. The organic layer was separated using a hydrophobic frit and concentrated in vacuo. The residue was purified by flash-silica gel column chromatography, eluting with a 0-70% gradient of ethyl acetate in hexane to yield 1 ,1-dimethylethyl {2- [(2,3-dichlorophenyl)amino]ethyl}carbamate (1.52 g) as a pale yellow oil.

(ii) 1 ,1-Dimethylethyl {2-[(2,3-dichlorophenyl)amino]ethyl}carbamate (1.52 g, 4.97 mmol) was dissolved in dichloromethane (20 ml). Triethylamine (0.76 ml, 5.47 mmol) was added and the solution cooled to O⁰C. Methyl chlorooxoacetate (0.51 ml, 5.47 mmol) was added and the yellow solution was allowed to warm to room temperature and stirred for 3 hours. The reaction mixture was washed with water (10 ml), the organic layer separated using a hydrophobic frit and concentrated in vacuo. The residue was purified by flash-silica gel column chromatography, eluting a 0-35% gradient of ethyl acetate in hexane to methyl {(2,3-dichlorophenyl)[2-({[(1 ,1- dimethylethyl)oxy]carbonyl}amino)ethyl]amino}(oxo)acetate (0.64 g) as an orange oil.

(iii) Methyl {(2,3-dichlorophenyl)[2-({[(1 ,1-dimethylethyl)oxy]carbonyl}amino)ethyl] amino}(oxo)acetate (0.64 g, 1.64 mmol) was dissolved in 4 Molar hydrochloric acid in dioxane (4 ml, 16.0 mmol) and stirred at room temperature for 2 hours. The reaction mixture was concentrated in vacuo to yield an orange oil, which was dissolved in dichloromethane (20 ml) and triethylamine (0.46 ml, 3.28 mmol) was added. The solution was stirred for 90 minutes at room temperature and then concentrated in vacuo. The residue was purified by flash-silica gel column chromatography, eluting with 10% methanol in dichloromethane to yield crude 1-(2,3-dichlorophenyl)-2,3- piperazinedione (0.453 g) as a pale orange solid, which was used without further purification.

Examples 115-118 (E115-E118)

In a manner analogous to that described for Example 114 above the compounds tabulated below (Table 1 1 ) were prepared by substituting the appropriate benzyl bromide for the 1-(bromomethyl)-2-methylbenzene used in the above procedure. The benzyl bromides that may be used to make the compounds shown in Table 1 1 are available from commercial sources or can be prepared using routes described previously in the chemical literature or analogous methods.

Table 1 1

Example 119

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(2-methyl-3-pyridinyl)-2,3- piperazinedione (E119)

1-(2-Methyl-3-pyridinyl)-2,3-piperazinedione (0.11 g, 0.53 mmol) was dissolved in N,N-dimethylformamide (5 ml) and cooled to O⁰C. Sodium hydride (0.025 g, 0.63 mmol) was added and the mixture stirred for 10 minutes, until the fizzing stopped. 1- (bromomethyl)-2-chloro-4-fluorobenzene (0.18 g, 0.79 mmol) was added and the reaction warmed to room temperature, before being heated to 50⁰C with stirring for 3 hours. The reaction mixture was concentrated in vacuo and the residue partitioned between dichloromethane (15ml) and water (10ml). The organic layer was separated using a hydrophobic frit and the aqueous layer washed with further dichloromethane (10ml). The combined organic layers were concentrated in vacuo and purified by mass-directed automated HPLC. The product was dissolved in methanol (~15ml) and passed down a pre-conditioned 10g aminopropyl cartridge. The cartridge was washed with methanol (40 ml) and the solution concentrated under vacuum to yield 1-[(2-chloro-4-fluorophenyl)methyl]-4-(2-methyl-3-pyridinyl)-2,3-piperazinedione (0.043 g) as a yellow solid. LC/MS [M+H]⁺ = 348, retention time = 1.83 minutes.

The 1-(2-methyl-3-pyridinyl)-2,3-piperazinedione used in the method described above was prepared as follows:

(i) 3-Amino-2-methylpyridine (0.61 g, 5.65 mmol) was dissolved in dichloromethane (25 ml). 4A molecular sieves (0.2 g), 1 ,1-dimethylethyl(2-oxoethyl)carbamate (1.0 g, 6.28 mmol), acetic acid (0.54 ml, 9.42 mmol) and sodium triacetoxyborohydride (2.0 g, 9.42 mmol) were added and the reaction was stirred at room temperature for approximately 65 hours. The reaction was quenched with saturated aqueous sodium bicarbonate (15 ml) and stirred for 5 minutes. The organic layer was separated using a hydrophobic frit and concentrated in vacuo to give 1 ,1-dimethylethyl {2-[(2-methyl- 3-pyridinyl)amino]ethyl}carbamate (1.56 g) as an orange gum, which was used without purification.

(ii) 1 ,1-Dimethylethyl {2-[(2-methyl-3-pyridinyl)amino]ethyl}carbamate (1.56 g, 6.20 mmol) was dissolved in dichloromethane (30 ml) and cooled to O⁰C. Triethylamine (0.86 ml, 6.20 mmol) and methyl chlorooxoacetate (0.69 ml, 7.43 mmol) were added and the reaction was warmed to room temperature and stirred for 3 hours. The reaction mixture was washed with water, the organic layer was separated using a hydrophobic frit and concentrated in vacuo. The residue was purified by flash silica gel chromatography, eluting with a 5-100% gradient of ethyl acetate in hexane to yield methyl [[2-({[(1 ,1-dimethylethyl)oxy]carbonyl}amino)ethyl](2-methyl-3- pyridinyl)amino](oxo)acetate (0.55 g) as a yellow gum. (iii) Methyl [[2-({[(1 ,1-dimethylethyl)oxy]carbonyl}amino)ethyl](2-methyl-3- pyridinyl)amino](oxo)acetate (0.55 g, 1.62 mmol) was suspended in 4 Molar hydrochloric acid in 1 ,4-dioxane (4.0 ml, 16.0 mmol) and stirred at room temperature for 2 hours. The reaction mixture was concentrated in vacuo and the solid residues suspended in dichloromethane (50 ml). To this was added triethylamine (0.45 ml, 3.23 mmol) and the mixture stirred at room temperature for 3 hours. After this time a small amount of undissolved material remained, so methanol (3ml) was added and the solution stirred for a further 1 hour. The reaction mixture was concentrated in vacuo and the residue dissolved in dichloromethane (-5OmI). This was washed with saturated aqueous sodium bicarbonate (~30ml). The organic layer was separated using a hydrophobic frit and concentrated in vacuo to give crude 1-(2-methyl-3- pyridinyl)-2,3-piperazinedione (0.11 g) as an orange solid, which was used without purification.

Example 120

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(3-methyl-4-pyridinyl)-2,3- piperazinedione (E120)

N-[(2-chloro-4-fluorophenyl)methyl]-N'-(3-methyl-4-pyridinyl)-1 ,2-ethanediamine (0.39 g, 1.33 mmol) was dissolved in dichloromethane (12 ml) and cooled to 0 ⁰C. Oxalyl chloride (0.23 ml, 2.66 mmol) was added dropwise and the resulting suspension stirred for 1 hour. Triethylamine (0.19 ml, 1.33 mmol) was then added and the reaction warmed to room temperature and stirred for a further 2 hours. The reaction was quenched by adding saturated aqueous sodium bicarbonate (20 ml) and the mixture extracted with dichloromethane (50 ml). The organic layer was separated using a hydrophobic frit and concentrated in vacuo. The residue was purified by flash-silica gel column chromatography, eluting a 0-10% gradient of 2M ammonia/methanol solution in dichloromethane, followed by trituration with diethyl ether (2 x 10 ml) to yield 1-[(2-chloro-4-fluorophenyl)methyl]-4-(3-methyl-4-pyridinyl)- 2,3-piperazinedione (0.1121 g) as a white solid.

LC/MS [M+H]⁺ = 348, retention time = 1.78 minutes. The N-[(2-chloro-4-fluorophenyl)methyl]-N'-(3-methyl-4-pyridinyl)-1 ,2-ethanediamine used in the method described above was prepared as follows:

(i) 4-chloro-3-methylpyridine (0.44 g, 2.65 mmol) and ethylenediamine (0.90 ml, 13.3 mmol) were dissolved in 1-methyl-2-pyrrolidone (2 ml) in a microwave vial. The vessel was sealed and heated to 220 ⁰C for 1 hr in the microwave. The reaction mixture was diluted with methanol (20 ml) and loaded onto 2x1 Og pre-conditioned

SCX cartridges. These were washed with methanol (2x40 ml) followed by 2 Molar ammonia in methanol (40 ml). The ammonia fractions were concentrated in vacuo to N-(3-methyl-4-pyridinyl)-1 ,2-ethanediamine (0.37 g) as a yellow solid.

(ii) N-(3-methyl-4-pyridinyl)-1 ,2-ethanediamine (0.37 g, 2.43 mmol) was suspended in dichloromethane (50 ml). 4A molecular sieves (0.20 g), 2-chloro-4- fluorobenzaldehyde (0.46 g, 2.92 mmol), sodium triacetoxyborohydride (0.77 g, 3.65 mmol) and acetic acid (0.42 ml, 7.30 mmol) were added and the reaction stirred at room temperature for 20 hours. The reaction was quenched with saturated aqueous sodium bicarbonate (30 ml) and stirred for 10 minutes. The organic layer was separated using a hydrophobic frit and concentrated in vacuo to give a yellow oil. This was dissolved in methanol and loaded onto a pre-conditioned 10 g SCX cartridge. The cartridge was then flushed with methanol (80 ml) followed by 2 Molar ammonia in methanol (40 ml). The ammonia fractions were concentrated in vacuo to yield crude N-[(2-chloro-4-fluorophenyl)methyl]-N'-(3-methyl-4-pyridinyl)-1 ,2- ethanediamine (0.391 ) as an orange oil, which was used without further purification.

Example 121

1 -[(2-Chloro-4-fluorophenyl)methyl]-4-(3,6-dimethyl-2-pyridinyl)-2,3- piperazinedione (E121)

N-[(2-Chloro-4-fluorophenyl)methyl]-N'-(3,6-dimethyl-2-pyridinyl)-1 ,2-ethanediamine (1.02 g, 3.31 mmol) was dissolved in dichloromethane (30 ml) and cooled to 0⁰C. Oxalyl chloride (0.58 ml, 6.63 mmol) was added dropwise and the reaction was stirred for 1 hour. Triethylamine (0.46 ml, 3.31 mmol) was then added, the reaction warmed to room temperature and stirred for a further 2 hours. The reaction was quenched by adding saturated aqueous sodium bicarbonate (20 ml) and the mixture extracted with dichloromethane (50 ml). The organic layer was separated using a hydrophobic frit and concentrated in vacuo. The residue was purified by flash-silica gel chromatography, eluting with a 0-10% gradient of 2 Molar ammonia in methanol solution in dichloromethane. The product fractions were concentrated in vacuo. The solids were triturated with diethyl ether (2 x 50 ml) and pale pink solids were filtered off. This material was suspended in methanol (15 ml) and 2 Molar hydrochloric acid was added (approx 5 ml). The mixture was concentrated in vacuo and purified by mass-directed automated HPLC. The product was dissolved in methanol and passed down a pre-conditioned 10 g aminopropyl cartridge. The cartridge was washed with methanol (50 ml) and the solution was concentrated to give 1-[(2-chloro- 4-fluorophenyl)methyl]-4-(3,6-dimethyl-2-pyridinyl)-2,3-piperazinedione (0.136 g) as a white solid. LC/MS [M+H]⁺ = 361 , retention time = 2.46 minutes.

The N-[(2-Chloro-4-fluorophenyl)methyl]-N'-(3,6-dimethyl-2-pyridinyl)-1 ,2-ethane- diamine used in the method described above was prepared in an analogous manner to the N-[(2-chloro-4-fluorophenyl)methyl]-N'-(3-methyl-4-pyridinyl)-1 ,2-ethane- diamine described in Example 120 above, by substituting the appropriate chloropyridine for the 4-chloro-3-methylpyridine used in the above procedure.

Example 122

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(3-methyl-2-pyridinyl)-2,3- piperazinedione (E122)

N-[(2-chloro-4-fluorophenyl)methyl]-N'-(3-methyl-2-pyridinyl)-1 ,2-ethanediamine (0.80 g, 2.72 mmol) was dissolved in dichloromethane (25 ml) and cooled to 0⁰C. Oxalyl chloride (0.48 ml, 5.43 mmol) was added dropwise and the reaction was stirred for 1 hour. Triethylamine (0.38 ml, 2.72 mmol) was added, the reaction was warmed to room temperature and stirred for a further 2 hours. The reaction was quenched by adding saturated aqueous sodium bicarbonate (20 ml) and the mixture extracted with dichloromethane (50 ml). The organic layer was separated using a hydrophobic frit and concentrated in vacuo. The residue was purified by flash-silica gel chromatography, eluting with a 0-10% gradient of 2 Molar ammonia in methanol solution in dichloromethane. The product fractions were concentrated in vacuo and purified by mass-directed automated HPLC. The product was then dissolved in methanol and passed through a pre-conditioned 1 O g aminopropyl cartridge. The column was washed with further methanol (40 ml) and the solution concentrated in vacuo. The oily residue was triturated with diethyl ether (2 x 20 ml) and the resultant white precipitate filtered off to yield 1-[(2-Chloro-4-fluorophenyl)methyl]-4-(3-methyl- 2-pyridinyl)-2,3-piperazinedione (0.077 g) as a white solid. LC/MS [M+H]⁺ = 348, retention time = 2.33 minutes.

The N-[(2-chloro-4-fluorophenyl)methyl]-N'-(3-methyl-2-pyridinyl)-1 ,2-ethanediamine used in the method described above was prepared in an analogous manner to the N- [(2-chloro-4-fluorophenyl)methyl]-N'-(3-methyl-4-pyridinyl)-1 ,2-ethanediamine described in Example 120 above, by substituting the appropriate chloropyridine for the 4-chloro-3-methylpyridine used in the above procedure.

Example 123

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[3-(1H-imidazol-1-yl)-2-methylphenyl]-2,3- piperazinedione (E123)

1-(3-Bromo-2-methylphenyl)-4-[(2-chloro-4-fluorophenyl)methyl]-2,3-piperazinedione (0.085 g, 0.200 mmol, prepared as described earlier), imidazole (0.0204 g, 0.300 mmol), potassium carbonate (0.0552 g, 0.399 mmol), copper (I) iodide (0.0038 mg, 0.020 mmol) and trans-N,N'-dimethyl-cyclohexane-1 ,2-diamine (6.30 μl, 0.040 mmol) were suspended in N-methyl-2-pyrrolidone (2 ml) and heated at 180 ⁰C for 1 hour in a microwave reactor.

In parallel, 1-(3-bromo-2-methylphenyl)-4-[(2-chloro-4-fluorophenyl)methyl]-2,3- piperazinedione (0.085 g, 0.200 mmol), imidazole (0.0204 g, 0.300 mmol), potassium carbonate (0.0552 g, 0.399 mmol), copper (I) iodide (0.0038 mg, 0.020 mmol) and trans-N,N'-dimethyl-cyclohexane-1 ,2-diamine (6.30 μl, 0.040 mmol) were suspended in N,N-dimethylformamide (2 ml) and heated at 180 ⁰C for 1 hour in a microwave reactor.

The two mixtures were combined and partitioned between ethyl acetate (25ml) and water (25 ml). Extracted in to ethyl acetate (3x25 ml) and the combined extracts were washed with 50% brine (3x25ml), brine (25ml) and dried over anhydrous sodium sulfate. This was concentrated in vacuo and purified by mass-directed automated HPLC to afford 1-[(2-chloro-4-fluorophenyl)methyl]-4-[3-(1 H-imidazol-1- yl)-2-methylphenyl]-2,3-piperazinedione (0.016 g). LC/MS [M+H]⁺ = 412.96, retention time = 1.73 minutes.

Example 124 1 -[(2-Chloro-4-fluorophenyl)methyl]-4-[2-methyl-3-(1 -pyrrolidinyl)phenyl]-2,3- piperazinedione (E124)

Palladium (II) acetate (0.004 g, 0.018 mmol), 2,2'-bis(diphenylphosphino)-1 ,1 '- binaphthalene (0.016 g, 0.026 mmol) and cesium carbonate (0.086 g, 0.264 mmol) were combined in tetrahydrofuran (5 ml) and stirred under argon at room temperature for 20 minutes. 1-(3-bromo-2-methylphenyl)-4-[(2-chloro-4-fluorophenyl)methyl]-2,3- piperazinedione (0.075 g, 0.176 mmol, prepared as described earlier) and pyrrolidine (0.044 ml, 0.529 mmol) were added and the reaction heated to reflux at 85 ⁰C under argon for 16 hours. The reaction mixture was diluted with ethyl acetate (10ml) and the insoluble material filtered off. The filtrate was concentrated in vacuo and purified by mass-directed automated HPLC. Product fractions were concentrated in vacuo to yield a pale yellow oil. The oil was dissolved in methanol (~10ml) and loaded onto a pre-conditioned 10g NH2 cartridge. The cartridge was washed with methanol (60ml) and the solution concentrated in vacuo to yield 1-[(2-chloro-4-fluorophenyl)methyl]-4- [2-methyl-3-(1-pyrrolidinyl)phenyl]-2,3-piperazinedione (0.0264 g) as a white solid. LC/MS [M+H]⁺ = 416, retention time = 2.17 minutes.

Example 125

(5S)-1-[(2,4-Dichlorophenyl)methyl]-5-methyl-4-phenyl-2,3-piperazinedione (E125)

(5S)-1-[(2,4-Dichlorophenyl)methyl]-5-methyl-2,3-piperazinedione (0.100 g, 0.348 mmol) was suspended in toluene (5 ml) and phenylboronic acid (0.085 g, 0.697 mmol) was added. 4 Angstrom molecular sieves (powdered) (0.500 g, 0.348 mmol), pyridine (0.056 ml, 0.697 mmol), pyridine N-oxide (0.0364 g, 0.383 mmol) and copper (II) acetate (0.0316 g, 0.174 mmol) were then added and the reaction mixture was heated at 90 ⁰C for a total of 5 hours, before being left to stand at room temperature for -72 hrs. The mixture was diluted with methanol (~10mL) and then filtered through a pad of celite, washing with more methanol. The filtrate was evaporated in vacuo and then the residue was dissolved in dichloromethane (-20 ml) and washed sequentially with 2N aqueous hydrochloric acid, saturated sodium bicarbonate, and then brine (-20 ml. of each). The dichloromethane layer was filtered through a hydrophobic frit, evaporated and purified by mass-directed automated HPLC to give (5S)-1-[(2,4-dichlorophenyl)methyl]-5-methyl-4-phenyl-2,3-piperazinedione (0.025 g) as a white solid (N3937-7-A1 ), 25mg.

LC/MS [M+H]⁺ = 362.9, retention time = 2.84 minutes.

The (5S)-1-[(2,4-Dichlorophenyl)methyl]-5-methyl-2,3-piperazinedione used in the method described above was prepared as follows:

(i) 1 ,1-Dimethylethyl [(1 S)-1-methyl-2-oxoethyl]carbamate (1 g, 5.77 mmol) was dissolved in dichloromethane (50 ml). 4 Angstrom molecular sieves (0.3 g) were added and the solution cooled to O⁰C. [(2,4-dichlorophenyl)methyl]amine (0.769 ml, 5.77 mmol) and sodium triacetoxyborohydride (1.835 g, 8.66 mmol) were added and the mixture warmed to room temperature. The reaction was stirred for -16 hrs then diluted with saturated aqueous sodium hydrogen carbonate (-50 ml). The dichloromethane layer was separated, washed with brine (-50 ml), and then filtered through a hydrophobic frit and evaporated to give a slightly opaque colourless oil. The oil was purified by flash-silica gel chromatography, eluting with a 0-100% gradient of ethyl acetate in isohexane, to give 1 ,1-dimethylethyl ((1S)-2-{[(2,4- dichlorophenyl)methyl]amino}-1-methylethyl)carbamate (1.57 g) as a clear pale- yellow oil.

(ii) 1 ,1-Dimethylethyl ((1 S)-2-{[(2,4-dichlorophenyl)methyl]amino}-1-methylethyl) carbamate (1.57 g, 4.71 mmol) was dissolved in dichloromethane (20 ml). Triethylamine (0.689 ml, 4.95 mmol) was added and the solution cooled to O⁰C. Methyl chlorooxoacetate (0.459 ml, 4.95 mmol) was added dropwise, the mixture was allowed to warm to room temperature and stirred for 4 hours. The mixture was diluted with water (-40 ml) and stirred for 5 minutes, then the dichloromethane layer was separated and dried by filtering through a hydrophobic frit. Evaporation gave crude methyl {[(2,4-dichlorophenyl)methyl][(2S)-2-({[(1 ,1-dimethylethyl)oxy]carbonyl} amino)propyl]amino}(oxo)acetate (1.85 g) as a white foam, which was used without purification.

(iii) Methyl {[(2,4-dichlorophenyl)methyl][(2S)-2-({[(1 ,1-dimethylethyl)oxy]carbonyl} amino)propyl]amino}(oxo)acetate (1.85 g, 4.41 mmol) was dissolved in 4 Molar hydrochloric acid in dioxane (20 ml, 80 mmol) and stirred at room temperature for 30 minutes. The reaction mixture was concentrated in vacuo to give a white foam. This was dissolved in dichloromethane (20 ml) and triethylamine (1.845 ml, 13.24 mmol) was added. The solution was stirred for 2 hrs at room temperature. The mixture was washed with 2 Molar aqueous hydrochloric acid and then the dichloromethane layer was separated, filtered though a hydrophobic frit, and evaporated to give a pale orange oil which solidified on standing. The solid was triturated with diethyl ether, filtered and dried in vacuo to give (5S)-1-[(2,4-dichlorophenyl)methyl]-5-methyl-2,3- piperazinedione (1.12 g) as a white solid.

Example 126 (5/?)-1-[(2,4-Dichlorophenyl)methyl]-5-methyl-4-phenyl-2,3-piperazinedione (E126)

(5R)-1-[(2,4-Dichlorophenyl)methyl]-5-methyl-2,3-piperazinedione (0.10 g, 0.348 mmol) was suspended in toluene (5 ml) and phenyl boronic acid (0.085 g, 0.697 mmol) was added. 4A molecular sieves (powdered), pyridine (0.056 ml, 0.697 mmol), pyridine N-oxide (0.0364 g, 0.383 mmol) and copper (II) acetate (0.0316 g, 0.174 mmol) were then added and the reaction mixture was heated at 90⁰C for 2 hours then left to stand at room temperature overnight. Additional aliquots of copper (II) acetate (0.0316 g, 0.174 mmol), phenyl boronic acid (0.085 g, 0.697 mmol), pyridine (0.056 ml, 0.697 mmol) and pyridine N-oxide (0.0364 mg, 0.383 mmol) were added to the mixture and the mixture was again heated at 9O⁰C for -4 hours. The mixture was then diluted with methanol (-1OmL) and filtered through a pad of celite, washing with more methanol. The filtrate was evaporated in vacuo and then the residue was dissolved in dichloromethane (-20 ml) and washed sequentially with 2N aqueous hydrochloric acid, saturated aqueous sodium bicarbonate and then brine (-20 ml. of each). The dichloromethane layer was filtered through a hydrophobic frit, evaporated and purified by mass-directed automated HPLC. The resultant oil was triturated with diethyl ether to yield (5R)-1-[(2,4-dichlorophenyl)methyl]-5-methyl-4- phenyl-2,3-piperazinedione (0.028 g) as a white solid. LC/MS [M+H]⁺ = 362.9, retention time = 2.85 minutes.

The (5R)-1-[(2,4-dichlorophenyl)methyl]-5-methyl-2,3-piperazinedione used in the method described above was prepared in an analogous manner to the (5S)-1-[(2,4- dichlorophenyl)methyl]-5-methyl-2,3-piperazinedione used in Example 125.

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

5μm.

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).

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 1100 Gradient Pump Agilent 1100 Autosampler Agilent 1100 DAD Detector Agilent 1100 Degasser Agilent 1100 Oven Agilent 1100 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. PHARMACOLOGICAL DATA

Compounds or salts of the invention may be tested for in vitro biological activity at the P2X7 receptor in accordance with the following studies:

Eithidium Accumulation Assay

Studies were performed using NaCI assay buffer of the following composition: 14OmM NaCI, 10 mM HEPES [4-(2-hydroxyethyl)-1-piperazine-1-ethanesulfonic acid], 5 mM Λ/-methyl-D-glucamine, 5.6 mM KCI, 10 mM D-glucose, 0.5 mM CaCI₂(PH 7.4).

Human Embryonic Kidney (HEK) 293 cells, stably expressing human recombinant P2X7 receptors, were grown in poly-D-lysine pretreated 96 well plates for 18-24 hours. (The cloning of the human P2X7 receptor is described in US 6,133,434, e.g. see Example 3 therein). The cells were washed twice with 350 μl of the assay buffer, before addition of 50 μl of the assay buffer containing the putative P2X7 receptor antagonist compound. (A small amount of dimethyl sulfoxide, for initially dissolving the compound, is optionally used and present in this 50μl test compound sample.) The cells were then incubated at room temperature (19-21 ⁰C) for 30 min before addition of ATP and ethidium (100 μM final assay concentration). The ATP concentration was chosen to be close to the ECβo f°^r 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 25 μl of 1.3M sucrose containing 4 mM 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 620 nm) from below the plate with a Canberra Packard Fluorocount (14 Station Road, Pangbourne, Reading, Berkshire RG8 7AN, United Kingdom) or a FlexStation Il 384 from Molecular Molecular Devices (660-665 Eskdale Road, Wokingham, Berkshire RG41 5TS, United Kingdom). Antagonist PIC50 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 for human P2X7: 137 mM NaCI; 20 mM HEPES [4-(2-hydroxyethyl)-1-piperazine-1- ethanesulfonic acid]; 5.37 mM KCI; 4.17 mM NaHCC>3; 1 mM CaC^; 0.5 mM MgSO4; and 1g/L of D-glucose (pH 7.4).

Human Embryonic Kidney (HEK) 293 cells, stably expressing human recombinant P2X7 receptors, were grown in poly-D-lysine pretreated 384 well plates for 24hours at room temperature (for a time sufficient for growth of a homogeneous layer of cells at the bottom of the wells). Alternatively, human osteosarcoma (U-2OS) cells (commercially available), transduced with modified Baculovirus (BacMam) vector to deliver the gene coding for human P2X7 receptor (i.e. transiently expressing human recombinant P2X7 receptors), were grown in substantially the same conditions as for the HEK293 cells except that the well plates were not pre-treated with poly-D-lysine. (The cloning of the human P2X7 receptor is described in US 6,133,434, e.g. see Example 3 therein). The cells were washed three times with 80μl of assay buffer, loaded for 1 h at 37°C with 2μM Fluo4-AM [4-(6-acetoxymethoxy-2,7-difluoro-3-oxo-9- xanthenyl)-4'-methyl-2,2'-(ethylenedioxy)dianiline-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl) ester], a Ca2⁺-sensitive, cell-permeable, fluorescent dye (T ef Labs. Inc., 9415 Capitol View Drive, Austin, TX 78747, USA), washed three times again (3 x 80μl), and left with 30μl buffer before the addition of 10μl of the assay buffer containing the putative P2X7 receptor antagonist compound, the compound being added at 4x the final assay concentration chosen. The solution of the putative P2X7 receptor antagonist compound was created by (i) dissolving the compound in dimethyl sulfoxide (DMSO) to create a stock solution in DMSO at 20Ox the final assay concentration, and (ii) mixing 1 μl of the stock solution of the compound in DMSO with 50μl of the assay buffer to create a solution at about 4x the final assay concentration. The cells were then incubated at room temperature for 30 mins before addition (online, by FLIPR384 or FLIPR3 instrument (Molecular Devices, 131 1 Orleans Drive, Sunnyvale, CA 94089-1136, USA)) of 10μl of the assay buffer containing benzoylbenzoyl-ATP (BzATP) such as to create a 60μM final assay concentration of BzATP (BzATP was added at 5x this final concentration). The BzATP concentration was chosen to be close to the ECβo for the receptor type.

Incubations and reading were continued for 90 sec, and intracellular calcium increase was determined by measuring fluorescence (excitation wavelength of 488nm and emission wavelength of 516nm) from below the plate, with FLIPR charged-coupled device (CCD) camera. Antagonist PIC50 values for blocking BzATP responses were determined using iterative curve fitting techniques. The compounds of Examples 1-126 were tested in the FLIPR Ca Assay and/or the Ethidium Accumulation Assay for human P2X7 receptor antagonist activity and were found to have plC50 values > 4.6 in the FLIPR Ca Assay and/or plC50 values > 5.1 in the Ethidium Accumulation Assay.

In particular, the compounds of Examples 1 , 3-9, 1 1 , 14, 18, 22-31 , 33, 35-41 , 43, 46-50, 52-53, 55-61 , 64-73, 75-80, 86-94, 96-100, 102-105, 107-115, 118, 121 and 123-126 were found to have plC50 values >6.5 in the Ethidium Accumulation Assay.

Claims

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

(I) wherein:

A represents an aryl, heteroaryl or heterocyclyl group; and any ring or ring system of said aryl or heteroaryl is optionally substituted with 1 to 3 substituents, which may be the same or different, selected from the group consisting of halogen, C_1-6 alkyl, -CF₃, -

OCF₃, cyano, C_1-6 alkoxy, -NR¹⁰R¹¹, -X-aryl, -X-heteroaryl and -X-heterocyclyl;

R¹, R², R³, R⁴ and R⁵ independently represent hydrogen, fluorine, chlorine, -CF₃, cyano or Ci_-6 alkyl, such that at least one of R¹, R², R³, R⁴ and R⁵ is other than hydrogen;

R⁶, R⁷, R⁸, R⁹, R¹⁰ and R¹¹ independently represent hydrogen or Ci_-6 alkyl;

2. A compound or salt as claimed in claim 1 , wherein A represents phenyl optionally substituted by 1 to 3 substituents, which may be the same or different, selected from fluorine, chlorine, bromine, iodine, methyl, -CF₃, cyano, methoxy, -morpholinyl, -(CH₂)-morpholinyl, -(CH₂)₂-morpholinyl, and -O-(CH₂)₂-morpholinyl.

3. A compound or salt as claimed in claim 1 or 2, wherein R¹ represents hydrogen, fluorine, chlorine, -CF₃ or methyl.

4. A compound or salt as claimed in claim 3, wherein R¹ represents chlorine.

5. A compound or salt as claimed in claim 1 , 2, 3 or 4, wherein R² represents hydrogen, fluorine, chlorine, Or -CF₃.

6. A compound or salt as claimed in claim 5, wherein R² represents hydrogen or chlorine.

7. A compound or salt as claimed in any one of the preceding claims, wherein R³ represents hydrogen, fluorine or chlorine.

8. A compound or salt as claimed in any one of the preceding claims, wherein R⁴ represents hydrogen.

9. A compound or salt as claimed in any one of the preceding claims, wherein R⁵ represents hydrogen, fluorine, chlorine, -CF₃ or methyl.

10. A compound or salt as claimed in claim 9, wherein R⁵ represents hydrogen.

11. A compound or salt as claimed in any one of the preceding claims, wherein R², R⁴ and R⁵ represent hydrogen, R¹ represents chlorine, and R³ represents fluorine or chlorine.

12. A compound as claimed in claim 1 , or a pharmaceutically acceptable salt thereof, which is:

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(1-naphthalenyl)-2,3-piperazinedione (E1 ); 1-[(2-Chloro-6-fluorophenyl)methyl]-4-(1-naphthalenyl)-2,3-piperazinedione (E2); 1-[1-(2,4-Dichlorophenyl)ethyl]-4-(2-methylphenyl)-2,3-piperazinedione (E3); 1-[(2,4-Dichlorophenyl)methyl]-4-(2-methylphenyl)-2,3-piperazinedione (E4); 1-[(2-Chloro-4-fluorophenyl)methyl]-4-(2-methylphenyl)-2,3-piperazinedione (E5); 1-[(2-Chlorophenyl)methyl]-4-(2-methylphenyl)-2,3-piperazinedione (E6); 1-(2-Methylphenyl)-4-[(2,3,4-trifluorophenyl)methyl]-2,3-piperazinedione (E7); 1-[(2,3-Dichlorophenyl)methyl]-4-(2-methylphenyl)-2,3-piperazinedione (E8); 1-{[4-Fluoro-2-(trifluoromethyl)phenyl]methyl}-4-(2-methylphenyl)-2,3-piperazinedione (E9);

2-{[4-(2-Methylphenyl)-2,3-dioxo-1 -piperazinyl]methyl}benzonitrile (E10); 1-{[2-Chloro-3-(trifluoromethyl)phenyl]methyl}-4-(2-methylphenyl)-2,3- piperazinedione (E1 1 ); 1-[(2-Fluorophenyl)methyl]-4-(2-methylphenyl)-2,3-piperazinedione (E12);

1-[(2-Chloro-6-fluorophenyl)methyl]-4-(2-methylphenyl)-2,3-piperazinedione (E13);

1-[(2,4-Dichloro-6-methylphenyl)methyl]-4-(2-methylphenyl)-2,3-piperazinedione

(E14);

1-(2-Methylphenyl)-4-[(2-methylphenyl)methyl]-2,3-piperazinedione (E 15);

1-[(4-Chlorophenyl)methyl]-4-(2-methylphenyl)-2,3-piperazinedione (E 16);

1-[(2,5-Dichlorophenyl)methyl]-4-(2-methylphenyl)-2,3-piperazinedione (E17);

1-[(3-Chlorophenyl)methyl]-4-(2-methylphenyl)-2,3-piperazinedione (E19);

1-(2-Methylphenyl)-4-{[3-(trifluoromethyl)phenyl]methyl}-2,3-piperazinedione (E20);

1-[(2,5-Dimethylphenyl)methyl]-4-(2-methylphenyl)-2,3-piperazinedione (E21 );

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(2,6-dimethylphenyl)-2,3-piperazinedione

(E22);

1-(2_!6-Dimethylphenyl)-4-[(2_!3_!4-trifluorophenyl)methyl]-2,3-piperazinedione (E25);

1-(2_!6-Dimethylphenyl)-4-{[4-fluoro-2-(trifluoromethyl)phenyl]methyl}-2,3- piperazinedione (E26);

1-[(2,4-Dichlorophenyl)methyl]-4-(2,6-dimethylphenyl)-2,3-piperazinedione (E27);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(4-fluoro-2_!6-dimethylphenyl)-2,3- piperazinedione (E28);

(E29);

1-{[2-Chloro-3-(trifluoromethyl)phenyl]methyl}-4-(4-fluoro-2_!6-dimethylphenyl)-2,3- piperazinedione (E30);

1-[(2,3-Dichlorophenyl)methyl]-4-(4-fluoro-2,6-dimethylphenyl)-2,3-piperazinedione

(E31 );

(E32);

1-(4-Fluoro-2,6-dimethylphenyl)-4-{[4-fluoro-2-(trifluoromethyl)phenyl]methyl}-2,3- piperazinedione (E33);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-phenyl-2,3-piperazinedione (E34);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(2,3-dimethylphenyl)-2,3-piperazinedione

(E36); 1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-(methyloxy)phenyl]-2,3-piperazinedione

(E37);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[3-(methyloxy)phenyl]-2,3-piperazinedione

(E39);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(2,4-dimethylphenyl)-2,3-piperazinedione

(E40);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(2,5-dimethylphenyl)-2,3-piperazinedione

(E41 );

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(3,5-dimethylphenyl)-2,3-piperazinedione

(E42);

(E43);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(3-iodophenyl)-2,3-piperazinedione (E44);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(3,4-dimethylphenyl)-2,3-piperazinedione

(E45);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(5-fluoro-2-methylphenyl)-2,3-piperazinedione

(E47);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-methyl-5-(methyloxy)phenyl]-2,3- piperazinedione (E49);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[5-fluoro-2-(methyloxy)phenyl]-2,3- piperazinedione (E50);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-(4-morpholinylmethyl)phenyl]-2,3- piperazinedione hydrochloride (E51 );

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-(1-piperidinyl)phenyl]-2,3-piperazinedione hydrochloride (E52);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-(1-pyrrolidinyl)phenyl]-2,3-piperazinedione hydrochloride (E53);

(E54);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-(1 /-/-pyrrol-1-yl)phenyl]-2,3-piperazinedione

(E55); 1-[(2-Chloro-4-fluorophenyl)methyl]-4-(4-fluoro-2_!3-dimethylphenyl)-2,3- piperazinedione (E56);

1-(4-Bromo-2-methylphenyl)-4-[(2-chloro-4-fluorophenyl)methyl]-2,3-piperazinedione (E57);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(5_!6_!7_!8-tetrahydro-1-naphthalenyl)-2,3- piperazinedione (E58);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[3-(4-morpholinyl)phenyl]-2,3-piperazinedione (E59);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[3-(4-morpholinylmethyl)phenyl]-2,3- piperazinedione (E62);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-{2-[2-(4-morpholinyl)ethyl]phenyl}-2,3- piperazinedione hydrochloride (E63);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-methyl-3-(4-morpholinylmethyl)phenyl]-2,3- piperazinedione hydrochloride (E64); 1-[(2-Chloro-4-fluorophenyl)methyl]-4-(2-methyl-3-{[2-(4- morpholinyl)ethyl]oxy}phenyl)-2,3-piperazinedione hydrochloride (E65); 1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-methyl-3-(methyloxy)phenyl]-2,3- piperazinedione (E66);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-methyl-3-(4-morpholinyl)phenyl]-2,3- piperazinedione (E67);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(1 /-/-indol-4-yl)-2,3-piperazinedione (E68); 1-[5-Bromo-2-(methyloxy)phenyl]-4-[(2-chloro-4-fluorophenyl)methyl]-2,3- piperazinedione (E69);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(1 /-/-indol-7-yl)-2,3-piperazinedione (E70); 1-[(2-Chloro-4-fluorophenyl)methyl]-4-[3-chloro-2-(methyloxy)phenyl]-2,3- piperazinedione (E71 );

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-(4-morpholinyl)-5-(trifluoromethyl)phenyl]- 2,3-piperazinedione (E74); 1-[3-Bromo-2-(methyloxy)phenyl]-4-[(2-chloro-4-fluorophenyl)methyl]-2,3- piperazinedione (E75);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[5-fluoro-2-(4-morpholinyl)phenyl]-2,3- piperazinedione (E79);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-(methyloxy)-5-(4-morpholinyl)phenyl]-2,3- piperazinedione (E80);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(2-pyrimidinyl)-2,3-piperazinedione (E83);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(2-pyridinyl)-2,3-piperazinedione (E84);

(E85);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-(1-methylethyl)phenyl]-2,3-piperazinedione

(E87);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(2_!3-dihydro-1 _!4-benzodioxin-5-yl)-2,3- piperazinedione (E88);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(2-fluoro-3-methylphenyl)-2,3-piperazinedione

(E90);

(E91 );

1-(2-Biphenylyl)-4-[(2-chloro-4-fluorophenyl)methyl]-2,3-piperazinedione (E92);

(E93);

1-(3-Bromo-2-methylphenyl)-4-[(2-chloro-4-fluorophenyl)methyl]-2,3-piperazinedione

(E94);

(E97);

1-(2-Chloro-4-fluorophenyl)-4-[(2-chloro-4-fluorophenyl)methyl]-2,3-piperazinedione

(E99);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(2-iodophenyl)-2,3-piperazinedione (E100);

(E101 );

2-{4-[(2-Chloro-6-fluorophenyl)methyl]-2,3-dioxo-1-piperazinyl}benzonitrile (E102);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(2-chlorophenyl)-2,3-piperazinedione (E103);

(E104);

1-(2-Bromophenyl)-4-[(2-chloro-4-fluorophenyl)methyl]-2,3-piperazinedione (E 105);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(5-quinolinyl)-2,3-piperazinedione (E106);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(4-isoquinolinyl)-2,3-piperazinedione (E107);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-{2-[(trifluoromethyl)oxy]phenyl}-2,3- piperazinedione (E109);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-chloro-3-(methyloxy)phenyl]-2,3- piperazinedione (E1 10);

2,3-piperazinedione (E111 );

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[2-(trifluoromethyl)phenyl]-2,3-piperazinedione

(E1 12);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[4-(diethylamino)-2-methylphenyl]-2,3- piperazinedione (E1 13);

1-(2,3-Dichlorophenyl)-4-[(2-methylphenyl)methyl]-2,3-piperazinedione (E1 14);

1-[(2-Chlorophenyl)methyl]-4-(2,3-dichlorophenyl)-2,3-piperazinedione (E115);

1-[(3-Chlorophenyl)methyl]-4-(2,3-dichlorophenyl)-2,3-piperazinedione (E116);

1-[(4-Chlorophenyl)methyl]-4-(2,3-dichlorophenyl)-2,3-piperazinedione (E117);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-(2,3-dichlorophenyl)-2,3-piperazinedione

(E1 18);

(E1 19); 1-[(2-Chloro-4-fluorophenyl)methyl]-4-(3-methyl-4-pyridinyl)-2,3-piperazinedione

(E120);

(E121 );

(E122);

1-[(2-Chloro-4-fluorophenyl)methyl]-4-[3-(1 /-/-imidazol-1-yl)-2-methylphenyl]-2,3- piperazinedione (E123);

or a pharmaceutically acceptable salt thereof.

13. A pharmaceutical composition which comprises a compound or salt as defined in any one of claims 1 to 12, and a pharmaceutically acceptable carrier or excipient.

14. A pharmaceutical composition according to claim 13, which further comprises an additional therapeutic agent.

15. A compound or a pharmaceutically acceptable salt thereof as defined in any one of claims 1 to 12, for use in therapy.

16. A method of treatment or prophylaxis of a human or animal subject suffering from or susceptible to pain, inflammation, or a neurodegenerative disease, or epilepsy and/or seizures, which method comprises administering to said subject an effective amount of a compound of formula (I) as defined in any one of claims 1 to 12.

17. Use of a compound or a pharmaceutically acceptable salt thereof as defined in any one of claims 1 to 12 for the manufacture of a medicament for the treatment or prophylaxis of pain, inflammation, or a neurodegenerative disease, or epilepsy and/or seizures.

18. A compound or pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 12, for use in the treatment or prophylaxis of pain, inflammation, or a neurodegenerative disease, or epilepsy and/or seizures.

19. Use as claimed in claim 17 or a compound or pharmaceutically acceptable salt thereof as claimed in claim 18, wherein the medicament is for the treatment or prophylaxis of inflammatory pain, neuropathic pain, visceral pain, rheumatoid arthritis or osteoarthritis.

20. Use as claimed in claim 17 or a compound as claimed in claim 18, for the treatment or prophylaxis of degenerative dementia including Alzheimer's disease, Parkinson's disease including dementia in Parkinson's disease, vascular dementia including multi-infarct dementia, dementia with Lewy bodies, Huntingdon's chorea or mild cognitive impairment (MCI) including MCI associated with ageing such as age associated memory impairment.