WO2009156724A2

WO2009156724A2 - Novel compounds

Info

Publication number: WO2009156724A2
Application number: PCT/GB2009/001582
Authority: WO
Inventors: Grant Churchill; Raman Parkesh; Arasu Ganesan
Original assignee: Isis Innovation Limited
Priority date: 2008-06-25
Filing date: 2009-06-24
Publication date: 2009-12-30
Also published as: WO2009156724A3; GB0811587D0

Abstract

A compound according to formula (I), (IA), (II)^a or (II)^band uses thereof.

Description

NOVEL COMPOUNDS

FIELD OF THE INVENTION

The invention relates to novel compounds, to pharmaceutical compositions comprising said compounds and to the use of said compounds in the manufacture of a medicament for the treatment of a disease modulated by nicotinic acid adenine dinucleotide phosphate mediated intracellular calcium release.

BACKGROUND OF THE INVENTION

Ca²⁺ signalling is responsible for a wide variety of functions within a cell. Such signalling typically occurs via a messenger molecule which is produced within cells in response to external stimuli (Yamasaki, M et al (2004) J. Biol. Chem. 279, 7234-7240; Cancela, JM (2002) EMBO J. 21 , 909-919). Inositol 1,4, 5-trisphosphate (IP3) and cADP-ribose (cADPr) are well-known examples of such messenger molecules for Ca²⁺ release from intracellular stores. Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent intracellular calcium mobilizing agent in important mammalian cells and tissues (Dowden et al (2006) Chemistry and Biology 13, 1-7).

NAADP synthesis involves a modification of the enzymatic reaction, which results in the exchange of nicotinamide for nicotinic acid on the unphσsphorylated ribose ring of NADP. This latter mechanism preferably occurs at low pHs (e.g. those related to endosomal compartments where CD38 is likely to- reside during recycling from the cell surface). Replacement of an uncharged amide in NADP for a negatively charged carboxyl function in NAADP confers on the latter a potent (nanomolar affinity) capacity to mobilise Ca²⁺ from responsive stores. Studies have shown that NAADP is crucial in cholecystokinin-mediated Ca²⁺ signalling in pancreatic acinar cells (Cancela, JM et al (1999) Nature 398, 74-76). In pancreatic β cells, glucose-mediated signals are affected by NAADP and endogenous levels of NAADP have been shown to increase in response to the glucose stimulus, confirming a Ca²⁺ messenger role (Yamasaki, M et al (2004) J. Biol. Chem. 279, 7234-7240; Johnson, JD and Misler, S (2002) Proc. Natl. Acad. Sci. USA 99, 14566-14571 ; Masgrau, R et al (2003) Curr. Biol. 13, 247-251) .

NAADP has also been implicated in the regulation of Ca²⁺ signalling in T cells (Berg, I et al (2000) J. Cell. Biol. 150, 581-588) , heart myocytes Bak, J et al (2001) Curr. Biol. 11 , 987-990) and neurons (Bak, J et al (1999) Curr. Biol. 9, 751-754; Patel, S et al (2000) J. Biol. Chem. 275, 36495-36497; Brailoiu, E et al (2005) J. Biol. Chem. 280, 5646-5650) .

Ca²⁺ release has been found to be observed at nanomolar concentrations of NAADP, however, a thousand-fold increase in NAADP concentration causes no Ca²⁺ release and actually inactivates the entire Ca²⁺ store (Cancela, JM et al (1999) Nature 398, 74-76; Masgrau, R et al (2003) Curr. Biol. 13, 247-251; Berg, I et al (2000) J. Cell. Biol. 150, 581- 588) .

NAADP mediated calcium release has been linked with a number of differing disorders. For example, multiple sclerosis (Guse, A. CD38 Meeting, Torino, June 8-10, 2006) , asthma and hypertension (Boittin,-FX et al. (2002) Circ. Res. 91 , 1168-1175; Deshpande, OA et-at. (2005) Am. J. Physiol. 288, L1-L16) , diabetes (Mas-grau, R et al. (2003) Curr. Biol. 13, 247-251) , male infertility ('€fϊurchill, G et al. (Curr. Biol. 13, 125- 128) , cardiac disorders (Macgregor, A et al. (2007) J. Biol. Chem. 282, 15302-11) and obesity (Senior, K (2005) Drug Discovery Today 10, 810) . WO 2005/054198 (University of Bath) describes a series of pyridinium derivatives for use in modulating the release of intracellular calcium from a store controlled by NAADP.

Dowden et al (2006) Chem. Biol. 13, 659-665 describe a partial agonist of NAADP mediated calcium release (CMA08) and this compound is demonstrated to be active at 30 micromolar. Billington, RA & Genazzani, AA (2007) Cell Calcium 41, 505-511 describe a further antagonist of NAADP mediated calcium release (PPADS) and this compound is indicated to be impermeable to the cell membrane and have micromolar potency.

There is therefore a great need for effective and potent inhibitors of NAADP mediated calcium release.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided a pharmaceutical composition comprising a compound of formula (I) :

wherein R¹ represents a halogen atom, or a pharmaceutically acceptable salt or solvate thereof, in admixture with a pharmaceutically acceptable excipient, with the proviso that R¹ does not represent 2-fluorine.

According to a second aspect of the invention, there is provided a compound of formula (II) ^a:

(II)^a wherein R¹ represents a halogen atom, or a pharmaceutically acceptable salt or solvate thereof.

According to a further aspect of the invention, there is provided a compound of formula (H)":

(ii)^b wherein R¹ represents a halogen atom, or a pharmaceutically acceptable salt or solvate thereof.

According to a further aspect of the invention, there is provided a use of a compound of formula (I) , (IA) , (II)^a or (II)^b, or a .pharmaceutically acceptable salt or solvate thereof, in therapy.

According to a further aspect of the invention, there is provided the use of a compound of formula (I) with or without the proviso, (II)" or (II)" or a pharmaceutically acceptable salt or solvate thereof in the manufacture of a medicament for the treatment of a disease modulated by nicotinic acid adenine dinucleotide phosphate mediated intracellular calcium release.

According to a further aspect of the invention, there is a compound of formula (I) with or without the proviso, (II)^a or (II)^b or a pharmaceutically acceptable salt or solvate thereof for use in tbe""treatment of a disease modulated by nicotinic acid adenine dinucleotide phosphate mediated intracellular calcium release.

According to a further aspect of the invention, there is provided a method of treating a disease modulated by nicotinic acid adenine dinucleotide phosphate mediated intracellular calcium release which comprises administration of a therapeutically effective amount of a compound of formula (I) with or without the proviso, (II)³ or (II)" or a pharmaceutically acceptable salt or solvate thereof.

According to a further aspect of the invention, there is provided a pharmaceutical composition comprising a compound of formula (I) with or without the proviso, (II)^a or (II)" or a pharmaceutically acceptable salt or solvate thereof for use in the treatment of a disease modulated by nicotinic acid adenine dinucleotide phosphate mediated intracellular calcium release.

According to a further aspect of the invention, there is provided a process for preparing a compound of formula (I) , (IA), (II)^a or (II) ^b which comprises one of the following processes (a) to (c) :

(a) reacting a compound of formula (III)

(III) wherein L¹ represents a suitable leaving group, such as a halogen atom (e.g. chlorine) and P¹ represents a suitable protecting group such as C,.₆ alkyl (e.g. methyl), with a compound of formula (IV)

(IV) wherein R¹ is the R¹ group required in the compound of formula (I) , (IA) , (II)^a or (II)^b; or (b) deprotecting a protected derivative of a compound of formula (I) , (IA), (II)^a or (II)^b; or (c) interconversion of a compound of formula (I) , (IA) , (II)^a αr-^v(II)^b. Process (a) typically comprises reaction of a compound of formula (III) with a compound in formula (IV) in the presence of a suitable solvent (such as dichloromethane) .

In process (b), examples of protecting groups and the means for their removal can be found in T. W. Greene "Protective Groups in Organic Synthesis" (J Wiley and Sons, 1991) . For example, in process (b), when the protected derivative is protected with a group P¹, and P¹ represents a C, to C₄ alkyl group, for example a methyl group, deprotection may typically comprise a standard hydrolysis reaction in the presence of an acid (such as Ambersep™ 900) or base. The acid may be a strong acid. The base may be a strong base. The acid or base may be provided in the form of an acidic or basic ion-exchange resin. In the protection derivative the protecting groups P¹ may replace the hydrogen atom in the COOH group of the compounds of formulae (I), (IA), (II)^a or (II)^b

Process (c) may be performed using conventional interconversion procedures such as epimerization, oxidation, reduction, alkylation, nucleophilic or electrophilic aromatic substitution, ester hydrolysis, amide bond formation or transition metal mediated coupling reactions.

Compounds of formula (III) may be prepared in accordance with the following scheme:

Scheme 1

wherein Ll and Pl are as defined hereinbefore. Stereocentres are indicated by the asterisk on appropriate carbon atoms.

Step (i) typically comprises a Pictet-Spengler reaction between a compound of formula (V) and a compound of formula (VI) in the presence of [BnNEt3]Cl-AlC13 and a suitable solvent (such as dichloromethane) .

Compounds of formula (IV) , (V) and (V-If are either known or may be prepared in accordance with known procedures. [BnNEt3]Cl-AlC13 may be prepared by reaction of benzyltriethylammonium chloride with anhydrous aluminium chloride as herein defined.

According to a further aspect of the invention, there is provided a pharmaceutical composition comprising a compound of formula (II)^a or (II)^b or a pharmaceutically acceptable salt or solvate thereof, in admixture with a pharmaceutically acceptable excipient.

According to a further aspect of the invention, there is provided a method of screening for an agent which effectively modulates nicotinic acid adenine dinucleotide phosphate mediated intracellular calcium release which comprises the steps of:

(a) contacting a compound of formula (I) , (IA), (II)^a or (II)^b with an

NAADP receptor; (b) measuring the level of intracellular calcium release, known as the control level;

(c) contacting an agent with an NAADP receptor;

(d) measuring the level of intracellular calcium release, known as the test level; and (e) comparing the test level with the control level, such that an effective modulator will be an agent demonstrating a lower test level than the control level.

According to a further aspect of the invention, there is provided an agent identifiable by the method of screening hereinbefore defined.

BRIEF DESCRIPTION OF THE FIGURES

Figures 1 and 2 describe a selectivity analysis on NAADP mediated calcium release with the compound of formula (IA) . Figures 3 and 4 describe a concentration analysis on NAADP mediated calcium release with El and related compounds.

Figure 5 describes a competition analysis of NAADP-induced calcium release.

Figure 6 describes an excitation and emission analysis of NAADP- induced calcium release with a compound of formula (IA).

Figure 7 describes an imaging analysis of NAADP-induced calcium release with a compound of formula (IA) .

Figure 8 demonstrates that a compound of formula (IA) (referred to in the figure as Ned-19) blocks collagen-related peptide-induced human platelet aggregation in a concentration-dependent manner.

Figure 9 demonstrates that a compound of formula (IA) (referred to in the figure as Ned-19) blocks histamine-induced bronchoconstriction in a concentration-dependent manner.

Figure 10 demonstrates that a compound of formula (IA) (referred to in the figure as Ned-19) blocks oxytocin-induced uterine contraction in a concentration-dependent manner.

DETAILED DESCRIPTION OF THE INVENTION

References to the term "halogen" include references to fluorine, chlorine, bromine or iodine. In one embodiment, R¹ represents fluorine (e.g. 2-fluorine, 3-fluorine or. 4-fluorine). In a further embodiment, R¹ represents 2-fluorine, i.e. a compound of formula (IA) :

(IA)

In an alternative embodiment, R¹ represents 3-fluorine.

References to the term "modulation" include references to antagonism, agonism, inverse agonism and the like. In one embodiment, modulation is antagonism.

The term "pharmaceutically acceptable salt" refers to a salt, for example an acid addition salt or, in certain cases salts of an organic and inorganic base such as carboxylate, sulphonate and phosphate salt. All such salts are within the scope of this invention, and references to compounds of the formula (I) include the salt forms of the compounds. Examples of pharmaceutically acceptable salts are provided in Berge et al. , 1977, "Pharmaceutically Acceptable Salts, " J. Pharm. ScL, Vol. 66, pp. 1-19. The salts of the present invention can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods such as methods described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are used.

Examples of acid addition salts include salts formed with an acid selected from the group consisting of acetic, 2,2-dichloroacetic, adipic, alginic, ascorbic (e.g. L-ascorbic) , L-aspartic, benzenesulphonic, benzoic, 4- acetamidobenzoic, butanoic, ( + ) camphoric, camphor-sulphonic, ( + )- (15) -camphor- 10-sulphonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulphuric, ethane- 1 ,2-disulphonic, ethanesulphonic, 2- hydroxyethanesulphonic, formic, fumaric, galactaric, gentisic, glucoheptonic, D-gluconic, glucuronic (e.g. D-glucuronic) , glutamic (e.g. L-glutamic), α-oxoglutaric, glycolic, hippuric, hydrobromic, hydrochloric, hydriodic, isethionic, lactic (e.g. ( + )-L-lactic, ( ± )-DL- lactic), lactobionic, maleic, malic, (-)-L-malic, malonic, ( ± )-DL- mandelic, methanesulphonic, naphthalenesulphonic (e.g.naphthalene-2- sulphonic) , naphthalene- 1 ,5-disulphonic, l-hydroxy-2-naphthoic, nicotinic,, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, L-pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulphuric, tannic, ( + )-L-tartaric, thiocyanic, toluenesulphonic (e.g. /7-toluenesulphonic) , undecylenic and valeric acids, as well as acylated amino acids and cation exchange resins. The compounds of the invention may exist as mono- or di-salts depending upon the pKa of the acid from which the salt is formed.

An anionic functional group (e.g. , -COOH may be -COO ), may form a salt with a suitable cation. Non-limiting examples of suitable inorganic cations include alkali metal ions such as Na⁺ and K⁺ , alkaline earth metal cations such as Ca²⁺ and Mg²⁺ , and other cations such as Al³⁺ . Non- limiting examples of suitable organic cations include ammonium ion (i.e. , NH₄ ⁺) and substituted ammonium ions (e.g. , NH₃R⁺, NH₂R₂ ⁺, NHR₃ ⁺ , NR₄ ⁺) .

Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH₃)₄ ⁺ .

Compounds of formula (I) , (IA) , (II)^a or (II)" are claimed as solids in either amorphous or crystalline form, including all polymorphic forms.

Crystalline forms may be prepared by recrystallisation of the compounds from appropriate solvents. Amorphous forms may be prepared e.g. by spray drying a solution of the compounds. Examples of polymorphic forms include solvates (e.g. hydrates), complexes (e.g. inclusion complexes or clathrates with compounds such as cyclodextrins, or complexes with metals of the compounds) , and pro-drugs of the compounds.

Examples of suitable excipients include carriers, diluents, binders, lubricants, preservatives, stabilizers, dyes, antioxidants, suspending agents, coating agents, solubilising agents and flavouring agents. Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like.

Examples of suitable diluents include ethanol, glycerol, water and the like.

Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol.

Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.

Examples of suitable preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid and the like.

Certain compounds described herein demonstrate inhibitory activity against NAADP-mediated calcium release.

The ability of a given compound to inhibit NAADP-mediated calcium release can be determined using the "Sea urchin egg homogenates assay" given in the Examples below.

"inhibition of NAADP-mediated calcium release" means it an IC50 value of less than 1 millimolar in the sea urchin egg homogenates assay, preferably less than 5 micromolar especially less than 1 micromolar. Examples of diseases which may be modulated by nicotinic acid adenine dinucleotide phosphate mediated intracellular calcium release include: macrophage inhibitory and/or T cell inhibitory activity and thus, anti- inflammatory activity; anti-immune activity, i. e. inhibitory effects against a cellular and/or humoral immune response, including a response not associated with inflammation; inhibit the ability of macrophages and T cells to adhere to extracellular matrix components and fibronectin, as well as up-regulated fas receptor expression in T cells; inhibit unwanted immune- reaction and inflammation including arthritis, including rheumatoid arthritis, inflammation associated with hypersensitivity, allergic reactions, asthma, systemic lupus erythematosus, collagen diseases and other autoimmune diseases, inflammation associated with atherosclerosis, arteriosclerosis, atherosclerotic heart disease, reperfusion injury, cardiac arrest, myocardial infarction, vascular inflammatory disorders, respiratory distress syndrome or other cardiopulmonary diseases, inflammation associated with peptic ulcer, ulcerative colitis and other diseases of the gastrointestinal tract, hepatic fibrosis, liver cirrhosis or other hepatic diseases, thyroiditis or other glandular diseases, glomerulonephritis or other renal and urologic diseases, otitis or otheroto- rhino-laryngological diseases, dermatitis or other dennal diseases, periodontal diseases or other dental diseases, orchitis or epididimo- orchitis, infertility, orchidal trauma or other immune-related testicular diseases, placental dysfunction, placental insufficiency, habitual abortion, eclampsia, pre-eclampsia and other immune and/or inflammatory-related gynaecological diseases, posterior uveitis, intermediate uveitis, anterior uveitis, conjunctivitis, chorioretinitis, uveoretinitis, optic neuritis, intraocular inflammation, e. g. retinitis or cystoid macular oedema, sympathetic ophthalmia, scleritis, retinitis pigmentosa, immune and inflammatory components of degenerative fondus disease, inflammatory components of ocular trauma, ocular inflammation caused- by infection, proliferative vitreo-retinopathies, acute ischa^:emic optic neuropathy, excessive scarring, e. g. following glaucoma filtration operation, immune and/or inflammation reaction against ocular implants and other immune and inflammatory-related ophthalmic diseases, inflammation associated with autoimmune diseases or conditions or disorders where, both in the central nervous system (CNS) or in any other organ, immune and/or inflammation suppression would be beneficial, Parkinson's disease, complication and/or side effects from treatment of Parkinson's disease, AIDS-related dementia complex HIV-related encephalopathy, Devic's disease, Sydenham chorea and Alzheimer's disease.

In one embodiment, the disease which may be modulated by nicotinic acid adenine dinucleotide phosphate mediated intracellular calcium release includes treating an autoimmune disease (such as thyroiditis, insulitis, multiple sclerosis, invectitis, orchitis, myasthenia gravis, rheumatoid arthritis or lupus erythematosis) or graft rejection, or Type II diabetes, or cardiac disorders (e.g. cardiac arrhythmia) , or treating or preventing an immune disorder in a human or animal or obesity or hypertension.

In a further embodiment, the disease modulated by nicotinic acid adenine dinucleotide phosphate mediated intracellular calcium release is selected from multiple sclerosis, asthma and hypertension, diabetes (e.g. Type II diabetes) , male infertility, cardiac disorders (e.g. cardiac arrhythmia) and obesity.

The compounds of the invention may be administered orally, topically, parenteral/, by inhalation spray or rectally in dosage unit formulations containing non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. The term parenteral as used herein includes subeu-t'aneous injections, intravenous, intramuscular, intrasteraaϊ injection or infusion techniques. In addition to the treatment of- warm-blooded animals such as mice, rats, horses, cattle, sheep, dogs, cats etc, the compounds of the invention are effective in the treatment of humans. The term inhalation as used herein also includes intranasal delivery.

The pharmaceutical composition containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavouring agents, colouring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with nontoxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc.

The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated by the" techniques described in the US Patents 4,256, 108; 4, 166,45'2; and 4,265,874, to form osmotic therapeutic tablets foe -control release.

Formulations for oral use may also be presented as hard gelatin capsules where in the active ingredient is mixed with an inert solid diluent, for example calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate polyvinyl -pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such a polyoxyethylene with partial esters derived from fatty acids and hexitol anhydrides, for example polyoxyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl, p-hydroxybenzoate, one or more colouring agents, one or more flavouring agents, and one or more sweetening agents, such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl a demulcent, a preservative and flavouring and colouring agents. The pharmaceutical compositions may be in the fornt_^of a sterile injectable aqueous or oleagenous suspensions This suspension may be formulated according to the- known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be in a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The compounds of the invention may also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials are cocoa butter and polyethylene glycols.

For topical use, creams, ointments, jellies, solutions or suspensions, etc containing the compounds of the invention are employed. For the purposes of this specification, topical application includes mouth washes and gargles.

Dosage levels of the order of from about 0.05 mg to about 140 mg per kilogram of body weight per day are useful in the treatment of the above- indicated conditions (about 2.5 mg to about 7 g per patient per day) . For example, inflammation may be effectively treaϊed'by the administration of from about 0.01 to 50 mg of the compound per kilogram of body weight per day (about 0.5-mg to about 3.5 g per patient per day) .

The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, a formulation intended for the oral administration of humans may vary from about 5 to about 95% of the total composition. Dosage unit forms will generally contain between from about 1 mg to about 500 mg of active ingredient.

It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

The invention will now be illustrated with reference to the following non- limiting examples.

EXAMPLES

General Methods All reagents were purchased from Aldrich and used without further purification. Dichloromethane was distilled over calcium hydride. ¹H and ¹³C NMR spectra were recorded using a BRUKER DPX300 Spectrometer (300 MHz and 75 MHz respectively), and ¹⁹F NMR. ¹³C NMR were recorded as "J modulated spin echo experiment" (Jmod) where the quaternary and CH₂ carbons give -ve responses and CH and CH₃ give + ve responses. IR spectra^ were taken using a Biorad 135 Spectrophotometer. TLC was carried out with ALUGRAM® SIL GAtFv'254 TLC plates (0.20mm thickness) . Besides UV visualisation, Dragendorff's spray reagent was used for Pictet-Spengler reaction products. Microwave assisted reactions were conducted in a Smith Synthesizer™. Intermediate 1

Preparation of benzyltriethylammonium chloride. AlCl₃, N= 0.5 (Dl)

To a dry two-necked roundbottom flask with a stopper and septum was added benzyltriethylammonium chloride (2.27g, lOmmol) with stirring while maintaining the flask under inert atmosphere by purging with nitrogen. Dichloromethane (25 ml) was added with gentle stirring. When all the benzyltriethylammonium chloride was dissolved completely, the flask was cooled (5-10⁰C) and anhydrous aluminium chloride granules (1.33 g, lOmmol) were added while maintaining the reaction flask under nitrogen. The reaction mixture was stirred under nitrogen for 30 min (5- 10⁰C) and then allowed to stir at room temperature for 12 h .The resultant reaction mixture on careful evaporation furnished a white solid, which was further dried under high vacuum for 6 h. The solid was transferred to an airtight container and should be stored in desiccators.

Yield: 3.50 g (97%); NMR in CD₂Cl₂: ¹H δ 7.45-7.60 (m, 5H, Ar), 4.35 (s, 2H, -CHa-Ph), 3.21-3.27 (q, 6H, 3-N-CH₂-CH₃, J = 7.18Hz) , 1.46- 1.50 (t, 9H, 3-N-CH₂-CH₁, J = 7.04Hz) ; ¹³C δ 132.87, 132.19, 132.19, 130.70, 126.52, 61.85, 53.67, 8.75; ²⁷Al δ 103.11.

Intermediate 2

Preparation of L-tryptophan methyl ester HCl salt (D2)

To a solution of L-tryptophan methyl ester HCl salt (1.0 g, 3.9 mmol) in MeOH (10 ml) was added Ambersep 900 OH resin (5.0 g) . The mixture stirred at room temperature for 15 min. The resin was then filtered and washed with MeOH, and filtrate upon evaporation provided the pure product. Yield: 0.86 g (quant) .

Intermediate 3

Preparation of cis/trans-l-(4-methoxy-3-((4-phenylpiperazin-l- yl)methyl)phenyl)-2,3,4,9-tetrahydro-lH-pyrido[3,4-b]indoIe-3- carboxylic acid (D3)

The L-tryptophan methyl ester (218 mg, 1.0 mmol; which may be prepared as described in D2) and [BnNEt₃]Cl-AlCl₃, N = 0.5, (36.1 mg, 10 mol%; which may be prepared as described in Dl) were placed in a microwave vial followed by the addition of dichloromethane (3 ml) . The vial was carefully capped and flushed with nitrogen before addition of the aldehyde (222 mg, 1.2 mmol) . Reactions with L-tryptophan methyl ester were heated at 100 °C for 30 min in the Smith Synthesizer™ focused microwave instrument. After cooling, the contents of the vial were diluted with water followed by the addition of 2% NaOH solution (5 ml). The resultant reaction mixture was extracted (3x5 ml ethyl acetate) and washed (water, brine) . After drying, excess aldehyde was scavenged by stirring with 400 mg of aminomethylated polystyrene VHL resin (Novabiochem, 1.2-1.6 mmol/g) for 10-12 h (TLC monitoring) . The resin was then filtered and washed (3x2 ml dichloromethane), and the filtrate upon evaporation provided the pure product (263 mg, 69%) . A mixture of diastereomers was obtained, 1 :1 cis/trans ratio by NMR analysis.

Intermediate 3A (cis/trans mixture) c/s/frα«s-methyl-l-(3-(chloromethyl)-4-methoxyphenyl)-2, 3,4,9- tetrahydro-lH-pyrido[3,4-b]indoIe-3-carboxyIate (D3A)

¹H NMR (CDCl₃)rδ^~ 9.88 (br s, IH, indole NH) , 7.52-7.07 (m, IH) , 6.84 (d, IH, J = 8.4 Hz, ArH) , 5.33 (s, IH, C₁-H, trans) , 5.21 (s, IH, C₁-H, cis) , 4.60 (d, 2H, J = 2.6 Hz, ArCH₂Cl, cis) , 4.58 (d, 2H, J = 6.6 Hz, ArCH₂Cl, trans) , 3.96 (dd, IH, J = 11.0, 4.7 Hz, C₃-H), 3.86 (s, 3H, OCH₃, cis) , 3.85 (s, 3H, OCH₃, trans) , 3.80 (s, 3H, -CO₂CH₃, cis) , 3.77 (s, 3H, -CO₂CH₃, trans) , 3.45 (br s, IH, N₂-H) , 3.22 (ddd, IH, J = 15.0, 4.7, 2.1 Hz, C₄-H₃) , 3.02 (ddd, IH, J = 15.0, 11.0, 2.4 Hz, C₄-Hj [3.85-2.90 (m, 2H, C₄-H)] ; ¹³C NMR (CDCl₃) δ 172.9, 157.7, 136.2, 134.2, 130.9, 130.4, 127.0, 126.3, 122.0, 119.6, 118.2, 111.2, 111.0, 108.9, 58.0 (C₁) , 56.9 (OCH₃) , 55.8 (C₃), 55.3, 52.2 (OCH₃, cis), 52.1 (OCH₃, trans), 41.4 (ArCH₂Cl, trans), 41.3 (ArCH₂Cl, cis), 25.5 (C₄) .

Intermediate 3B (c/s-isomer) c/s-methyl-l-(3-(chloromethyl)-4-methoxyphenyl)-2,3,4,9-tetrahydro- lH-pyrido[3,4-b]indole-3-carboxylate (D3B)

¹H NMR (CDCl₃) δ 7.54 (br s, IH, indole NH) , 7.52-7.07 (m, IH) , 6.84 (d, IH, J = 8.4 Hz, ArH) , 5.21 (s, IH, C₁-H) , 4.58 (d, 2H, J = 3.3 Hz, ArCH₂Cl) , 3.96 (dd, IH, J = 11.0, 4.7 Hz, C₃-H) , 3.86 (s, 3H, OMe) , 3.77 (s, 3H,.-CO₂CH₃) , 3.45 (br s, IH, N₂-H) , 3.22 (ddd, IH, J = 15.0, 4.7, 2.1 Hz, C₄-H₃), 3.02 (ddd, IH, J = 15.0, 11.0, 2.4 Hz, C₄-Hj ; ¹³C NMR (CDCl₃) δ 172.9, 157.7, 136.2, 134.2, 130.9, 130.4, 127.0, 126.3, 122.0, 119.6, 118.2, 111.2, 111.0, 108.9, 57.9 (C₁) , 56-8"^'(OCH₃) , 55.7 (C₃) , 55.3, 52.2 (OCH₃) , 41.3 (ArCH₂Cl) , 25.5 (C₄) ; MS (ESI) mlz 385.5 ([M + H]⁺).

Intermediate 4

Preparation of c/s-methyl-l-(3-((4-(2-fluorophenyl)piperazin-l- yl)methyl)-4-methoxyphenyl)-2,3,4,9-tetrahydro-lH-pyrido[3,4- b]indole-3-carboxylate (D4)

To a solution of c/s-methyl l-(3-(chloromethyl)-4-methoxyphenyl)- 2,3,4,9-tetrahydro-lH-pyrido[3,4-b]indole-3-carboxylate (55 mg 0.14 mmol; which may be prepared as described in D3B) in dichloromethane (5 ml) was added l-(2-fluorophenyl)-piperazine (31.1 mg, 0.17 mmol) and then stirred with 500 mg of diethylaminomethyl polystyrene resin (Fluka, 3.2 mmol/g). After 12h at room temperature, the resin was then filtered and washed (3x3 ml dichloromethane : methanol = 1 : 1) , and the filtrate upon evaporation provided the yellow mixture. The residue was submitted to chromatography (Silica-gel, eluent dichloromethane:Methanol = 90: 10, v/v) , to give a yellow product. ¹H NMR (CDCl₃) δ 7.60 (br s, IH, indole NH) , 7.52 (dd, IH, J = 5.8, 2.9 Hz), 7.37 (d, IH, J = 1.8 Hz), 7.22-6.83 (m, 9H), 6.95 (td, IH, J = 7.5, 0.7 Hz) , 5.28 (s, IH, N₂-H), 5.15 (S, IH, C₁-H), 3.96 (dd, IH, J = 11.1 , 4.2 Hz, C₃-H) , 3.82 (s, 3H, OCH₃) , 3.80 (s, 3H, -CO₂CH₃) , 3.63 (s, 2H, Ar-CH₂-NR₂) , 3.24-3.18 (m, IH, C₄-H) , 3.06 (t, 4H, J = 4.2 Hz, (CH₂)₂N-Ar) , 3.01-2.95 (m, IH, C₄-H) , 2.67 (br d, 4H J = 2.2 Hz, - N(CH₂)₂-) ; ¹³C NMR (CDCl₃) δ 173.2, 158.1 (d), 136.1 , 135.1 , 132.5, 131.1 , 128.3, 127.2, 124.3, 122.3 122.2, 121.8, 119.3, 118.9, 118.1, 116.1 , 115.8, 110.9, 110.8, 108.8, 58.0 (C₁), 56.9 (OCH₃) , 55.8, 55.6 (C₃), 53.0, 52.1 (OCH₃), 50.4, 25.7 (C₄); ¹⁹F NMR (CDCl₃) δ -122.9; MS (ESI) mlz 529.5 ([M + H] ⁺) .

Example 1

Preparation of c/s-l-(3-((4-(2-fluorophenyl)piperazin-l-yl)methyl)-4- methoxyphenyl)-2,3_»4,9-tetrahydro-lH-pyrido[3,4-b]indole-3- carboxylic acid (El)

To a solution of c/5'-methyl-l-(3-((4-(2-fluorophenyl)piperazin-l- yl)methyl)-4-methoxyphenyl)-2,3,4,9-tetrahydro-lH-pyrido[3,4-b]indole- 3-carboxylate (200 mg, 0.92 mmol; which may be prepared as described in D4) in THF (3 ml) was added 3.0 g of Ambersep 900 OH (Fluka, 3.2 mmol/g) , and stirred for 1 d at room temperature. The resin was then filtered and washed (3x3 ml dichloromethane : methanol = 1 :1), and the filtrate upon evaporation provided the pure product (1.6 mg) . 1H NMR (CDCl₃) δ 7.60 (br s, IH, indole NH) , 7.52 (dd, IH, J = 5.8, 2.9 Hz), 7.37 (d, IH, J = 1.8 Hz), 7.22-6.83 (m, 9H) , 6.95 (td, IH, J = 7.5, 0.7 Hz), 5.28 (s, IH, N₂-H), 5.15 (S, IH, C₁-H) , 3.96 (dd, IH, J = 11.1, 4.2 Hz, C₃-H) , 3.82 (s, 3H, OCH₃) , 3.63 (s, 2H, Ar-CH₂-NR₂), 3.24-3.18 (m, IH, C₄-H) , 3.06 (t, 4H, J = 4.2 Hz, (CH₂)₂N-Ar), 3.01- 2.95 (m, IH, C₄-H) , 2.67 (br d, 4H J = 2.2 Hz, -N(CH₂)₂-) ; ¹⁹F NMR (CDCl₃) δ -122.9; MS (ESI) mlz 515.4 ([M + H] ⁺) .

Biological Assays

Inhibition of NAADP-mediated calcium release in sea urchin egg homogenates

A sea urchin homogenate was used to assess inhibition of NAADP- induced calcium release. The intracellular stores in the sea urchin homogenate were loaded with calcium by incubating homogenates for 3 h at 17°C in intracellular medium containing the additions for ATP- regenerating system, mitochondrial inhibitors (1 mg/ml oligomycin and antimycin, 1 mM sodium azide) and protease inhibitors-. To monitor calcium concentration, the calcium-reporting dye fluo-3 (3 μM) was added. Calcium was measured at^* 17°C using 500 μl of homogenate in a stirring Perkin-BFmer LS-50B/PTI fluorimeter at 505 nm excitation and > ' 515 nm, emission wavelengths. Additions of compounds to the homogenate in the cuvette were made in 5 μl volumes, and changes in relative fluorescence units, which correspond to changes in calcium concentration were determined. The test compounds were dissolved in pure, dry DMSO to create 10 mM stock solutions. The final DMSO in the homogenate in the cuvette never exceeded 1 percent by volume.

A) Selectivity analysis on NAADP mediated calcium release Calcium release was initiated by adding 5 μL of a 100-fold concentrated stock solution of messenger (NAADP (30 nM) , inositol 1 ,4,5- trisphosphate (1 μM) and cyclic ADP-ribose (60 nM)) to a cuvette containing 500 μL of homogenate. After the calcium released by a messenger was re-sequestered (10-20 minutes) , the next messenger was added. The experiment was then repeated using the compound of formula (IA) at 100 μM to assess the effect of this compound on calcium release mediated by the calcium-releasing messengers.

The left hand panel of Figure 1 shows that the sea urchin egg homogenate releases calcium in response to all three calcium-releasing messengers: NAADP, inositol 1 ,4,5-trisphosphate (IP₃) and cyclic ADP-ribose.

The right hand panel of Figure 1 shows that the compound of formula (IA) selectively inhibited NAADP-mediated calcium release but not IPs- mediated or cyclic ADP-ribose-mediated calcium release.

5 μL of test compound was added to achieve a given final concentration in the homogenate in the cuvette. After 3-15 minutes, a test concentration of NAADP was* added to assess whether or not the test compound inhibited (antagonized) the NAADP-mediated calcium release. The test concentration of NAADP was 30 nM, which is its half maximal effective' concentration. A single cuvette was therefore used to obfcfin one result on the dose-inhibition curve. For example-, 1 μM of tfie compound of formula

(IA) followed by NAADP at 30 nM would provide one point on the dose- response curve. This was repeated 4 times for each concentration. To determine selectivity of the NAADP inhibition relative to other calcium- releasing messengers, dose-response curves in the presence of the compound of formula (IA) were performed for inositol 1 ,4,5- trisphosphate and cyclic ADP-ribose (Figure 2) .

Figure 2 shows that the compound of formula (IA) inhibits NAADP- mediated calcium release with a half-maximal inhibitor concentration of 2 μM. In contrast, even when present at 100 μM, the compound of formula (IA) has minimal effects on c ADPR- and IP₃-mediated calcium release. The implication is that the selectivity of the compound of formula (IA) for NAADP-mediated calcium release is at least 1000-fold greater than c ADPR- and IP₃-mediated calcium release.

B) Concentration analysis on NAADP mediated calcium release with El and related compounds

A wide range of concentrations of test compounds were assessed to produce the resulting dose-response curves shown in Figures 3 and 4.

In Figure 3, the relative activities of two of four possible diastereomers were quantitatively assessed for their ability to inhibit NAADP-mediated calcium release in sea urchin egg homogenates. Dose-inhibition curves were developed as described hereinbefore. For comparison, the mixture containing all 4 diastereomeric forms of the compound of formula (IA) had an IC₅₀ of 3 μM. The pure cis compound (El) revealed single-binding site inhibition curve with a half maximal inhibitory concentration (IC₅₀) of 644 nM. A mixture of 2 diastereomers of formula (IA), the cis-compound and the trans compound, revealed a two-binding site inhibition curve; the first had an IΘ₅₀ of 170 nM (statistically equal to 644 nM) and the second had an IC₅₀ of 3 nM. The results of Figure 3 demonstrate that a mixture of the cis/trans isomers (compound of formula (IA)) is more active than a pure composition of the cis isomer (El) . Moreover, El is consistent with a single binding site, whereas the cis/trans mixture is consistent with two binding sites. One site is consistent with the half-inhibitory concentration of cis, and the other is of greater potency. These results imply that the trans isomer of the compound of formula (IA) is more potent than the cis isomer.

In Figure 4, the relative activities of the following 5 fragments and analogues of the compound of formula (IA) were quantitatively assessed for their ability to inhibit NAADP-mediated calcium release in sea urchin egg homogenates:

Dose-inhibition curves were developed as described hereinbefore. For comparison, the diastereomeric mixture of formula (IA) had an IC₅₀ of 3 μM. All fragments and the methyl ester (on the carboxylic acid) of formula (IA) were significantly less potent than the parent molecule formula (IA) . This is based on right-shifted (less potent) dose-inhibition curves that yielded IC₅₀'s that were all significantly higher (less inhibition) than the parent structure of formula (IA). The interpretation of these results is that there is no one crucial part of the molecule that provides high potency. Each part of the structure of formula (IA) synergistically contributes to NAADP binding and inhibition.

C) Competition analysis on NAADP mediated calcium release

To determine whether the compound of formula (IA) competed with NAADP for binding to its receptor, radioactive NAADP binding was performed. [³²P]NAADP was synthesized in a two-step reaction. First, [³²P]NADP was synthesized by incubating [³²P]NAD (GE Healthcare, UK) with 0.5 U/ml human NAD kinase (Alexis, UK) , 5 mM MgATP, and 100 mM HEPES for 1 h. Nicotinic acid 100 mM and ADP-ribosyl cyclase 1 μg/ml (Sigma) were added to commence the second step, which was allowed to proceed for 1 h. The resulting mixture was pumped onto a HPLC column with a peristaltic pump. Separation was carried out on an anion-exchange resin (AGMPl , Biorad, USA) using a concave upwards gradient of trifluoroacetic acid (TFA) with detection using an in-line Geiger counter. The NAADP fraction was then collected and stored at 4°C for use in the assay.

Sea urchin egg homogenate was diluted to a final concentration of 1% (v/v) in 150 μL buffer. After incubation for 10 min at 25°C, [³²P]-NAADP in buffer was added to give a concentration of 0--.3 nM in a final volume of 250 μL. Samples were incubated for 15 minutes in the presence of a wide range of concentrations of the compound of formula (IA) . Bound [³²P]NAADP was then trapped onto Whatmann GF/B filter papers using a Brandel Cell Harvester. Washing was carried out using a buffer containing 20 mM HEPES, 500 mM potassium acetate, pH 7.4. Retained radioactivity was then quantified. The competition of NAADP and compound of formula (IA) for radioactive NAADP binding are shown in Figure 5.

Figure 5 shows that authentic NAADP competes for radioactive NAADP binding to sea urchin egg homogenate with a half-inhibitory concentration of 0.2 nM. The compound of formula (IA) can also compete for radioactive NAADP binding with a half-inhibitory concentration of 2 μM. These results imply that the compound of formula (IA) binds competitively to the NAADP binding site.

D) Excitation and Emission Analysis

To determine the excitation and emission spectrum of the compound of formula (IA) , a 1 mM solution in dimethylsulfoxide was placed in a quartz cuvette in a Perkin-Elmer spectrofluorimeter. The excitation scan was from 210-410 nm with emission collected at 430 nm. The emission scan was from 390-580 nm at an excitation of 370 nm. The resulting curves are shown in Figure 6.

Figure 6 shows that the compound of formula (IA) is fluorescent with maximum excitation 310 nm and 370 nm and maximum emission at 420 nm. These results imply that the compound of formula (IA) should be suitable for labelling receptors of the compound of formula (IA) in intact cells and tissues.

E) Fmaging Analysis To label and image NAADP receptors in intact cells, the compound of, formula (IA) was added to cells in culture at 100 μM. After'' loading for 30 min, the compound of formula (IA) was viewed with confocal microscopy (Zeiss) with an excitation of 360 nm and emission collected with a 440 band pass filter. The visible light image and fluorescent image of the compound of formula (IA) are shown in Figure 7.

Figure 7 shows that the compound of formula (IA) can label intact cells and be imaged by laser-scanning confocal microscopy. The compound of formula (IA) labels small vesicles within cells consistent with the known location of NAADP receptors in lysosome-related organelles. These results imply that the compound of formula (IA) can be used to label NAADP receptors in intact, living cells.

Inhibition of Platelet Aggregation

Human platelets were isolated from blood from healthy volunteers. Aggregation was determined with a light-scattering aggregometer. After establishing a stable base-line (1-3 min), collagen-related peptide was added and the effect quantified and normalized to 100 percent. In the experiments to evaluate the effect of a compound of the invention, a compound of formula (IA) or its vehicle (DMSO) was first added to the cuvette for 30 seconds and then collagen-related peptide was added. The resulting response is presented in Figure 8 as the percentage of the control (collagen-related peptide alone) . The results demonstrate that a compound of formula (IA) results in a concentration-dependent inhibition in collagen-related peptide-induced aggregation. The clinical implication is that a comparand according to the invention may be used to prevent thrσmtTosis.

Inhibition of Bronchial Smooth Muscle Contraction

Bronchia from guinela pigs were cut into rings and mounted in an organ bath with a force transducer to measure contraction. Histamine at increasing concentrations was then added to produce a cumulative concentration-response curve. DMSO was added to establish the control response. The entire histamine concentration-response curve was then repeated in the presence of 100 μM a compound of formula (IA). As illustrated in Figure 9 the compound of formula (IA) made the tissue less sensitive to histamine, thereby relaxing the tissue. The clinical implication is that compounds according to the invention may be used to relax airway smooth muscle cells during asthma.

Inhibition of Uterine Contraction

Uterine tissue from a rat was cut into strips and mounted in a force transducer in an organ bath. Oxytocin was then added at its saturating concentration 100 nM to establish a control contraction. Increasing concentrations of a compound of formula (IA) were then pre-added (15 minutes) before challenging the tissue with the 100 nM oxytocin. As depicted in Figure 10 the compound of formula (IA) resulted in a concentration-dependent decrease in the amplitude of contraction. The clinical implication is that compounds according to the invention may be used as a tocolytic.

Claims

1. A pharmaceutical composition comprising a compound of formula (I) :

(D wherein R¹ represents a halogen atom, or a pharmaceutically acceptable salt or solvate thereof, in admixture with a pharmaceutically acceptable excipient, with the proviso that R¹ does not represent 2-fluorine.

2. A compound of formula (II)^a:

3. A compound of formula (II) ^b:

4. A compound as defined in any of claims 2 or 3 wherein R¹ represents 2-fluorine.

5. A compound as defined in any of claims 1 , 2 or 3 wherein R¹ represents fluorine.

6. A pharmaceutical composition comprising a compound of formula (II)^a or (II)^b as defined in any of claims 2 to 5 or a pharmaceutically acceptable salt or solvate thereof, in admixture with a pharmaceutically acceptable excipient.

7. Use of a compound of formula (I) with or without the proviso, (IA), (II)^a or (II)\ or a pharmaceutically acceptable salt or solvate thereof, in therapy

8. Use of a compound of formula (I) with or without the proviso, (II) ^a or (H)" as defined in any of claims 1 to 5 or a pharmaceutically acceptable salt or solvate thereof in the manufacture of a medicament for the treatment of a disease modulated by nicotinic acid adenine dinucleotide phosphate mediated intracellular calcium release.

9. A compound of formula (I) with or without the proviso, (II)^a or (II)^b as defined in any of claims 1 to 5 or a pharmaceutically acceptable salt or solvate thereof for use in the treatment of a disease modulated by nicotinic acid adenine dinucleotide phosphate mediated intracellular calcium release.

10. A method of treating a disease modulated by nicotinic acid adenine dinucleotide^ phosphate mediated intracellular calcium release which comprises administration of a therapeutically effective amount of a compound of formula (I) with or without the proviso, (II)^a or (II)" as defined in any of claims 1 to 5 or a pharmaceutically acceptable salt or solvate thereof.

11. A pharmaceutical composition comprising a compound of formula (I) with or without the proviso, (II)^a or (II)" as defined in any of claims 1 to 5 or a pharmaceutically acceptable salt or solvate thereof for use in the treatment of a disease modulated by nicotinic acid adenine dinucleotide phosphate mediated intracellular calcium release.

12. Use, method or composition as defined in any of claims 7 to 11 wherein the disease modulated by nicotinic acid adenine dinucleotide phosphate mediated intracellular calcium release is selected from multiple sclerosis, asthma, hypertension, diabetes (e.g. Type II diabetes) , male infertility, cardiac disorders (e.g. cardiac arrhythmia) and obesity.

13. A process for preparing a compound of formula (I) , (II)^a or (II)^b which comprises one of processes (a) , (b) or (c) :

(a) reacting a compound of formula (III)

(III) wherein L¹ represents a suitable leaving group, such as a halogen atom (e.g. chlorine) and P¹ represents a suitable protecting group such as C₁^ alkyl (e.g. methyl) , with a compound of formula (IV)

(IV) wherein R¹ represents a halogen atom; or

(b) deprotecting a protected derivative of a compound of formula (I) , &" (II)^a or (II)^b; or

(c) interconversion of a compound of formula (I), (II)^a or (II)^b.

14. A method of screening for an agent which effectively modulates nicotinic acid adenine diriucleotide phosphate mediated intracellular5 calcium release which comprises the steps of: (a) contacting a compound of formula (I) , (II)^a or (II)^b as defined in any of claims 1 to 5 with an NAADP receptor;

(b) measuring the level of intracellular calcium release, known as the control level; (c) contacting an agent with an NAADP receptor;

(d) measuring the level of intracellular calcium release, known as the test level; and

(e) comparing the test level with the control level, such that an effective modulator will be an agent demonstrating a lower test level than the control level.

15. An agent identifiable by the method of screening as defined in claim 14.