WO2004014425A1

WO2004014425A1 - Activators of small conductance calcium activated potassium channels and use thereof

Info

Publication number: WO2004014425A1
Application number: PCT/EP2003/008752
Authority: WO
Inventors: Timothy James Dale; David John Davies; Derek John Trezise
Original assignee: Glaxo Group Limited
Priority date: 2002-08-07
Filing date: 2003-08-05
Publication date: 2004-02-19
Also published as: AU2003253390A1; GB0218326D0

Abstract

The invention relates to activators of small conductance calcium potassium channels of formula (I) and their use in the treatment of disorders responsive to enhanced calcium activated potassium channel activity.

Description

ACTIVATORS OF SMALL CONDUCTANCE CALCIUM ACTIVATED POTASSIUM CHANNELS AND USE THEREOF

The invention relates to activators of small conductance calcium activated potassium channels and their use in the treatment of disorders responsive to enhanced calcium activated potassium channel activity.

Calcium activated potassium (K¹) channels play an important functional role in many cell types including neurones, epithelial cells, T-lymphocytes and skeletal muscle. These channels open in response to a rise in intracellular Ca²⁺ and thereby link cellular excitation/activation to membrane K⁺ permeability. In some cell types (e.g. neurones) the hyperpolarisation that results from K⁺ efflux curtails cellular excitation by preventing opening of voltage-gated Ca²⁺ and Na⁺ channels. In others (e.g. lymphocytes) the increased electrical driving force for other ions to flow can enhance cell activation. Thus, Ca ⁺-activated K⁺ channels are a key determinant of the excitability of cells in which they are found.

Calcium activated potassium channels can be divided into three main classes: large conductance (BK), intermediate conductance (IK) and small conductance (SK). To date three types of SK channels termed SK-1, SK-2 and SK-3 have been identified. The cloning of IK and SK-1, SK-2 and SK-3 channels has been described in WO 98/11139. SK channels are comprised of four α-subunits each of which has 6 putative transmembrane domains that are tightly coupled to calmodulin which acts as the Ca²⁺ sensor. These channels have a small single channel conductance for K⁺ of less than 25 pS and are voltage-independent. The bee venom peptide toxin, apamin is a highly potent and specific blocker of SK channels.

From molecular distribution, electrophysiology and pharmacological studies an array of functions for SK channels have been proposed. SK channels are widespread in the central and peripheral nervous systems notably in the hippocampus, cortex, midbrain dopaminergic neurones, dorsal root and superior cervical ganglia. In these excitable cells, activation of SK channels contributes to the after-hyperpolarization (AHP) which is a key determinant ofthe interspike- and interburst-interval. Neuronal SK channels are thus implicated in functions ranging from long term potentiation, synaptic enhancement, learning and memory through to sensory and visual processes and sympathetic control of blood pressure. SK-3 channels are also highly expressed in denervated skeletal muscle, liver and the interstitial cells of Cajal in the gastointestinal tract whilst SK-2 is found in the adrenal medulla, retina and T-lymphocytes. The SK-1 channel appears to be neuronal specific. There is evidence for heteromeric assembly between subunits such that SK-1 and SK-2 may co-assemble to form channels with properties that are different from those of the homomerically assembled proteins. Where subunits are co-localised, such as SK-1 and SK-2 in hippocampal pyramidal neurones, it is possible that the native channel is a heterotetramer.

The IK channel has a greater single channel conductance for K⁺ ranging from 25-60pS and a different pharmacology to SK channels. Specifically, hIK channels are potently blocked by charybdotoxin, an extract from scorpion venom, and by the antifungal agent clotrimazole. Structurally IK channels share a similar topology and architecture to SK channels, but at the amino acid level are less than 40% homologous. In particular, IK channels possess a truncated N- terminal region compared to SK channels. IK channels are expressed in erythrocytes, lung and colonic epithelial cells, and T-lymphocytes. The invention provides the use of compounds of formula (I):

wherein

X represents O, NH or CH₂; one of W, Y and Z represents N, the other two represent CH_2;

R¹ independently represents an optionally substituted methoxy;

R² represents H; R³ represents COOR⁴, COSR⁴, COR⁶, S0₂-R⁷ or CONHR⁵;

R⁴ represents optionally substituted (Cι_₈)alkyl, optionally substituted (C .₆)cycloalkyl, optionally substituted (C_2-g)alkenyl or aryl;

R⁵ represents H, optionally substituted, (Cι_-g)alkyl, optionally substituted (C₄.₆)cycloalkyl or aryl;

R⁶ represents optionally substituted (Cι_-6)alkyl, aryl or furanyl; R⁷ represents optionally substitued aryl or thienyl; n is an interger from 0 to 2; y is an interger from 0 to 2; m is 0 or 1;

W can not be N when m is 0; and pharmaceutically acceptable derivatives thereof; as activators of a SK channel.

A further aspect ofthe invention provides compounds of formula (la);

(1a)

wherein

R¹ independently represents an optionally substituted methoxy;

R² represents H; R³ represents H, COOR⁴, COSR⁴, COR⁶, S0₂-R⁷ or CONHR⁵;

R⁴ represents optionally substituted (C_1-8)alkyl, optionally substituted (C _-6)cycloalkyl, optionally substituted (C_2-8)alkenyl or aryl;

R⁵ represents H, optionally substituted, (C_1-S)alkyl, optionally substituted (C .₆)cycloalkyl or aryl;

R⁶ represents optionally substituted (C_!-6)alkyl, aryl or furanyl; R⁷ represents optionally substitued aryl or thienyl; n is an interger from 0 to 2; y is an interger from 0 to 2; m is 0 or 1;

W can not be N when m is 0; and pharmaceutically acceptable derivatives thereof; with the proviso that the compound is not:

[ 1 -[ 1 -(3 ,4-dichlorobenzyl)pyrrolidin-3 -ylmethyl] -3 -(4-methoxyphenyl) urea; urea N-[[l-[(3,4-dichlorophenyl)methyl-3-pyrrolidinyl]methyl]-N'-(2-methoxyphenyl).

[l-[l-(3,4-dichlorobenzyl)pyrrolidin-3-ylmethyl]-3-(4-methoxyphenyl) urea and urea N- [[l-[(3,4-dichlorophenyl)methyl-3-pyrrolidinyl]methyl]-N'-(2-methoxyphenyl) are disclosed in WOOO/31032 as compounds which are capable of inhibiting the binding of eotaxin to the CCR-3 receptor and thereby providing a means of combating induced diseases such as asthma.

A further aspect ofthe invention provides compounds of formula (II);

wherein X represents O, NH or CH₂;

R¹ independently represents an optionally substituted methoxy;

R² represents H;

R³ represents H, COOR⁴, COSR⁴, COR⁶, S0₂-R⁷ or CONHR⁵;

R⁴ represents optionally substituted (C-_..₈)alkyl, optionally substituted (C .₆)cycloalkyl, optionally substituted (C_2-8)alkenyl or aryl;

R⁵ represents H, optionally substituted (Cι_-8)alkyl, optionally substituted (G-^cycloalkyl or aryl;

R⁶ represents optionally substituted

aryl or furanyl;

R⁷ represents optionally substituted aryl or thienyl; n is an interger from 0 to 2; y is an interger from 0 to 2; m is 0 or 1 ; and pharmaceutically acceptable derivatives thereof; with the proviso that the compound is not:

[ 1 -[ 1 -(3 ,4-dichlorobenzyl)pyrrolidin-3 -ylmethyl] -3 -(4-methoxyphenyl) urea; ureaN-[[l-[(3,4-dichlorophenyl)methyl-3-pyrrolidinyl]methyl]-N'-(2-methoxyphenyl).

Preferably R¹ is an optionally substituted 0-(C-_..₅)alkyl. Preferably X is O. Preferably n is 1. Preferably y is 1 or 2.

Preferably R³ is H or COOR⁴, more preferably COOR⁴.

Preferably R⁴ represents optionally substituted (Cι„₈)alkyl, optionally substituted ( ,

₆)cycloalkyl or aryl, more preferably R⁴ is an optionally substituted (C-..₈) alkyl or optionally substituted (C₄.₆)cycloalkyl.

Compounds wherein R³ as H are useful as intermediates in the preparation of compounds of formula (I).

Illustrative compounds ofthe invention are: tert-Butyl 4-[({[(2-methoxyphenyl)amino]carbonyl}oxy)methyl]piperidine-l-carboxylate; tert-Butyl 3 -[( { [(2-methoxyphenyl)amino] carbonyl } oxy)methyl]pyrrolidine- 1 -carboxylate; tert-Butyl-4-({[(2-methoxyphenyl)amino]carbonyl}oxy)piperidine-l-carboxylate; tert-Butyl 4-[2-({[(2-methoxyphenyl)amino]carbonyl}oxy)ethyl]piperidine-l-carboxylate; tert-Butyl 4-[( { [(2,4-dimethoxyphenyl)amino] carbonyl } oxy)methyl]piperidine- 1 -carboxylate; tert-Butyl 4-{3-[(2-methoxyphenyl)amino]-3-oxopropyl}piperidine-l-carboxylate; tert-Butyl 4-{3-[(2,4-dimethoxyphenyl)amino]-3-oxopropyl}piperidine-l-carboxylate;

Piperidin-4-ylmethyl 2-methoxyphenylcarbamate;

Piperidin-4-ylmethyl 2,4-dimethoxyphenylcarbamate;

Pyrrolidin-3 -ylmethyl 2-methoxyphenylcarbamate;

N-(2-Methoxyphenyl)-3-piperidin-4-ylpropanamide; Cyclopentyl 4-[({[(2-methoxyphenyl)amino]carbonyl}oxy)methyl]piperidine-l-carboxylate;

[l-(2,2-Dimethylpropanoyl)piperidin-4-yl]methyl 2-methoxyphenylcarbamate;

Neopentyl 4-[({[(2-methoxyphenyl)amino]carbonyl}oxy)methyl]piperidine-l-carboxylate; tert-Butyl 4-[( { [(2,5 -dimethoxyphenyl)amino] carbonyl} oxy)methyl]piperidine- 1 -carboxylate;

Cyclopentyl 4-[({[(2,4-dimethoxyphenyl)amino]carbonyl}oxy)methyl]piperidine-l-carboxylate; tert-Butyl 4-[( { [(2-methoxyphenyl)amino]carbonyl} amino)methyl]piperidine-l -carboxylate;

Isopropenyl 4-[({[(2,4-dimethoxyphenyl)amino]carbonyl}oxy)methyl]piperidine-l-carboxylate;

S-Ethyl 4-[({[(2,4-dimethoxyphenyl)amino]carbonyl}oxy)methyl]piperidine-l-carbothioate;

2-Chlorobenzyl 4-[({[(2,4-dimethoxyphenyl)amino]carbonyl}oxy)methyl]piperidine-l- carboxylate; Benzyl 4-[( { [(2,4-dimethoxyphenyl)amino]carbonyl} oxy)methyl]piperidine- 1 -carboxylate;

[l-(Thien-2-ylsulfonyl)piperidin-4-yl]methyl 2,4-dimethoxyphenylcarbamate; isopropyl 4-[({[(2,4-Dimethoxyphenyl)amino]carbonyl}oxy)methyl]piperidine-l-carboxylate;

Cyclopentyl 4-{3-[(2-methoxyphenyl)ammo]-3-oxopropyl}piperidine-l-carboxylate;

Cyclopentyl 3-[({[(2-methoxyphenyl)amino]carbonyl}oxy)methyl]pyrrolidine-l-carboxylate; [1 -(3 ,3 -Dimethylbutanoyl)piperidin-4-yl]methyl 2-methoxyphenylcarbamate;

[l-(Cyclopentylacetyl)piperidin-4-yl]methyl 2-methoxyphenylcarbamate;

[ 1 -(Cyclohexylcarbonyl)piperidin-4-yl]methyl 2-methoxyphenylcarbamate;

[l-(Cyclohexylacetyl)piperidin-4-yl]methyl 2-methoxyphenylcarbamate;

Cyclopentyl 3-[( { [(2-methoxyphenyl)amino]carbonyl} oxy)methyl]pyrrolidine- 1 -carboxylate; tert-Butyl 3 - [( { [(2-methoxyphenyl)amino] carbonyl } oxy)methyl]pyrrolidine- 1 -carboxylate; and pharmaceutically acceptable derivatives thereof. As used herein the term: "Ci-g alkyl" whether alone or part of another group means an alkyl group having from 1 to 8 carbon atoms. "Cι_-6 alkyl" whether alone or part of another group means an alkyl group having from 1 to 6 carbon atoms. Such a group may be a linear, branched chain or cyclic alkyl group or a mixture thereof e.g. CH₂-cyclopentane. Examples of suitable alkyl groups are preferably methyl, ethyl, propyl, prop-2-yl, butyl, but-2-yl, 2-methylprop-2-yl, pentyl or hexyl. Alkyl groups are most preferably methyl or ethyl. Alkyl groups can be substituted by OH, halo or optionally substituted aryl groups. Alkyl groups can additionally be substituted by (C .₆)cycloalkyl groups.

"C_2-8 alkenyl" whether alone or part of another group means a hydrocarbon chain which contains one or more carbon-carbon double bonds, for example ethenyl, propenyl, butenyl, pentenyl or hexenyl.

"(C .₆)cycloalkyl" means a cycloalkyl group having 4, 5 or 6 carbon atoms, for instance cyclopropyl, cyclobutyl or cyclohexyl. Preferably it is cyclopropyl. Cycloalkyl groups can additionally be substituted by straight or branched alkyl groups.

"Aryl" means a 5- or 6- membered aromatic ring for example, phenyl or a 7- to 12- membered bicyclic aromatic ring for example, napthyl. Aryl groups can be substituted by halo.

"Halogen" or "halo" means fluorine, chlorine, bromine or iodine, preferably fluorine, chlorine or bromine.

"Pharmaceutically acceptable derivative" means any other pharmaceutically acceptable derivative of a compound ofthe present invention, for example a salt, ester or salt of such ester, which upon administration to the recipient, such as a human, is capable of providing (directly or indirectly) the said compound or an active metabolite thereof.

It will be appreciated that compounds ofthe invention can exist in various geoisometric forms or mixtures of geoisomers, including the individual E and Z isomers ofthe compounds of the invention as well as mixtures of such isomers, in any proportions. Preferred compounds of formula (I) are those wherein the group adjacent to the exo double bond and the carbonyl group are on opposite sides ofthe exo double bond. The compounds ofthe invention may exist in forms wherein one or more carbon centre is/are chiral. The present invention includes within its scope each possible optimal isomer substantially free, i.e., associated with less than 5% of any other optimal isomer(s), as well as mixtures of one or more optical isomers in any proportion, including racemic mixtures thereof.

Pharmaceutically acceptable salts are within the scope ofthe invention and are particularly suitable for medical applications because of their greater aqueous solubility relative to the parent (i.e. basic) compounds. Such salts must clearly have a pharmaceutically acceptable anion or cation. Suitable pharmaceutically acceptable base salts include ammonium salts, alkali metal salts, such as sodium or potassium salts, and alkaline earth salts, such as magnesium and calcium salts. Salts having a non-pharmaceutically acceptable anion are also within the scope of the invention as useful intermediates for the preparation or purification of pharmaceutically acceptable salts in the process of manufacturing a medicament.

Compounds of formula (A), (B), (C) and (D):

can be formed by reacting compounds of formula (E) with the appropriate sulphonyl chloride, chloroformate, acid, or isocyanate under standard conditions.

Compounds of formula (E) are prepared by acid treatment of compounds of formula (F), which are themselves available by reaction of compounds of formula (G), with a range of anilines at 0°C using triphosgene or by direct reaction with the appropriate isocyanate.

N-Boc protection to give compounds of type (G) is achieved by reaction ofthe relevant amino alcohol with Boc anhydride in the presence of triethylamine using literature procedures.

Compounds of formula (H) can be obtained by coupling the relevant N-Boc cycloalkyl carboxylic acid with the required aniline using HATU/DIPEA DMF at 80°C. Removal ofthe Boc protecting group under acid conditions, followed by reaction ofthe liberated amine yields the desired compounds of formula (J).

Compounds of formula (K) and (L) can be obtained by alkylation of compounds of formula (F) and (H) using potassium bis(trimethylsilyl)amide and the appropriate alkyl halide.

Compounds of formula (M) can be prepared by reaction between amines of formula (N) and the required anilines using triphosgene.

Chiral compounds (such as those where m = 0) were either prepared from the commercially available chiral starting materials or the enantiomers obtained by high pressure chromatographic separation using a chiral stationary phase.

Radiolabelled compounds bearing ¹²⁵I can be prepared from the appropriately labelled anilines and compounds of formula (O) using the triphosgene method. The anilines themselves can be prepared by procedures reported in the literature. In addition, radiolabelled Carbon or Tritium can be introduced to compounds bearing an aromatic methoxyl substituent such as (P) by reaction of radiolabelled alkyl halides with the appropriate phenol using literature procedures.

Figures 1 describes the activation of hSK-1 K⁺ channels expressed in HEK293T cells by a compound of Example 1.

Figure 2 shows selective activation of SK K⁺ channels by a compound of Example 1. Figure 3 illustrates how synergy between a compound ofthe invention and CCI7950, a riluzole-like SK channel activator (2-amino-4,7-dichloro-benzothiazole) indicates binding to different sites.

A compound ofthe invention typically acts as a SK channel opener, preferably an SK-1 channel opener. A SK channel opener is a compound which activates SK channels. An SK channel is activated when it is open to allow potassium ions to flow through the channel.

Preferably a compound ofthe invention activates SK channels in a Ca²⁺ sensitive manner. A compound that activates SK channels in a Ca²⁺ sensitive manner typically activates K⁺ channel activity ofthe channel a greater amount in the presence of greater amounts of Ca²⁺.

A compound ofthe invention may activate SK-1, SK-2 and SK-3 channels or may selectively activate one or more SK channel subtype. For example a compound ofthe invention may selectively activate SK channels. A compound which selectively activates SK channels will activate SK channels but will not activate or will activate less strongly IK channels. Preferably a compound ofthe invention will have no activity at IK channels. A compound may selectively activate a subtype of SK channels. For example a compound which selectively activates SK-1 channels will activate SK-1 channels but will not activate or will activate only to a lesser extent SK-2 and SK-3 channels.

Preferably compounds ofthe invention are selective for SK-1 over SK-2 or SK-3.

Preferably a compound ofthe invention will activate SK-1 channels, more preferably a compound ofthe invention will selectively activate SK-1 channels.

In a preferred embodiment a compound ofthe invention will have no activity at SK-2 or SK-3 channels.

The present invention provides a method of treating a patient afflicted with a disorder responsive to enhanced SK channel activity, which method comprises administering a therapeutically effective amount of a compound of formula (I). A patient in need of treatment will typically be afflicted with a urinogenital disorder such as bladder hyperexcitability, MED or urinary incontinence, a respiratory disorder such as asthma, chronic obstructive pulmonary disease (COPD) or cystic fibrosis, a cardiovascular disorder such as hypertension, angina pectoris, ischaemic heart disease or stroke (e.g. cerebral ischaemia), a neurological or psychiatric disorder such as bipolar disorder, psychosis or sleeping disorders, a disorder ofthe central nervous system such as cognitive dysfunction or epilepsy, a pain condition such as neuropathic pain or inflammatory pain, a gastro-intestinal disorder such as inflammatory bowel disease (IBD), irritable bowel syndrome (IBS) or collitis. Preferably the disorder is epilepsy, IBS, stroke, cognitive dysfunction, neuropathic pain or inflammatory pain.

Administration of a compound of formula (I) to a patient afflicted with a disorder responsive to enhanced SK channel activity will typically improve the condition of a patient afflicted with the disorder and/or alleviate the symptoms ofthe disorder.

The invention also provides the use of a compound of formula (I) in the manufacture of a medicament for the treatment of a disorder responsive to enhanced SK channel activity.

Use of a SK-1 ion channel activator, in the manufacture of a medicament for the treatment of pain and/or irritable bowel and or epilepsy is provided. Figure 3 shows the synergistic effect ofthe compound of example 1 and a riluzole-like SK channel opener indicating the binding ofthe compounds ofthe invention to a different binding site from the riluzole-like compounds (see example 35).

Preferably a compound ofthe invention will activate SK channels independently of other known SK channel openers such as riluzole-like compounds. More preferably a compound ofthe invention will potentiate the activity of a riluzole-like SK channel activator in a synergistic manner.

An further embodiment ofthe invention comprises a radiolabel assay for identification of a compound which selectively activates SK channels by; determining the displacement of a labelled compound of formula (I) by an unlabelled candidate compound, and optionally, comparing this to the displacement of a labelled compound of formula (D by an unlabelled compound of formula (I). Without wishing to be bound by any theory, it is believed that the compounds ofthe present invention are selective because they bind to a previously unrecognised binding site, which is different to that to which previously known SK-1 activators bind.

Compounds that displace the labelled compound of formula (I) bind to the same or a similar site to compounds of formula (I) and show selectivity for SK channels.

The invention further provides a compound identified by the radiolabel assay described above. In a further embodiment, a compound identified by this radiolabel assay could be used in the preparation of a medicament for the treatment of a urinogenital disorder such as bladder hyperexcitability, MED or urinary incontinence, a respiratory disorder such as asthma, chronic obstructive pulmonary disease (COPD) or cystic fibrosis, a cardiovascular disorder such as hypertension, angina pectoris, ischaemic heart disease or stroke (e.g. cerebral ischaemia), a neurological or psychiatric disorder such as bipolar disorder, psychosis or sleeping disorders, a disorder ofthe central nervous system such as cognitive dysfunction or epilepsy, a pain condition such as neuropathic pain or inflammatory pain, a gastro-intestinal disorder such as inflammatory bowel disease (IBD), irritable bowel syndrome (IBS) or collitis. Preferably the disorder is epilepsy, IBS, stroke, cognitive dysfunction, neuropathic pain or inflammatory pain.

Pharmaceutical compositions comprising compounds of also referred to herein as active ingredients, may be administered for therapy by any suitable route including oral, rectal, topical and parenteral (including subcutaneous, transdermal, intramuscular and intravenous). It will also be appreciated that the preferred route will vary with the conditions and age ofthe recipient and the chosen active ingredient.

The amount required ofthe individual active ingredient for the treatment ofthe disorder of course depends upon a number of factors including the severity of the symptoms of the disorder and the identity ofthe recipient and will ultimately be at the discretion ofthe attendant physician.

In general, for the foregoing conditions a suitable dose of a compound ofthe invention is in the range of from 0.05 to lOOmg per kilogram body weight ofthe recipient per day, preferably in the range of from 0.1 to 50mg per kilogram body weight of the recipient per day, most preferably in the range of from 0.5 to 20mg per kilogram body weight ofthe recipient per day and optimally from 1 to lOmg per kilogram body weight per day. The desired dose is preferably presented as two, three, four, five, six or more sub-doses administered at appropriate intervals during the day. These sub-doses may be administered in unit dosage forms, for example, containing from 1 to 1500mg, preferably from 5 to lOOOmg and most preferably from 10 to 700mg of active ingredient per unit dosage form.

While it is possible for the active ingredient to be administered alone it is preferable to present it as a pharmaceutical composition. The compositions ofthe present invention comprise at least one active ingredient as defined above together with one or more acceptable carriers therefor and optionally other therapeutic agents. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients ofthe composition and not injurious to the recipient. The test agents may be formulated with standard carriers and/or excipients as is routine in the pharmaceutical art, and as fully described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Eastern Pennsylvania 17^th Ed. 1985.

Compositions include those suitable for oral, rectal, topical or parenteral (including subcutaneous, intramuscular, transdermal and intravenous) administration. The compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general the compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.

Compositions ofthe present invention suitable for oral administration may be presented as discreet units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as powder or granules; as a solution or suspension in an aqueous or non- aqueous liquid; or as an oil-in-water liquid emulsion or in a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.

A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g. povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g. sodium starch glycollate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose) surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of powdered compound moistened with an inert liquid diluent. The tablets may be optionally coated or scored and may be formulated so as to provide slow or controlled release ofthe active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile.

Tablets may optionally be provided with an enteric coating, to provide release in parts ofthe gut other than the stomach.

Compositions suitable for oral use as described above may also include buffering agents designed to neutralise stomach acidity. Such buffers may be chosen from a variety of organic or inorganic agents such as weak acids or bases admixed with their conjugated salts.

Compositions for rectal administration may be presented as a suppository with a suitable base comprising for example cocoa butter or a salicylate.

Compositions suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the composition isotonic with the blood ofthe intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, as liposomes or other microparticulate systems which are designed to target the compounds to one or more organs. The compositions may be presented in unit-dose or multi- dose sealed containers, for example ampoules or vials, and may be stored in a freeze dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets ofthe kind previously described. Compositions suitable for transdermal administration may be presented as discreet patches adapted to remain in intimate contact with the epidermis ofthe recipient for a prolonged period of time. Such patches typically contain the active compound as an optionally buffered aqueous solution of, for example, from 0.1 to 0.2M concentration with respect to the said compound. As one particular possibility, the active compound, may be delivered from the patch by ionophoresis as generally described in Pharmaceutical Research 3(6), 318 (1986).

Preferred unit dosage compositions are those containing a daily dose or unit, daily sub- dose, as herein above recited, or an appropriate fraction thereof, of an active ingredient.

It should be understood that in addition to the ingredients particularly mentioned above the compositions of this invention may include other agents conventional in the art having regard to the type of composition in question, for example, those suitable for oral administration may include such further agents as sweeteners, thickeners and flavouring agents.

The following Examples illustrate the preparation of pharmacologically active compounds ofthe invention. The Procedures illustrate generic procedures used to prepare compounds ofthe invention.

Abbreviations used are as follows: DCM represents dichloromethane. THF represents tetrahydrofuran. TFA represents trifluoroacetic acid. DIPEA represents di-isopropylethylamine.

HATU represents 0-(7-azabenzotriazol-lyl)-l,l,3,3-tetramethyluronium hexafluorophosphate.

TEA represents triethylamine.

BOC represents N-tert-butoxycarbonyl.

Procedure A

The N-Boc cycloalkanemethanol (1 mM) was dissolved in anhydrous DCM. (40mL/g) and cooled to 0°C under a nitrogen atmosphere. Sodium hydride (1.05mM) was added portionwise over 20 minutes and the reaction then stirred at 0°C for 1 hr. The isocyanate (1 mM) was then added portionwise over 15 minutes, the cooling bath subsequently removed and stirring continued for 18hrs at ambient temperature. The reaction was quenched with saturated aq. sodium bicarbonate solution, diluted with an equal volume of DCM and washed with saturated aq. sodium bicarbonate solution. The organic layer was separated, dried over anhydrous MgS0 and evaporated in vacuo. The crude product was purified by mass directed autopreparative HPLC.

Procedure B

The N-Boc cycloalkanemethanol (lOmM) was dissolved in anhydrous THF (25mL/g) and cooled to 0°C under a nitrogen atmosphere. Sodium hydride (10.5mM) was added portionwise over 20 minutes and the reaction then stirred at 0°C for 2 hrs. A solution of triphosgene (4mM) in anhydrous THF (40mL/g) was then added dropwise over 30 minutes, the cooling bath subsequently removed and the reaction allowed to reach room temperature with stirring over 1 hr. The reaction was then cooled down to 0°C and a solution of the aniline (lOmM) in anhydrous THF (40mL/g) added dropwise over 2 hrs. The cooling bath was then removed and stirring continued for 18hrs at ambient temperature. The reaction was quenched with saturated aq. sodium bicarbonate solution and the THF removed by evaporation in vacuo. The residue was extracted with dichloromethane (2 x 100 mL) and the combined organic layers washed with saturated sodium bicarbonate (2 x 40 mL), 2M citric acid (2 x 40 mL) and brine (1 x 40 mL). The organic layer was separated, dried over anhydrous MgS0₄ and evaporated in vavuo. The crude product was purified by Biotage chromatography, eluting with cyclohexane: ethyl acetate 9:1, or by mass directed autopreparative HPLC.

Procedure C

The N-Boc carbamate (4mM) was dissolved in 19 mL of 4M HC1 in dioxan and 1ml of water at 0°C. Stirred for 1 hr and then partially evaporated in vacuo. The remaining solution was then lyophilised and either used without further purification or purified using an SCX cartridge with methanol washing and elution with 2M ammonia in methanol.

Procedure D

The N-Boc carbamate (4mM) was dissolved in 6 mL of 50%TFA in DCM and 0.15mL of anisole. Stirred for 2 hr at ambient temperature and then partially evaporated several times in vacuo. The residue was the dissolved in methanol and purified using an SCX cartridge with methanol washing and elution with 2M ammonia in methanol.

Procedure E

The amine (ImM) was dissolved in anhydrous dichloromethane (30mL/g) and DΪPEA (2mM) added. The solution was cooled to 0°C and the chloroformate (ImM) added dropwise. Cooling was removed and the reaction stirred at ambient temperature for 2-18 hrs, then quenched by the addition of aq. saturated sodium bicarbonate. The organic layer was separated, dried and evaporated in vacuo. The crude product was purified by mass directed HPLC and the required fractions recovered by lyophilisation.

Procedure F

The amine (ImM) was dissolved in anhydrous dichloromethane (30mL/g) and DIPEA (2mM) added. The solution was cooled to 0°C and a solution ofthe acid chloride (ImM) in anhydrous dichloromethane (lOmL/g) added dropwise. Cooling was removed and the reaction stirred at ambient temperature for 2-18 hrs, then quenched by the addition of aq. saturated sodium bicarbonate. The organic layer was separated, dried and evaporated in vacuo. The crude product was purified by mass directed HPLC and the required fractions recovered by lyophilisation.

Procedure G The amine (ImM) was dissolved in anhydrous dichloromethane (30mL/g) and DIPEA (2mM) added. The solution was cooled to 0°C and a solution ofthe sulphonyl chloride (ImM) in anhydrous dichloromethane (lOmL/g) added dropwise. Cooling was removed and the reaction stirred at ambient temperature for 2-18 hrs, then quenched by the addition of aq. saturated sodium bicarbonate. The organic layer was separated, dried and evaporated in vacuo. The crude product was purified by mass directed HPLC and the required fractions recovered by lyophilisation.

Procedure H

The acid (ImM) was dissolved in anhydrous dimethylformamide (20mL/g) and DIPEA (2mM) added followed by HATU (ImM). After 2 minutes this solution was added to a solution ofthe amine (ImM) in anhydrous dimethylformamide (5mL/g). The reaction was stirred at ambient temperature for 18 hrs and then evaporated in vacuo. The residue was partitioned between dichloromethane (15mL) and aq. saturated sodium bicarbonate (5mL) and the organic layer washed with aq. saturated sodium bicarbonate (5 mL), 2M citric acid (2 x 5 mL) and brine (1 5 mL). The organic layer was separated, dried and evaporated in vacuo. The crude product was purified by mass directed HPLC and the required fractions recovered by lyophilisation.

Procedure I

The amine (ImM) was dissolved in anhydrous THF (25mL/g) and cooled to 0°C under a nitrogen atmosphere. DIPEA (1.1 mM) was added and the reaction then stirred at 0°C for 5 mins. A solution of triphosgene (0.4 mM) in anhydrous THF (40mL/g) was then added dropwise over 10 minutes, the cooling bath subsequently removed and the reaction allowed to reach room temperature with stirring over 1 hr. The reaction was then cooled down to 0°C and a solution of the aniline (lOmM) in anhydrous THF (40mL/g) added dropwise over 20 mins. The cooling bath was then removed and stirring continued for 18 hrs at ambient temperature. The reaction was quenched with saturated aq. sodium bicarbonate solution and the THF removed by evaporation in vacuo. The residue was extracted with dichloromethane (2 x 10 mL) and the combined organic layers washed with saturated sodium bicarbonate (2 x 4 mL), 2M citric acid (2 x 4 mL) and brine (1 x 4 mL). The organic layer was separated, dried over anhydrous MgS0 and evaporated in vavuo. The crude product was purified by mass directed autoprep.

Procedure J

The carbamate (0.5mM) was dissolved in anhydrous THF (30mL/g) at 0°C under an atmosphere of nitrogen and a solution of potassium bis(trimethylsilyl)amide (0.75mM of an 0.5M solution in toluene) added via syringe. The reaction was stirred at 0°C for 30 minutes and then a solution of methyl iodide (0.5mM) in dry THF (30 mL/g) added dropwise. Stirring was continued at 0°C for 30 minutes and then cooling was removed and the reaction left at ambient temperature for 18 hours. The reaction mixture was then evaporated in vacuo and the residue partitioned between ethyl acetate (20mL) and IN HC1 (10 mL). The organic layer was washed with saturated aq. sodium, bicarbonate (2 x 10 mL) and brine (1 x 10 mL), dried and evaporated in vacuo. The crude product was then purified by mass directed HPLC and the required product recovered by lyophilisation.

Procedure K

The aminoalcohol (0.1M) was dissolved in anhydrous dioxan (lOmL/g) at 0°C under an atmosphere of nitrogen and TEA (0.3M ) added. A solution of Boc anhydride (0.12M) in dioxan (1.5mL/g) was added dropwise over 30 mins and the cooling bath subsequently removed. The reaction was then stirred at ambient temperature for 18 hrs and then evaporate in vacuo. The residue was dissolved in DCM, washed with 0.5M citric acid (2 x 50 mL), saturated aq. sodium bicarbonate (2 x 50 mL) and water (1 x 50 mL). The organic layer was then dried over anhydrous magnesium sulphate and evaporated in vacuo. The product was generally suitable for use without further purification.

Procedure L

The acid (ImM) was dissolved in anhydrous dimethylformamide (20mL/g) and DIPEA (2mM) added followed by HATU (ImM). After 2 minutes this solution was added to a solution ofthe amine (ImM) in anhydrous dimethylformamide (5mL/g). The reaction was stirred at 80°C for 2 hrs and then evaporated in vacuo. The residue was dissolved in ethyl acetate and then washed with aq. saturated sodium bicarbonate (2 x), 0.5M hydrochloric acid (2 x) and brine (1 x). The organic layer was separated, dried and evaporated in vacuo. The crude product was purified by mass directed HPLC and the required fractions recovered by lyophilisation.

Procedure M

The aniline (ImM) and triethylamine (1.05mM) were dissolved in anhydrous THF (50mL/g of aniline) and cooled to 0°C under a nitrogen atmosphere. Triphosgene (0.4mM) was added portionwise over 20 minutes and the reaction then stirred for a further 30 mins at 0°C. A solution of the primary amine (ImM) in anhydrous THF (40mL/g) was then added dropwise over 15 minutes, the cooling bath subsequently removed and the reaction allowed to reach ambient temperature. Stirring was then continued for 18hrs and then quenched by the addition of ice. The reaction mixture was partially evaporated in vacuo to remove THF and then extracted into dichloromethane. The organic layer was washed with saturated aq. sodium bicarbonate solution (2x), and brine (2x), dried over anhydrous magnesium sulphate and evaporated in vacuo. The residue contained a mixture of the required product and symmetrical urea side products and required mass directed autopreparative HPLC for purification.

Example 1: tert-Butyl 4-[({[(2-methoxyphenyl)amino]carbonyl}oxy)methyl]piperidine-l- carboxylate

The title compound was prepared from N-Boc piperidinemethanol and 2 methoxy isocyanate by procedure A or on a larger scale (> 0.1M) by using 2-methoxy aniline and procedure B. The sample was purified in small quantities by mass directed HPLC, or in larger quantity by biotage chromatography, eluting with cyclohexane:ethyl acetate 9:1. The product was obtained as a colourless oil which slowly crystallised to a white solid on standing. MH÷ 365 (100%), MΝH4⁺ 382 (100%), 265 (40%).

Example 2: tert-Butyl 3-[({[(2-methoxyphenyl)a ino]carbonyl}oxy)methyl]pyrrolidine-l- carboxylate

The title compound was prepared from 3 hydroxymethyl- 1-boc-pyrrolidine and 2 methoxy isocyanate by procedure A or on a larger scale (> 0.1M) by using 2-methoxy aniline and procedure B. The sample was purified in small quantities by mass directed HPLC, or in larger quantity by biotage chromatography, eluting with cyclohexane: ethyl acetate 9:1. The product was obtained as a colourless oil. MH⁴ 351 (100%)

Example 3; tert-Butyl 4-({[(2-methoxyphenyl)amino]carbonyl}oxy)piperidine-l-carboxylate

The title compound was prepared from tert butyl-4-hydroxy-l-piρeridinecarboxylate and 2- methoxy isocyanate by procedure A. The sample was purified by mass directed HPLC. The product was obtained as a white solid by lyophilisation from 1,4-dioxan. MH⁺ 351 (20%), 295 (100%), 251 (20%). Example 4: tert-Butyl 4-[2-({[(2-methoxyphenyl)amino]carbonyl}oxy)ethyI]piperidine-l- carboxylate

The title compound was prepared from N-Boc 4-piperidineethanol and 2-methoxy isocyanate by procedure A. The sample was purified by mass directed HPLC. The product was obtained as a white solid by lyophilisation from 1,4-dioxan. MH⁺ 379 (100%), 323 (100%), 279 (80%).

Example 5: tert-Butyl 4-[({[(2,4-dimethoxyphenyl)amino]carbonyl}oxy)methyl]piperidine-l- carboxylate

The title compound was prepared from N-Boc piperidinemethanol and 2,4-dimethoxy aniline using procedure B. The sample was purified in small quantities by mass directed HPLC, or in larger quantity by biotage chromatography, eluting with cyclohexane: ethyl acetate 9:1. The product was obtained as a white solid. MH⁺ 395(90%), 339 (100%), 295 (60%).

Example 6: tert-Butyl 4-{3-[(2-methoxyphenyl)amino]-3-oxopropyl}piperidine-l-carboxylate

The title compound was prepared from N-boc-4-piperidinepropionic acid and 2-methoxy aniline using procedure L. The crude sample was purified by mass directed HPLC and the required product obtained by lyophilisation from 1,4-dioxan. MH^1" 363 (90%), 307 (100%), 263 (80%). Example 7: tert-Butyl 4-{3-[(2,4-dimethoxyphenyl)amino]-3-oxopropyI}piperidine-l- carboxylate

The title compound was prepared from N-boc-4-piperidinepropionic acid and 2,4-dimethoxy aniline using procedure L. The crude sample was purified by mass directed HPLC and the required product obtained by lyophilisation from 1,4-dioxan. MH÷ 393 (100%), 337 (90%), 293 (40%).

Example 8: Piperidin-4-ylmethyl 2-methoxyphenylcarbamate

The title compound was prepared from the compound of Example 1 using procedure C. The crude residue was dissolved in methanol and purified using an SCX cartridge (methanol washing and elution with 2M ammonia in methanol). The eluted product was evaporated in vacuo and lyophilised to give a colourless gum which was purified by mass directed HPLC in small quantities or used without further purification for subsequent synthesis. MH⁺ 265 (100%), 2MH⁺ 529 (20%).

Example 9: Piperidin-4-ylmethyI 2,4-dimethoxyphenylcarbamate

2,4-Dimethoxyphenyl carbamic acid piperidinemethanol ester was prepared from Example 5 using procedure C. The crude residue was dissolved in methanol and purified using an SCX cartridge (methanol washing and elution with 2M ammonia in methanol). The eluted product was evaporated in vacuo and lyophilised to give a colourless gum which was purified by mass directed HPLC in small quantities or used without further purification for subsequent synthesis. MET 295 (100%).

Example 10: Pyrrolidin-3-ylmethyl 2-methoxyphenylcarbamate

2-Methoxyphenyl carbamic acid pyrrolidinemethanol ester (26) was prepared from Example 2 using procedure C. The crude residue was dissolved in methanol and purified using an SCX cartridge (methanol washing and elution with 2M ammonia in methanol). The eluted product was evaporated in vacuo and lyophilised to give a colourless gum which was purified by mass directed HPLC in small quantities or used without further purification for subsequent synthesis. MH⁺ 251 (100%).

Example 11: N-(2-Methoxyphenyl)-3-piperidin-4-ylpropanamide

The title compound was prepared from Example 6 using procedure C. The crude residue was dissolved in methanol and purified using an SCX cartridge (methanol washing and elution with 2M ammonia in methanol). The eluted product was evaporated in vacuo and lyophilised to give a colourless gum which was purified by mass directed HPLC in small quantities or used without further purification for subsequent synthesis. MH⁺ 263 (100%).

Example 12: Cyclopentyl 4-[({[(2-methoxyphenyl)amino]carbonyl}oxy)methyl]piperidine-l- carboxylate

The title compound was prepared from compound of Example 8 and cyclopentyl chloroformate using procedure E. The crude product was purified by mass directed HPLC and the required fractions recovered by lyophilisation as a colourless oil. MET^1" 377 (100%), MNH4⁺ 394 (100%).

Example 13: [l-(2,2-Dimethylpropanoyl)piperidin-4-yl]methyl 2-methoxyphenylcarbamate

The title compound was prepared from Example 8 and 2,4-tert butylcarbonyl chloride using procedure F. The crude sample was purified by mass directed HPLC. MH⁺ 349 (100%), 284 (50%).

Example 14: Neopentyl 4-[({[(2-methoxyphenyl)amino]carbonyl}oxy)methyl]piperidine-l- carboxylate

The title compounds was prepared from Example 8 and neopentyl chloroformate using procedure E. The crude product was purified by mass directed HPLC and the required fractions recovered by lyophilisation. MH⁺ 379 (100%), 212 (40%).

Example 15: tert-Butyl 4-[({[(2,5-dimethoxyphenyl)amino]carbonyI}oxy)methyl]piperidine- 1-carboxylate

The title compound was prepared from N-boc-4-piperidinemethanol and 2,5-dimethoxy aniline using procedure B. The crude sample was purified by mass directed HPLC and the required product obtained by lyophilisation from 1 ,4-dioxan. Mϊ 395 (50%), 339 (100%), 295 (40%).

Example 16: Cyclopentyl 4-[({[(2,4- dimethoxyphenyl)amino]carbonyI}oxy)methyl]piperidine-l-carboxylate

The title compound was prepared from 2,4-dimethoxyphenyl carbamic acid piperidinemethanol ester (Example 5) and cyclopentyl chloroformate using procedure E. The crude product was purified by mass directed HPLC and the required fractions recovered by lyophilisation. MH" 407 (100%), 339 (60%), 295 (40%).

Example 17: tert-Butyl 4-[({[(2-methoxyphenyl)amino]carbonyl}amino)methyI]piperidine-l- carboxylate

The title compound was prepared from 2-methoxyaniline and Boc-4-(aminoethyl) piperidine using procedure M. The crude product was purified by mass directed HPLC and the required fractions recovered by lyophilisation. MHT 394 (80%), 338 (100%), 294 (20%). Example 18: Isopropenyl 4- [({[(2,4- dimethoxyphenyl)amino]carbonyl}oxy)methyI]piperidine-l-carboxyIate

The title compound was prepared from 2,4-dimethoxyphenyl carbamic acid piperidinemethanol ester (Example 9) and isopropenyl chloroformate using procedure E. The crude product was purified by mass directed HPLC and the required fractions recovered by lyophilisation. MH⁺ 379 (100%), 339 (100%), 321 (20%).

Example 19: S-ethyl 4-[({[(2,4-dimethoxyphenyI)amino]carbonyl}oxy)methyI]piperidine-l- carbothioate

The title compound was prepared from 2,4-dimethoxyphenyl carbamic acid piperidinemethanol ester (Example 9) and ethyl thiolchloroformate using procedure E. The crude product was purified by mass directed HPLC and the required fractions recovered by lyophilisation. MH^1" 383 (100%)

Example 20: 2-ChIorobenzyl 4-[({[(2,4- dimethoxyphenyl)amino]carbonyl}oxy)methyl]piperidine-l-carboxylate

The title compound was prepared from 2,4-dimethoxyphenyl carbamic acid piperidinemethanol ester (Example 9) and 2-chlorobenzyl chloroformate using procedure E. The crude product was purified by mass directed HPLC and the required fractions recovered by lyophilisation. MH 463 (100%).

Example 21: Benzyl 4-[({[(2,4-dimethoxyphenyl)amino]carbonyl}oxy)methyI]piperidine-l- carboxylate

The title compound was prepared from 2,4-dimethoxyphenyl carbamic acid piperidinemethanol ester (Example 9) and benzyl chloroformate using procedure E. The crude product was purified by mass directed HPLC and the required fractions recovered by lyophilisation. MH⁺ 429 (100%).

Example 22: [l-(Thien-2-ylsulfonyl)piperidin-4-yl]methyl 2,4-dimethoxyphenylcarbamate

The title compound was prepared from 2,4-dimethoxyphenyl carbamic acid piperidinemethanol ester (Example 9) and 2-thiophenesulphonyl chloride using procedure G. The crude product was purified by mass directed HPLC and the required fractions recovered by lyophilisation. MH⁺ 441 (100%).

Example 23: Isopropyl 4-[({[(2,4-dimethoxyphenyl)amino]carbonyl}oxy)methyl]piperidine- 1-carboxylate

The title compound was prepared from 2,4-dimethoxyphenyl carbamic acid piperidinemethanol ester (Example 9) and isopropyl chloroformate using procedure E. The crude product was purified by mass directed HPLC and the required fractions recovered by lyophilisation. MH⁺ 381 (100%), 295 (10%), 184 (70%).

Example 24: Cyclopentyl 4-{3-[(2-methoxyphenyl)amino]-3-oxopropyI}piperidine-l- carboxylate

The title compound was prepared from Example 11 and cyclopentyl chloroformate using procedure E. The crude product was purified by mass directed HPLC and the required fractions recovered by lyophilisation. MH⁺ 375 (100%), 307 (30%), 263 (80%).

Example 25: Cyclopentyl 3-[({[(2-methoxyphenyl)amino]carbonyI}oxy)methyl]pyrrolidine- 1-carboxylate

The title compound was prepared from 2-methoxyphenyl carbamic acid pyrrolidmemethanol ester (Example 10) and cyclopentyl chloroformate using procedure E. The crude product was purified by mass directed HPLC and the required fractions recovered by lyophilisation. MH⁺ 363 (100%), 295 (10%).

Example 26: [l-(3,3-Dimethylbutanoyl)piperidin-4-yl]methyl 2-methoxyphenylcarbamate

The title compound was prepared from the product of Example 8 and t-butylacetyl chloride using procedure F. The crude product was purified by mass directed HPLC and the required fractions recovered by lyophilisation. MH⁺ 363 (100%). Example 27: [l-(Cyclopentylacetyl)piperidin-4-yl]methyl 2-methoxyphenylcarbamate

The title compound was prepared from the product of Example 8 and cyclopentylacetylcarbonyl chloride using procedure F. The crude product was purified by mass directed HPLC and the required fractions recovered by lyophilisation. MH⁺ 375 (100%).

Example 28: [l-(CyclohexylcarbonyI)piperidin-4-yl]methyl 2-methoxyphenylcarbamate

The title compound was prepared from the product of Example 8 and cyclohexylcarbonyl chloride using procedure F. The crude product was purified by mass directed HPLC and the required fractions recovered by lyophilisation. MH⁺ 375 (100%).

Example 29: [l-(Cyclohexylacetyl)piperidin-4-yl]methyl 2-methoxyphenylcarbamate

The title compound was prepared from the product of Example 8 and cyclohexylacetyl chloride using procedure F. The crude product was purified by mass directed HPLC and the required fractions recovered by lyophilisation. MET 389 (100%).

Example 30a) and b): Cyclopentyl 3-[({[(2- methoxyphenyl)amino]carbonyl}oxy)methyl]pyrrolidine-l-carboxylate

The title compounds were prepared by chiral chromatographic separation ofthe product of Example 25 using a chiralcel OD column and eluting with Ethanol/Heptane. Evaporation ofthe required eluent fractions gave the products as colourless oils. MH÷ 363 (100%)

Example 31a) and b) tert-Butyl 3-[({[(2- methoxyphenyl)amino]carbonyl}oxy)methyl]pyrrolidine-l-carboxylate

The title compounds were prepared by chiral chromatographic separation of Example 2 using a chiralcel OD column and eluting with Ethanol/Heptane. Evaporation ofthe required eluent fractions gave the products as colourless oils.

MH⁺ 351 (100%) - -

Example 32: Cloning of SK/IK channels & generation of stable cell lines

The full length hSK-1, 2, 3 and IK clones were obtained either by PCR and 5 '-RACE from a human cDNA library (Genbank accession numbers hSKl U69883; hSK-2 unpublished) or from the Incyte or dbEST databases (Incyte Pharmaceuticals, Inc.; hSK3 AJ251016, hlK

AF022797). An optimal Kozak sequence was added to the initiation site and the cDNA was cloned into Bluescript and subsequently the IRES vector pCTN5 for mammalian expression.

All SK/IK stable cell lines were generated in either CHO-K1 or HEK293T using the pCIN5 construct, under selection for resistance to 500 μg/ml geneticin Five μg of the purified plasmid DNA was used to transfect 5x10⁶ CHO-K1 cells (ATCC N° CRL 9618) by electroporation (standard protocol with BIORAD Gene Pulser IT). After two days of growth in

Petri dishes in alpha-MEM (Gibco BRL, Cat. No. 22571) and 10% FBS (Gibco BRL), chemical selection was initiated by addition of the antibiotic G418-sulfate (Calbiochem) at a final concentration of 0.5 mg/ml. After another two weeks of culture, antibiotic-resistant clones were isolated, expanded under antibiotic selection and subsequently stored frozen in liquid nitrogen until further analysis.

Channel activity was determined either using electrophysiology (see later) or with a microtitre plate assay using membrane potential-sensitive fluorescence dyes. This involved reading DiBAC₄(3) fluorescence on a fluorometric imaging plate reader (FLIPR®, Molecular Devices) or fluorescence resonance energy transfer (FRET) between a phospholipid anchored coumarin donor (CC2-DMPE) and oxonol acceptor (DisBAC2(3)) on a voltage ion probe reader (VTPR, Aurora Biosciences). For the FLIPR assays, cells were seeded in black walled clear bottom sterile 96 well plates at 50K cells per well and grown at 37°C, 5% C0₂ for 2-3 days. After removal of media, cells were then loaded with 5μM DiBAC₄ for 45 min at 37°C prior to addition of Ca²⁺ mobiliser (e.g. UTP) or ionomycin (lμM) using the FLIPR pipettor head. A reduction in fluorescence is observed on opening the channel, equating to membrane hyperpolarisation. The cell line with the highest activity (eg greatest hyperpolarisation) for each channel was further dilution cloned, and again selected for channel activity this time be elecfrophysiology. The presence of the correct channels was determined by RT-PCR using gene-specific primers and immunoblotting with specific antibodies. Stable cell lines were cultured under selection for resistance to 500 μg/ml geneticin in α-MEM (Gibco BRL, Cat.No 22571) with 10% heat inactivated foetal bovine serum (Gibco BRL, Cat.No 10108-165) at 37°C and 5% CO, 2.

Example 33: Membrane potential assays for channel activators

For pharmacological studies on the VIPR, hSK-1 or hIK expressing cells are plated in black walled clear bottom sterile 96 well plates (Costar, cat. No. 3603) at a density of 50K cells per well well and grown at 37°C, 5% C0₂ for 2-4 days. Membrane potential assays are performed at room temperature by first washing the cells with buffer A (145mM NaCl, 5mM KCI, 2mM CaCl₂, ImM MgCl₂, 20mM HEPES, lOmM glucose, pH 7.4) and then incubating with the coumarin dye (CC2-DMPE, Aurora, diluted in buffer A, 8μM) for 30min. Cells are then washed to remove unbound coumarin prior to addition ofthe oxonol DisBAC₂ (3) (Aurora, buffer A, 8μM, 45min) and ESS Cy4 a background signal fluorescence quenching reagent. The coumarin fluorophore is excited at 405nM using a Xenon lamp and dual emission is measured every second at 460nM (coumarin) and 580nM (oxonol) using the VIPR photomultiplier tubes. The signal ratio at the two wavelengths is calculated as a measure ofthe FRET between the two fluorophores. After 5 consecutive baseline readings test compounds are added online using the VIPR pipetting head. Fluorescence is monitored for up to a further 2min. To determine final RfZRi values fluorescence values from a cell free microtitre plate were first subtracted. Typically channel activators produce a concentration-dependent reduction in the ratio by up to 0.5 units and are active at hSK-1 at final concentrations of lOOnM-lOOμM. Compounds of Examples 1 to 31 have potencies in the range of lOOnm to lOOμM.

Example 34: Patch clamp electrophysiology analysis

Standard whole cell patch clamp electrophysiology methods are used to record membrane currents from single cells expressing either SK or IK channels. Cells are plated on to glass coverslips and placed in a small (volume <500μl) chamber on the platform of an inverted microscope (Nikon Diaphot 200). A gravitational flow system is used to perfuse the cells (2ml min^"1) with an external salt solution containing 144mM KCI, ImM MgCl₂, lOmM HEPES, 2mM CaCl₂ buffered to pH 7.4 (osmolarity 290-3 lOmOsm). All experiments are performed at room temperature (20-22°C). Membrane currents are amplified and recorded with an Axopatch 200A/B or HEKA EPC-9 patch clamp amplifier coupled to a Pentium microprocessor via an analog- digital interface. Patch pipettes are pulled (Sutter model 97) from 1.5mm outside diameter borosilicate glass (Clark Electromedical) and fire polished (Narishige Microforge) to give final tip resistances of 2-4MΩ. Pipettes are backfilled with an internal solution containing a 144mM KCI, 5mM EGTA, lOmM HEPES, ImM MgCl₂ and concentrations of CaCl₂ calculated to give free Ca²⁺ concentrations ranging from 30nM to lμM. A silver/silver chloride pellet is used as the bath (reference) electrode. Signals are pre-filtered at bandwidth 2kHz and sampled at 4kHz. Capacitance transients and series resistance errors are compensated for (80-85%) using the amplifier circuitry.

Voltage ramp protocols (-lOOmV for 50ms followed by a 200ms ramp to +70mV) from a holding potential of OmV are applied to the cell to frack the membrane current. Ramps are applied every 10s. Non-specific' leak current' is defined by the residual current at OmV when the external solution is replaced by one containing zero K⁺ (144mM NaCl, ImM MgCl₂, lOmM HEPES, 2mM CaCl₂ buffered to pH 7.4 (osmolarity 290-3 lOmOsm)). Once a stable Ca²⁺-activated K⁺ current is recorded (always <150pA in amplitude, 3-5min after whole cell recording configuration is achieved) drug solutions are applied to the cell via a 'U-tube' system. This is comprised of a fine microfil tube positioned within lOOμm of the cell, attached to two solenoid valves and a reservoir of drug containing solution.

Application of drugs typified by a, b, c, and d at concentrations of lOμM increased the whole cell K⁺ current in SK cells but not in untransfected cells, indicating that they are specific SK openers. The effects of these agents are concentration-related, typically between 30nM- lOOμM. Generally the effects could be reversed on washout. Compounds of Example 1 and analogues had no effect in hIK channel expressing cell lines indicating that these agents are selective for SK channels. In some experiments the effect of internal Ca²⁺ concentrations on the potency of drugs typified by a,b,c and d was explored by comparing responses between cells recorded with either 30nM, lOOnM or 300nM free Ca²⁺ in the pipette solution. Typically, lower concentrations of openers were required to activate channels when Ca²⁺ concentrations were high i.e. the activity of these agents may be Ca²⁺-sensitive. Representative data sets are shown in Figures 2 and 3.

Example 35: Binding site analysis/identification

Combination experiments with SK channel openers from other structural classes (e.g. riluzole-like compounds; Grunnet et al., 2001, Neuropharmacol., 40, 879-887) indicate that compounds from the chemical series of the invention bind to a distinct or overlapping site on the channel. Figure 3 illustrates such an experiment. Figure 3(a) shows Ca²⁺-dependence of responses to a compound of the invention

(compound of example 1). The Concentration-response curves for CHO-hSKl whole cell currents evoked by the compound are shown under conditions of low (30nM) and intermediate (300nM) free internal Ca²⁺. Currents were recorded at V_h -lOOmV. The x-axis shows log μM concentration of the compound. The y-axis shows current amplitude in pA. Note the small increase in potency (EC₅₀ lμM compared to 4.1μM) and large augmentation of the response (3700pA compared to 460pA at 30μM) to the compound in the presence of higher Ca²⁺. This indicates that the effects of compound of example 1 are Ca²⁺ and/or open channel dependent. The concentration of compound selected for the interaction studies in panel B (lOμM) produced an equivalent response to 300nM Ca ⁺ alone

Figure 3(b) shows interaction experiments between a compound of the invention (compound of example 1) and CCI7950, a riluzole-like SK channel activator (2-amino-4,7- dichloro-benzothiazole). Concentration-response curves for CHO-hSKl whole cell currents evoked by CCI7950 are shown under conditions of low (30nM) and intermediate (300nM) free internal Ca²⁺. Also shown is the effect of CCI7950 in the presence of 30nM Ca²⁺ and a fixed concentration (lOμM) of (externally applied) compound of example 1. The response to the compound of example 1 alone was 460pA - panel A. Note the leftward shift ofthe concentration response curve to CCI7950 in the presence ofthe compound ofthe invention, indicating that these two agents synergise and thereby interact with different sites. Concentrations of drug were applied directly to the cell using a U-tube delivery system. Each point represents the mean of >3 cells, and the error bars the s.e.m.

In the presence of a low concentration of intracellular Ca²⁺ (30nM), the potency of CCI7950, is weak (EC₅₀=14μM [7-29μM]; n=3.). Partially activating the channel by increasing [Ca²⁺]i to 300nM markedly potentiates CCI7950, such that the EC₅₀ value is reduced to 153nM [58-407nM]; n=4. If one substitutes the Ca²⁺ increase in this experiment with the compound of example 1 to produce equivalent partial channel activation, potentiation of CCI7950 still occurs. This can be seen in fig. 3b where the compound of example 1 acts to decrease the EC₅₀ value of CCI7950 from 14μM to 1.7μM [0.8-3.6]; n=4; PO.05. This synergy between the compounds of the invention and CCI7950 indicates that the two agents must bind to distinct or possibly overlapping binding sites to produce channel activation.

Example 36: Radiolabel Assay By attaching a radioisotope to an appropriate compound in the chemical series of the invention, radioligand binding assays can be established to identify novel channel modulators. Radiolabelled compounds bearing ¹²⁵I can be prepared from the appropriately labelled anilines and compounds of formula O (defined above) using the triphosgene method. The anilines themselves can be prepared by procedures reported in the literature. In addition, radiolabelled Carbon or Tritium can be introduced to compounds bearing an aromatic methoxyl substituent such as those of formula P (defined above) by reaction of radiolabelled alkyl halides with the appropriate phenol using literature procedures.

In order to identify a candidate compound which selectively activates SK channels, the displacement of a labelled compound of formula (I) by an unlabelled candidate compound should be determined, and optionally, as a control, compared to the displacement of a labelled compound of formula (I) by an unlabelled compound of formula (I). Without wishing to be bound by any theory, it is believed that the compounds of the present invention are selective because they bind to a previously unrecognised binding site which is different to that to which previously known SK-1 activators bind. Therefore, compounds that displace the labelled compound of formula (I) bind to the same or a similar site to compounds of formula (I) and show selectivity for SK channels.

Radioligand binding assays may also be used to characterise the binding sites for different channel modulators. In order to do this, a radiolabelled compound of example 1 is employed in a competitive assay with a riluzole-like compound, and the results compared with those from an assay in which the labelled riluzole-like compound is in binding competition with a second unlabelled riluzole-like compound. As a further demonstration, labelled and un-labelled compounds of the present invention may be used in the same competitive radioligand binding assay.

Thus, the unlabelled riluzole-like compound will displace the labelled riluzole-like compound in the binding assays because they will bind to the same binding site. Similarly the labelled and un-labelled compounds of example 1 will displace one another. However the labelled compound of example 1 will show a different displacement profile when in binding competition with the unlabelled riluzole-like compound, because, as example 36 indicates, the riluzole-like compound binds to a distinct or overlapping site on SK-1 ion channels from the compound of example 1.

Quantification of radioligand binding by scintillation proximity assay (SPA) is a long- established principle. Briefly, the affinity of compounds for a receptor is assessed by the specific competition between known quantities of radiolabelled ligand and compound for that receptor. Increasing concentrations of compound reduce the amount of radiolabel that binds to the receptor. This gives rise to a diminishing scintillation signal from SPA beads coated with membranes that bear the channel. The signal may be detected with a suitable scintillation counter and the data generated may be analysed with suitable curve-fitting software, (see Udenfriend et al., 1987 Anal Biochem. 161, 494-500 and Carpenter et al., 2002, Methods Mol Biol 190, 31-49).

As an example, membrane fractions are prepared from hSK-1 expressing HEK cells by centrifuging for 6min at 500g, 37°C and then lysing by addition of a suitable ice cold hypotonic buffer such as one containing lOmM HEPES and ImM EGTA, pH 7.40. The lysate is then centrifuged (10⁵ x g, 4°C, 60min) and the resulting membrane pellet is resuspended in buffer (e.g. HEPES 5, KCI 5, NaCl 140, pH 7.4, 4°C) at approx 3mg ml^"1 protein concentration (determined according to Lowry et al., 1951 J. Biol Chem 193, 265-275). Membrane suspensions such as that described may be stored at -80°C until used. For assay, membranes expressing hSKl channels are diluted in a pH-buffered medium and mixed with SPA beads coated with a suitable substance to facilitate the adhesion of membranes to the beads. The concentrations of membrane protein and SPA beads chosen should result in SPA binding signal of at least 300 corrected counts per minute (CCPM) when tritiated radioligand at a concentration close to its K_d (affinity value) is combined with the mixture. Non-specific binding (nsb) may be determined by competition between the radiolabelled ligand and a saturating concentration of unlabelled ligand. In order to quantify the affinity of the compound of the invention binding site ligands, compounds are diluted in a stepwise manner across the wells of a 96-well plate. Radioligand, compound, and unlabelled ligand are then added to a 96-well plate suitable for the measurement of SPA binding signals prior to the addition of bead / membrane mixture to initiate the binding reaction. Equilibrium may be achieved by incubation at room temperature for 120 minutes prior to scintillation counting. The data so generated may be analysed by means of a computerised curve-fitting routine in order to quantify the concentration of compound that displaces 50% of the specific radioligand binding (IC₅₀). The affinity (pK*) of the compound may be calculated from the IC₅₀ by application of the Cheng-Prusoff correction. Suitable reagents and protocols are: reaction buffer containing NaCl 140mM, KCI 5mM HEPES 5mM MgCl ImM EGTA 5mM CaCl 2mM pH adjusted to 7.4 with KOH; SPA beads coated with wheatgerm agglutinin; 1.25nM [³H]- radioligand; lOμM unlabelled ligand; a three-fold dilution series of compound starting at lOμM and ending at 0.3nM is adequate.

The application of which this description and claims forms part may be used as a basis for priority in respect of any subsequent application. The claims of such subsequent application may be directed to any feature or combination of features described herein. They may take the form of product, composition, process, or use claims and may include, by way of example and without limitation the following claims:

Claims

1. A compound which selectively activates SK channels. 2. A compound according to claim 1 which has no activity at IK channels.

3. A compound according to claim 1 or claim 2 which selectively activates SKI channels.

4. A compound according to any one of claims 1 to 3 which has no activity at SK2 or SK3 channels.

5. A compound which potentiates the activity of a riluzole-like SK channel activator in a synergistic manner.

6. Use of a compound of formula (I):

wherein

R¹ independently represents an optionally substituted methoxy;

R² represents H;

R³ represents COOR⁴, COSR⁴, COR⁶, S0₂-R⁷ or CONHR⁵;

R⁴ represents optionally substituted (C]_-8)alkyl, optionally substituted (C_4-6)cycloalkyl, optionally substituted (C₂.₈)alkenyl or aryl;

R⁵ represents H, optionally substituted, (Cι_-8)alkyl, optionally substituted (C .₆)cycloalkyl or aryl;

R⁶ represents optionally substituted (Cι_-6)alkyl, aryl or furanyl;

R⁷ represents optionally substitued aryl or thienyl; n is an interger from 0 to 2; y is an interger from 0 to 2; m is 0 or 1 ;

W can not be N when m is 0; and a pharmaceutically acceptable derivative thereof; as an activator of a SK channel.

7. A compound of formula (la);

(1a)

wherein

R¹ independently represents an optionally substituted methoxy;

R² represents H;

R³ represents H, COOR⁴, COSR⁴, COR⁶, S0₂-R⁷ or CONHR⁵;

R⁴ represents optionally substituted (C₁_₈)alkyl, optionally substituted (C₄.₆)cycloalkyl, optionally substituted (C_2-8)alkenyl or aryl;

R⁶ represents optionally substituted (C-._₆)alkyl, aryl or furanyl;

R⁷ represents optionally substitued aryl or thienyl; n is an interger from 0 to 2; y is an interger from 0 to 2; m is 0 or 1;

W can not be N when m is 0; and pharmaceutically acceptable derivatives thereof; with the proviso that the compound is not: [l-[l-(3,4-dichlorobenzyl)pyrrolidin-3-ylmethyl]-3-(4-methoxyphenyl) urea; urea N-[[l-[(3,4-dichlorophenyl)methyl-3-pyrrolidinyl]methyl]-N'-(2-methoxyphenyl).

A compound as claimed in claim 7 wherein the compound is of formula (IT);

wherein X represents O, NH or CH₂;

R^! independently represents an optionally substituted methoxy;

R² represents H;

R³ represents H, COOR⁴, COSR⁴, COR⁶, S0₂-R⁷ or CONHR⁵; R⁴ represents optionally substituted (C_]-8)alkyl, optionally substituted (C .₆)cycloalkyl, optionally substituted (C_2-8)alkenyl or aryl;

R⁵ represents H, optionally substituted (C_1-8)alkyl, optionally substituted (C_4-6)cycloalkyl or aryl; R⁶ represents optionally substituted (Cι.₆)alkyl, aryl or furanyl; R⁷ represents optionally substituted aryl or thienyl; n is an interger from 0 to 2; y is an interger from 0 to 2; m is O or 1; and pharmaceutically acceptable derivatives thereof; with the proviso that the compound is not:

[ 1 -[ 1 -(3 ,4-dichlorobenzyl)pyrrolidin-3 -ylmethyl] -3 -(4-methoxyphenyl) urea; or urea N-[[l -[(3 ,4-dichlorophenyl)methyl-3 -pyrrolidinyl]methyl]-N'-(2-methoxyphenyl) .

9. A compound as claimed in any one of claims 7 to 8 wherein X is O.

10. A compound as claimed in any one of claims 7 to 9 wherein n is 1.

11. A compound as claimed in any one of claims 7 to 10 wherein y is 1 or 2.

12. A compound as claimed in any one of claims 7 to 11 wherein R³ is H or COOR⁴.

13. A compound as claimed in any one of claims 7 to 12 wherein R⁴ is an optionally substituted (C_1-8) alkyl or optionally substituted (C₄.₆)cycloalkyl.

14. A radiolabel assay for identification of a compound which selectively activates SK channels by; determining the displacement of a labelled compound of example 1 by an unlabelled candidate compound and optionally, comparing this to the displacement of a labelled compound of formula (I) by an unlabelled compound of formula (I).

15. A compound identified by the assay of claim 14.

16. Use of a compound according to claim 15 in the preparation of a medicament for the treatment of a urinogenital disorder such as bladder hyperexcitability, MED or urinary incontinence, a respiratory disorder such as asthma, chronic obstructive pulmonary disease (COPD) or cystic fibrosis, a cardiovascular disorder such as hypertension, angina pectoris, ischaemic heart disease or stroke (e.g. cerebral ischaemia), a neurological or psychiatric disorder such as bipolar disorder, psychosis or sleeping disorders, a disorder ofthe central nervous system such as cognitive dysfunction or epilepsy, a pain condition such as neuropathic pain or inflammatory pain, a gastro-intestinal disorder such as inflammatory bowel disease (DBD), irritable bowel syndrome (IBS) or collitis.