WO2005054198A2

WO2005054198A2 - Therapeutics use of pyridinium compounds to modulate naadp activity

Info

Publication number: WO2005054198A2
Application number: PCT/GB2004/005109
Authority: WO
Inventors: Barry Victor Lloyd Potter; James Dowden; Antony Galione
Original assignee: University of Bath
Current assignee: University of Bath
Priority date: 2003-12-05
Filing date: 2004-12-06
Publication date: 2005-06-16
Anticipated expiration: 2006-06-05
Also published as: JP2007516964A; AU2004295178A1; GB0328314D0; CA2547791A1; WO2005054198A3; EP1689714A2

Abstract

The present invention relates to the use of a compound of formula (I); wherein: R1 comprises a carbonyl group and R2 is a hydrocarbyl group; optionally wherein said ring is further substituted; or a pharmaceutically acceptable salt thereof; in the manufacture of a medicament for use in one or more of: modulating the release of intracellular calcium from a store controlled by nicotinic acid adenine dinucleotide phosphate; modulating calcium spikes in mammalian cells; treating diseases in one or more of brain, heart, pancreatic cells (e.g. pancreatic acinar and pancreatic beta cells), immune cells, T-cells, haemopoietic cells including phagocytes; treating diseases in one or more of brain, heart, pancreatic cells (e.g. pancreatic acinar and pancreatic beta cells), immune cells, T-cells, haemopoietic cells including phagocytes by modulating the release of intracellular calcium from a store controlled by nicotinic acid adenine dinucleotide phosphate; treating diseases in one or more of brain, heart, and T-cells by modulating calcium spikes in mammalian cells.

Description

THERAPEUTICS

FIELD OF THE INVENTION

The present invention relates to therapeutics.

In particular, but not exclusively, the present invention relates to therapeutics - such as compounds and compositions - for modulating the release of intracellular calcium from a store controlled by nicotinic acid adenine dinucleotide phosphate; modulating calcium spikes in mammalian cells; treating diseases in one or more of brain, heart, pancreatic cells (e.g. pancreatic acinar and pancreatic beta cells), immune cells, T-cells, haemopoietic cells including phagocytes; treating diseases in one or more of brain, heart, pancreatic cells (e.g. pancreatic acinar and pancreatic beta cells), immune cells, T-cells, haemopoietic cells including phagocytes by modulating the release of intracellular calcium from a store controlled by nicotinic acid adenine dinucleotide phosphate; and/or treating diseases in one or more of brain, heart, pancreatic cells (e.g. pancreatic acinar and pancreatic beta cells), immune cells, T-cells, haemopoietic cells including phagocytes by modulating calcium spikes in mammalian cells.

BACKGROUND TO THE INVENTION

Calcium plays a pivotal role within the cell from initial fertilisation of an egg, through every day functions, to cell death. Exactly how calcium exerts such remarkable specificity is the subject of intense study.

Inositol 1,4,5-trisphosphate (IP₃) and cADP-ribose (cADPr) are well-accepted as mediators of Ca²⁺ release from intracellular stores. However, the role of a third putative Ca²⁺-mobilizing molecule, nicotinic acid-adenine dinucleotide phosphate (NAADP), has until recently been more controversial. Of all the Ca²⁺ mobilising molecules considered as intracellular messengers, NAADP is the most potent. Typically, nanomolar concentrations in the cytoplasm lead to the release of Ca²⁺ from intracellular stores. By way of example, evidence in the sea urchin egg has been obtained to show that the major NAADP-sensitive Ca -pool is non-endoplasmic reticular in nature.

NAADP was first discovered as a contaminant in NADP that has Ca²⁺-releasing activity on sea urchin egg microsomes. Synthesis of NAADP involves a modification of the enzymatic reaction, resulting in the exchange of nicotinamide for nicotinic acid on the unphosphorylated ribose ring of NADP. The latter mechanism is favoured at low pHs such as those pertaining in an endosomal compartments where CD38 may reside during recycling from the cell surface. Replacement of an uncharged amide in NADP for a negatively charge carboxyl function in NAADP confers on the latter a potent (nanomolar affinity) capacity to mobilise Ca²⁺ from responsive stores.

NAADP mobilises intracellular Ca²⁺ stores in several cell types. For example, NAADP mobilises Ca²⁺ in a number of systems from higher organisms, including mammalian T- lymphocytes, pancreatic , kidney and heart cells. The pancreatic acinar cells were the first reported, where NAADP was found to be responsible for initiating the cholecystokinin- induced Ca²⁺ signals. Likewise, in pancreatic 3-cells, NAADP-sensitive Ca²⁺ stores also play important roles in mediating Ca²⁺ signalling activated by insulin and glucose.

It is also known that NAADP+ specifically and dose-dependently stimulates Ca²⁺ signalling in human T cells (WO 02/11736).

Evidence suggests that NAADP activates intracellular Ca²⁺ channels distinct from those that are sensitive to inositol trisphosphate and ryanodine/cyclic ADP-ribose. Recent studies in intact cells have demonstrated functional coupling between Ca²⁺ release pathways mediated by NAADP, inositol trisphosphate and cyclic ADP-ribose. Thus, NAADP is an important determinant in shaping cytosolic Ca²⁺ signals. A specific binding site for NAADP has been identified in sea urchin egg preparations (J. Biol. Chem (1996) 271 8531-8516; Biochem J. (2000) 352, 725-729) and evidence that this binding site is related to NAADP induced Ca²⁺ mobilisation has also been obtained.

The role of NAADP receptors in the co-ordination of calcium signalling has been reviewed in Trends Biochem Sc/.(2001) 26(8):482-9 and Cell Calcium (2002) 32(5-6):343-54.

Despite the importance of NAADP as a Ca²⁺-mobilizing molecule, there are at present no small molecules that selectively affect the NAADP binding site. Consequently there are no examples of any compounds that exploit such activity as therapeutic agents.

SUMMARY OF THE INVENTION

The present invention is based in part upon the development of chemical entities that modulate the release of intracellular calcium from a specific store controlled by NAADP. Typically, these molecules are small molecules with a RMM of <500 and are cell permeable. Advantageously, these and related compounds may find application as novel therapeutic agents and as probes for biological assays.

These molecules will help to solve the technical problems associated with further characterisation of this biological pathway by providing readily available small chemical tools. To date, no cell permeable molecules have been available to scientists working in the field and this has hampered biological studies.

Aspects and embodiments of the present invention are presented in the accompanying claims and in the following description and discussion. These aspects are presented under separate section headings. However, it is to be understood that the teachings under each section heading are not necessarily limited to that particular section heading. ASPECTS OF THE INVENTION

Aspects of the present invention include:

The use of a compound of formula (I):

(I)

wherein: Rl comprises a carbonyl group R2 is a hydrocarbyl group; optionally wherein said ring is further substituted; or a pharmaceutically acceptable salt thereof; in the manufacture of a medicament for use in one or more of: modulating the release of intracellular calcium from a store controlled by nicotinic acid adenine dinucleotide phosphate modulating calcium spikes in mammalian cells treating diseases in one or more of brain, heart, pancreatic cells (e.g. pancreatic acinar and pancreatic beta cells), immune cells, T-cells, haemopoietic cells including phagocytes treating diseases in one or more of brain, heart, pancreatic cells (e.g. pancreatic acinar and pancreatic beta cells), immune cells, T-cells, haemopoietic cells including phagocytes by modulating the release of intracellular calcium from a store controlled by nicotinic acid adenine dinucleotide phosphate treating diseases in one or more of brain, heart, pancreatic cells (e.g. pancreatic acinar and pancreatic beta cells), immune cells, T-cells, haemopoietic cells including phagocytes by modulating calcium spikes in mammalian cells.

Examples of optional ring substituents (sometimes referred to as R3) include one or more H, a substituted or unsubstituted aryl (e.g. Cl-15 aryl), Cl-20 alkyl, X (e.g. F, Cl, Br, I), OY, NY_n (n = 1, 2, or 3), SY, COY, wherein Y is H, substituted or unsubstituted aryl (e.g. Cl-15 aryl), Cl-20 alkyl. Thus, in the formulae presented in the claims, R3 represents one or more substituents, the or each substituent being independently selected from H, substituted or unsubstituted aryl, Cl-20 alkyl, F, Cl, Br, I, OY, NY_n (n = 1 , 2, or 3), SY, COY, CONY₂ (z = 2), C(O)OY, wherein the or each Y is independently selected from H, a substituted or unsubstituted aryl, and a Cl-20 alkyl group. R3 may be selected from one or more H, Cl-15 aryl, Cl-20 alkyl, X (e.g. F, Cl, Br, I), OY, NY_n (n = 1, 2, or 3), SY, COY, wherein Y is H, Cl-15 aryl, Cl-20 alkyl.

The optional ring substituents, R3, may comprise a substituted or unsubstituted aryl group. A substituted aryl group comprises one or more independently selected substituents on an aryl ring system. In particular, the substituted aryl group may comprise one or more hydroxy; alkyl, especially lower (C]_.-C₆) alkyl (e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert- butyl, n-pentyl and other pentyl isomers, and n-hexyl and other hexyl isomers); alkoxy, especially lower (Cj_.-C₆) alkoxy (e.g. methoxy, ethoxy, propoxy etc.); alkinyl (e.g. ethinyl); or halogen (e.g. fluoro, chloro, bromo, iodo) substituents. Preferably the aryl ring system is a C3- C15 aryl ring system. Preferably the aryl ring system is a C6-C15 aryl ring system. A pharmaceutical composition comprising a compound as defined herein or a pharmaceutically acceptable salt thereof admixed with a pharmaceutically acceptable carrier, diluent or excipient.

A. compound of formula (I) or a pharmaceutically acceptable salt thereof as defined herein for use in medicine.

A. compound of formula (1) or a pharmaceutically acceptable salt thereof. medicament comprising a compound as defined herein.

An assay method for identifying an agent that modulates intracellular calcium release comprising the steps of: (a) providing an agent; (b) providing an NAADP receptor; (c) contacting said agent with an NAADP receptor; and (d) measuring the level of intracellular calcium release; wherein a difference between (i) the level of intracellular calcium release in the presence of the agent; and (ii) the level of intracellular calcium release in the absence of the agent is indicative of an agent that modulates intracellular calcium release and may be useful in one or more of: modulating the release of intracellular calcium from a store controlled by nicotinic acid adenine dinucleotide phosphate; modulating calcium spikes in mammalian cells; treating diseases in one or more of brain, heart, pancreatic cells (e.g. pancreatic acinar and pancreatic beta cells), immune cells, T-cells, haemopoietic cells including phagocytes; treating diseases in one or more of brain, heart, pancreatic cells (e.g. pancreatic acinar and pancreatic beta cells), immune cells, T-cells, haemopoietic cells including phagocytes by modulating the release of intracellular calcium from a store controlled by nicotinic acid adenine dinucleotide phosphate; and treating diseases in one or more of brain, heart, pancreatic cells (e.g. pancreatic acinar and pancreatic beta cells), immune cells, T-cells, haemopoietic cells including phagocytes by modulating calcium spikes in mammalian cells.

An assay method comprising the steps of:

(a) performing the assay method as defined herein;

(b) identifying one or more agents capable of modulating intracellular calcium release; and

(c) preparing a quantity of those one or more identified agents.

A method comprising the steps of:

(a) performing the assay method as defined herein;

(c) preparing a pharmaceutical composition comprising those one or more identified agents.

An agent identifiable, preferably, identified by the assay method as defined herein.

A pharmaceutical composition prepared by the method as defined herein.

A method of treating and/or preventing a disease in a human or animal patient in need of same which method comprises administering to the patient an effective amount of a compound as defined herein, a composition as defined herein, or a medicament as defined herein.

A process of preparing a pharmaceutical composition, said process comprising admixing one or more of the compounds as defined herein with a pharmaceutically acceptable diluent, excipient or carrier. A pharmaceutical pack comprising one or more compartments, wherein at least one compartment comprises one or more of the compounds as defined herein, a composition as defined herein, or a medicament as defined herein.

A container comprising a compound as defined herein, a composition as defined herein, or a medicament as defined herein, wherein said container is labelled for use in one or more of: modulating the release of intracellular calcium from a store controlled by nicotinic acid adenine dinucleotide phosphate modulating calcium spikes in mammalian cells treating diseases in one or more of brain, heart, pancreatic cells (e.g. pancreatic acinar and pancreatic beta cells), immune cells, T-cells, haemopoietic cells including phagocytes treating diseases in one or more of brain, heart, pancreatic cells (e.g. pancreatic acinar and pancreatic beta cells), immune cells, T-cells, haemopoietic cells including phagocytes by modulating the release of intracellular calcium from a store controlled by nicotinic acid adenine dinucleotide phosphate treating diseases in one or more of brain, heart, pancreatic cells (e.g. pancreatic acinar and pancreatic beta cells), immune cells, T-cells, haemopoietic cells including phagocytes by modulating calcium spikes in mammalian cells.

The use of a compound of formula (I) in the manufacture of a medicament for use in one or more of: modulating the release of intracellular calcium from a store controlled by nicotinic acid adenine dinucleotide phosphate modulating calcium spikes in mammalian cells treating diseases in one or more of brain, heart, pancreatic cells (e.g. pancreatic acinar and pancreatic beta cells), immune cells, T-cells, haemopoietic cells including phagocytes treating diseases in one or more of brain, heart, pancreatic cells (e.g. pancreatic acinar and pancreatic beta cells), immune cells, T-cells, haemopoietic cells including phagocytes by modulating the release of intracellular calcium from a store controlled by nicotinic acid adenine dinucleotide phosphate treating diseases in one or more of brain, heart, pancreatic cells (e.g. pancreatic acinar cells)and T-cells by modulating calcium spikes in mammalian cells.

wherein said compound is cell permeable; wherein said compound has a relative molecular mass of less than about 500; wherein said compound is a mimetic of the nicotinic group of NAADP, wherein said NAADP has the formula (II):

(II) fr a further aspect, said compound, that is a mimetic of the nicotinic group of NAADP, has the formula (A):

(A) wherein Rl and R2 are ring substituents; wherein Rl and R2 are as defined for compound of formula (I); and wherein R3 is as defined herein. h addition to compounds of formula (A), the present invention also relates to pharmaceutically acceptable salts thereof.

SOME PREFERRED ASPECTS OF THE INVENTION

Preferably, said compound is cell permeable.

Preferably, said compound has a relative molecular mass (RMM) of less than about 500.

Preferably, Rl is -C(O)R4 wherein R4 is -OH, -O-hydrocarbyl, or -O-N(R5)(R6); wherein each of R5 and R6 is independently selected from H or a hydrocarbyl.

Preferably, Rl is a hydrocarbyl group comprising a carbonyl group, or COOH.

Preferably, R2 is hydrocarbyl, hydrocarbyl-N, hydrocarbyl-N(R7)(R8), or hydrocarbyl-C(O)- N(R9)(R10); wherein each of R7, R8, R9 and R10 is independently selected from H or a hydrocarbyl. Preferably, R2 is a hydrocarbyl group comprising a carbonyl group.

Preferably, R2 is a hydrocarbyl group comprising an amide group.

Preferably, R2 is a group comprising -hydrocarbyl-C(O)N(H or hydrocarbyl)(H or hydrocarbyl).

Preferably, R2 is a group comprising - CH₂C(O) O)N(H or hydrocarbyl) (H or hydrocarbyl).

Preferably, R2 is a group comprising - CH₂C(O)NH₂.

Preferably, said compound is a mimetic of nicotinic group of NAADP, wherein said NAADP has the formula (II):

(U)

Preferably, the present invention relates to the use of said compounds in the manufacture of a medicament for use in one or more of treating an autoimmune disease (such as thyroiditis, insulitis, multiple sclerosis, invectitis, orchitis, myasthenia gravis, rhematoid arthritis or lupus erythematosis) or graft rejection, or Type II diabetes, or cardiac arrhythmia, or treating or preventing an immune disorder in a human or animal.

Preferably, the present invention relates to the use of said compounds in the manufacture of a medicament for use in one or more of treating thyroiditis, insulitis, multiple sclerosis, invectitis, orchitis, myasthenia gravis, rhematoid arthritis, lupus erythematosis, graft rejection, Type II diabetes or cardiac arrhythmia.

SOME ADDITIONAL ASPECTS OF THE COMPOUNDS OF THE INVENTION

The ring structure of the compound of formula (1) may be further substituted - such as with another hydrocarbyl group. By way of example, such substituents may be a halo group and/or a hydrocarbyl group.

SOME EXEMPLARY COMPOUNDS OF THE INVENTION

Exemplary compounds of the present invention include the following (which are shown as salt forms - however the non-salt forms are also covered as well as other salt forms):

ADNANTAGES

The present invention has a number of advantages. These advantages will be apparent in the following description.

By way of example, the present invention is advantageous since it provides commercially useful compounds, compositions and methods.

By way of example, the present invention is advantageous since it provides commercially useful compounds, compositions and methods that selectively affect the NAADP binding site.

By way of example, the present invention is advantageous since it provides commercially useful compounds, compositions that may be used as therapeutic agents.

DESCRIPTION OF THE FIGURES

Figure 1

[³²P]NAADP binding to 0.5% sea urchin egg homogenate.

Competitive CMA008 (l-Carbamoylmethyl-3-carboxy-pyridinium iodide) displacement of [³²P]NAADP (0.2nM). Samples diluted in GluLM (intracellular medium), pre-incubated with indicated concentration of CMA008 for lOminutes, then 0.2nM [³²PJNAADP added and incubated at room temperature for a further 15mins. Samples filtered through Whatman GF/B filters to separate bound and free [³²P]NAADP ligand. Non specific binding is defined by incubation of the homogenate in the presence of lOμM NAADP. n=2. Data expressed as a fraction of total binding. The inset in Figure 1 shows that adding nicotinic acid (up to ImM) was without effect on [³²P]NAADP binding to egg membranes. Figure 2

CMA008 (l-Carbamoylmethyl-3-carboxy-pyridinium iodide) inhibits NAADP-mediated calcium release in sea urchin egg homogenate.

Samples diluted to 2.5% in GluIM in the presence of regenerating system and kept at 17°C with agitation for 3 hours to facilitate calcium uptake into stores. Calcium release was determined by measuring increase in Fluo-3 fluorescence at 526nm. Data expressed as % of 25ΘnM NAADP calcium release in the absence of CMA008 (l-Carbamoylmethyl-3-carboxy- pyridinium iodide). CMA008 inhibits NAADP calcium mobilisation with an ICso= 15.13μM. CMA008 has no effect on the total calcium mobilisation by IP₃ or cADPR (insert) indicating the selectivity of this compound on the NAADP-sensitive release mechanism.

Figure 3

Effects of CMA008 (l-Carbamoylmethyl-3-carboxy-pyridinium iodide) on the competition of [³²P]NAADP in the presence of increasing concentrations of unlabelled NAADP.

2.5% sea urchin egg homogenate was diluted into GluIM pre-incubated with indicated concentration of CMA008 for lOminutes at room temperature (A=100nM, B=lnM, C=10μM). Subsequently, [³²P]NAADP was added and samples incubated for a further 15minutes at room temperature. Samples were filtered through Whatman GF/B filters to separate bound and free [³²P]NAADP ligand. CMA008 reduces the B_max from 68.65% ± 2.7 total specific binding in the absence of CMA008 to 46.28% ± 2.61 in the presence of lOμM CMA008. InM and lOOnM CMA008 had no effect on the B_max. There is no effect on the IC₅₀ for NAADP at any of the tested concentrations, n is between 2 and 6 for each point.

Figure 4

Effect of CMA008 (l-Carbarnoylmethyl-3-carboxy-pyridinium iodide) on CCK-induced oscillations of Ca²⁺ in pancreatic acinar cells Pancreatic acinar cells were seeded onto poly-lysine-coated number 1 glass coverslips and loaded by incubating cells with 1-5 μM fura-2 acetoxymethylester (Molecular Probes; Leiden, Holland) for 60 min at room temperature. After the loading period, cells were subsequently washed and maintained in buffer at room temperature and used immediately. Cells were excited alternately with 340 and 380 nm light (emission 510 nm), and ratio image of clusters were recorded every 4-5 s, using a 12-bit CCD camera (MicroMax; Princeton Instruments, NJ). All experiment were conducted at room temperature. CMA008 (l-Carbamoylmethyl-3- carboxy-pyridinium iodide) is dissolved in 50%DMSO. Final concentration of DMSO is 0.5% in the solution (composition in mM:140 NaCl, 4.7 KCl, 1 CaC12, 1.13 MgC12, 10 HEPES, 10 Glucose, pH adjusted to 7.2)

Figure 5

HPLC of the pyridinium salts.

Figure 6

Abolition of calcium release in response to uncaging of caged NAADP in intact sea urchin eggs (loaded with the calcium reporter dye calcium green dextran) with external application of CMA008.

Sea urchin eggs were microinjected with a solution containing the calcium reporter dye, calcium green dextran, together with caged NAADP. Fluorescence was then imaged on a Leica confocal microscope using an excitation wavelength of 488 nm. In the control egg [see Figure 6 A] a brief UN flash photolysed a proportion of the caged ΝAADP and evoked a large calcium transient. However, when eggs were incubated with sea water containing CMA008 (10 mM) [see Figure 6 B], liberation of ΝAADP by photolysis failed to induce and calcium release. This indicates that CMA008 permeates the cell membrane and inhibits ΝAADP- induced calcium release.

Figure 7 Cholecystokinin (CCK)-evoked calcium oscillations (dependent on ΝAADP signalling) in isolated mouse pancreatic cells are inhibited or reduced by external application of CM008. Traces show the Ca²⁺ dye fluorescence.

Pancreatic acinar cells were isolated from mice and dispersed by coUagenase treatment. Cells were incubated with fura-2 AM for 30 min and washed before being imaged on a Metafluor system. The cells were alternately excited at 34O/380 nm and emitted light collected at around 510 nm. The ratio of the intensities of emitted light at the two excitation wavelengths were calculated, converted to free calcium concentrations, and plotted again time.

In Figure 7 A, the peptide cholecystokinin (CCK; 5 pM) was added to the cells and resulted in a robust series of calcium spikes in the continued presence of the agonist. However, when CMA008 (1 mM) was added to the bathing solution (i.e. post-CCK addition), it resulted in the inhibition of CCK-evoked calcium spiking. In Figure 7 B, the CMA008 (1 mM) was added first to the medium (i.e. pre-CCK addition) with no apparent effect on resting calcium levels. However, now on application of CCK, a much smaller response to CCK was seen, with a greatly reduced frequency. Since it has been shown that NAADP is a critical messenger for CCK-mediated calcium signalling, it appears that CMA008 is able to enter pancreatic acinar cells where it may block NAADP-evoked calcium release.

DETAILED DESCRIPTION OF THE INVENTION

SUBSTITUENTS

The compounds of the present invention may have substituents other than those of the ring systems shown herein. Furthermore the ring systems herein are given as general formulae and should be interpreted as such. The absence of any specifically shown substituents on a given ring member indicates that the ring member may be substituted with any moiety of which H is only one example. The ring system may contain one or more degrees of unsaturation, for example in some aspects one or more rings of the ring system is aromatic. The ring system may be carbocyclic or may contain one or more hetero atoms. Preferably, the compounds of the present invention have the structures shown herein.

The compound of the invention, in particular the ring system compound of the present invention may contain substituents other than those shown herein. By way of example, these other substituents may be one or more of: one or more halo groups, one or more O groups, one or more hydroxy groups, one or more amino groups, one or more sulphur containing grouρ(s), one or more hydrocarbyl group(s) - such as an oxyhydrocarbyl group.

In general terms the ring system of the present compounds may contain a variety of non- interfering substituents. In particular, the ring system may contain one or more hydroxy, allcyl especially lower (Cj_.-C₆) alkyl, e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert- butyl, n-pentyl and other pentyl isomers, and n-hexyl and other hexyl isomers, alkoxy especially lower (CrC₆) alkoxy, e.g. methoxy, ethoxy, propoxy etc., alkinyl, e.g. efriinyl, or halogen, e.g. fluoro substituents.

COMPOUND

The tenn "compound" is intended to encompass isomeric forms (such as stereoisomers and/or geometric and/or optical isomers, and mixtures thereof), chemical derivatives, mimetics, solvates and salts of the compounds.

HYDROCARBYL

In the context of the present invention, the term "hydrocarbyl group" as used herein means a group comprising at least C and H and may optionally comprise one or more other suitable substituents. Examples of such substituents may include halo, alkoxy, nitro, an alkyl group, a cyclic group etc. In addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group. If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the hydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur, nitrogen and oxygen. A non-limiting example of a hydrocarbyl group is an acyl group.

A typical hydrocarbyl group is a hydrocarbon group. Here the ter "hydrocarbon" means any one of an alkyl group, an alkenyl group, an alkynyl group, which groups may be linear, branched or cyclic, or an aryl group. The term hydrocarbon also includes those groups but wherein they have been optionally substituted. If the hydrocarbon is a branched structure having substituent(s) thereon, then the substitution may be on either the hydrocarbon backbone or on the branch; alternatively the substitutions may be on the hydrocarbon backbone and on the branch.

In some aspects of the present invention, one or more hydrocarbyl groups is independently selected from optionally substituted alkyl group, optionally substituted haloalkyl group, aryl group, alkylaryl group, alkylarylalkyl group, and an alkene group.

In some aspects of the present invention, one or more hydrocarbyl groups is independently selected from Cι-C₁₀ alkyl group, such as Ci-C₆ alkyl group, and Cj,-C₃ alkyl group. Typical alkyl groups include Ci alkyl, C₂ allcyl, C₃ alkyl, C₄ alkyl, C₅ alkyl, C alkyl, and C₈ alkyl.

For some aspects, preferably, the hydrocarbyl group comprises a carbonyl group. More preferably, the carbonyl group has the formula COOH.

For some aspects, preferably, the hydrocarbyl group comprises an amide group. More preferably, the amide group has the formula CH₂C(O)NH₂.

More preferably, the R2 group of the compound of Formula (I) is a hydrocarbyl group comprising an amide group.

More preferably, the Rl group of the compound of Formula (I) is a hydrocarbyl group comprising a carbonyl group. Most preferably, the R2 group of the compound of Formula (I) is a hydrocarbyl group comprising an amide group of the formula CH₂C(O)NH₂.

Most preferably, the Rl group of the compound of Fomvula (I) is a hydrocarbyl group comprising a carbonyl group of the formula COOH.

In some aspects of the present invention, one or more hydrocarbyl groups may be independently selected from one or more oxyhydrocarbyl groups.

OXYHYDROCARBYL

The term "oxyhydrocarbyl" group as used herein means a group comprising at least C, H and O and may optionally comprise one or more other suitable substituents. Examples of such substituents may include halo-, alkoxy-, nitro-, an alkyl group, a cyclic group etc. In addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group. If the oxyhydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the oxyhydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur and nitrogen.

h one embodiment of the present invention, the oxyhydrocarbyl group is a oxyhydrocarbon group.

Here the term "oxyhydrocarbon" means any one of an alkoxy group, an oxyalkenyl group, an oxyalkynyl group, which groups may be linear, branched or cyclic, or an oxyaryl group. The term oxyhydrocarbon also includes those groups but wherein they have been optionally substituted. If the oxyhydrocarbon is a branched structure having substituent(s) thereon, then the substitution may be on either the hydrocarbon backbone or on the branch; alternatively the substitutions may be on the hydrocarbon backbone and on the branch. STEREO AND GEOMETRIC ISOMERS

Some of the compounds/agents of the present invention may exist as stereoisomers and/or geometric isomers - e.g. they may possess one or more asymmetric and/or geometric centres and so may exist in two or more stereoisomeric and or geometric forms. The present invention contemplates the use of all of the individual stereoisomers and geometric isomers of those compounds, and mixtures thereof. The terms used in the claims encompass these forms, provided said forms retain the appropriate functional activity (though not necessarily to the same degree).

SOLVATES

The present invention also includes the use of solvate forms of the compounds/agents of the present invention. The terms used in the claims encompass these forms.

PRO-DRUG

As indicated, the present invention also includes the use of pro-drug forms of the compounds/agents of the present invention. The terms used in the claims encompass these forms. Examples of prodrugs include entities that have certain protected group(s) and which may not possess pharmacological activity as such, but may, in certain instances, be administered (such as orally or parenterally) and thereafter metabolised in the body to form the compounds of the present invention which are pharmacologically active.

It will be further appreciated that certain moieties known as "pro-moieties", for example as described in "Design of Prodrugs" by H. Bundgaard, Elsevier, 1985 (the disclosure of which is hereby incorporated by reference), may be placed on appropriate functionalities of the compounds. Such prodrugs are also included within the scope of the invention.

An example of a prodrug according to the present invention is:

This prodrug requires light for activation.

MIMETIC

In one embodiment of the present invention, the compound/agent may be a mimetic.

As used herein, the term "mimetic" relates to any chemical which includes, but is not limited to, a peptide, polypeptide, antibody or other organic chemical which has the same qualitative activity or effect as a reference agent.

In a preferred embodiment, the compound is a mimetic of nicotinic group of NAADP, wherein said NAADP has the formula (II)

CHEMICAL DERIVATIVE

In one embodiment of the present invention, the compound/agent may be a derivative.

The term "derivative" as used herein includes chemical modification of a compound/agent. Illustrative of such chemical modifications would be replacement of hydrogen by a halo group, an alkyl group, an acyl group or an amino group. CHEMICAL MODIFICATION

In one embodiment of the present invention, the compound/agent may be a chemically modified compound/agent.

The chemical modification of a compound/agent may either enhance or reduce hydrogen bonding interaction, charge interaction, hydrophobic interaction, van der Waals interaction or dipole interaction between the agent and the target.

GENERALASSAYTECHNIQUES

h one aspect, the identified compounds/agents according to the present invention may act as a model (for example, a template) for the development of other compounds. The compounds/agents employed in such a test may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The abolition of activity or the formation of binding complexes between the compound and the agent being tested may be measured.

The assay of the present invention may be a screen, whereby a number of agents are tested. In one aspect, the assay method of the present invention is a high through put screen.

Techniques for drug screening may be based on the method described in Geysen, European Patent Application 84/03564, published on September 13, 1984. In summary, large numbers of different small peptide test compounds are synthesised on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with a suitable compound or fragment thereof and washed. Bound entities are then detected - such as by appropriately adapting methods well known in the art. A purified compound can also be coated directly onto plates for use in a drug screening techniques. Alternatively, non-neutralising antibodies can be used to capture the peptide and immobilise it on a solid support. This invention also contemplates the use of competitive drug screening assays in which neutralising antibodies capable of binding a compound/agent specifically compete with a test compound for binding to a compound according to the present invention.

Another technique for screening provides for high throughput screening (HTS) of compounds/agents having suitable binding affinity to the substances and is based upon the method described in detail in WO 84/03564.

It is expected that the assay methods of the present invention will be suitable for both small and large-scale screening of test compounds as well as in quantitative assays.

HOST CELLS

The term "host cell" - in relation to the present invention includes any cell that could comprise the target - such as the NAADP receptor - for the compound/agent of the present invention.

Thus, host cells may be transformed or transfected with a polynucleotide that is or expresses the target of the present invention. Preferably said polynucleotide is carried in a vector for the replication and expression of polynucleotides that are to be the target or are to express the target. The cells will be chosen to be compatible with the said vector and may for example be prokaryotic (for example bacterial), fungal, yeast or plant cells.

The gram negative bacterium E. coli is widely used as a host for heterologous gene expression. However, large amounts of heterologous protein tend to accumulate inside the cell. Subsequent purification of the desired protein from the bulk oϊE.coli intracellular proteins can sometimes be difficult.

In contrast to E.coli, bacteria from the genus Bacillus are very suitable as heterologous hosts because of their capability to secrete proteins into the culture medium. Other bacteria suitable as hosts are those from the genera Streptomyces and Pseudomonas. Depending on the nature of the polynucleotide encoding the polypeptide of the present invention, and/or the desirability for further processing of the expressed protein, eukaryotic hosts such as yeasts or other fungi may be preferred. In general, yeast cells are preferred over fungal cells because they are easier to manipulate. However, some proteins are either poorly secreted from the yeast cell, or in some cases are not processed properly (e.g. hyperglycosylation in yeast). In these instances, a different fungal host organism should be selected.

Examples of suitable expression hosts within the scope of the present invention are fungi such as Aspergillus species (such as those described in EP-A-0184438 and EP-A-0284603) and Trichoderma species; bacteria such as Bacillus species (such as those described in EP-A- 0134048 and EP-A-0253455), Streptomyces species and Pseudomonas species; and yeasts such as Kluyveromyces species (such as those described in EP-A-0096430 and EP-A- 0301670) and Saccharomyces species. By way of example, typical expression hosts may be selected from Aspergillus niger, Aspergillus niger var. tubigenis, Aspergillus niger var. awamori, Aspergillus aculeatis, Aspergillus nidulans, Aspergillus orvzae, Trichoderma reesei, Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Kluyveromyces lactis and Saccharomyces cerevisiae.

The use of suitable host cells - such as yeast, fungal and plant host cells - may provide for post-translational modifications (e.g. myristoylation, glycosylation, truncation, lapidation and tyrosine, serine or threonine phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the present invention.

ORGANISM

The term "organism" in relation to the present invention includes any organism that could comprise the target according to the present invention and/or products obtained therefrom. Examples of organisms may include a fungus, yeast or a plant.

The term "transgenic organism" in relation to the present invention includes any organism that comprises the target according to the present invention and/or products obtained.

TRANSFORMATION OF HOST CELLS/HOST ORGANISMS

As indicated earlier, the host organism can be a prokaryotic or a eukaryotic organism. Examples of suitable prokaryotic hosts include E. coli and Bacillus subtilis. Teachings on the transformation of prokaryotic hosts is well documented in the art, for example see Sambroolc et al (Molecular Cloning: A Laboratory Manual, 2nd edition, 1989, Cold Spring Harbor Laboratory Press) and Ausubel et al., Current Protocols in Molecular Biology (1995), John Wiley & Sons, Inc.

If a prokaryotic host is used then the nucleotide sequence may need to be suitably modified before transformation - such as by removal of introns.

In another embodiment the transgenic organism can be a yeast. In this regard, yeast have also been widely used as a vehicle for heterologous gene expression. The species Saccharomyces cerevisiae has a long history of industrial use, including its use for heterologous gene expression. Expression of heterologous genes in Saccharomyces cerevisiae has been reviewed by Goodey et al (1987, Yeast Biotechnology, D R Berry et al, eds, pp 401-429, Allen and Unwin, London) and by King et al (1989, Molecular and Cell Biology of Yeasts, E F Walton and G T Yarronton, eds, pp 107-133, Blackie, Glasgow).

For several reasons Saccharomyces cerevisiae is well suited for heterologous gene expression. First, it is non-pathogenic to humans and it is incapable of producing certain endotoxins. Second, it has a long history of safe use following centuries of commercial exploitation for various purposes. This has led to wide public acceptability. Third, the extensive commercial use and research devoted to the organism has resulted in a wealth of knowledge about the genetics and physiology as well as large-scale fermentation characteristics of Saccharomyces cerevisiae.

A review of the principles of heterologous gene expression in Saccharomyces cerevisiae and secretion of gene products is given by E Hinchcliffe E Kenny (1993, "Yeast as a vehicle for the expression of heterologous genes", Yeasts, Vol 5, Anthony H Rose and J Stuart Harrison, eds, 2nd edition, Academic Press Ltd.).

Several types of yeast vectors are available, including integrative vectors, which require recombination with the host genome for their maintenance, and autonomously replicating plasmid vectors.

In order to prepare the transgenic Saccharomyces, expression constructs are prepared by inserting the nucleotide sequence into a construct designed for expression in yeast. Several types of constructs used for heterologous expression have been developed. The constructs contain a promoter active in yeast fused to the nucleotide sequence, usually a promoter of yeast origin, such as the GAL1 promoter, is used. Usually a signal sequence of yeast origin, such as the sequence encoding the SUC2 signal peptide, is used. A terminator active in yeast ends the expression system.

For the transformation of yeast several transformation protocols have been developed. For example, a transgenic Saccharomyces according to the present invention can be prepared by following the teachings of Hinnen et al (1978, Proceedings of the National Academy of Sciences of the USA 75, 1929); Beggs, J D (1978, Nature, London, 275, 104); and Ito, H et al (1983, J Bacteriology 153, 163-168).

The transformed yeast cells are selected using various selective markers. Among the markers used for transformation are a number of auxotrophic markers such as LEU2, HIS4 and TRPl, and dominant antibiotic resistance markers such as amino glycoside antibiotic markers, e.g. G418.

Another host organism is a plant. The basic principle in the construction of genetically modified plants is to insert genetic information in the plant genome so as to obtain a stable maintenance of the inserted genetic material. Several techniques exist for inserting the genetic information, the two main principles being direct introduction of the genetic information and introduction of the genetic information by use of a vector system. A review of the general techniques may be found in articles by Potrykus (Annu Rev Plant Physiol Plant Mol Biol [1991] 42:205-225) and Christou (Agro-Food-Industry Hi-Tech March/April 1994 17-27). Further teachings on plant transformation may be found in EP-A-0449375.

Thus, the present invention also provides a method of transforming a host cell with a nucleotide sequence that is to be the target or is to express the target. Host cells transformed with the nucleotide sequence may be cultured under conditions suitable for the expression of the encoded protein. The protein produced by a recombinant cell may be displayed on the surface of the cell. If desired, and as will be understood by those of skill in the art, expression vectors containing coding sequences can be designed with signal sequences which direct secretion of the coding sequences through a particular prokaryotic or eukaryotic cell membrane. Other recombinant constructions may join the coding sequence to nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins (Kroll DJ et al (1993) DNA Cell Biol 12:441-53).

EXPRESSION VECTORS

The nucleotide sequence for use as the target or for expressing the target can be incorporated into a recombinant replicable vector.

The vector may be used to replicate and express the nucleotide sequence in and/or from a compatible host cell. Expression may be controlled using control sequences which include promoters/enhancers and other expression regulation signals. Prokaryotic promoters and promoters functional in eukaryotic cells may be used. Tissue specific or stimuli specific promoters may be used. Chimeric promoters may also be used comprising sequence elements from two or more different promoters described above.

The protein produced by a host recombinant cell by expression of the nucleotide sequence may be secreted or may be contained intracellularly depending on the sequence and/or the vector used. The coding sequences can be designed with signal sequences which direct secretion of the substance coding sequences through a particular prokaryotic or eukaryotic cell membrane.

FUSION PROTEINS

The target amino acid sequence may be produced as a fusion protein, for example to aid in extraction and purification. Examples of fusion protein partners include glutathione-S- transferase (GST), 6xHis, GAL4 (DNA binding and/or transcriptional activation domains) and (-galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences. Preferably the fusion protein will not hinder the activity of the target.

The fusion protein may comprise an antigen or an antigenic determinant fused to the substance of the present invention. In this embodiment, the fusion protein may be a non-naturally occurring fusion protein comprising a substance which may act as an adjuvant in the sense of providing a generalised stimulation of the immune system. The antigen or antigenic determinant may be attached to either the ammo or carboxy terminus of the substance.

In another embodiment of the invention, the amino acid sequence may be ligated to a heterologous sequence to encode a fusion protein. For example, for screening of peptide libraries for agents capable of affecting the substance activity, it may be useful to encode a chimeric substance expressing a heterologous epitope that is recognised by a commercially available antibody.

REPORTERS

A wide variety of reporters may be used in the assay methods (as well as screens) of the present invention with preferred reporters providing conveniently detectable signals (e.g. by spectroscopy). By way of example, a number of companies such as Pharmacia Biotech (Piscataway, NJ), Promega (Madison, Wl), and US Biochemical Corp (Cleveland, OH) supply commercial kits and protocols for assay procedures. Suitable reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles and the like. Patents teaching the use of such labels include US-A-3817837; US-A-3850752; US-A-3939350; US-A- 3996345; US-A-4277437; US-A-4275149 and US-A-4366241.

ASSAY METHOD

In a further aspect, the present invention relates to an assay method for identifying an agent that modulates intracellular calcium release comprising the steps of: (a) providing an agent; (b) providing an NAADP receptor; (c) contacting said agent with an NAADP receptor; and (d) measuring the level of intracellular calcium release; wherein a difference between (i) the level of intracellular calcium release in the presence of the agent; and (ii) the level of intracellular calcium release in the absence of the agent is indicative of an agent that modulates intracellular calcium release and may be useful in one or more of: modulating the release of intracellular calcium from a store controlled by nicotinic acid adenine dinucleotide phosphate; modulating calcium spikes in mammalian cells; treating diseases in one or more of brain, heart, pancreatic cells (e.g. pancreatic acinar and pancreatic beta cells), immune cells, T-cells, haemopoietic cells including phagocytes; treating diseases in one or more of brain, heart, pancreatic cells (e.g. pancreatic acinar and pancreatic beta cells), immune cells, T-cells, haemopoietic cells including phagocytes by modulating the release of intracellular calcium from a store controlled by nicotinic acid adenine dinucleotide phosphate; and treating diseases in one or more of brain, heart, pancreatic cells (e.g. pancreatic acinar and pancreatic beta cells), immune cells, T-cells, haemopoietic cells including phagocytes by modulating calcium spikes in mammalian cells.

AGENT

As described herein, the assay methods of the present invention may be used to identify one or more agents that modulates intracellular calcium release.

As used herein, the term "agent" may refer to a single entity or a combination of entities. The agent may be an organic compound or other chemical. The agent may be a compound, which is obtainable from or produced by any suitable source, whether natural or artificial.

The agent may be an amino acid molecule, a polypeptide, or a chemical derivative thereof, or a combination thereof.

The agent may even be a polynucleotide molecule - which may be a sense or an anti-sense molecule.

The agent may even be an antibody or a part or parts thereof.

The agent may be designed or obtained from a library of compounds, which may comprise peptides, as well as other compounds, such as small organic molecules.

By way of example, the agent may be a natural substance, a biological macromolecule, or an extract made from biological materials such as bacteria, fungi, or animal (particularly mammalian) cells or tissues, an organic or an inorganic molecule, a synthetic agent, a semi- synthetic agent, a structural or functional mimetic, a peptide, a peptidomimetics, a derivatised agent, a peptide cleaved from a whole protein, a peptide synthesised synthetically (such as, by way of example, either using a peptide synthesizer or by recombinant techniques) or combinations thereof, a recombinant agent, an antibody, a natural or a non-natural agent, a fusion protein or equivalent thereof and mutants, derivatives or combinations thereof.

The agent may be an organic compound. Typically the organic compounds may comprise two or more hydrocarbyl groups. Here, the term "hydrocarbyl group" means a group comprising at least C and H and may optionally comprise one or more other suitable substituents. Examples of such substituents may include halo-, alkoxy-, nitro-, an allcyl group, a cyclic group etc. In addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group. If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the hydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur, nitrogen and oxygen. The agent may comprise at least one cyclic group. The cyclic group may be a polycyclic group, such as a non-fused polycyclic group. The agent may comprise at least one of said cyclic groups linked to another hydrocarbyl group.

The agent may contain halo groups.

The agent may contain one or more of alkyl, alkoxy, alkenyl, alkylene and alkenylene groups - which may be unbranched- or branched-chain.

The agent may be in the form of a pharmaceutically acceptable salt - such as an acid addition salt or a base salt - or a solvate thereof, including a hydrate thereof. For a review on suitable salts see Berge et al, J. Pharm. Sci., 1977, 66, 1-19.

The agent may be capable of displaying other therapeutic properties.

The agent may be used in combination with one or more other pharmaceutically active agents.

In a highly preferred aspect, the agent is cell permeable; has a relative molecular mass of less than about 500; is a mimetic of the nicotinic group of NAADP, wherein said NAADP has the formula:

(II)

If combinations of active agents are administered, then they may be administered simultaneously, separately or sequentially.

PHARMACEUTICAL SALTS

The compounds and/or agents of the present invention may be administered as pharmaceutically acceptable salts. Typically, a pharmaceutically acceptable salt may be readily prepared by using a desired acid or base, as appropriate. The salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent.

Pharmaceutically-acceptable salts are well known to those skilled in the art, and for example include those mentioned by Berge et al, in J.Pharm.Sci. 66, 1-19 (1977). Suitable acid addition salts are formed from acids which form non-toxic salts and include the hydrochloride, hydrobromide, hydroiodide, nitrate, sulphate, bisulphate, phosphate, hydrogenphosphate, acetate, trifluoroacetate, gluconate, lactate, salicylate, citrate, tartrate, ascorbate, succinate, maleate, fumarate, gluconate, formate, benzoate, methanesulphonate, ethanesulphonate, benzenesulphonate and p-toluenesulphonate salts.

When one or more acidic moieties are present, suitable pharmaceutically acceptable base addition salts can be formed from bases which form non-toxic salts and include the aluminium, calcium, lithium, magnesium, potassium, sodium, zinc, and pharmaceutically- active amines such as diethanolamine, salts.

The compounds and/or agents of the present invention may exist in polymorphic form.

In addition, the compounds and/or agents of the present invention may contain one or more asymmetric carbon atoms and therefore exists in two or more stereoisomeric forms. Where a compound and/or agent contains an alkenyl or alkenylene group, cis (E) and trans (Z) isomerism may also occur. The present invention includes the individual stereoisomers of the compound and/or agent and, where appropriate, the individual tautomeric forms thereof, together with mixtures thereof.

Separation of diastereoisomers or cis and trans isomers may be achieved by conventional techniques, e.g. by fractional crystallisation, chromatography or H.P.L.C. of a stereoisomeric mixture of the agent or a suitable salt or derivative thereof. An individual enantiomer of the compound and/or agent may also be prepared from a corresponding optically pure intermediate or by resolution, such as by H.P.L.C. of the corresponding racemate using a suitable chiral support or by fractional crystallisation of the diastereoisomeric salts formed by reaction of the corresponding racemate with a suitable optically active acid or base, as appropriate.

The present invention also includes all suitable isotopic variations of the compound and/or agent or a pharmaceutically acceptable salt thereof. An isotopic variation of a compound and/or agent of the present invention or a pharmaceutically acceptable salt thereof is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature. Examples of isotopes that can be incorporated into the compound and/or agent and pharmaceutically acceptable salts thereof include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine and chlorine such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁷O, ¹⁸O, ³¹P, ³²P, ³⁵S, ¹⁸F and ³⁶C1, respectively. Certain isotopic variations of the compound and/or agent and pharmaceutically acceptable salts thereof, for example, those in which a radioactive isotope such as ³H or ¹⁴C is incorporated, are useful in drug and/or substrate tissue distribution studies. Tritiated, i.e., ³H, and carbon-14, i.e., ^I4C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with isotopes such as deuterium, i.e., ²H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements and hence may be preferred in some circumstances. Isotopic variations of the compound and/or agent of the present invention and pharmaceutically acceptable salts thereof of this invention can generally be prepared by conventional procedures using appropriate isotopic variations of suitable reagents.

The present invention also includes (wherever appropriate) the use of zwitterionic forms of the compounds and/or agents of the present invention.

The terms used in the claims encompass one or more of the forms just mentioned.

FORMULATION

The component(s) of the present invention may be formulated into a pharmaceutical composition, such as by mixing with one or more of a suitable carrier, diluent or excipient, by using techniques that are known in the art.

PHARMACEUTICAL COMPOSITIONS

The present invention provides a pharmaceutical composition comprising a therapeutically effective amount of one or more compounds and/or agents of the present invention and a pharmaceutically acceptable carrier, diluent or excipient (including combinations thereof). The pharmaceutical compositions may be for human or animal usage in human and veterinary medicine and will typically comprise any one or more of a pharmaceutically acceptable diluent, carrier, or excipient. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).

Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like. Examples of suitable diluents include ethanol, glycerol and water.

Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, com 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.

Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents maybe also used.

There may be different composition/formulation requirements dependent on the different delivery systems. By way of example, the pharmaceutical composition of the present invention may be formulated to be administered using a mini-pump or by a mucosal route, for example, as a nasal spray or aerosol for inhalation or ingestable solution, or parenterally in which the composition is formulated by an mjectable form, for delivery, by, for example, an intravenous, intramuscular or subcutaneous route. Alternatively, the formulation may be designed to be administered by a number of routes.

Where the composition is to be administered mucosally through the gastrointestinal mucosa, it should be able to remain stable during transit though the gastrointestinal tract; for example, it should be resistant to proteolytic degradation, stable at acid pH and resistant to the detergent effects of bile.

Where appropriate, the pharmaceutical compositions can be administered by inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intravenously, intramuscularly or subcutaneously. For parenteral administration, the compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood. For buccal or sublingual administration the compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.

For some embodiments, one or more compounds and/or agents may also be used in combination with a cyclodextrin. Cyclodextrins are known to form inclusion and non- inclusion complexes with drug molecules. Formation of a drug-cyclodextrin complex may modify the solubility, dissolution rate, bioavailability and/or stability property of a drug molecule. Drug-cyclodextrin complexes are generally useful for most dosage forms and administration routes. As an alternative to direct complexation with the drug the cyclodextrin may be used as an auxiliary additive, e.g. as a carrier, diluent or solubiliser. Alpha-, beta- and gamma-cyclodextrins are most commonly used and suitable examples are described in WO-A- 91/11172, WO-A-94/02518 and WO-A-98/55148. The pharmaceutical composition may comprise one or more additional pharmaceutically active compounds and/or agents.

CHEMICAL SYNTHESIS METHODS

The compounds and/or agents of the invention may be prepared by chemical synthesis techniques.

It will be apparent to those skilled in the art that sensitive functional groups may need to be protected and deprotected during synthesis of a compound and/or agent of the invention. This may be achieved by conventional techniques, for example as described in "Protective Groups in Organic Synthesis" by T W Greene and P G M Wuts, John Wiley and Sons h e. (1991), and by PJ.Kocienski, in "Protecting Groups", Georg Thieme Verlag (1994).

It is possible during some of the reactions that any stereocentres present could, under certain conditions, be epimerised, for example if a base is used in a reaction with a substrate having an having an optical centre comprising a base-sensitive group. It should be possible to circumvent potential problems such as this by choice of reaction sequence, conditions, reagents, protection/deprotection regimes, etc. as is well-known in the art.

The compounds/agents and salts of the invention may be separated and purified by conventional methods.

THERAPY

As with the term "treatment", the term "therapy" includes curative effects, alleviation effects, and prophylactic effects.

Preferably, the term therapy includes at least curative treatment and/or palliative treatment.

The therapy may be on humans or animals. In one aspect, the present invention relates to the use of compound (I) wherein: Rl comprises a carbonyl group; R2 is a hydrocarbyl group; optionally wherein said ring is further substituted; or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of a disease.

COMBINATION PHARMACEUTICAL

The compound/agents of the present invention may be used in combination with one or more other active agents, such as one or more other pharmaceutically active agents.

ADMINISTRATION

The components of the present invention may be administered alone but will generally be administered as a pharmaceutical composition - e.g. when the components are is in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.

For example, the composition can be administered (e.g. orally or topically) in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed-, modified-, sustained-, pulsed- or controlled-release applications.

If the pharmaceutical composition is a tablet, then the tablet may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably com, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included. Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the compounds/agents may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.

The routes for adrniriistration (delivery) include, but are not limited to, one or more of: oral (e.g. as a tablet, capsule, or as an ingestable solution), topical, mucosal (e.g. as a nasal spray or aerosol for inhalation), nasal, parenteral (e.g. by an mjectable form), gastrointestinal, intraspinal, intraperitoneal, intramuscular, intravenous, intrauterine, intraocular, intradermal, intracranial, intratracheal, intravaginal, intracerebroventricular, intracerebral, subcutaneous, ophthalmic (including intravitreal or intracameral), transdermal, rectal, buccal, vaginal, epidural, sublingual.

Where the composition comprises more than one compound/agent, it is to be understood that not all of the components of the pharmaceutical need be administered by the same route. Likewise, if the composition comprises more than one active component, then those components may be administered by different routes.

If a component of the present invention is administered parenterally, then examples of such administration include one or more of: intravenously, intra-arterially, intraperitoneally, intrathecally, intraventricularly, intraurethrally, intrasternaHy, intracranially, intramuscularly or subcutaneously administering the component; and/or by using infusion techniques.

For parenteral administration, the component is best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art. As indicated, the component(s) of the present invention can be administered intranasally or by inhalation and is conveniently delivered in the form of a dry powder inhaler or an .aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluoroalkane such as 1,1,1,2-tetrafluoro ethane (UFA 134A™) or 1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA™), carbon dioxide or other suitable gas. In the case of a pressurised aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active compound, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of the agent and a suitable powder base such as lactose or starch.

Alternatively, the component(s) of the present invention can be administered in the form of a suppository or pessary, or it may be applied topically in the form of a gel, hydrogel, lotion, solution, cream, ointment or dusting powder. The component(s) of the present invention may also be dermally or transdermally admimstered, for example, by the use of a skin patch. They may also be administered by the pulmonary or rectal routes. They may also be administered by the ocular route. For ophthalmic use, the compounds/agents can be formulated as micronised suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride. Alternatively, they may be formulated in an ointment such as petrolatum.

For application topically to the skin, the component(s) of the present invention can be formulated as a suitable ointment containing the one or more active compounds/agents suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, it can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

In a preferred embodiment of the invention, the pharmaceutical composition is admimstered orally.

DOSE LEVELS

Typically, a physician will determine the actual dosage which will be most suitable for an individual subject. The specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific one or more compounds/agents employed, the metabolic stability and length of action of that compound/agent, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy.

The routes of administration and dosages described are intended only as a guide since a skilled practitioner will be able to determine readily the optimum route of administration and dosage for any particular patient depending on, for example, the age, weight and condition of the patient.

OTHER THERAPIES

It is also to be understood that the compounds/agents/compositions of the present invention may have other important medical implications.

For example, the compounds, compositions or agents of the present invention may be useful in the treatment of the disorders listed in WO-A-99/52890.

In addition, or in the alternative, the compounds, compositions or agents of the present invention may be useful in the treatment of the disorders listed in WO-A-98/05635. For ease of reference, part of that list is now provided: diabetes including Type II diabetes, obesity, cancer, inflammation or inflammatory disease, dermatological disorders, fever, cardiovascular effects, haemorrhage, coagulation and acute phase response, cachexia, anorexia, acute infection, HIV infection, shock states, graft-versus-host reactions, autoimmune disease, reperfusion injury, meningitis, migraine and aspirin-dependent anti-thrombosis; tumour growth, invasion and spread, angiogenesis, metastases, malignant, ascites and malignant pleural effusion; cerebral ischaemia, ischaemic heart disease, osteoarthritis, rheumatoid arthritis, osteoporosis, asthma, multiple sclerosis, neurodegeneration, Alzheimer's disease, atherosclerosis, stroke, vasculitis, Crohn's disease and ulcerative colitis; periodontitis, gingivitis; psoriasis, atopic dermatitis, chronic ulcers, epidermolysis bullosa; comeal ulceration, retinopathy and surgical wound healing; rhinitis, allergic conjunctivitis, eczema, anaphylaxis; restenosis, congestive heart failure, endometriosis, atherosclerosis or endosclerosis.

In addition, or in the alternative, the compounds, compositions or agents of the present invention may be useful in the treatment of disorders listed in WO-A-98/07859. For ease of reference, part of that list is now provided: cytokine and cell proliferation/differentiation activity; immunosuppressant or immunostimulant activity (e.g. for treating immune deficiency, including infection with human immune deficiency virus; regulation of lymphocyte growth; treating cancer and many autoimmune diseases, and to prevent transplant rejection or induce tumour immunity); regulation of haematopoiesis, e.g. treatment of myeloid or lymphoid diseases; promoting growth of bone, cartilage, tendon, ligament and nerve tissue, e.g. for healing wounds, treatment of bums, ulcers and periodontal disease and neurodegeneration; inhibition or activation of follicle-stimulating hormone (modulation of fertility); chemotactic/chemokinetic activity (e.g. for mobilising specific cell types to sites of injury or infection); haemostatic and thrombolytic activity (e.g. for treating haemophilia and stroke); antiirjflarrrmatory activity (for treating e.g. septic shock or Crohn's disease); as antimicrobials; modulators of e.g. metabolism or behaviour; as analgesics; treating specific deficiency disorders; in treatment of e.g. psoriasis, in human or veterinary medicine. In addition, or in the alternative, the composition of the present invention may be useful in the treatment of disorders listed in WO-A-98/09985. For ease of reference, part of that list is now provided: macrophage inliibitory 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 uro logic diseases, otitis or other oto-rhino-laryngological diseases, dermatitis or other demial 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 inflarnn ation caused by infection, proliferative vitreo-retinopathies, acute ischaemic 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 HTN-related encephalopathy, Devic's disease, Sydenham chorea, Alzheimer's disease and other degenerative diseases, conditions or disorders of the CNS, inflammatory components of stokes, post-polio syndrome, immune and inflammatory components of psychiatric disorders, myelitis, encephalitis, subacute sclerosing pan-encephalitis, encephalomyelitis, acute neuropathy, subacute neuropathy, chronic neuropathy, Guillaim- Barre syndrome, Sydenham chora, myasthenia gravis, pseudo-tumour cerebri, Down's Syndrome, Huntington's disease, amyotrophic lateral sclerosis, inflammatory components of CNS compression or CNS trauma or infections of the CNS, inflammatory components of muscular atrophies and dystrophies, and immune and inflammatory related diseases, conditions or disorders of the central and peripheral nervous systems, post-traumatic inflammation, septic shock, infectious diseases, inflammatory complications or side effects of surgery, bone marrow transplantation or other transplantation complications and/or side effects, inflammatory and/or immune complications and side effects of gene therapy, e.g. due to infection with a viral carrier, or inflammation associated with AIDS, to suppress or inhibit a humoral and/or cellular immune response, to treat or ameliorate monocyte or leukocyte prohferative diseases, e.g. leukaemia, by reducing the amount of monocytes or lymphocytes, for the prevention and/or treatment of graft rejection in cases of transplantation of natural or artificial cells, tissue and organs such as cornea, bone marrow, organs, lenses, pacemakers, natural or artificial skin tissue.

In a preferred aspect, the condition or disease is selected from the list consisting of: modulating the release of intracellular calcium from a store controlled by nicotinic acid adenine dinucleotide phosphate; modulating calcium spikes in mammalian cells; treating diseases in one or more of brain, heart, pancreatic cells (e.g. pancreatic acinar and pancreatic beta cells), immune cells, T-cells, haemopoietic cells including phagocytes; treating diseases in one or more of brain, heart, pancreatic cells (e.g. pancreatic acinar and pancreatic beta cells), immune cells, T-cells, haemopoietic cells including phagocytes by modulating the release of intracellular calcium from a store controlled by nicotinic acid adenine dinucleotide phosphate; and treating diseases in one or more of brain, heart, pancreatic cells (e.g. pancreatic acinar and pancreatic beta cells), immune cells, T-cells, haemopoietic cells including phagocytes by modulating calcium spikes in mammalian cells.

In a preferred aspect, the condition or disease is selected from the list consisting of: treating an autoimmune disease (such as thyroiditis, insulitis, multiple sclerosis, invectitis, orchitis, myasthenia gravis, rhematoid arthritis or lupus erythematosis) or graft rejection, or Type II diabetes, or cardiac arrhythmia, or treating or preventing an immune disorder in a human or animal.

GENERAL RECOMBINANT DNA METHODOLOGY TECHNIQUES

The present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular

Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory

Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B. Roe, J. Crabfree, and A.

Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; J. M.

Polak and James O'D. McGee, 1990, In Situ Hybridization: Principles and Practice; Oxford

University Press; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach,

Irl Press; and, D. M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic

Press. Each of these general texts is herein incorporated by reference. The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention.

EXAMPLES

Material and Methods

Thin layer chromatography (TLC) was performed on precoated plates (Merck TLC aluminum sheets silica 60 F254) with eluants as indicated. The compounds were detected using a UV lamp at 254nm. Flash chromatography was carried out using Sorbisil c60 silica gel. H and ¹³C NMR spectra were recorded on either a JEOL GX270 or 400 spectrometer. Unless otherwise stated chemical shifts were measured in parts per million (ppm) relative to residual protonated solvent.

Melting points were determined using a Reichert Jung Thermo Galen Kofler block and are unconected. Mass spectra were recorded at the University of Bath Mass Spectrometry Service using positive and negative fast atom bombardment (FAB) with 3-nitro benzyl alcohol (NBA) as a matrix carrier.

Intracellular calcium measurements

Subcellular fractions : calcium concentrations are measured with fluo-3 (3 μM) at physiological temperatures, using cell homogenates or subcellular fractions in a fluorimeter (Perkin-Elmer LS-50B) at 506 nm excitation and 526 nm emission. Intact cells: Injected or hydrolysed ester derivatives of calcium sensitive dye are imaged in intact cells by laser-scanning confocal microscopy (TCS NT, Leica) Images were processed with the software NIH Image to create a self-ratio by dividing each image (F) by an image acquired before stimulation (Fo). General reaction schemes

Preparation of nicotnic acid derivatives

B - Preparation of alkylating agents

Alkylation

D - Zincke

wherein Rl, R2 and R3 are as defined herein. I Preparation of Nicotinic acid Derivatives

Nicotinic acid (5 g, 40.6 mmol) in methanol (85 mL) and concentrated sulfuric acid (17 mL) was refluxed for 14 hours. Water (50 mL) was then added and the aqueous solution was then neutralised with saturated NaHCO₃ (~300 mL), extracted with chloroform (3 x 50 mL), dried (Na₂SO₄), filtered and evaporated under reduced pressure to afford a pale yellow liquid which crystallise on standing as a white solid (4.8 g, 88%) m.p. 34-36°C which showed 5H (400 MHz, CDC1₃) 9.23 (IH, d, J 1.6, H2), 9.78 (IH, dd, J4.9 and 1.6, H4), 8.29 (IH, m, H6), 7.40 (IH, m, H5) and 3.96 (3H, s, CH₃).

5-Bromo-nicotinic acid methyl ester (Example 1)

5-Bromo-nicotinic acid (300mg, 1.5m ol) was added methanol (6ml) and concentrated H₂S0₄ (1.1ml). This reaction mixture was then heated at reflux for overnight. After cooling down, about 5ml of concentrated NaHCO₃ solution was added to adjust the PH to 7-8. The product was then extracted with DCM (20ml). Removal of the solvent gave the product as white solid in 81% yield. TR: 3450, 3047, 1722, 1577, 1419, 1311, 1107, 1016, 955, 764, 688; 1H NMR (270 MHz, CDC1₃): 9.12 (s, IH, H-2), 8.85 (m, IH, H-6), 8.44 (m, IH, H-4), 3.99 (s, 3H, CH₃); ¹³C NMR (100.4 MHz, CDC1₃): 164.4 (CO), 154.4 (C2), 148.7 (C6), 139.6 (C4), 127.4 (C3), 120.7 (C5), 52.9 (CH₃); MS: m/z (FAB⁺) 216.2 (M⁺).

Nicotinic acid l-(2-nitro-phenyl)-ether ester

To a solution of l-(2-nitrophenyl)ethanol (618 mg, 3.7 mmol) in dry dichloromethane (18 mL) was added nicotinic acid (500 mg, 4.1 mmol), dicyclohexylcarbodiimide (838 mg, 4.1 mmol) and DMAP (45 mg, 0.37 mmol). The reaction was stured at room temperature overnight after which the urea was filtered. The filtrate was diluted in chloroform (20 mL), extracted with saturated NaHCO₃ (15 mL) and brine (15 mL). The combined extracts were dried Na₂S0₄), filtered and evaporated under reduced pressure. The residue was purified on silica gel (hexane : EtOAc - 7 : 3) to afford the desired ester as a pale yellow oil (910 mg, 81%) which showed δ_H (270 MHz, CDC1₃) 9.16 (IH, d, J 1.5, H2), 8.70 (IH, dd, J 4.8 and 1.8, H6), 8.21 (IH, m, H), 7.88 (IH, dd, J 8.4 and 1.1, CH), 7.65 (IH, dd, J 8.1 and 1.8, H4), 7.56 (IH, dt, J 7.5 and 1.1, CH), 7.40-7.30 (2H, m, 2 x CH), 6.51 (IH, q, J 6.4, CH) and 1.71 (3H, t, J 6.4, CH₃). δ_c (65 MHz, CDC1₃) 164.3 (CO), 153.2, 150.7 (both CH), 137.3 (C), 137.0, 133.6, 128.5, 126.9 (all CH), 125.6 (C), 124.4, 123.3 (both CH), 69.2 (CH) and 21.9 (CH₃). 1 C hidden, m/z [FAB⁺] 273.1 (M⁺+H, 100%) [found 273.0871 M⁺+H. C₁₄H₁₃N₂O₄ requires 273.0873].

5-(4-Methoxy-phenyl)-nicotinic acid methyl ester

5-Bromo-nicotinic acid methyl ester (153mg, 0.7mmol) was added toluene (1. -ml) and 2M Na₂C0₃ (0.7ml), To this suspension, Pd(PPh₃)₄ (24mg) was added under N₂. Then a solution of p-methoxyphenylboronic acid (127.6mg) in methanol (0.35ml) was added. This reaction mixture was vigorously stkred and heated to 80 °C for 6h. After cooling, the crude product was partitioned between DCM (7ml) and 2M Na₂C0₃ (3.5ml +0.35ml NH₃). Removal of the organic layer gave light yellow crude product, which was then purified by silica gel column, eluted with DCM. White solid was given in 41% yield.

TR: 3036, 2956, 2844, 1716, 1609, 1518, 1443, 1427, 1309, llll, 1026, 841; ^!H NMR (270 MHz, CDC1₃): 9.13 (d, J=1.9, IH, H-2), 8.95 (d, J=2.4,1H, H-6), 8.43 (m, IH, H-4), 7.55 (d, J-5.4, 2H, ArH-2 and 6), 7.03 (d, J=5.4, 2Η, ArH-3 and 5), 3.97 (s, 3H, CH₃ 3.86 (s, 3H, CHs); ¹³C NMR (100.4 MHz, CDC1₃): 165.6 (CO), 160.1 (ArC4), 151.4 (C6), 148.7 (C2), 136.1 (C4), 134.7 (C5), 129.0 (ArCl), 128.3 (ArC2 and C6), 125.9 (C3), 114.7 (ArC3 and C5), 55.6 (OCH₃), 52.7 (CH₃-CO); MS: m/z (FAB ⁺) 244.3 (M⁺+l).

Nicotinic acid (100 mg, 0.812 mmol) and organic halide (0.812 mmol) were mixed in dry DMF (1.5 mL) and stirred at 50°C in the dark overnight after which time DMF was evaporated under reduced pressure. The resulting product was then recrystallised to afford the conesponding pyridinium salt.

II Preparation of (bromoacetylamide alkylating agents.

Amine (3ml, 24mmol) was added dropwise to a stirring solution of 2-bromoacetylbromide (10 ml, 12mmol) at -10 °C, such that the temperature did not rise above 0 °C. When the reaction was complete, cold water was added to dissolve the precipitated salt. The organic layer was separated and washed successively with aqueous acetic acid (20 ml, IM), aqueous NaOH (20 ml, IM) and finally with saturated brine. After removal of the ether, the crude product was used for alkylation directly without further purification. Analytical data was given by recrystalization or flash chromatography. 2-Bromo-N, N-diethyl-acetamide (2)

Colorless oil was given after working up. 1H NMR (CDC1₃): δ 3.73 (s, 2H, -CH₂CO) 3.25 (q, 4H, J = 7.5 Hz, -CH₂ CH₃), 1.24(t, 3H, J= 7.5 Hz, -CH₃), 1.03(t, 3H, J= 7.5 Hz, -CH₃). Yield 60%. Data is identical to the literature [1].

2-Bromo-N-butyl-acetamide (3)

This product is a solid at room temperature. Mp: 30°C. IHNMR (CDC1₃): δ 6.65 (brs, IH, NH), 3.81 (s, 2H, -CH2CO), 3.19 (m, 2H, N-CH2), 1.41(m, 2H, -CH2 CH2), 1.25 (m, 2H, - CH2 CH3), 0.78 (t, 3H, -CH3). Yield 76%. Data is identical to the literature [1].

2-Bromo-N-phenyl-acetamide (4)

This product is a yellow solid. Mp: 128-131. IR: 3296, 3203, 3146, 3099, 1657, 1618, 1555, 1486, 1445, 1335, llll; ¹HNMR (CDC1₃): 8.13 (brs, IH, NH) 7.52 (d, 2h, J= 2.7 Hz, ArH2, 6), 7.34 (m, 2H, ArH3, 5), 7.17(m, IH, ArH4), 4.00(s, 2H, -CH₂CO). MS: m/z (FAB⁺) 213.9 (M⁺+l).

2-Bromo-N-cyclohexyl-acetamide (5)

This product is a white fine powder. Yield 90%. Mp: 106-108. IR: 3284, 3072, 2937, 2919, 2853, 1645, 1551, 1446, 1328, 1210, 1155, 1089; 1H NMR (CDC1₃): 6.33 (brs, IH, NH), 3.84(s, 2H, -CH₂CO), 3.74(m, IH, N-CH), 1.10-1.92 (m, 10H, ring-H). ¹³C NMR (CDC1₃): 164.7 (CO), 49.6 (CH), 33.4 (2CH₂), 30.2 (CH₂-Br), 26.1 (2CH₂), 25.3 (CH₂). MS: m/z (FAB⁺) 220 (M⁺+1). 2-Bromo-N- (4-ethyl-phenyl)-acetamide (6)

This product is yellow solid. Mp: 131-132. 1H NMR (CDC1₃): 8.07 (br, IH, NH), 7.40 (d, 2H, J= 8.4 Hz, ArH), 7.15 (d, 2H, J= 8.4 Hz, ArH), 4.00 (s, 2H, -CH₂CO), 2.59 (q, 2H, J= 7.67 Hz, -CH₂ CH₃), 1.18 (t, 3H, J= 7.67 Hz, -CH₃). MS: m/z (FAB⁺) 242.0 (M⁺+l).

2-Bromo-N-(2-Bromophenyl)-acetamide (7)

Crude product was subjected to silica gel column, eluted with DCM: Hexane 10: 1. Yellow solid was given in 52% yield.

Mp: 96-98. LR: 3184, 3107, 1653, 1609, 1591, 1542, 1472, 1422, 1315, 700; 1H IMMR (CDC1₃): 8.15 (s, br, IH, NH), 7.77 (m, IH, H-3), 7.43 (d, J= 7.83, IH, H-5), 7.23 (m, 2H, H- 2, 3), 4.00 (s, 2H, CH₂); ¹³C NMR (CDC1₃): 164.3 (C=O), 138.1 (C-l), 130.3 (C-3), 128.2 (C- 5), 123.0 (C-4), 122.6 (C-2), 118.5 (C-6); MS (FAB⁺): m z 293 (M⁺+l).

2-Bromo-N,N-dibutylacetamide (8)

Mp: oil. IR: 2959, 2932, 2872, 1648, 1460, 1376, 1209, 1098; 1H NMR (CDC1₃): 3.77 (s, 2H, CO-CH₂), 3.23 (m, 4H, 2NCH₂), 1.48 (m, 4H, 2CH₂), 1.25 (m, 4H, 2CH₂), 0.86 (m, 6H, 2CH₃); ¹³C NMR (CDC1₃): 166.3 (CO), 48.8 (CH₂), 46.2 (CH₂), 31.5 (CH₂-Br), 29.7 (CH₂), 27.0 (CH₂), 20.5 (2CH₂), 14.27 (CH₃), 14.19 (CH₃); MS m/z (FAB⁺) 250 (M⁺+l), 170 (M⁺- Br), 128 (M⁺-BrCOCH₂).

2- Bromo-N, N-dicyclohexylacetamide (9)

The crude product was purified by Flash chromatography (DCM: hexane 12:1). White solid was obtained in 40% yield. Analytical data was given by crystallization from DCM. Mp: 122-124, IR: 3466, 2933, 2869, 2857, 1637, 1467, 1371, 1113, 1000; 1H NMR (CDC1₃): 3.80 (s, 2H, CO-CH₂), 2.41 (m, 2H, 2CH), 1.17-1.86 (m, 20H, ring-H); ¹³C NMR (CDC1₃): 162.6 (CO), 56.3 (N-C), 31.0 (CH₂), 29.4 (CH₂), 29.1(Br-CH₂), 26.5-25.2 (ring-C). MS: m/z (FAB ; 302.0 (M*).

2- Bromo-N-decylacetamide (10)

Colourless oil was given after purified by Flash chromatography (DCM: hexane 10: 1). Yield 81%.

Mp: oil, IR: 3282, 2920, 2852, 1640, 1560, 1471, 1436, 1214; 1H NMR (CDC1₃): 6.49 (s, brs, IH, NH), 3.86 (s, 2H, CH₂-Br), 3.25 (m, 2H, CH₂), 2.15 (s, acetone peak), 1.52 (m, 2H, CH₂), 1.24 (m, 14H, 7CH₂), 0.86 (m, 3H, CH₃); ¹³C NMR (CDC1₃): 165.4 (CO), 40.7 (CH₂-Br), 32.3 (CH₂-N), 29.7-29.9 (6CH₂), 27.2 (CH₂), 23.1 (CH₂), 14.6 (CH₃);

2- Bromo-N-methyl-N-phenylactamide (11)

Yellow solid was given after working up. Yield 80%. Mp: 43-45. IR: 1646, 1595, 1498, 1385, 1298, 1120, 1043, 882, 771; 1H NMR (CDC1₃): 7.28 (m, 5H, Ar-H), 3.87 (s, 2H, CH₂), 3.29 (s, 3H, CH₃). MS: m/z (FAB⁺) 228 (M⁺+l).

2- Bromo-N-(3-methoxybenzyl)acetamide (12)

The crude product was subjected to silica gel column, eluted with DCM: hexane 10:1. Yellow solid was given in 72% yield.

Mp: 79-80. IR: 3289, 3048, 1641, 1543, 1489, 1441, 1296, 1200, 1158, 1049, 999, 890, 784, 697; 1H NMR (CDC1₃): 7.24-6.83 (m, 4H, Ar-H), 4.40 (s, 2H,Br-CH₂), 3.86 (s, 2H, CO-CH₂), 3.77 (s, 3H, CH₃). ¹³C NMR (CDC1₃): 165.8 (CO), 159.9 (C-O), 139.1 (C), 130.0 (C5), 120.O (C6), 113.6 (C2), 113.3 (C4), 55.6 (OCH₃), 44.4 (COCH₂), 29.4 (Br-CH₂); MS m/z (FAB^"'^") 257.9 OV +1), 178.0 (M⁺-Br). 2- Bromo-N, N-dipropylacetamide (13)

Yellow oil was given after working up in 65% yield.

TR: 3451, 2966, 2876, 1645, 1456, 1381, 1302, 1207, 1098, 896,744; 1H NMR (CDC1₃): 3.81 (s, 2H, CH₂-Br), 3.24 (m, 4H, 2CH₂), 1.55 (m, 4H, 2CH₂), 0.86 (m, 6H, 2CH₃); ¹³C NMR (CDC1₃): 166.5 (CO), 50.3 (CH₂), 47.7 (CH₂), 26.2 (CH₂-Br), 22.2 (CH₂), 20.4 (CH₂), 11.2 (2CH₃). MS m/z (FAB ⁺) 222 (M⁺+1).

2- Bromo-N, N-dioctylacetamide (14)

Crude product was purified by Flash chromatography (DCM: hexane 9:1). Yellow oil was given in 81% yield.

IR: 2925, 2855, 1649, 1460, 1376, and 1101. 1H NMR (CDC1₃): 3.80 (s, 2H, CH₂-Br), 3.26

(m, 4H, 2CH₂), 1.54 (m, 4H, 2CH₂), 1.25 (m, 20H, CH₂), 0.84 (m, 6H, 2CH₃); ¹³C NMR

(CDC1₃): 166.3 (CO), 49.1 (CH₂), 46.5 (CH₂), 27.6 (CH₂-Br), 32.2-23.0 (CH₂), 14.6 (2CH₃).

Ms m/z (FAB ; 362 (M⁺+l).

[Cl N-Alkylation of nicotinic acid methyl ester (Example 2)

Nicotinic acid methyl ester (500 mg, 3.65 mmol), the halide (3.65 mmol) and sodium iodide (3.65 mmol) were stirred in DMF or acetonitrile in the dark at 50°C for 24 hours. The solvent was then evaporated under reduced pressure and crystallised from methanol/ether.

l-[(2-Bromo-phenylcarbamoyl)-methyl]-3-methoxycarbonyl-pyridinium (25)

MP: 171-172, IR: 3225, 3173, 3057, 3015, 1743, 1687, 1596, 1549, 1307, 780, 740; 1H NMR (DMSO): 10.97 (s, brs, IH, NH), 9.68 (s, IH, H-2), 9.26 (d, J= 6.2, IH, H-6), 9.11 (d, J= 8.1,

IH, H-4), 8.38 (dd, J= 6.2 and 8.1, IH, H-5), 7.92 (s, IH, H-3'), 7.32-7.50 (m, 3H, AH), 5.78 (s, 2H, CH₂), 3.99 (s, 3H, CH₃); ¹³C NMR (DMSO): 164.1 (COOMe), 162.6 (CO-N), 150.4 (C-2), 148.1 (C-6), 146.6 (C-4), 131.7 (C-N), 129.8 (C-3'), 128.5 (C-5'), 127.3 (C-2'), 122.3 (C-5), 122.2 (C-6'), 63.2 (CH₂-CO), 54.4 (CH₃); MS: m/z (FAB^"1") 351.1 (M⁺+l).

DiethyIcarbamoylmethyl-3-methoxycarbonyl-pyridinium; iodide (Example 3)

Nicotinic acid methyl ester and 2-chloro-N,N'-diethylacetamide were reacted in DMF under the standard protocol to afford a bright yellow solid (363 mg, 26%) m.p. 163-165°C which showed δ_H (270 MHz, D₂0) 9.30 (IH, s, H2), 9.07 (IH, d, J 8.2, H6), 8.90 (IH, d, J 6.2, H4), 8.19 (IH, m, H5), 5.75 (2H, s, py-CH₂), 3.96 (3H, s, OCH₃), 3.41 (2H, q, J 7.2, CH₂), 3.84 (2H, q, J 7.2, CH₂), 1.23 (3H, t, J 7.2, CH₃) and 1.05 (3H, t, J 7.2, CH₃). δ_c (65 MHz, D₂O) 164.3 (C=O), 149.2, 147.6, 146.8 (all CH), 130.6 (C), 128.3 (CH), 62.1 (py-CH₂), 54.1 (OCH₃), 42.3, 41.9 (both CH₂), 13.2 and 12.1 (both CH₃). m/z [FAB⁺] 251.1 (M⁺, 100%) [found 251.1405 M+. C₁₃H₁₉Ν₂O₃I requires 251.1395]. IR (KBr) 1731 (CO ester) and 1653 (CO amide).

l-(2-Carbamoyl-ethyl)-3-methoxycarbonyl-pyridinium; iodide (Example 4)

Nicotinic acid methyl ester and 3-chloropropionamide were reacted in acetonitrile under the standard protocol to afford a pale yellow solid (266 mg, 22%) m.p. 98-100°C which showed δ_H (270 MHz, D₂O) 9.48 (IH, s, H2), 9.08 (IH, d, J 6.2, H6), 9.03-8.99 (IH, m, H4), 8.21- 8.16 (IH, m, H5), 4.94 (2H, t, J6.4, py-CH₂), 4.01 (3H, s, OCH₃) and 3.09 (2H, t, J6.4, CH₂- CO). δ_c (65 MHz, D₂O) 173.7, 168.5 (both CO), 148.1, 146.2 (both CH), 131.0 (C), 130.9, 128.6 (both CH), 58.2 (py-CH₂), 54.3 (OCH₃) and 35.4 (CH₂). m/z [FAB⁺] 209.1 (M⁺, 100%) [found 209.0935 M+. C₁₀H₁₃N₂O₃I requires 209.0926]. IR (KBr) 1736 (CO ester) and 1674 (CO amide).

l-(3-Cyano-propyl)-3-methoxycarbonyl-pyridinium; iodide (Example 5)

Nicotinic acid methyl ester and 4-chlorobutyronitrile were reacted in DMF under the standard protocol to afford a pale yellow solid (219 mg, 18%) m.p. 167-170°C which showed δπ (270 MHz, D₂O) 9.48 (IH, s, H2), 9.08 (IH, d, J 6.4, H6), 9.01 (IH, dt, J 8.2 and 1.5, H4), 8.20- 8.17 (IH, m, H5), 4.79 (2H, t, J7.4, py-CH₂), 3.98 (3H, s, OCH₃), 2.64 (2H, t, J7.4, CH₂-CN) and 2.40 (2H, q, J 7.4, CH₂). 163.4 (CO), 147.9, 146.4, 146.2 (all CH), 131.3 (C), 129.1 (CH), 60.9 (py-CH₂), 54.2 (OCH₃), 26.3 and 14.1 (both CH₂). m/z [FAB⁺] 205.0 (M⁺, 100%) [found 205.0977 M+. C_nH₁₄N₂O₂I requires 205.0972].

l-(2-Diethylamino-ethyl)-3-methoxycarbonyI-pyridinium; iodide (Example 6)

Nicotinic acid methyl ester and diethylaminoethylchloride hydrochloride were reacted in DMF under the standard protocol to afford a pale yellow solid (60 mg, 5%) m.p. 172-174°C which showed δ_H (270 MHz, D₂0) 9.53 (IH, s, H2), 9.13 (IH, d, J6.2, H6), 9.05 (IH, d, J 8.2, H4), 8.25-8.25 (IH, m, H5), 5.12 (2H, t, J 7.7, py-CH₂), 3.95 (3H, s, OCH₃), 3.82 (2H, t, J 7.7, CH₂-N), 3.30 (4H, q, J 7.4, 2 x CH₂-Me) and 1.25 (6H, t, J 7.4, 2 x CH₃). m/z [FAB⁺] 237.2 (M⁺, 100%) [found 237.1601 M+. C₁₃H₂₁N₂0₂I requires 237.1603].

l-(2-Cyano-ethyI)-3-methoxycarbonyl-pyridinium; iodide (Example 7)

Nicotinic acid methyl ester and 3-chloropropionitrile were reacted in DMF under the standard protocol to afford a pale yellow solid (88 mg, 8%) which showed δ_H (270 MHz, D₂0) 9.54 (IH, s, H2), 9.11 (IH, d, J6.2, H6), 9.05 (IH, d, J 8.2, H4), 8.25-8.20 (IH, m, H5), 4.99 (2H, t, J6.4, py-CH₂), 3.97 (3H, s, OCH₃) and 3.29 (2H, t, J6.4, CH₂-CN). m/z [FAB⁺] 191.0 (M⁺, 100%) [found 191.0820 M+. CιoHπN₂0₂I requires 191.0821].

l-(l-Carbamoyl-ethyl)-3-methoxycarbonyl-pyridinium; iodide (Example 8)

Nicotinic acid methyl ester and 2-chloro propionamide were reacted in DMF under the standard protocol to afford a yellow solid (363 mg, 26%) which showed δ_H (270 MHz, D₂O) 9.34 (IH, s, H2), 9.03-8.96 (2H, m, H6 and H4), 8.16-8.11 (IH, m, H5), 5.66 (IH, q, J7.2, py- CH), 3.91 (3H, s, OCH₃) and 1.88 (3H, d, J7.2, CH₃). δ_c (65 MHz, D₂O) 163.1 (CO), 147.5, 147.0, 145.8 (all CH), 130.7 (C), 128.5 (CH), 69.2 (py-CH), 54.2 (OCH₃) and 18.2 (CH₃). m/z [FAB⁺] 209.0 (M⁺, 100%) [found 209.0929 M+. Cι₀H₁₃N₂O₃I requires 209.0927]. l-Cyclohexyl-3-methoxycarbonyl-5- (4-methoxy-phenyl)-pyridinium

5-(4-Methoxy-phenyl)-nicotinic acid methyl ester (40) (50mg, 0.2mmol) was added 2-bromo- N-cyclohexylacetamide (115mg, 0.5mmol). To this mixture was added DMF (1ml) under N₂ and then heated at 60 °C overnight. After removal of the solvent, the residual was dissolved in methanol (0.5ml) and then ether (20ml) was added to crystallize the product. Yellow oil was given (40mg).

IR: 3436, 2929, 1738, 1652, 1557, 1450, 1346, 1260; 1H NMR (270 MHz, CDC1₃): 9.66 (s, IH, H-2), 9.46 (m, IH, H-6), 9.15 (m, IH, H-4), 8.62 (m, IH, NH), 7.93 (m, 2H, ArH-2 and 6), 7.16 (m, 2H, ArH-3 and 5), 5.58 (s, 2H, CH₂-CO), 4.01 (s, 3Η, CH₃), 3.85 (s, 3H, CH₃- CO), 1.79-1.22 (m, 11H, ring-H)

Hvdrolvsis of N-alkylated nicotinic acid methyl ester (Example 9)

The conesponding nicotinic acid methyl ester (0.4 mmol) was dissolved in 48% aqueous HBr (0.2 mL) and stined at 60°C overnight after which it was then evaporated under reduced pressure. To the brown residue was added acetonitrile. The precipitate was filtered and washed with acetonitrile leaving the desired N-alkylated nicotinic acid.

3-Carboxy-l-diethylcarbamoylmethyl-pyridinium; iodide (Example 10)

Diethylcarbamoylmethyl-3-methoxycarbonyl-pyridinium; iodide was reacted under the general protocol to afford the desired product as a white solid (104 mg, 74%) m.p. 97-100 °C which showed δ_H (270 MHz, D₂0) 9.19 (IH, s, H2), 8.99 (IH, d, J8.2, H6), 8.82 (IH, d, J6.2, H4), 8.13 (IH, m, H5), 5.71 (2H, s, py-CH₂), 3.37 (2H, q, J 7.2, CH₂), 3.32 (2H, q, J 7.2, CH₂), 1.20 (3H, t, J 7.2, CH₃) and 1.02 (3H, t, J 7.2, CH₃). δ_c (65 MHz, D₂0) 164.4 (CO), 148.9, 147.7, 147.1 (all CH), 131.7 (C), 128.3 (CH), 62.1 (py-CH₂), 42.3, 41.9 (both CH₂), 13.1 and 12.1 (both CH₃). m/z [FAB⁺] 237.1 (M^1", 100%) [found 237.1250 M+. C₁₂Hj₇Ν₂0₃I requires 237.1239]. IR (KBr) 3150 (OH) and 1652 (CO amide). l-(2-Carbamoyl-ethyl)-3-carboxy-pyridinium; iodide (Example 11)

l-(2-Carbamoyl-ethyl)-3-methoxycarbonyl-pyridinium; iodide was reacted under the general protocol to afford the desired product as a white solid (120 mg, 84%) m.p. 170-173°C which showed δ_H (270 MHz, D₂0) 9.38 (IH, s, H2), 9.02 (IH, d, J 6.2, H6), 8.91 (IH, d, J7.9, H4), 8.12-8.06 (IH, m, H5), 4.87 (2H, t, J 6.2, py-CH ) and 3.13 (2H, t, J 6.2, CH₂-CO). δ_c (65 MHz, D₂O) 173.7, 164.8 (both CO), 147.8, 146.6, 146.4 (all CH), 132.4 (C), 128.5 (CH), 57.5 (py-CH₂) and 34.5 (CH₂). m/z [FAB⁺] 196.1 (M^1", 100%) [found 196.0611 M+. C₉H₁₁N₂O₃I requires 196.0609]. IR (KBr) 3154 (OH) and 1648 (CO amide).

3-Carboxy-l-(2-diethyIamino-ethyl)-pyridinium; iodide (Example 12)

l-(2-Diethylamino-ethyl)-3-methoxycarbonyl-pyridinium; iodide was reacted under the general protocol to afford the desired product as a white solid (41 mg, 84%) m.p. 210-212°C which showed δ_H (270 MHz, D₂O) 9.60 (IH, s, H2), 9.06 (IH, d, J6.2, H6), 8.97 (IH, d, J8.2, H4), 8.20-8.15 (IH, m, H5), 5.11 (2H, t, J7.7, py-CH₂), 3.83 (2H, t, J 7.7, CH₂-N), 3.33 (4H, q, J7.4, 2 x CH₂-Me) and 1.26 (6H, t, J7.4, 2 x CH₃). δ_c (65 MHz, D₂O) 164.8 (CO), 147.3, 146.4, 146.4 (all CH), 132.4 (C), 129.2 (CH), 55.6 (py-CH₂), 50.3, 48.2 (both CH₂) and 34.5 (CH₃).

[Dl Alkylation of nicotinic acid (Example 13)

Nicot ic acid (100 mg, 0.812 mmol) and organic halide (0.812 mmol) were mixed in dry DMF (1.5 mL) and stined at 50°C in the dark overnight after which time DMF was evaporated under reduced pressure. The resulting product was then recrystallised to afford the conesponding pyridinium salt.

3-Carboxy-l-diethylcarbamoylmethyl-pyridinium (15)

Yellow oil was obtained after crystallization from methanol/ether. Yield 21%. IR (KBr) J 3429, 3072, 2981, 2934, 1729, 1659, 1219, 1135, 1036, 811, 748 cm^'1 1H NMR (D₂O): 9.06 (d, IH, J = 7.5 Hz, H2), 8.87 (m, IH, H6), 8.74 (m, IH, H4), 8.03 (m, IH, H5), 5.68 (s, 2H, -CH₂CO), 3.33 (q, 4H, J =7.5 Hz, -CH₂ CH₃), 1.19 (t, 3H, J= 7.5Hz, -CH₃), 1.00 (t, 3H, J= 7.5Hz, -CH₃). MS: m/z (FAB⁺) 237 (M⁺+l).

l-Butylcarbamoylmethyl-3-carboxy-pyridinium (16)

Mp: 190-194. TR (KBr) * 3235, 3078, 2958, 2931, 2873, 2750, 2494, 1727, 1672, 1558, 1394, 1351, 1131, 848 cm^"1 _; 1H NMR (D₂0): 9.25 (s, IH, H2), 8.96 (d, IH, J=5.3 Hz, H6), 8.85 (d, IH, J= 5.9 Hz, H4), 8.13 (m, IH, H5), 5.43 (s, 2H, -CH₂CO), 3.16 (m, 2H, N-CH₂), 1.40 (m, 2H, -CH₂ CH₂), 1.20 (m, 2H, -CH₂ CH₃), 0.77 (t, 3H, -CH₃). ¹³C NMR (D₂0): 167.9 (CO-O), 165.2 (CO-N), 148.1 (C3), 147.0 (C6), 146.7 (C2 and C4), 128.1 (C5), 62.2 (CH₂- TST¹ , 39.9 (CH₂), 30.5 (CH₂), 19.7 (CH₂), 13.2 (CH₃); MS: m/z (FAB⁺) 237.0 (M⁺+l).

3-Carboxyl-l-phenylcarbamoylmethyl-pyridinium (17)

Mp: 148-149. IR (KBr) v_max 3393, 3277, 3080, 3019, 1699, 1637, 1599, 1558, 1498, 1446, 1350, 1311, 1258, 737, 690 cm^"1; 1H NMR (D₂O): 9.18 (s, IH, H2), 8.90 (d, IH, J= 6.6 Hz, H6), 8.83 (d, IH, J= 5.4 Hz, H4), 8.07 (m, IH, H5), 7.25 (m, 5H, ArH), 5.59 (s, 2H, -CH₂CO). ¹³C NMR (D₂0): 163.8 (CO-O), 163.5 (CO-N), 148.2 (C6), 147.9 (C2 and C4), 146.2 (C3), 127.8 (C5), 138.9 (ArCl), 129.6 (ArC3 and C5), 119.8 (ArC2 and C6), 124.6 (ArC4); MS: m/z (FAB⁺) 257.1 ^+1).

3-Carboxy-l-cycIohexylcarbamoylmethyl-pyridinium (18)

Mp: 188-192. IR (KBr) J 3244, 3073, 2931, 1709, 1679, 1530, 1389, 1240, 1210, 1120, 837 cm^"1 1H NMR (D₂0) 9.32.(s, IH, H2), 9.00 (d, IH, J= 5.2 Hz, H6), 8.84 (d, IH, J= 4.5 Hz, H4), 8.23(m, IH, H5), 5.50 (s, 2H, -CH₂CO), 3.65 (m, IH, N-CH), 1.87-1.25 (m, 11H, ring- H). ¹³C NMR (D₂0): 163.6 (CO-O), 163.5 (CO-N), 149.7 (C6), 147.9 (C2), 146.4 (C4), 131.0 (C3), 128.3 (C5), 62.5 (CH₂-CO), 39.7 (CH), 33.1 (2CH₂), 25.9 (CH₂), 25.2 (2CH₂). MS: m/z (FAB⁺) 263.3 (M⁺+l). 3-Carboxy-l-[(4-ethyl-phenylcarbamoyl)-methyl]-pyridinium (19)

Mp: 210-214. IR (KBr)j>_max: 3424, 3243, 3190, 3063, 2962, 2930, 1717, 1697, 1608, 1543, 1392, 1257, 1125, 834, 670 cm^"1. 1H NMR (D₂O): 9.29.(s, 3.52, H2), 8.98 (d, IH, J= 8.7, H6), 8.91 (d, IH, J= 6.5, H4), 8.14 (m, IH, H5), 7.28 (d, 2H, J= 8.1 Hz, ArH2, 6), 7.18 (d, 2H, J= 8.1 Hz, ArH3, 5), 5.60 (s, 2H, -CH₂CO), 2.49 (q, 2H, J= 7.67 Hz, -CH₂ CH₃), 1.06 (t, 3H, J= 7.67 Hz, -CH₃). ¹³C NMR (D₂O): 163.6 (CO-O), 163.4 (CO-N), 149.9 (C6), 148.3 (C2), 146.7 (C4), 140.0 (C3), 136.5 (ArCl), 131.1 (ArC4), 128.4 (C5), 128.8 (ArC3 and C5), 119.8 (ArC2 and C6), 63.1 (CH₂-CO), 28.5 (CH₂), 16.5 (CH₃). MS: m/z (FAB⁺) 285.1 (M⁺+l).

3-Carboxy-l-[(2-bromo-phenylcarbamoyl)-methyl]-pyridinium (20)

Mp: 216-218. TR: 3177, 3055, 2743, 1727, 1687, 1608, 1588, 1477, 1253, 1130, 850; h Η NMR (D₂0): 9.36 (s, IH, H-2), 9.04 (d, J= 8.1, IH, H-6), 8.99 (d, J=5.6, IH, H-4), 8.23 (m, IH, H- 5), 7.68 (s, IH, H-3'), 7.31 (m, 3H, H-4', 5', 6'), 5.70 (s, 2H, CH₂-CO), ¹³C NMR (DMSO) 163.5 (CO-OH), 163.0 (CO-NH), 147.5 (C-2), 145 (C-6), 139.3 (C-3), 131.1 (C-4), 130.0 (ArCl), 127.8 (C-5), 126.6 (ArC3), 121.6 (ArC2), 120.9 (ArC4), 116.0 (ArC6); MS m z (FAB⁺) 335.1 (M⁺+l).

3-Carboxy-l-dibutylcarbamoylmethyl-pyridinium (21)

MP: Not crystal, IR: 2924, 2854, 1720, 1459, 1377, 1215; 1H NMR (D₂0) 9.26 (s, IH, H-2), 9.10 (m, IH, H-6), 8.90 (m, IH, H-4), 8.24 (m, IH, H-5), 5.82 (s, 2H, COCH₂), 3.44 (m, 4H, 2CH₂), 1.72 (m, 2H, CH₂), 1.54 (m, 2H, CH₂), 1.40 (m, 2H, CH₂), 1.30 (m, 2H, CH₂), 0.92 (m, 6H, 2CH₃); ¹³C NMR (D₂0): 164.3 (CO-O), 163.6 (CO-N), 149.9 (C-6), 148.2 (C-2), 146.5 (C-4), 131.0 (C-3), 127.8 (C-5), 62.0 (CH₂-CO), 47.3 (CH), 46.6 (CH), 30.9 (CH₂), 30.1 (CH₂), 20.5 (CH₂), 20.4 (CH₂), 14.7 (CH₃), 14.6 (CH₃); MS: m/z (FAB ⁺) 293 (M⁺+l). 3-Carboxy-l - [(3-methoxy-b enzylcarb amoyl)-methyl] -pyridinium (22)

Mp: 194-196. IR (KBr): 3217, 1722, 1675, 1588, 1556, 1397, 1259, 1129, 713.3; 1H NMR (D₂0): 9.33 (s, IH, H-2), 9.07 (d, J=7.8, IH, H-6), 8.96 (d, J=6.2, IH, H-4), 8.24 (m, IH, H- 5), 7.37 (m, IH, ArH-2), 6.98 (m, 3H, ArH-4, 5, 6), 5.62 (s, 2H, CH₂-CO), 4.47 (s, 2H, CH₂- N), 3.84 (s, 3H, CH₃); ¹³C NMR (D₂0): 164.2 (CO-O), 163.1 (CO-N), 158.2 (ArC3), 148.9 (C6), 147.4 (C2), 145.9 (C4), 139.0 (C3), 130.4 (ArCl), 129.4 (ArC5), 127.7 (C5), 119.5 (ArC6), 113.2 (ArC4), 112.3 (ArC2); MS: m/z (FAB⁺) 301.0 (M⁺+l).

3-Carboxy-l-dipropylcarbamoylmethyl-pyridinium (23)

Yellow oil was given after crystallization from methanol and ether.

Mp: Oil. IR: 3392, 2964, 2875, 1729, 1649, 1458, 1430, 1385, 1243, 1207; 1H NMR (D₂0): 9.23 (s, IH, H-6), 9.04 (d, J=8.1, IH, H-2), 8.87 (d, J=5.1, IH, H-4), 8.20 (m, IH, H-5), 5.80 (s, 2H, CH₂-CO), 3.34 (m, 4H, 2CH₂), 1.71 (m, 2H, CH₂), 1.54 (m, 2H, CH₂), 0.92 (t, J=7.4, CH₃), 0.82 (t, J=7.4, CH₃). ¹³C NMR (D₂0): 164.7 (CO-O), 164.6 (CO-N), 148.6 (C6), 147.4 (C2), 146.8 (C4), 132.1 (C3), 128.1 (C5), 62.1 (CH₂-CO), 49.4 (CH₂), 48.9 (CH₂), 21.4 (CH₂), 20.4 (CH₂), 10.9 (CH₃), 10.7 (CH₃); MS: m/z (FAB⁺) 266.3 (M⁺+l).

3-Carb oxy-1 -decylcarb amoylmethyl-pyridinium (24)

Mp: 185-186. IR: 3229, 3079, 2924, 1726, 1679, 1554, 1394, 1225, 1130, 846, 723; 1H NMR (DMSO): 9.53 (s, IH, H-2), 9.19 (d, J= 5.4, IH, H-6), 9.03 (d, J=8.1, IH, H-4), 8.70 (s, brs, IH, NH), 8.31 (m, IH, H-5), 5.57 (s, 2H, CH₂-CO), 3.13 (m, 2H, CH₂-N), 1.24-1.61 (m, 16H, 8CH₂), 0.83 (m, 3H, CH₃); ¹³C NMR (DMSO): 164.5 (CO-O), 163.6 (CO-N), 149.7 (C-6), 147.9 (C-2), 146.4 (C-4), 131.1 (C-3), 128.8 (C-5), 62.4 (CH₂-CO), 32.2 (CH₂-N), 29.9 (CH₂), 29.8 (CH₂), 29.7 (CH₂), 29.6 (2CH₂), 27.2 (CH₂), 23.0 (CH₂), 14.9 (CH₃); MS m/z l-Allyl-3-carboxy-pyridinium bromide (Example 14)

Nicotinic acid (100 mg, 0.812 mmol) and allyl bromide (70 μL, 0.812 mmol) were reacted under the general protocol to afford a yellow solid which was recrystallised from methanol to afford the desired quarternised product as a white solid (165 mg, 83%) which showed δπ (400 MHz, D₂0) 9.20 (IH, s, H2), 8.81 (2H, d, J 5.8, 2 x CH), 8.0 (IH, d, J 8.2, H5), 5.91-6.01 (IH, m, :CH), 5.37 (2H, app t, J 10.5, :CH₂) and 5.11 (2H, d, J 6.2, CH₂-N). δ_c (100 MHz, D₂0) 161.0 (CO), 142.7, 141.9, 141.6 (all CH), 128.8 (C-C0₂H), 125.5, 124.2 (both CH), 119.4 (:CH₂) and 59.7 (CH₂). m/z [ES] 164.0 (M⁺, 55%).

l-Propyl-3-carboxy-pyridinium iodide (Example 15)

Nicotinic acid (100 mg, 0.812 mmol) and 2-iodopropane (80 μL, 0.812 mmol) were reacted under the general protocol to afford a yellow solid which was recrystallised from methanol to afford the desired quarternised product as a white solid (181mg, 76%) m.p. 162-165°C which showed δ_H (400 MHz, D₂0) 9.30 (IH, s, H2), 8.91-8.96 (2H, m, 2 x CH), 8.10-8.17 (IH, m, H5), 4.61 (2H, t, J 7.4, CH₂-N), 2.03 (2H, sextet, J7.4, CH₂) and 0.92 (3H, t, J 7.4, CH₃). δ_c (100 MHz, D₂0) 165.1 (CO), 146.2, 144.8, 142.9 (all CH), 133.1 (C-CO₂H), 127.7 (CH), 63.0 (CH₂-N), 23.5 (CH₂) and 8.9 (CH₃). v_m cm ^l 3396 (CO₂H) and 1636 (CO), m/z [ES] 167 (M\ 80%). l-Benzyl-3-carboxy-pyridmium bromide (Example 16)

Nicotinic acid (100 mg, 0.812 mmol) and benzyl bromide (0.1 mL, 0.812 mmol) were reacted under the general protocol to afford a yellow solid which was recrystallised from water to afford the desired quarternised product as a white solid (219 mg, 92%) m.p. 203-205°C which showed δ_H (400 MHz, D₂0) 9.30 (IH, s, H2), 8.80 (IH, d, J5.9, H4), 8.75 (IH, d, J 8.2, H6), 8.01 (IH, m, H5), 7.43 (5H, m, ArH) and 5.82 (2H, s, CH₂). δ_c (100 MHz, D₂0) 166.1 (CO), 146.3, 145.9, 145.5 (all CH), 132.6 (C-C0₂H), 130.2, 129.8, 129.3, 128.5 (all CH) and 65.0 (CH₂). V_max/cm^"1 3416 (C0₂H) and 1668 (CO), m/z [FAB⁺] 214.2 (M⁺+H, 100%).

l-Carbamoylmethyl-3-carboxy-pyridinium iodide (Example 17)

Nicotinic acid (100 mg, 0.812 mmol) and 2-iodoacetamide (150 mg, 0.812 mmol) were reacted under the general protocol to afford a yellow oil which was recrystallised from dichloromethane to afford the desired quarternised product as a yellow solid (175 mg, 70%>) m.p 223-225°C which showed δ_H (400 MHz, D₂0) 9.24 (IH, s, H2), 9.0 (IH, d, J 8.2, H6), 8.89 (IH, d, J 4.8, H4), 8.14-8.20 (IH, m, H5) and 5.56 (2H, s, CH₂). δ_c (100 MHz, D₂0) 169.5, 167.3 (both OO), 149.3, 148.6, 148.1 (all CH), 135.6 (C-C0₂H), 129.5 (CH) and 63.2 (CH₂). v^_ax/cm^"1 3379 (C0₂H), 1701 (CO-NH₂) and 1665 (CO), m/z [FAB⁺] 181.2 (M⁺+H, 100%).

3-Carbamoyl-l-carbamoylmethyl-pyridinium; iodide (Example 18)

Nicotinamide (500 mg, 4.06 mmol) and iodoacetamide (764 mg, 4.06 mmol) were stined in DMF at 50°C overnight. The solvent was then evaporated and the residue crystallised from methanol/ether to afford a yellow solid which showed δ_H (270 MHz, D₂O) 9.24 (IH, s, H2),

8.95-8.93 (2H, m, H4 and H6), 8.23-8.17 (IH, m, H5) and 5.56 (2H, s, py-CH₂). δ_c (65 MHz, D₂0) 167.3 (CO), 148.2, 146.1, 145.2 (all CH), 133.9 (C), 128.3 (CH) and 62.0 (CH₂). m/z [FAB⁺] 180.0 (M⁺, 100%) [found 180.0780 M+. C₆H₁₀N₂O₃I requires 180.0781].

5-Bromo-l-carbamoylmethyl-3-carboxy-pyridinium iodide (Example 19)

5-Bromonicotinic acid (50 mg, 0.247 mmol) and iodoacetamide (45 mg, 0.247 mmol) were stined in DMF (ImL) in the dark at 50°C overnight. The solvent was evaporated under reduced pressure and the residue was crystallised from MeOH7Et₂θ to afford the desired product as a brown gum (48 mg, 51%) which showed δ_H (270 MHz, D₂0) 9.66 (IH, s, H2), 9.46 (IH, m, H6), 9.15 (IH, m, H4) and 5.58 (2H, s, CH₂). m/z [FAB⁺] 260.0 (M⁺, 60%) [found 260.0634 M⁺. C₈H₈ ⁷⁹BrN₂O₃ requires 260.0648].

l-Carbamoylmethyl-3-[l-(2-nitrophenyl)-ethoxycarbonyl]-pyridinium iodide (Example 20)

Nicotinic acid l-(2-nitro-phenyl)-ether ester (300 mg, 1.1 mmol) and iodoacetamide (204 mg, 1.1 mmol) were stined in the dark at 50°C overnight. The solvent was then evaporated under reduced pressure ans the residue recrystallised from MeOH/ether to afford the desired product as a yellow solid which showed δ_H (270 MHz, D₂O) 9.46 (IH, s, H2), 9.14 (IH, d, J 8.1, CH), 9.06 (IH, d, J 5.9, CH), 8.31-8.26 (IH, m, CH), 8.0 (IH, d, J 8.1, CH), 7.85 (IH, d, J 8.1, CH), 7.76-7.70 (IH, m, H4), 7.57-7.52 (IH, m, CH), 6.56 (IH, q, J 6.3, CH-Me) 5.64 (2H, s, CH₂) and 1.82 (3H, t, J 6.3, CH₃). δ_c (65 MHz, CDC1₃) 161.8 (CO), 149.2, 147.3, 146.8 (all CH), 137.3 (C), 135.7 (C), 134.5, 129.5, 128.3, 127.6, 124.7 (all CH), 72.1, 61.9 and 20.9 (CH₃). m/z [FAB⁺] 273.1 (IV^+H, 100%) [found 273.0871 M⁺+H. C₁₄H₁₃N₂0₄ requires 273.0873].

[El Synthesis of pyridinium salts by Zincke reaction

3-Carbamoyl-l- (2,4-dinitro-phenyl)-pyridinium chloride (Zincke salt)

Nicotinamide (0.5g, 4.0mmol) was mixed with 2,4-dinitrochlorobenzene (2.5g, 12mmol). This mixture was then heated at 90 °C for 2h. After cooling down, the residual was dissolved in methanol (3ml) and then was added ether (40ml). The solid that followed was filtered and redissolved again in methanol (3ml) and then ether (40ml), this procedure was repeated four times. The solid was filtered and dried in vacuum. Orange colour foam was given in 76% yield.

IR: 1690, 1616, 1543, 1347, 1202, 704; 1H NMR (270 MHZ, CD₃OD): 9.74 (s, IH, H2), 9.39 (dd, J6.2 and 1.3, IH, H4), 9.25 (dd, J8.4 and 1.3, IH, H6), 9.22 (d, J2.6, IH, ArH), 8.86 (dd, J 8.8 and 2.6, IH, ArH), 8.42 (dd, J 8.4 and 6.2, 1H, H5) and 8.30 (d, J 8.8, IH, ArH). ¹³C NMR (100.4 MHz, D₂0): 165.0 (CO), 149 (C-2'), 147 (C-4'), 147.5 (C-4'), 145.7 (C-6'), 142.9 (C-2'), 138 (C-3'), 134.2 (C-l'), 131.4 (C-5), 131.0 (C-6'), 128 (C-5'), 122.9 (C-3'); MS: m/z (FAB⁺) 289 (M⁺+l), 273 (M⁺-NH₂).

Procedure for Zincke reaction

Amines (118mg, 0.8mmol) were dissolved in dry methanol (20ml). To this solution, Zincke salt (26) (270mg, 0.83mmol) was added. The following dark red solution was stined at room temperature for 5h (color from dark red to yellow). Crude products were given after removal of the solvent which were then purified by flash chromatography or crystallization. 1 -Butyl-3-carb amoyl-py ridinium

Crude product was crystallized from methanol/ether. Yellow solid was given in 75% yield. Mp: 205-207°C (crystallized from methanol/ether); TR: 1669, 1647.8, 1457, 1403, 1204; 1H NMR (270 MHz, D₂0): 9.30 (s, IH, H-2), 9.00 (d, J= 8.1, IH, H-6), 8.87 (d, J= 8.2, IH, H-4), 8.17 (m, IH, H-5), 4.66 (m, 2H, CH₂), 2.01 (m, 2H, CH₂), 1.38 (m, 2H, CH₂), 0.93 (m, 3H, CH₃); MS: m/z (FAB⁺) 179.0 (M⁺+l). M/z calcd for C₁₀H₁₅N₂O 179.1184 found 179.1178.

3-Carbamoyl-l-cyclohexyl-pyridinium

Crude product was crystallized from methanol and ether. Yellow solid was given in 67% yield.

Mp: 275-276°C (crystallized from methanol/ether); IR: 3278, 3137, 2857, 1695, 1644, 1590, 1512, 1453, 1407, 1142, 679; 1H NMR (270 MHz, D₂0): 9.12 (s, IH, H-2), 8.95 (d, J= 8.1, IH, H-6), 8.77 (d, J=8.2, IH, H-4), 8.01 (m, IH, H-5), 1.13-2.05 (m, 11Η, ring-H) ¹³C NMR (100.4 MHz, D₂0): 163.3 (CO), 145.6 (C-4), 144.6 (C-6), 143.9 (C-2), 134.5 (C-3), 128.6 (C- 5), 72.0 (C-1'), 33.1 (C-2' and C-6'), 25.8 (C-3' and C-5'), 24.9 (C-4'); MS: m/z (FAB ⁺) 205.0 (M⁺+l).M/z calcd for C₁₂H₁₇N₂0 205.1341 found 205.1339.

3-Carboxy-l-phenyl-pyridinium

The crude product was purified by Flash chromatography, eluted with DCM: methanol 5:2. Yellow solid was given in 83% yield. Analytical data was obtained by crystallization from methanol and ether.

Mp: 235-237°C (crystallized from methanol/ether), IR: 3370, 3140, 1687, 1491, 1398, 1256, 766, 680; 1H NMR (270 MHz, D₂0): 9.55 (s, IH, H-2), 9.27 (d, J= 5.5, IH, H-6), 9.06 (d, J= 8.1, IH, H-4), 8.34 (m, IH, H-5), 7.75 (s, 5R, ArH); ¹³C NMR (100.4 MHz, D₂0): 165.7 (CO), 146.7 (C-4), 144.9 (C-6), 144.5 (C-2), 134.0 (ArCl), 132.0 (C-5), 130.6 (ArC2 and C6), 128.5 (ArC4), 124.2 (ArC3 and ArC5); MS: m/z (FAB ⁺) 199.1 (M⁺+l). C₁₂H_πN₂0 199.0871 found 199.0871. 3-Carbamoyl-l-decyl-pyridinium

The cruded product was crystallized from methanol and ether. Yellow oil was given which was further purified by Silica column (DCM: methanol 5:1). Yield 66%.

IR: 3257, 3075, 2920, 2852, 1703, 1512, 1451, 1407, 1335, 1144, 805, 679; 1H NMR (270 MHz, D₂0): 9.29 (s, IH, H-2), 9.00 (d, J=6.0, IH, H6), 8.88 (d, J=8.3, IH, H4), 8.17 (dd, J=8.3, 6.0, IH, H5), 4.78 (m, 2H, CH₂-N), 3.33 (MeOΗ), 2.05 (m, 2Η, CH₂), 1.23 (m, 14H, 7CH₂), 0.83 (m, 3Η, CH₃); ¹³C NMR (100.4 MHz, D₂0): 165.3 (CO), 146.6 (C4), 144.3 (C2), 143.9 (C6), 134.0 (C3), 128.6 (C5), 62.7 (CH₂-CO), 31.7 (CH₂), 31.2 (CH₂), 29.3 (CH₂), 29.2 (CH₂), 29.1 (CH₂), 28.8 (CH₂), 25.8 (CH₂), 22.6 (CH₂), 13.9 (CH₃); MS: m/z (FAB⁺) 263.0 (M⁺+l). M/z calcd for C₁₆H₂₇N₂0 263.2123 found 263.2126.

3-Carbamoyl-l-(2-hydroxy-l-hydroxymethyl-2-phenyl-ethyl)-pyridinium

Yellow oil was obtained after flash chromatography (DCM: methanol 5:1) in 100% yield. IR: 3350, 3158, 1702, 1646, 1448, 1401, 1289, 1019, 678; 1H NMR (270 MHz, D₂0): 9.27 (s, IH, H2), 9.04 (d, J=6.2, IH, H-6), 8.94 (d, J=8.4, IH, H-4), 8.18 (m, IH, H-5), 7.35 (m, 5H, ArH), 5.45 (m, IH, CH-N), 5.12 (m, 1Η, CH-OΗ), 4.14 (m, 2Η, CH₂-OH); ¹³C NMR (100.4 MHz, D₂0): 165.7 (CO), 146.4 (C4), 144.9 (C2), 144.1 (C6), 138.3 (ArCl), 133.7 (C3), 129.2 (ArC3 and C5), 128.2 (C5), 126.1 (ArC2 and C6), 126.7 (ArC4); MS: m/z (FAB ⁺) 273.1 (M⁺+l). HPLC: 10.20 (RP-18, WL 254 Acetonitrile/water gradient 5-5%). M/z calcd for C₁₅H₁₇N₂0₃ 273.1239 found 273.1238.

Procedure for 3-carboxamide hydrolysis

3-Carbamoyl-l-(2-hydroxy-l-hydroxymethyl-2-phenyl-ethyl)-pyridinium (200mg, 1.02mmol) was added concentrated HCI (3ml). This reaction mixture was then heated to reflex for 2h and then kept at r.t for overnight. Removal of solvent gave crude product, which was crystallized from acetone to give yellow solid in over 80% yield. 3-Carboxy-l-(2-hydroxy-l-hydroxymethyl-2-phenyl-ethyl)-pyridinium

IR: 1731, 1636, 1402, 1298, 1173, 1H NMR (270 MHz, D₂0): 9.27 (s, IH, H-2), 9.02 (m, 2H, H-6 and 4), 8.16 (m, IH, H-5), 7.54-7.30 (m, 5H, ArH), 5.48 (m, IH, CH-N), 5.12 (m, 1Η, CH-OΗ), 4.12 (m, 2Η, CH₂-OH); ¹³C NMR (100.4 MHz, D₂0): 165.6 (CO), 146.6 (C4), 146.5 (C2), 145.3 (C6), 138.3 (ArCl), 129.2 (ArC3 and C5), 128.1 (C5), 126.1 (ArC2 and C6) 79.2 (CH-N), 69.8 (CH₂-OH), 60.3 (CH-OH); MS: m/z (FAB⁺) 274 (M^.m/z calcd for C₁₅H₁₆N0₄ 274.1074 found 274.1077. HPLC: 7.45min (RP-18, WL 254 Acetonitrile/water gradient 5-50%).

3-Carboxy-l-cyclohexyl-pyridinium

Yellow solid was given after crystallized from acetone. Yield 80%.

Mp: 235-237, 1H NMR (270 MHz, D₂0): 9.56 (s, IH, H-2), 9.49 (d, J= 5.5, IH, H-6), 8.94 (d, J= 8.1, IH, H-4), 8.28 (m, IH, H-5), 4.86 (m, IH, CH-1 2.14-1.07 (m, 1 IH, ring-H); ¹³C NMR (100.4 MHz, D₂0): 163.6 (CO), 146.5 (C-4), 145.9 (C-6), 145.6 (C-2), 131.8 (C-3), 129.1 (C-5), 71.9 (C-l'), 33.1 (C-2' and C-6'), 25.8 (C-3' and C-5'), 24.8 (C-4'); MS: m/z (FAB⁺) 206.1 (M⁺+l).

Synthesis of Pyridinium Salt in Solvent System (Example 21)

model compoimd (III).

(III) The synthesis of pyridinium salts was shown in (a).

(a) Synthesis of Pyridinium Salts in Solvent System.

The Bromoacetylamides were prepared [1] in ether at -10 °C in a yield at 60-80% and the following alkylation reaction was carried out in DMF at 60-70 °C in the dark.

(b) List of amides

(b) List of Pyridinium salts.

HPLC of the pyridinium salts (Example 22).

The purity of the pyridinium salts was determined by HPLC [2] (RP-18; WL 254, Flow rate: lml/niin; acetonitrile/water 5-50% gradient). Some of the results are shown in Figure 5.

Biological Data (Example 23)

Biological data is presented in Figures 1 to 4. These experiments were carried out as described above in the description of the figures.

The effect of externally applied CMA008 on calcium release in response to photolysis of NPE-NAADP in intact sea urchin eggs (Example 24)

Sea urchin eggs of Lytechinus pictus were obtained by intracoelomic injection of 0.5 M KCl shed into artificial sea water (in mM, NaCl 435, MgCl₂40, MgS04 15, CaCl₂ 11, KCl 10, NaHCO₃ 2.5, EDTA 1), dejellied by passing through 90-mm nylon mesh, and then washed twice by centrifugation. Eggs were transfened to polylysine-coated glass coverslips for microinjection and microscopy. Oregon Green 488 BAPTA (l,2-bis(2-aminophenoxy)ethane- N,N,N9,N9-tetraacetic acid dextran; Molecular Probes) was pressure-microinjected (Picospritzer; World Precision Instruments). The calcium-sensitive dye was imaged by laser- scanning confocal microscopy (Leica model TCS NT) using the 488-nm line of an argon ion laser for excitation, and the emission was long passfiltered (515 nm) and detected with a photomultiplier tube. Caged NAADP (²⁹P-(l-(2-nitrophenyl)efhyl) NAADP; Molecular Probes) was purified further by high performance liquid chromatography to remove small amounts of contaminating free NAADP. Caged NAADP were photolyzed with ultraviolet light (351- and 364-nm lines) from an argon ion laser (Enterprise model 651; Coherent) that was directed into the scanning head by a quartz fiber optic cable. The spatial location of photolysis was controlled via a shutter that was placed in the light path of the ultraviolet laser. This resulted in a band of UN across the image with the position and width of the band being controllable. The confocal images were processed with the software Hϊ Image to create a self ratio by dividing the intensity (F) of each image on a pixel by pixel basis by the intensity of an image acquired before stimulation (Fo). Time courses of F/Fo are plotted against time. Results are shown of the effect of externally applied CMA008 on the effect of photolysing ΝPE-ΝAADP. CMA0O8 (10 mM) blocks the effect of photolysis of ΝPE-ΝAADP (1 μM) on calcium release in intact sea urchin eggs, supporting the notion of membrane permeance of CMA008. The biological data for these experiments is presented in Figure 6 (see also the description of the figures above).

Effect of externally applied CMA008 (l-Carbamoylmethyl-3-carboxy-pyridinium iodide) on CCK-induced oscillations of Ca2+ in pancreatic acinar cells from single cell imaging measurements. (Example 25)

Pancreatic acinar cells were seeded onto poly-lysine-coated number 1 glass coverslips and loaded with calcium indicator by incubating cells with 1-5 mM fura-2 acetoxymethylester (Molecular Probes; Leiden, Holland) for 60 min at room temperature. After the loading period, cells were subsequently washed and maintained in buffer at room temperature and used immediately. Cells were excited alternately with 340 and 380 nm light (emission 510 nm), and ratio image of clusters 5 were recorded every 4-5 s, using a 12-bit CCD camera (MicroMax; Princeton Instruments, NJ).

All experiments were conducted at room temperature. CMA008 (l-Carbamoylmethyl-3- carboxy-pyridinium iodide) was dissolved in 50%DMSO. Final concentration of DMSO was 0.5% in the solution (composition in mM:140 NaCl, 4.7 KCl, 1 CaCl₂, 1.13 MgCl₂, 10 HEPES, 10 Glucose, pH adjusted to 7.2). The inhibition of CCK-evoked calcium spiking, both pre- and post- applied with respect to CCK (5 pM), was consistent with inhibition of NAADP- induced calcium release by CMA008 (1 m ). The biological data for these experiments is presented in Figure 7 (see also the description of the figures above).

SUMMARY

Novel chemical entities that modulate the release of intracellular calcium by a novel mechanism from a specific store controlled by nicotimc acid adenine dinucleotide phosphate are described. These small molecules are cell permeable and have been shown to ter alia modulate calcium spikes in mammalian beta cells. NAADP mediated calcium stores are found in a wide range of mammalian cells including brain, heart, pancreatic acinar and T-cells. These and related compounds may thus find application as novel therapeutic agents and as probes for biological assays.

Nicotmic acid adenine dinucleotide phosphate (NAADP) displays a carboxylate at the 3- position of the pyridinium, unlike the carboxamide displayed by the related biological co- substrate nicotinamide adenine dinucleotide phosphate (NADP). It has recently emerged that low concentrations of NAADP (approx. lOOnM) causes release of calcium from a discrete intracellular store that is not addressed by any other second chemical messenger, such as cADPR or l,4,5-InsP₃. Despite being a close analogue NADP is not active. Studies on closely related NAADP analogues confirmed strong specificity at the nicotmic acid position. Strong specificity is often accompanied by sub-sites that are responsible for tight binding interactions with the ligand and the inventors reasoned that small molecule analogues might bind at such a sub-site and by competing with the natural ligand, potentially act as modulators of this novel biological mechanism.

Simple pyridinium salts of nicotinic acid have been prepared and have been shown to modulate calcium release in model systems, such as sea urchin homogenate, as well as mammalian pancreatic cells. The compounds were applied outside of the cell, yet demonstrated potent activity so confirming that they are cell permeable.

It is believed that these novel molecules act by binding to the NAADP receptor at a sub-site responsible for binding the nicotinic acid portion of the ligand. Such compounds provide a powerful basis for the development of novel therapeutics that act by modulating calcium signals critical to controlling a number of important biological processes, such as fertility, insulin production, T-cell activation, controlling the frequency of heart muscle contractions and the activity of brain cells.

The chemical entities described herein may be used either for assays, or themselves developed into novel pharmaceutical agents that intercept and control this important biological pathway. Examples of diseases that feature abenant intracellular calcium signalling are manifold and include diabetes, while the role of calcium in the activation of T-cells offers the prospect of control of the immune system. That NAADP receptors have been shown to be active in the brain suggests a possible role for controlling neurological diseases.

The chemical entities described will potentially be applicable for the modulation of any disease due to abenant NAADP induced calcium release. The general structure of the compounds may find application for other biological targets that feature related binding sites for nicotinic acid/amide derivatives.

REFERENCES

[1] T. Hamas, D. A. Culkin, J. F. Rartwig, J. Am. Chem. Soc, 2003, 125 (37), 11176-11177. [2] M. A. Lago, T. T. Nguyen, P. Bhatnagar, Tetrahedron Lett. 1998, 39, 3885-3888.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific prefened embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry, molecular biology or related fields are intended to be within the scope of the following claims.

Claims

CLAΓMS

1. The use of a compound of formula (I) :

wherein:

Rl comprises a carbonyl group

R2 is a hydrocarbyl group;

optionally wherein said ring is further substituted;

or a pharmaceutically acceptable salt thereof;

in the manufacture of a medicament for use in one or more of: modulating the release of intracellular calcium from a store controlled by nicotinic acid adenine dinucleotide phosphate modulating calcium spikes in mammalian cells treating diseases in one or more of brain, heart, pancreatic cells (e.g. pancreatic acinar and pancreatic beta cells), immune cells, T-cells, haemopoietic cells including phagocytes treating diseases in one or more of brain, heart, pancreatic cells (e.g. pancreatic acinar and pancreatic beta cells), immune cells, T-cells, haemopoietic cells including phagocytes by modulating the release of intracellular calcium from a store controlled by nicotinic acid adenine dinucleotide phosphate treating diseases in one or more of brain, heart, pancreatic cells (e.g. pancreatic acinar and pancreatic beta cells), immune cells, T-cells, haemopoietic cells including phagocytes by modulating calcium spikes in mammalian cells.

2. Use according to claim 1 wherein said compound is cell permeable.

3. Use according to claim 1 or claim 2 wherein said compound has a relative molecular mass (RMM) of less than about 500.

4. Use according to any preceding claim wherein R2 is a hydrocarbyl group comprising a carbonyl group.

5. Use according to any preceding claim wherein R2 is a hydrocarbyl group comprising an amide group.

6. Use according to any preceding claim wherein R2 is CH₂C(0)NH2.

7. Use according to any preceding claim wherein Rl is COOH.

8. Use according to any preceding claim wherein said compound is a mimetic of nicotinic group of NAADP, wherein said NAADP has the formula:

(II)

9. A pharmaceutical composition comprising a compound as defined in any of the preceding claims or a pharmaceutically acceptable salt thereof admixed with a pharmaceutically acceptable carrier, diluent or excipient.

10. A pharmaceutical composition accordmg to claim 9 wherein said composition comprises one or more additional pharmaceutically active compounds.

11. A compound of formula (I) or a pharmaceutically acceptable salt thereof as defined in any one of claims 1 to 8 for use in medicine.

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

13. A medicament comprising a compound according to any of claims 1-8.

14. An assay method for identifying an agent that modulates intracellular calcium release comprising the steps of:

(a) providing an agent;

(b) providing an NAADP receptor;

(c) contacting said agent with an NAADP receptor; and

(d) measuring the level of intracellular calcium release;

wherein a difference between (i) the level of intracellular calcium release in the presence of the agent; and (ii) the level of intracellular calcium release in the absence of the agent is indicative of an agent that modulates intracellular calcium release and may be useful in one or more of: modulating the release of intracellular calcium from a store controlled by nicotinic acid adenine dinucleotide phosphate; modulating calcium spikes in mammalian cells; treating diseases in one or more of brain, heart, pancreatic cells (e.g. pancreatic acinar and pancreatic beta cells), immune cells, T-cells, haemopoietic cells including phagocytes; treating diseases in one or more of brain, heart, pancreatic cells (e.g. pancreatic acinar and pancreatic beta cells), immune cells, T-cells, haemopoietic cells including phagocytes by modulating the release of intracellular calcium from a store controlled by nicotinic acid adenine dinucleotide phosphate; and treating diseases in one or more of brain, heart, pancreatic cells (e.g. pancreatic acinar and pancreatic beta cells), immune cells, T-cells, haemopoietic cells including phagocytes by modulating calcium spikes in mammalian cells.

15. An assay method according to claim 14 wherein said agent is cell permeable.

16. An assay method according to claim 14 or claim 15 wherein said agent has a relative molecular mass (RMM) of less than about 500.

17. An assay method according to any of claims 14-16 wherein said agent is a mimetic of nicotinic group of NAADP, wherein said NAADP has the formula:

(II)

18. An assay method comprising the steps of:

(a) performing the assay method according to any of claims 14-17; (b) identifying one or more agents capable of modulating intracellular calcium release; and

(c) preparing a quantity of those one or more identified agents.

19. A method comprising the steps of:

(a) performing the assay method according to any of claims 14-17;

20. An agent identifiable, preferably, identified hy the assay method according to claim any of claims 14-17.

21. A pharmaceutical composition prepared by the method of claim 19.

22. A method of treating and/or preventing a disease in a human or animal patient in need of same which method comprises administering to the patient an effective amount of a compound as defined in any of claims 1-8 or 11-12, a composition according to claims 9 or 10, or a medicament according to claim 13.

23. A process of preparing a pharmaceutical composition, said process comprising admixing one or more of the compounds defined in any of claims 1-8 or 11-12 with a pharmaceutically acceptable diluent, excipient or carrier.

24. A pharmaceutical pack comprising one or more compartments, wherein at least one compartment comprises one or more of the compounds defined in any of claims 1-8 or 11-12 , a composition according to claims 9 or 10, or a medicament according to claim 13.

25. A container comprising a compound according to any of claims 1-8 or 11-12, a composition according to claims 9 or 10, or a medicament according to claim 13, wherein said container is labelled for use in one or more of: modulating the release of intracellular calcium from a store controlled by nicotinic acid adenine dinucleotide phosphate modulating calcium spikes in mammalian cells treating diseases in one or more of brain, heart, pancreatic cells (e.g. pancreatic acinar and pancreatic beta cells), immune cells, T-cells, haemopoietic cells including phagocytes treating diseases in one or more of brain, heart, pancreatic cells (e.g. pancreatic acinar and pancreatic beta cells), immune cells, T-cells, haemopoietic cells including phagocytes by modulating the release of intracellular calcium from a store controlled by nicotinic acid adenine dinucleotide phosphate treating diseases in one or more of brain, heart, pancreatic cells (e.g. pancreatic acinar and pancreatic beta cells), immune cells, T-cells, haemopoietic cells including phagocytes by modulating calcium spikes in mammalian cells.

26. The use of a compound of formula (I) in the manufacture of a medicament for use in one or more of: modulating the release of intracellular calcium from a store controlled by nicotinic acid adenine dinucleotide phosphate modulating calcium spikes in mammalian cells treating diseases in one or more of brain, heart, pancreatic cells (e.g. pancreatic acinar and pancreatic beta cells), immune cells, T-cells, haemopoietic cells including phagocytes treating diseases in one or more of brain, heart, pancreatic cells (e.g. pancreatic acinar and pancreatic beta cells), immune cells, T-cells, haemopoietic cells including phagocytes by modulating the release of intracellular calcium from a store controlled by nicotinic acid adenine dinucleotide phosphate treating diseases in one or more of brain, heart, pancreatic cells (e.g. pancreatic acinar cells)and T-cells by modulating calcium spikes in mammalian cells.

wherein said compound is cell permeable; wherein said compound has a relative molecular mass of less than about 500; wherein said compound is a mimetic of the nicotinic group of NAADP, wherein said NAADP has the formula:

(ID

27. The use of a compound in the manufacture of a medicament for use in one or more of: modulating the release of intracellular calcium from a store controlled by nicotinic acid adenine dinucleotide phosphate modulating calcium spikes in mammalian cells treating diseases in one or more of bram, heart, pancreatic cells (e.g. pancreatic acinar and pancreatic beta cells), immune cells, T-cells, haemopoietic cells including phagocytes treating diseases in one or more of brain, heart, pancreatic cells (e.g. pancreatic acinar and pancreatic beta cells), immune cells, T-cells, haemopoietic cells including phagocytes by modulating the release of intracellular calcium from a store controlled by nicotinic acid adenine dinucleotide phosphate treating diseases in one or more of brain, heart, pancreatic cells (e.g. pancreatic acinar cells)and T-cells by modulating calcium spikes in mammalian cells.

wherein said compound is cell permeable;

wherein said compound has a relative molecular mass of less than about 500;

wherein said compound is a mimetic of the nicotinic group of NAADP, wherein said NAADP has the formula:

and wherein said compound has the formula (A):

(A) wherein Rl and R2 are ring substituents; wherein Rl and R2 are as defined for compound of formula (I); and wherein R3 represents one or more substituents, the or each substituent being independently selected from H, substituted or unsubstituted aryl, Cl-20 alkyl, F, Cl, Br, I, OY, NY_n (n = 1, 2, or 3), SY, COY, CONY_z (z = 2), C(0)OY, wherein the or each Y is independently selected from H, a substituted or unsubstituted aryl, and a Cl-20 alkyl group.

28. A compound for use in medicine

wherein said compound is cell permeable;

wherein said compound has a relative molecular mass of less than about 500;

(II)

and wherein said compound has the formula (A):

29. A compound, or a pharmaceutical composition comprising same, wherein said compound is cell permeable;

wherein said compound has a relative molecular mass of less than about 500;

(II) and wherein said compound has the formula (A):