The adenosine-A1 receptor (Al-AR) is a GPCR which is found in a variety of tissues including brain, heart, adipose tissue and muscle, and has been implicated in the pathophysiology of a number of conditions (Ralevic, V. and Bumstock, J (1998) Pharmacol. Rev. 50,415).

Currently the study of Al-AR pharmacology can only be performed well in cells which can be grown in large numbers using for example techniques such as radioligand binding. Autoradiography enables single cell studies but does not allow direct reading of binding and can take up to 4-6 weeks to develop the film to obtain results of binding. To overcome this problem, a very few fluorescent ligands have been adapted for use in visualising receptors and obtaining quantitative receptor- ligand binding data in single cells, using confocal microscopy (CSLM), confocal plate readers, fluorescence polarisation plate readers, and fluorescence correlation spectroscopy (FCS). Confocal microscopy allows visualisation of a section through a cell, concentration of fluorophore at the cell edges indicates membrane receptor binding. FCS analyses the diffusion characteristics of fluorescent species, fast- diffusing free ligand can be distinguished from slowly-diffusing receptor-bound ligand and quantified simultaneously when the volume is localised to the cell membrane.

McGrath et al TiPS November 1996 (Vol 17) 393-399 reviews the possibilities for using fluorescent ligands in place of more traditional radioactive ligands in the study of cell receptors to report the amount of ligand-receptor complex indicating the number of receptors, using confocal spectroscopy and fluorescence activated cell sorting (FACS). He states that many attempts have been made at conjugating fluorescent molecules to receptor ligands in the hope of identifying their binding sites, aimed mainly at localisation of the receptors rather than studying their properties. Some compounds are reported that fluoresce when bound to a receptor but which give low background noise in the aqueous phase. A reported objective was to produce a fluorescent drug which would remain fluorescent when bound to the receptor and would remain bound when unbound drug was washed away. Therefore

there was a need for very high receptor binding affinity. Reviewed work includes fluorescent ligand binding to nicotinic receptors, beta adrenoceptors, opioid GPCR type receptors, histamine, neurotensin and alpha-adrenoceptors. The publication also reviews benefits of confocal microscopy. Efforts made to study the pharmacological properties of the ligands are reported in only a few of the above cases.

However very few efforts to visualise receptors or classes of receptors have been shown to work. Pharmacological properties are usually to some extent affected by linking of a fluorophore to any receptor binding ligand, and include change in binding affinity, and in activation or otherwise of receptor, ie agonist or antagonist properties. It is important that the pharmacology of the fluorescent ligand is known in any studies in order to quantify the binding results observed.

In fact the synthesis of non-peptide fluorescent ligands for GPCRs presents serious problems. The few commercial non peptide fluorescent ligands for cell surface receptors that have been synthesised include histamine-BODIPY FL and (pictured below) CGP12177-BODIPY TMR (Molecular Probes): The BODIPY (BDI) fluorophores were initially designed for attaching to proteins which present a much more uniform prospect for attaching : kits are available comprising a fluorophore and a set of reagents for universally attaching to most proteins. These give non specific attachments to any reactive site on the protein of interest and usually there is no need to know the nature or location of the attachment.

However these proteins are larger molecules than the non-peptide ligands, including drugs such as XAC (xanthine amine congener) etc envisaged in the present invention. The ligand binding site for the many GPCR receptors is also usually deep within the transmembrane regions of the receptor and thus the challenge is to attach to the fluorophore in such a way as to retain pharmacological activity. None of these BDI fluorophores are concerned with the specific design of fluorescent agonists/antagonists with defined properties at GPCR's but rather with the fluorophore as an"add-on"probe.

In summary therefore the availability of fluorescent ligands and in particular non- peptide fluorescent ligands suitable for FCS and CM binding studies is virtually non- existent. The preparation of such compounds is far from routine and few efforts have been made to establish pharmacology. McGrath above only looked at a few of the receptor types studied.

There is moreover no unified approach in much of the prior art. Individual research has addressed fluorescent ligand systems which are limited to specific drug classes and or to the use of specific fluorophores. Such systems are limiting in both the information which can be obtained and in the number of systems which can be investigated.

Accordingly there is a need for novel selective fluorescent ligands for binding at desired receptors giving reliable and effective receptor visualisation and receptor selectivity with established pharmacology in terms of both affinity and agonist and antagonist properties.

We have now applied a multidisciplinary approach to fluorescent ligand design to provide a library of rationally designed fluorescently tagged ligands and a process for preparation thereof that may be used in a method for selection of a fluorescently tagged ligand which is selective to a desired GPCR, having required defined pharmacological characteristics.

The library is obtained from preferably non-peptide ligand precursors comprising chemical functionality for linking to any fluorophore to provide known or novel fluorescent ligands with linking at a desired site enabling selection of a fluorescent ligand providing retention of receptor binding capability and linking in manner not to interfere with receptor binding capability, or to modify binding capability in known manner. The linker precursors may also provide improved properties such as water solubility, on linking to a fluorescent moiety or any other desired non-hydrophilic probe.

In the broadest aspect of the invention there is provided a library comprising a plurality of tagged non-peptide ligands of formula I (Lig JL) m L (JT Tag) , (JT L (JL Lig) m) p including salts thereof comprising one or a plurality of same or different ligand moieties Lig each linked to a one or a plurality of same or different tag moieties Tag via same or different linker moieties L and same or different linking site or linking functionality JT and JL wherein Lig comprises a GPCR ligand, an inhibitor of an intracellular enzyme or a substrate or inhibitor of a drug transporter; L is a single bond or is any linking moiety selected from a heteroatom such as N, O, S, P, branched or straight chain saturated or unsaturated, optionally heteroatom containing, Cl-6oo hydrocarbyl and combinations thereof, which may be monomeric, oligomeric having oligomeric repeat of 2 to 30 or polymeric having polymeric repeat in excess of 30 up to 300 ; Tag is any known or novel tagging substrate; m are each independently selected from a whole number integer from 1 to 3 ; p isOto3

characterised in that linking is at same or different linking sites in compounds comprising different Lig, JL, L JT and/or-Tag and is at different linking sites in compounds comprising same Lig, JL, L JT and/or-Tag.

Preferably the library does not comprise as Lig NECA, as Tag dansylamide or NBD, as each J a single bond and as L a methylene chain of 3-12.

The innovation of the present invention relates to the design of specifically tagged ligands or"drugs"eg fluorescent ligands or"drugs"with known or selectable pharmacological properties. A key to this success is that each tag or fluorophore has a specific influence on the pharmacology of the resulting product, and it is incorrect to assume that the compound will retain the properties of the precursor drug.

Preferably the library is constructed by the rational design of library members representing modifications in linking sites and ligand moieties, which can be used as a basis for selection of a tagged or fluorescent ligand retaining the properties of the precursor ligand. Preferably the library comprises a plurality of defined and well characterized tagged ligands, having verified properties corresponding to those of the non-tagged ligand.

A GPCR ligand may be selected from any compound which is effective as an agonist or antagonist for an adenosine receptor, a beta-adrenoceptor, a muscarinic receptor, a histamine receptor, an opiate receptor, a cannabinoid receptor, a chemokine receptor, an alpha-adrenoceptor, a GABA receptor, a prostanoid receptor, a 5-HT (serotonin) receptor, an excitatory aminoacid receptor (e. g. glutamate), a dopamine receptor, a protease-activating receptor, a neurokinin receptor, an angiotensin receptor, an oxytocin receptor, a leukotriene receptor, a nucleotide receptor (purines and pyrimidines), a calcium-sensing receptor, a thyroid-stimulating hormone receptor, a neurotensin receptor, a vasopressin receptor, an olfactory receptor, a nucleobase receptor (e. g. adenosine), a lysophosphatidic acid receptor, a sphingolipid receptor, a tyramine receptor (trace amines), a free-fatty acid receptor and a cyclic nucleotide receptor or the like, preferably for a GPCR receptor for example a) an adenosine receptor antagonist b) an adenosine receptor agonist c) a beta-adrenoceptor agonist and d) a beta-adrenoceptor antagonist. Preferably a ligand is a non-peptide ligand.

An inhibitor of intracellular enzymes is preferably e) an inhibitor of an intracellular enzyme such as an inhibitor of cyclic nucleotide phosphodiesterases; or a derivative or analogue thereof.

A substrate of a drug transporter is any drug that is transported into or out of the cell via the transporter. An inhibitor of a drug transporter is any compound which binds to the transporter and prevents a substrate being transported. Thus, a tagged inhibitor can be used to bind to the transporter and localise it. A tagged substrate of the invention could be used to follow transport into or out of the cell and to test whether inhibitor drugs can prevent the transport of the tagged substrate. A substrate or inhibitor is preferably selected from a substrate or inhibitor of any equilibrium based drug transporters or ATP driven pumps such as a catecholamine transporter, a nucleoside transporter, an ATP-binding cassette transporter, a cyclic nucleotide transporter or the like.

Preferably the library provides tagged ligands which are suited for surface cell receptor binding or for intracellular binding, or for penetrating or exiting live cells.

Accordingly the library represents the rational design of compounds which are predicted to have retained pharmacology and properties suitable for specific binding applications.

Each Tag may be independently selected from any entity which is known in the art of tagging molecules to form a marker or reporter group for detecting molecules and which may be used in analytical studies relating to the ligand, particularly for visualisation, and includes but is not limited to fluorophore tags as known in the art.

An additional Tag may be present and may perform a function in situ, eg may be any laser activated Tag which is activated to have a local or targetted therapeutic or destructive effect. This allows in a first stage visualising the compound of formula I by means of a visualisation Tag, in a second stage activation of laser activated Tag, and optionally in a third stage visualising the compound of formula I or fragments thereof. For example a laser activated Tag may comprise malachite green which may be activated for targetted protein destruction.

In a particular advantage, in the case that Tag is a chemical entity which might be anticipated to inhibit receptor ligand binding or to inhibit intracellular enzyme or drug transporter inhibition in or by a compound of formula I such inhibition is negated or dispelled by the presence of group L and/or of each J or by the chosen site of linking in one or more library members.

Preferably one or more of each-Tag in one or more or each library compound is an entity-Fl and comprises any known or novel fluorophore, whereby the library comprises compounds of which one or more or all of which are of formula I' (LigJL) m L (JT F1) m (JT L (JlLig) m) P Preferably each compound of formula I or I'comprises one of a plurality of fluorophores and/or tags providing a library of differently fluorescently tagged ligands comprising one or a number of different fluorophores (preferably of different chemical composition, spectral characteristics etc); and/or providing a library of differently tagged ligands including at least one fluorescently tagged ligand; alternatively each compound of formula I or I'comprises one of a plurality of precursor ligands linked each to one or a plurality of different tags providing a library of same or differently tagged ligands of plural ligand type; alternatively each compound of formula I comprises one of a plurality of linkers linking a precursor ligand and at least one Tag at the same or different linking site; alternatively each compound of formula I comprises the same linker linking a precursor ligand and at least one Tag at different linking sites providing a library of differently linked tagged ligands of different conformation or anticipated pharmacology and binding.

In each case the library of the invention provides for the selection of a tagged ligand of desired binding affinity inhibition or transport at a desired receptor, intracellular enzyme or at or by a drug transporter with desired pharmacology, visualisation, mechanism or the like.

More preferably a library comprises a plurality of compounds of one or more of formula II to III": II (LigJL), L JT TagJT L (JL Lig) m where each m is as hereinbefore defined and is preferably 1 or 2, more preferably 1 III (LigJL) m L (JTTag) m where m in each is as hereinbefore defined and is preferably 1 and/or 2, more preferably Lig JL-L-JL Tag and/or wherein each JL and JT comprises J as hereinbefore defined and may be same or different and may derive from functionality originally present in Lig or L and Tag or L or a combination thereof, characterised in that linking is at same or different linking sites in compounds comprising different Lig, JL, L, JT and/or Tag, and is at different linking sites in the case of any two or more compounds comprising identical Lig, JL, L, JT and/or Tag.

In one preferred embodiment the invention comprises a library of compounds of formula I as hereinbefore defined wherein Lig, JL, L, JT and Tag are the same in all compounds, and wherein the compounds differ by site of linking thereof.

In a further preferred embodiment the invention comprises a library of compounds of formula I or I'as hereinbefore defined wherein Lig and JL are the same in all compounds and L and JT are the same or similar in all compounds and Tag is different in some or all compounds.

In a further preferred embodiment the invention comprises a library of compounds of formula I or I'as hereinbefore defined wherein Lig-and-Tag are the same in all compounds and-L-is different in all compounds.

The library may comprise from 3 to 250 tagged ligands. Preferably the library comprises from 1 to 10 families comprising 3 to 25 tagged ligands each family comprising a ligand moiety of a common ligand type and from 3 to 25 different tag moiety types at least one of which is a fluorescent tag, more preferably each of which is a different fluorescent tag; or the library comprises from 5 to 250 fluorescently tagged ligands of different ligand type and different fluorophore type.

A library providing fluorescent ligands comprising different Fl is useful to enable studying binding, inhibition or transport with different colour fluorescence for example to distinguish from same colour native fluorescence or to distinguish plural types of binding site, enzyme, transporter or the like.

It is known that ligands modified ie by linking to a fluorophore typically undergo a change in binding affinity, inhibition or transport and suitably the library of the invention comprises characterisation of the pharmacology of each compound including binding affinity or inhibition or transport for certain GPCRs, intracellular enzymes or drug transporters. Preferably the library includes information for each

tagged ligand comprised in the library, relating to the pharmacology for binding to or inhibition of a GPCR receptor or to inhibition of an intracellular enzyme such as cyclic nucleotide phosphodiesterases, or inhibition of or transport by a drug transporter including designation as agonist, antagonist, substrate or inhibitor and measure of affinity or inhibition etc, enabling quantification of results.

In the prior art methods of preparing ligands the linking sites have in many cases been non-specific or unknown, as in the case of Molecular Probes ligands, or at best have been specific or known but not predetermined, designed or rationalised for a desired effect. Preferably in the library of the invention tagged ligands comprise fluorophores linked at any of a number of linking sites at which ligand receptor binding, inhibition or transport is maintained to a greater extent or is modified or inhibited to a lesser extent. Preferably the library comprises tagged ligands designed from reaction of reactive precursor ligand (s) and reactive fluorophores having reactive site chemical functionality and suited for reaction with associated reagents, for site specific reaction and linking, wherein the design is the result of extensive investigation of all or many of the possible linking sites and the resulting pharmacological characteristics and selection of one or more linking combinations which provide favorable binding, inhibition or transport characteristics.

Preferably Lig is selected from a) xanthine like structures including XAC, theophylline, caffeine, theobromine, dyphilline, enprofylline and the like; or fused biaryl structures including papaverine, dihydroquinilones such as cilostamide, dipyridamole, vinpocetine and the like ; and analogues thereof; b) adenosine like structures including ADAC, NECA and analogues thereof; c) ethanolamine like structures including Salmeterol, salbutamol, terbutaline, <BR> <BR> quinprenaline, labetalol, sotalol, bambuterol, fenoterol, reprotolol, tulobuterol, clenbuterol and analogues thereof ; d) oxypropanolamine like structures including CGP12177, propranolol, practolol, acebutalol, betaxolol, ICI 118551, alprenolol, celiprolol (celectol), metoprolol (betaloc), CGP20712A, atenolol, bisoprolol, misaprolol, carvedilol, bucindolol, esmolol, nadolol, nebivolol, oxprenolol, xamoterol, pindolol, timolol and analogues thereof; e) xanthine like structures including XAC, theophylline, caffeine, theobromine, dyphilline, enprofylline, sildenafil, EHNA (erytliro-9- (2-hydroxyl-3-nonyl) adenine), zaprinast and the like; or spiro bicyclic structures including bypyridines such as amrinone, imidazolines such as CI930, dihydropyridazinones such as indolan, rolipram, SB207499, and the like ; or fused biaryl structures including papaverine, dihydroquinilones such as cilostamide, dipyridamole, vinpocetine and the like and analogues thereof.

Linker L may perform a number of functions including preventing loss of affinity of a ligand when modified to comprise a fluorescent moiety, by distancing the fluorophore moiety from the ligand structure, in cases that modifying by direct linking of Lig and Fl would interfere with ligand binding, inhibition or transport in which case a linker L may be designed as a short, medium or long chain structure as appropriate.

A library compound of formula I or I'may optionally comprise functionality J as hereinbefore defined derived from its synthesis by the reaction of one or more reactive group (s) of a linker precursor or its components, providing a linker moiety, with a reactive group of one or more ligand precursors providing a ligand moiety and reaction of one or more other reactive group (s) of the linker precursor with a reactive group of one or more tag precursors such as a fluorescent tag precursor providing a tag moiety.

In a particular advantage of the present invention linker L and/or linking site or functionality J facilitates linking of fluorescent moiety and ligand, in cases that groups of respective moieties are not reactive, or that stereochemistry or other effects would inhibit linking, or that reaction of existing reactive groups in commercially available precursor ligands and fluorophores would require the inclusion of protecting groups for functionalities present therein, in which case a linker is usually derived from a short, medium or long chain structure. In a further advantage linker- L-may be derived from a tri-, tetra-, penta-or hexa-functional precursor, linking 3 or more ligands Lig and tags Fl, enabling modified or more complex binding, inhibition or transport and associated pharmacology, for example binding to a plurality of receptor sites to explore receptor dimerisation such as homo or heterodimerisation. In a further advantage of the invention linker L may confer properties facilitating crossing the cell membrane, hydrophobicity, hydrophilicity and the like as required, in which case a linker is usually any functionalised structure.

Preferably L is selected from a saturated or unsaturated single or double bond,-0-,- S-, amine, COO-, amide,-NN-hydrazine ; and saturated or unsaturated, substituted or unsubstituted Cl 600, preferably Cl 3oo, more preferably Cl-loo branched or straight chain aliphatic, aromatic, alicyclic and combinations thereof, any of which may comprise one or more heteroatoms selected from N, O, S, P, wherein optional substituents are selected from any Cl 20 aliphatic, aromatic or alicyclic substituents any of which may comprise one or more heteroatoms as hereinbefore defined, hydroxy, thiol, halo, amine, hydrazine, oxo, cyano, carbonyl and the like.

More preferably L is selected from a single bond,-O-,-S-, amino; and branched or straight chain Cl 50 alkyl, alkenyl, alkynyl, alkoxy, amino, cycloalkyl, heterocyclic, aryl, heteroaryl, and combinations thereof such as aralkyl, aralkylamino, aralkylamido and the like, optionally comprising one or more heteroatoms wherein heteroatoms are as hereinbefore defined, optionally substituted as hereinbefore defined wherein substituents are selected from Cl 12 aliphatic, aromatic or alicyclic substituents as defined, hydroxy, thiol, halo, amine, oxo, carbonyl, and the like.

JL and JT may comprise functionality derived from a reactive group or site for linking to fluorophore and/or to ligand selected from a saturated or unsaturated single or double bond,-O-,-S-, amino, amido, hydrazine, carbonyl, oxo, alkyl, alkenyl, alkynyl, alkoxy, thioxy, and the like.

In the case that L comprises a single or double bond, JL and JT if present may comprise functionality derived from a reactive group or site for linking linker and fluorophore derived from the fluorescent moiety and/or the ligand moiety.

Preferably the moiety JL.. L Jim comprises a mono, di, tri, tetra, penta or hexa amino, alkylthio, alkoxy, carboxylic acid, and combinations thereof more preferably a mono, di or tri aminoalkylthio, amino alkoxy, alkoxy carboxylic acid, alkoxy amine and the like. Preferably JLm L Jim is selected from mono, di or tri amino menthane, amino ethane, thio ethane, ethane, amino acyl, from polypeptide, or from mono or polyether derivatives thereof eg diamine or dithio such as mono or polyethylene glycol di or tri amine or thio.

Preferably a linker moiety JLm L JTn, as hereinbefore defined comprises a single or double bond or a single atom or group as hereinbefore defined or comprises a mono-, di-, tri-or tetrafunctional linear or branched or cyclic substituted or unsubstituted hydrocarbyl of formula-L. I- J[A] qL RL [A'qL'J' ]m-1 A''qL''J'' wherein each of J to J'' is a linking site or functionality as hereinbefore defined independently selected from a single bond, methylene, alkyne, alkene, NR, O, NRCO, S, CO, NCO, CHHal, P and the like wherein R is H or Cl 8 alkyl or cycloalkyl or forms part of a cyclic ring with N, Hal is any halogen selected from chlorine, iodine, bromine ; and is present in any rational location in a group A to A" ; each of A to A"is a group selected from-O-,-C (=O)-, CI-12 alkoxy, alkoyl, cycloalkyl, heterocyclic, alkyl, alkenyl, aryl, arylamide, arylamine, amino, thioalkyl, heteroaryl as hereinbefore defined and combinations thereof and the like, optionally substituted by groups selected independently from Cl-3 alkyl, CI-5 alkoxy and the like ; each of qL to qL"are independently-selected from 0 or 1 or indicates an oligomeric repeat and is from 2 to 30, or indicates a polymeric repeat unit and is from 31 up to 300.

RL is a C, N or S atom or is a CRL, RL', alkyl, cycloalkyl, heterocyclic, aryl heteroaryl, amine or thio moiety and provides for branching when p is 1 or 2 ; wherein RL iS H or Cl 3 alkyl ; and p is as hereinbefore defined and is 0, 1 or 2.

Preferably each J, J'and J"independently is a single or double bond, NRL,-0 or - S or-C (O) or-NRC (O) or-C (O) NR, as hereinbefore defined A is alkoxy preferably CH2CH20 (PEG) and oligomers thereof or is aralkylamine aralkylamide, aralkyloxy, or is alkyl, preferably (CH2) 1- 12 RL is a Cl 5 alkyl chain comprising or containing a single or double branching C atom when p is 1 or 2; p is 0, 1 or 2 ; A'and A"are each selected from Cl_$ alkyl, amine, phenylamine, phenylamide ; and qL is 0,1, 2 to 30 or 31 to 300, and qL. and qL » are 0 or 1

More preferably JLm L Jim is a single bond or is of formula J AqL RL J wherein each of J and J"is amine or -O-, A is CH2CH2O, qL is 1-30 or 31 to 300 and RL is CH2CH2 or of formula J AqL RL (A'J') J" wherein each of J, J'and J"independently is amine,-0 or a single bond, qL is 1, 2 or 3-30 or 31 to 300 and A is CH2CH2O or HNCH2CO or qL is 1 and A is C (O) or (CH2) i-s or qL is 0, kL is CH or CH2CH, qL is 0 or qL'is 1 and A'is CH2 and qL is 0 preferably O (CH2CH20) qLCH2CH2NH, O (CH2CH20) qLCH2CH (CH2NH) NH, OCH (CH2NH) NH,-CH (CH2NH) NH, -C (O) NH-, -(CH2)1-8-, (-HNCH2CO-)1-3 (= - gly1-3-) - or the like.

More preferably each compound of formula I or I'as hereinbefore defined comprises a moiety Lig and L as hereinbelow defined: Wherein: Lig. am is suitably of the formula, in either of the following forms given, including any of its possible linking configurations or sites: Lig. a lm Wherein any or each of Ra to Ra4, Xl and X2 may comprise a linking site or functionality J as hereinbefore defined X1 and X2 are each independently selected from H, O, OR. a, NR. a, NHR. a; Xl and X2 are each preferably O ; each of R. a, R. al, R. a2 and R. a3 independently is selected from H or Ci-4 linear or branched alkyl, preferably H, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl or isobutyl optionally mono or multi hydroxy or halo substituted, such as CH2OH, CH2F or CH2CHOHCH20H; R. a4 is selected from a heteroatom O, S or substituted or unsubstituted amine or saturated or unsaturated, substituted or unsubstituted C1-20 branched or straight chain aliphatic, aromatic, alicyclic and combinations thereof, any of which may comprise one or more heteroatoms selected from N, O, S, P ; wherein optional substituents are selected from any CI-12 aliphatic, aromatic or alicyclic substituents any of which may comprise one or more heteroatoms as hereinbefore

defined, hydroxy, thiol, halo, amine, hydrazine, oxo, cyano, and the like; preferably R. a4 is selected from optionally substituted aryl, cycloalkyl, alkyl, ketone, (di) amine, (di) amide, more preferably optionally substituted alkoxy, cycloalkyl, amine, amide, carboxylic acid or optionally o-, m- or p-substituted phenyl wherein substituents include aryl, alkyl, cycloalkyl, heteroaryl or heteroalkyl, amine, amide, carboxyl, carbonyl etc, for example substituents include, or R. a4 comprises, cyclohexyl, cyclopentyl, ethoxy, (CH2) 2PhPh, CH2Ph, CONH (CH2) nCONH, CH2CONH (CH2) 2NH, CH2PhNHCOCH2, CH2CH20COCH2, succinimidyl ester, NHCOCH2, CH2 (CH3) NCOCH2, H2N (CH2) 2NHCOCH2, H2N (CH2) 8NHCOCH2, H2NNHCOCH2, CH2CONH (CH2) 2NHCOCH2, HOPhCH2N (CH2CH3. HOAc) (CH2) 2NHCOCH2, heterocyclic- (CH2) 4CONH (CH2) 2NHCOCH2, heterocyclic-NHCON (heterocyclic) COCH2 and the like; or Lig. a is of the formula Lig. a2- wherein any or each of Ras to Ra6, or a cyclic C or heteroatom may comprise a linking site or functionality J as hereinbefore defined each of C. AI and C. A2 is independently selected from 5-6 aryl, heteroaryl, cycloalkyl and heterocyclic, more preferably from phenyl, or aryl containing 1 or 2 ring heteroatoms, or heterocyclic containing 1 ring heteroatom and/or 1 ring-C=C-group ; Each of up to seven R. as is a substituent of a ring carbon or a ring heteroatom and: is independently selected from H, halo, hydroxy, thiol, amine, COOH, hydrazine, cyano, saturated or unsaturated, substituted or unsubstituted Cl 20 branched or straight chain aliphatic, aromatic, alicyclic and combinations thereof, any of which may comprise one or more heteroatoms selected from N, O, S, P, and wherein optional substituents are selected from any CI-12 aliphatic, aromatic or alicyclic substituents any of which may comprise one or more heteroatoms as hereinbefore defined, hydroxy, thiol, halo, amine, hydrazine, oxo, cyano, and the like, such as =O, OCH3, CH2Ph (OCH3) 2, O (CH2) 3CON (CH3) c. hex, N (CH2CH20H) 2, c. hex, COOCH2CH3, CH2CH3 ; or any two or more of R. as form a one, two or three ring fused cyclic structure, preferably comprising a fused 3 ring aryl, 5-heterocyclic, 6-heterocyclic structure having 4 ring atoms common with the fused bicyclic Lig. 2structure ; and R. a6 is a moiety as defined for R. as above; and L. a is as hereinbefore defined for L or JL L JT and is suitably of formula L. I or subformulae as hereinbefore defined, more preferably is selected from a single bond,

amino acid or amide such as a peptide or polypeptide for example gly or gly3, alkyl of formula- (CH2) n where n is 3 to 8, preferably 3,4 or 6, optionally including one or more heteroatoms or unsaturated groups, such as-O-or-S-or-CH=CH-and the like: Lig. b is suitably of the formula Lig. b including any of its possible linking configurations or sites: Lig. b wherein any or each of Rbl to Rb5 or Xbl to Xb3 may comprise a linking site or functionality J as hereinbefore defined ring substituents X. bl and X. b2 are independently selected from hydrocarbon such as alkyl or SRx, NRx. 2 and ORx wherein (each) Rx is selected from H, Ci-salkyi, alkenyl ; ring heteroatom X. b3 is selected from-S-,-O-and-CH2-; Rbl is selected from saturated or unsaturated, substituted or unsubstituted C1-4 aliphatic, or Cl 3 alicyclic optionally including one or more heteroatoms N, 0, S, P, wherein substituent (s) are selected from one or more cycloalkyl, heterocyclic, hydroxy, oxo, halo, amine ; preferably R. bl comprises a carbonyl substituted by H, alkyl or a linear or cyclic primary, secondary or tertiary amine, substituted C1-3 alkyl, cycloalkyl or amide, more preferably cyclopropyl, or CONHC1- 3alkyl such as CONHEt or CH20H and each of R. b2 and R. b3 is selected from H, halo, hydroxy, thiol, amine, COOH, CHO, hydrazine, cyano or saturated or unsaturated, substituted or unsubstituted Cl 20 branched or straight chain aliphatic, aromatic, alicyclic and combinations thereof, any of which may comprise one or more heteroatoms selected from N, O, S, P; wherein optional substituents are selected from any Cl-l2 aliphatic, aromatic or alicyclic substituents any of which may comprise one or more heteroatoms as hereinbefore defined, hydroxy, thiol, halo, amine, hydrazine, oxo, cyano, and the like, preferably from H, halo or hydroxy, preferably H or Cl; Rb4 is H; Rb5 is H or alkyl L. b may comprise a linking site or functionality J as hereinbefore defined ; and

is as hereinbefore defined for L or its subformulae, more preferably is saturated and unsaturated substituted or unsubstituted C1-12 aliphatic or Cl 24 aromatic as defined for L preferably including one or more heteroatoms O, S or N, cyclic or heterocyclic groups, more preferably is of formula L. I or its subformulae as hereinbefore defined, most preferably is (CH2) m wherein m is 2 to 12, preferably 3,4, 6 or 8, or is (Ph-CH2CONH) 2 (CH2)2 ; Lig. c is suitably a non-peptide of the formula Lig. c including any of its possible linking configurations or sites: Where any or each of Rcl to Rc2 or OH, or a chain C or N may comprise a linking site or functionality J as hereinbefore defined * indicates an optically active centre and Wherein R. cl is C6 l4 aryl optionally including one or more heteroatoms selected from H, O, optionally substituted by OH, Hal eg Cl, NH2, NHC1- 3alkyl, sulphonamide, oxoamine (-CONH2) and the like, more preferably mono, di or tri substituted phenyl or quinoline wherein substituents include OH, Cl or NH2, more preferably m-CE2OH, p- OH phenyl, m-, p-dihydroxy phenol or m-, m-dihydroxyphenol, m-, m- diCl, p-NH2 phenol, p-OH, m-CONH2 phenol or 5-OH, 8-quinoline and the like, such as R. c2 is selected from saturated or unsaturated, substituted or unsubstituted Cl 20, preferably Cl l2, branched or straight chain aliphatic, aromatic, alicyclic and combinations thereof, any of which may comprise one or more heteroatoms selected from N, O, S, P ; wherein optional substituents are selected from any optionally substituted C1-12 aliphatic, aromatic or alicyclic substituents any of which may comprise one or more heteroatoms as hereinbefore defined, hydroxy, thiol, halo, amine, hydrazine, oxo, cyano, and the like and combinations thereof ; Preferably R. c2 is selected from C1-6 branched or straight chain aliphatic, C6-10 araliphatic optionally substituted by OH and optionally including heteroatoms selected from N, O, preferably including an ether O, such as selected from-(CH2) 60CH ((CH2) 3Ph), CHCH3 (CH2) 2Ph, CHCH3CH2PhOH, C (CH3) 2CH2 or from the structures:

L. c may be present as R. c2 or may comprise a linking site or functionality J as hereinbefore defined, and is as hereinbefore defined for L and is suitably of formula L. I or its subformulae as hereinbefore defined, more preferably is selected from CI-i2 alkyl, amide etc; Lig. d is suitably a non-peptide of the formula Lig. d including any of its possible linking configurations or sites: Lig. d R. dl OCH2C*HOHCH2NH-R. d2 where any or each of Rdl to Rd2 or OH, a chain C or N may comprise a linking site or functionality J as hereinbefore defined indicates an optically active centre Wherein R. dl is saturated or unsaturated, substituted or unsubstituted Cl 20 branched or straight chain aliphatic, aromatic, alicyclic and combinations thereof, any of which may comprise one or more heteroatoms selected from N, 0, S, P wherein optional substituents are selected from any Cl l2 aliphatic, aromatic or alicyclic substituents any of which may comprise one or more heteroatoms as hereinbefore defined, hydroxy, thiol, halo, amine, hydrazine, oxo, cyano, and the like; Preferably R. dl is substituted or unsubstituted CI-24 aralkyl or heteroaralkyl, including single ring and fused ring systems with (hetero) aryl or cycloalkyl rings, wherein optional substituents include Cl-6 alkyl, alkoxy, ether, carbonyl, alkenyl, amine, amide each optionally carbonyl, amide, halo or OH subtitited, or halo such as chloro or OH, preferably R. dl is unsubstituted or substituted alkyl, alkenyl, halo, amine, amide, carbonyl, ketone, ether substituted phenyl or naphthyl, illustrated as follows, most preferably mono-, di-, tri-or tetra substituted mono or polycyclic fused aryl or cycloaryl or heterocycloaryl such as phenyl, carbazole or structures shown below or spiro ring systems, most preferably mono-, di-, tri-or tetra alkoxyalkyl, alkoxyalkoxyalkyl or CF3 substituted phenyl or unsubstituted or monosubstituted naphthalene or 5,6 ring systems most preferably of the structures:

R. d is substituted or unsubstituted amine, saturated or unsaturated, substituted or unsubstituted Cl l2 branched or straight chain aliphatic, aromatic, alicyclic and combinations thereof, any of which may comprise one or more heteroatoms selected from N, O, S, P; wherein optional substituents are selected from any CI-12 aliphatic, aromatic or alicyclic substituents any of which may comprise one or more heteroatoms as hereinbefore defined, hydroxy, thiol, halo, amine, hydrazine, oxo, cyano, and the like, more preferably amine, C1-6 branched or straight chain alkyl optionally including ether 0, and optionally substituted by C6 l0 aryl, for example i. pr, i. bu, or of the formula: L. d may be present as R. c2 or may comprise a linking site or functionality J as hereinbefore defined and is as hereinbefore defined for L and its subformulae and is suitably of formula L. I and its subformulae as hereinbefore defined, more preferably is a single bond or is as hereinbefore defined for L. a; Lig. e comprises a cell permeant moiety or is associated with a cell permeat L or Fl moiety and is suitably of the formula, in either of the following forms given including any of its possible linking configurations or sites: Lig. el wherein any or each of Rel to Re4, X and a ring C or N may comprise a

linking site or functionality J as hereinbefore defined h is selected from each optionally substituted by lRe3-R. e4 wherein R. el-R. e4 are as R. al-R. a4 defined above or in which R. e3 is C5-glinear or branched alkyl, optionally mono or multi hydroxy or halo substituted or is aryl optionally substituted by alkoxy, sulfonyl and the like eg ortho-OEt, meta-S02N NCH3 each X is independently selected from H, O,-OR. e2, N, HN, NR. e5, HR. e6, and aryl optionally substituted by ether ; or X is aryl optionally alkyl or alkoxy substituted such as Ph-ortho-OCH2CH2CH3 ; and where R. e5 is as defined above for R. el above or forms a fused cyclic ring together with the adjacent ring N atom; preferably 1 or 2 fused 5 membered cyclic rings; and R. e6 is as defined above for R. el above or is selected from optionally substituted phenyl wherein optional substituents include ether such as o-ethoxy or o-propoxy, alkyl, OH and the like, sulphonyl, carbonyl and the like substituted by heterocyclic, or cyclic C5-8 alkyl such as methyl, piperazinyl, sulphonyl and the like; or Lig. e is of the formula Lig. e2

Wherein any or each free ring atom or their substituents may comprise a linking site or functionality J as hereinbefore defined each spiro ring optionally comprises zero or one or more heteroatoms h which are preferably N, more preferably (h) zero or 1 N heteroatom and 0 6 (h) comprises zero, 1 or 2 N heteroatoms and is unsaturated or comprises one or two - C=C-or-C=N-groups ; and wherein each ring is optionally substituted by one or more oxo, CO, COOH, Cl 6 alkyl or linear or cyclic alkoxy such as methoxy, ethoxy or cyclopentyloxy optionally substituted by one or more oxo, CO, COOH, CN, or C1 6 alicyclic or amine groups, amine or one or more spiro or fused heterocycles; or Lig. e is of the formula Lig. e3 Wherein any or each of Re to Rel2, or a ring C or heteroatom or ring substituent may comprise a linking site or functionality J as hereinbefore defined each of C. EI and C. E2 is independently selected from 5-6 aryl, heteroaryl, cyloalkyl and heterocyclic, more preferably from phenyl, or aryl containing 1 or 2 ring heteroatoms, or heterocyclic containing 1 ring heteroatom and/or 1 ring-C=C-group ; Each of up to seven R. tell ils a substituent of a ring carbon or a ring heteroatom and: is independently selected from saturated or unsaturated, substituted or unsubstituted Cl 20 branched or straight chain aliphatic, aromatic, alicyclic and combinations thereof, any of which may comprise one or more heteroatoms selected from N, O, S, P, and wherein optional substituents are selected from any Cl-12 aliphatic, aromatic or alicyclic substituents any of which may comprise one or more heteroatoms as hereinbefore defined, hydroxy, thiol, halo, amine, hydrazine, oxo, cyano, and the like, such as =O, OCH3, CH2Ph (OCH3) 2, O (CH2) 3CON (CH3) c. hex, N (CH2CH20H) 2, c. hex, COOCH2CH3, CH2CH3 ;

or any two or more of R. ell form a one, two or three ring fused cyclic structure, preferably comprising a fused 3 ring aryl, 5-heterocyclic, 6-heterocyclic structure having 4 ring atoms common with the fused bicyclic Lig. e3 structure; and R. el2 is a moiety as defined for R. ell above ; Preferably Lig. e is of the formula Lig. el as hereinbefore defined in particular where R. e2 and R. e3 are respectively propyl and butyl; L. e may comprise a linking site or functionality J as hereinbefore defined and is suitably as hereinbefore defined for L. a.

Linking sites J as hereinbefore defined are suitably of any nature and location, ie any sites, which do not inhibit binding, inhibition or transport. Receptor binding is complex, and may require a specific binding site to be available and/or require a specific fluorescent ligand conformation.

The fluorescent ligands of the library of the invention may be characterised by different linking sites linking ligand and fluorescent moiety as hereinbefore defined.

From a comprehensive knowledge of the binding, inhibition or transport behaviour and the specific target sites, which remain unchanged in the fluorescent ligands of the invention, we have been able to determine a method for selecting suitable linking sites for desired retention of binding, inhibition or transport and pharmacological properties. Preferably the compounds of formula I or I'include compounds representing all operative linking configurations exposing possible binding, inhibition or transport site options.

Fl may include any red, green, near ir, blue or the like absorbing dyes and other classes of dyes. Suitably Fl is selected from dyes in particular including fluorescein, fluorescein derivatives including FITC, and fluorescein-like molecules such as Oregon Green and its derivatives, Texas red, 7-nitrobenz-2-oxa-1, 3-diazole (NBD) and derivatives thereof, coumarin and derivatives, naphthalene including derivatives of dansyl chloride or its analogues or derivatives, Cascade Blue, EvoBlue and fluorescent derivatives thereof, pyrenes and pyridyloxazole derivatives, the cyanine dyes, the dyomics (DY dyes and ATTO dyes) and fluorescent derivatives thereof, the Alexafluor dyes and derivatives, BDI dyes including the comercially available Bodipy dyes, erythosin, eosin, pyrenes, anthracenes, acridines, fluorescent phycobiliproteins and their conjugates and fluoresceinated microbeads, Rhodamine and fluorescent derivatives thereof including Rhodamine Green including the tetramethylrhodamines, X-rhodamines and Texas Red derivatives, and Rhodol Green, coupled to amine groups using the isocyanate, succinimidyl ester or dichlorotriazinyl-reactive groups and other red, blue or green absorbing fluorescent dyes in particular red absorbing dyes as reviewed in Buschmann V et al, Bioconjugate Chemistry (2002), ASAP article.

More preferably Fl is selected from fluorescein derivatives and fluorescein-like molecules such as Oregon Green and its derivatives, Texas red, 7-nitrobenz-2- oxa-1, 3-diazole (NBD) and derivatives thereof, coumarin and derivatives,

naphthalene including derivatives of dansyl chloride or its analogues or derivatives, Cascade Blue, EvoBlue and fluorescent derivatives thereof, pyrenes and pyridyloxazole derivatives, the cyanine dyes, the dionics (DY dyes and ATTO dyes) and fluorescent derivatives thereof, the Alexafluor dyes and derivatives, BDI dyes including the commercially available Bodipy dyes, erythosin, eosin, FITC, pyrenes, anthracenes, acridines, fluorescent phycobiliproteins and their conjugates and fluoresceinated microbeads, Rhodamine derivatives thereof including Rhodamine Green including the tetramethylrhodamines, X-rhodamines and Texas Red derivatives, and Rhodol Green.

More preferably Fl comprises fluorescein, Texas Red , Cy5.5 or Cy5 or analogues thereof, BODIPYTM 630/650 and analogues thereof, DY-630, DY-640, DY-650 or DY-655 or analogues thereof, ATTO 655 or ATTO 680 or analogues thereof, EvoBlue 30 or analogues thereof, Alexa 647 or analogues thereof.

Suitably Fl is derived from any of the above commercially available fluorophores, comprising or modified to comprise a reactive group facilitating linking to a ligand by a moiety J as hereinbefore defined. Preferably Fl comprises any of the above commercially available fluorophores modified to form a derivative or group of derivatives suitable for visualising ligand binding, inhibition or transport in a library as hereinbefore defined comprising JT-t-F1 wherein JT is as hereinbefore defined and comprises functionality derived from linking to a precursor ligand as hereinbefore defined and may optionally comprise a linking group-t-which is a proximal unsaturated or aryl moiety, comprising a medial short, medium or long chain alkynyl or cycloalkyl moiety and comprising a moiety derived from linking via a reactive group as hereinbefore defined such as carboxyl, sulphonate or as a heteroatom such as O or S or methylene derived from linking at an alkylhalide such as methylbromide, haloacetamide, sulphonate ester or the like electrophilic group.

For example Fl may include a substituent-t-which performs a fluorescence modifying function, for example is a heteroaryl or alkenyl such as mono-, di-or tri- enyl group which shifts the fluorescence of the compound to the red part of the spectrum and raises the absorption max value, or performs a linking function.

Preferred BODIPYTM (4, 4-difluoro-4-bora-3a, 4a-diaz-s-indacene) fluorophores include those which span the visible spectrum and include those listed in U. S. Pat.

No. 4,774, 339; U. S. Pat. No. 5,187, 288 ; U. S. Pat. No. 5, 248, 782 ; U. S. Pat. No.

5,274, 113; U. S. Pat. No. 5,433, 896; U. S. Pat. No. 5,451, 663. A preferred member of this group is selected from any heteroaryl substituted BODIPY dyes as described in the above patents the contents of which are incorporated herein by reference.

Suitably JT-t-Fl comprising a BODIPY TM structure is characterised by a dipyrrometheneboron difluoride core, optionally modified by one or two fused rings, optionally substituted by one or several substituents such as alkyl, alkoxy, aryl, heterocyclic and the like, wherein one substituent-t-is adapted for linking as hereinbefore defined to a ligand precursor as hereinbefore defined, the substituent-t- optionally comprising a proximal unsaturated or aryl moiety, comprising a medial short, medium or long chain alkynyl or cycloalkyl moiety and comprising a moiety

derived from linking via a reactive group as hereinbefore defined such as carboxyl, sulphonate or as a heteroatom such as O or S or methylene derived from linking at an alkylhalide such as methylbromide, haloacetamide, sulphonate ester or the like electrophilic group.

Fl may include a substituent-t-as hereinbefore defined which is heteroaryl or alkenyl such as mono-, di-or tri-enyl group which shifts the fluorescence of the compound to the red part of the spectrum and raises the absorption max value as in US 5187288; or may include alkenyl substituent linked to one or more of an aryl, carbonyl or like group, preferably linked to a fatty acid sidechain comprising (CH- 2) nCOaH where n = 5-22 as in US 5330854, more preferably linked via an aryloxymethylene to a and carbonyl ; or may include an aryl alkenyl aryl group as in US 6005113.

More preferably-Fl is of the formula-Fll : Fll dipyrrometheneborondifluoride analogues including any of its possible linking configurations or sites: Wherein any or each of Rl to R7, or a ring atom may comprise a linking site or functionality J as hereinbefore defined R7 is N or C-R8 ; Substituents R1, R2, R3, R4, R5, R6 and R8 which may be the same or different are H, halogen, nitro, sulfo, cyano, aLkyl, perfluoroalkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, arylalkyl, or acyl wherein the alkyl portions of each contain fewer than 20 carbons ; or substituted or unsubstituted aryl or heteroaryl; preferably at least four of fRl to R8 are non-hydrogen, alternatively adjacent substituents Rl and R2 taken in combination and adjacent substituents R5 and R6 taken in combination form fused 6- membered (hetero) aromatic rings or including any of its possible linking configurations or sites: wherein any or each of R3, R4 or R7, or a ring atom may comprise a linking site or functionality J as hereinbefore defined each fused ring is optionally and independently substituted by H, halogen, nitro, sulfo, cyano, alkyl, perfluoroalkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, alkylthio, alkylamido, amino, (mono or dialkyl) amino (wherein the alkyl portions of each

contain fewer than 20 carbons), or substituted or unsubstituted aryl, heteroaryl, arylamido, heteroarylamido, aryloxy, heteroaryloxy, arylamino or heteroarylamino; or 1 to 2 additional fused benzo or heteroaromatic rings that are optionally substituted or unsubstituted.

Preferably any or all of R23 to R45 is heteroaryl, more preferably a single ring single heteroatom such as such as pyrrole, thiophene, furan or single ring di heteroatom structure such as oxazole, isoxazole, oxadiazole, imidazole, or multi ring such as benzoxazole, benzothiazole, benzimidazole, or multi ring one heteroatom structure such as benzofuran, indole, preferably thienyl.

More preferably Fl is selected from the BODIPY core structures of formulae FL. A1 or FL. A2 as shown below, in each case = indicating the attachment site of a sidechain and including any of its possible linking configurations or sites: Fl. Al preferably comprising or derived from BODIPY TMR or BODIPY FL (4,4-difluoro- 5,7dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-propionic acid) or BODIPY FL ethylene diamine including any of its possible linking configurations or sites:

BODIPY TMR BODIPY FL ethylene diamine (X is CONH (CH2) 2NH2) or BODIPY FL (X is COOH) Or Fl. A2 including any of its possible linking configurations or sites:

preferably comprising or derived from BODIPY 630/650 or BODIPY 630/650 methyl bromide including any of its possible linking configurations or sites: BODIPY 630/650 methyl bromide BODIPY 630/650 BODIPY 630/650 X

most preferably the succinimidyl esters thereof, for example BODIPY 630/650 X- SE.

In a further aspect of the invention there is provided a process for the preparation of a library as hereinbefore defined comprising the reaction of one or each of a plurality of ligand precursors and tag precursors comprising linker moieties or ligand, tag and

linker precursors wherein linking may be at same or different reactive sites in different compounds as hereinbefore defined.

Preferably the process is a combinatorial process. Preferably the process comprises the reaction of one or more ligand precursors of formula IV and/or IV' IV (LigJL) m-L-YLn IV'Lig YL ; gn comprising one or more or different reactive groups YL or YLjg forming a linking functionality J, JL or JT as hereinbefore defined with one or more of a plurality of analytical tagging substrates of formula V and/or <BR> <BR> V'<BR> <BR> <BR> <BR> <BR> <BR> V YTm Tag . YTm L (JTTag) m comprising one or more or different reactive groups YT forming a linking functionality J or JT as hereinbefore defined and optionally one or more linking species VI or VI'or VI" VI YLm L YLm wherein Lig, J, L, JT and Tag and each m is independently as hereinbefore defined wherein the or each compound of formula IV or IV'is capable of reaction with the or each compound of formula V or V', optionally via the or each species VI or VI'or VI"to form a plurality of compounds of formula I as hereinbefore defined.

Preferably in some or each compound of formula V or V', Tag is Fl as hereinbefore defined, whereby the process is a process for preparing a library comprising a plurality of compounds of which one or more or all of which are of formula I'as hereinbefore defined.

Suitably reactive groups YLjg, YL9 YT have suitable reactive group functionalities for linking, as hereinbefore defined, for example by substitution or by addition or addition-elimination reaction. Substitution reaction is suitably selected from reaction of electrophilic and nucleophilic reactive sites as hereinbefore defined such as : Electrophilic Nucleophilic Resulting covalent leaving Y Y Linkage, J groups Carboxylic acid alcohol ester-OH,-H Carboxylic acid amine carboxamide-OH, -H Carboxylic acid hydrazine hydrazide-OH, -H Alkyl halide alcohol ether-Hal,-H Alkyl halide thiol thioether-Hal,-H Alkyl halide amine alkylamine-Hal,-H Alkyl halide COOH ester-Hal,-H Haloacetamides thiols thioethers-Hal,-H Sulphonate esters amines alkyl amines RSO3-,-H Sulphonate esters alcohols ethers RS03-,-H Sulphonate esters thiols thioethers RSO3-,-H Sulphonyl halides amines sulphonamides-Hal, -H Sulphonyl halides alcohols sulphonate esters-Hal,-H

Succinimide ester alcohols esters-OSu*,-H Succinimide ester alkoxides esters-OSu*, HorS Succinimide ester thiols thioesters-OSu*, -H Succinimide ester amine carboxamide-OSu*, -H Succinimide ester hydrazine hydrazide-OSu*, -H Addition reaction is suitably selected from cycloaddition or addition-elimination reaction of electrophilic and nucleophilic reactive sites in IV and V as hereinbefore defined: Electrophile Nucleophile Covalent Leaving Y Y Linkage, J Group <BR> <BR> azide alkyne triazole l none 2-acyl cyclic mono-dinucleophile 6, 7-dihydro-lH-indazol-4 (5H)-one H20 /di-ketone eg hydrazine 4,5, 6, 7-tetrahydro-lH-indazole H20 (5 or 6 mem ring) 1, 4, 5, 6-tetrahydrocyclopenta [c] pyrazole H2O 5,6-dihydrocyclopenta [c]pyrazol-4(1H)-one H2O wherein * is [3+2] dipolar cycloaddition Preferably a compound of formula I or IV'comprises no protecting group and is capable of reaction with a compound of V or'optionally via a compound of VI, without degradation of functionality by choice of reaction and of respective reactive sites ; or a compound of formula IV or IV'comprises one or more protecting groups which are adapted for removal under ambient conditions, for example under neutral pH, room temperature or the like. Preferably the process comprises reaction wherein reactive groups Y are selected so as to enable reaction with a fully deprotected ligand ie without the need for protecting groups or so as to enable reaction with protecting groups present which may be removed under mild conditions, for example one of Yug or YL or YT comprises amine or alcohol or thiol and the other comprises succinimide ester.

In the case that choice of reactive groups requires protection of compounds of formula IV or IV', a protecting group is preferably such as to allow removal under mild conditions, preferably comprises benzyloxycarbonyl and the like which are removed at ambient conditions such as room temperature or under conditions which do not prejudice functional groups such as the glycosidic group in Lig. b.

The process of the invention is characterised by a high yield of compounds of formula I or I'as hereinbefore defined by use of chemoselectivity and is superior to

known methods which prejudice yields by use of non chemoselective reactive groups or protecting groups.

Preferably the compounds of formula I or I'are obtained by: reacting the unprotected primary alkyl amine group a compound of formula IV as hereinbefore defined with a compound of formula V comprising a reactive succinimidyl ester group in solvent at ambient temperature without need for subsequent deprotection. In a particular advantage of the invention the method provides greater yield than with the prior art processes.

Compounds of formula IV, IV', V', V'or VI may be commercially available or may be prepared by known means. A linker may be installed as an independent entity or may be constructed as part of a synthetic process as hereinbefore defined, preferably is synthesised as an additional substituent on the ligand moiety or fluorescent moiety prior to reaction thereof.

In a further aspect of the invention there is provided a process for the preparation of a compound of formula I as hereinbefore defined comprising the reaction of a compound of formula IV or IV'and a compound of formula V or V'and optionally additionally VI, as hereinbefore defined.

In a further aspect of the invention there is provided a process for the preparation of a compound of formula IV as hereinbefore defined comprising : obtaining where commercially available or preparing the ligand precursor Lig, by routes as known in the art, and reacting with linker precursor VI", if required, or components thereof, and/or generating one or more reactive sites Y or YLjg or YL. Protection of IV may be required in which case reaction is followed by removing any protecting group present during the reaction, optionally replacing with a protecting group which may be removed under ambient conditions. A reactive group Y or YLjg or YL is preferably selected from groups as hereinbefore defined.

Preferably the process comprises: a), e) ring closure of 5, 6-diamino-1, 3-dialkyl uracil with the appropriate substituted aldehyde under acid conditions with ferric chloride, b) reacting Lig. b- comprising a protected inosine derivative with chlorinating agent and linking the chloro derivative with the amine group of a suitably protected amine reactive linker H-L-PL wherein PL comprises N-benzyloxycarbonyl-to form Lig. b- L-PL and removing PL to generate Lig. b-L. b; preferably R. bl comprises a OH terminating group and protected inosine comprises Acyl protecting groups or R. bl comprises a stable group such as amine or amide and protected inosine comprises 2,2-dimethoxypropane protecting group; preferably the protected inosine is reacted with oxidising agent and protected alkylamine which is an N-alkylcarboxamide with removal of amine protecting group to generate a reactive ligand; c), d) reacting p-hydroxybenzaldehyde with formaldehyde under acid catalysis and protection of the resulting 4-hydroxy-3-hydroxymethylbenzaldehyde with dimethoxypropane to generate the resulting acetonide. Converting the Benzaldehyde to its corresponding epoxide and ring opening with a suitably protected linker such as

Boc-L. c-H supplies Ligm-L-PL. Finally, deprotection under acid conditions supplies Lig. cLc or Lig. dLd for coupling to an appropriate tag.

In a particular advantage of the present invention linker moiety L facilitates linking of fluorescent moiety and ligand moiety, in cases that moieties are not reactive, or that stereochemistry or other effects inhibit linking, or that reaction of existing reactive groups in commercially available compounds of formula IV or IV'and V or V'would require the inclusion of protecting groups for functionalities present therein, in which case a linker is usually a difunctional short, medium or long chain structure. In a further advantage of the invention linker L may confer properties facilitating crossing the cell membrane, hydrophobicity, hydrophilicity and the like as required, in which case a linker is usually any functionalised structure.

Preferably a linker precursor of formula VI as hereinbefore defined is selected from a heteroatomic species such as a species providing N, O, S, or P, or a branched or straight chain saturated or unsaturated, optionally heteroatom containing, Cl 600 reactive hydrocarbon and combinations thereof, which may be monomeric, oligomeric having oligomeric repeat of 2 to 30 or polymeric having polymeric repeat in excess of 30 up to 300 and comprises reactive groups or sites for linking to ligand and fluorophore selected from hydroxy, alkoxy, thiol, thioxy, amine, hydrazine, carbonyl and the like. In the case that a linker comprises a single bond, then a reactive site is usually present on the compound of formula IV', whereby is reactive with compound of formula V or V'.

Preferably a compound of formula VI comprises three, four, five or six reactive sites, for linking 3 or more ligands and tags of formula IV or V. Preferably a linker precursor is selected from any substrate which generates or donates a moiety L as hereinbefore defined.

Suitably a linker precursor of formula VI is a short, medium or long chain, comprising rationally designed functionality and comprising reactive sites providing functionality in moiety L as hereinbefore defined. Preferably a linker precursor of formula VI is a mono, di or mixed amine, hydroxy, thiol, carboxylic acid, acid chloride, acid fluoride, acid bromide, (acid halide), isocyanate NCO, isothiocyanate NCS, halide, alkylhalide, aldehyde, epoxide, sulphonyl chloride SOUCI or hydrazine NHNH2, more preferably is selected from mono, di or tri amino menthane, amino ethane, ethanethiol, hydroxy ethane, amino acid, from polypeptide, or from mono or polyether derivatives thereof eg diamine or dithiol such as mono or polyethylene glycol di or tri amine or thiol.

Preferably a linker precursor of formula VI is selected from any CI-12 substituted or unsubstituted alkylamine, aminoacid, cycloalkyl, aryl, heteroaryl, aralkyl, and the like providing one or more reactive end groups for linking to Fl, more preferably selected from (di) amine, comprising cyclic or linear amine, more preferably diamine menthane, or diamino ethylene, amino acid or polypeptide, or from mono or polyether diamine such as polyethylene glycoldiamine, more preferably from H2N (CH2) 4NHCO2CH2Ph, H2N (CH2) 5NHCO2CH2Ph, H2N ((CH2) 20) 2 (CH2) 2NHCO2CH2Ph and H2N (CH2) nNHBoc where n is 2 to 8.

Preferably a linker precursor comprises a linear or branched or cyclic substituted or unsubstituted alkyl having one, two or three reactive sites, of formula YLm L. I YLm wherein L. I is as hereinbefore defined Preferably each YLis independently selected from H, C02H, NH2, O, P, S and groups providing on reaction a single bond, alkyl such as methylene, alkyne, alkene, NH, NR, O, NRCO, S, CO, NCO, CHHal, P and the like wherein Hal is any halogen selected from chlorine, iodine, bromine, or wherein YL comprises protecting leaving groups ZL such as-NHCO2CH2Ph, H, OH, SH, halogen, amine, aliphatic, N-alkylcarboxamide, Boc and the like; In a further aspect of the invention there is provided a method for selecting a compound of formula I from a library as hereinbefore defined comprising the rational design of a library of compounds of formula I as hereinbefore defined using the process as hereinbefore defined, determining pharmacology for a plurality of or all compounds in the library and selecting a compound exhibiting desired pharmacology at a desired target.

Preferably the method comprises preparing a preliminary library of compounds, conducting screens to assess binding, inhibition, transport and the like, selecting compound identified in the screen as having beneficial properties, and modifying or functionalising by nature of moieties or linking location of linking on the basis of the indications from the screen to prepare an optimised library. In a particular advantage of the invention the molecular pharmacology and photochemistry from the screen feedback into the design of the library.

The linker strategy is in some cases specific for the tag to be used, whereby modifying the tag may require modifying the linker. We have surprisingly found that modifying a moiety without consequential modification of other moieties may result in an inactive compound which is for example incapable of binding.

In a further aspect of the invention there is provided a known or novel compound of formula I or I'as hereinbefore defined wherein the compound is associated with information relating to its pharmacological properties in the form of Spectral Properties given as Excitation Max and Emission Max, Fluorescence Lifetime and Emission quantum yield and Pharmacology defined in terms of cells expressing a GPCR receptor as hereinbefore defined or expressing an intracellular enzyme such as a cyclic nucleotide phosphodiesterase, or a drug transporter as hereinbefore defined and given as the Inhibition or Antagonism of receptor binding or of receptor functionality together with a value for the Inhibition (pKB) or Antagonism (pKI) binding constants, and optionally together with fluorescent images of the pharmacological binding in single living cells illustrating the defined inhibition or antagonism.

Preferably the compound is associated with information relating to its pharmacological properties wherein pharmacology is defined in terms of a cell or protein wherein the cell expresses a GPCR, intracellular enzyme or drug transporter

or the protein is a GPCR, intracellular enzyme or drug transporter preferably in terms of a CHO cell comprising GPCR receptors as hereinbefore defined, preferably selected from an adenosine receptor such as an A1-, A2A-, A2B-and A3-receptor, a beta-adrenoreceptor such as an pi, ß2- and P3-adrenoceptors or like receptor, or comprises an inhibitor of an intracellular enzyme such as cyclic nucleotide phosphodiesterases or a substrate or inhibitor of a drug transporter as hereinbefore defined; more preferably in terms of CHO-cells expressing human adenosine A1- receptor or beta-adrenoceptor or an inhibitor of an intracellular enzyme such as an inhibitor of intracellular phosphodiesterases. The pharmacological properties are given as ECso values for agonist stimulated-or pK ; values for antagonism of agonist stimulated second messenger generation, or substrate Km values or antagonist Ki values for stimulation or inhibition of intracellular enzymes or drug transporters.

Preferably a novel compound is of the formula I or I'as hereinbefore defined, more preferably is selected from formulae Lig. am L. a-Fl. an to Lig. em L. eFl. en as hereinbefore defined with the proviso that: a) when Lig is XAC ie in Lig. a when each of R. al and R. a is propyl, R. a3 is H and R. a4 is-Ph-OCH2CONH (CH2) 2NH-, and L is a single bond or-L is gly and n=3 or L is NCS, Fl is not fluorescein; or when Lig is XAC and L is a single bond or NCS, Fl is not fluorescein or NBD ; b) when Lig is adenosine Fl is not Fmoc (CA 134: 204756); or when Lig is ADAC, ie R. bl is CH2OH, R. b2 and R. b3 are H and L is- (Ph- CH2CONH) 2 (CH2) 2- or L is a single bond, Fl is not fluorescein, NBD or Rhodamine ; or when Lig is NECA (incorporating the moiety- (CH2) m) ie R. b2 and R. b3 are H and L is a single bond, or is- (CH2) m when m is 2, 4, 6,8 or 10 then Fl is not NBD, or when m is 3, 4, 6, 8, 10 or 12 then Fl is not dansyl ; or when Lig is N6-[2-(4-aminophellyl) ethylJadenosine and L is (CH2) 2PhNH, Fl is not FITC (CA 131: 56155 (8)) d) when Lig is CGP12177 and L (R. d2) is mono amine menthane, Fl is not BODIPY TMR ; or when Lig is CGP12177 and L is 1,1, 4, 4-tetramethyl butylamine, i. e C (CH3) 2 (CH2) 2C (CH3) 2NH- Fl is not BODIPY@ FL, or when L is C (CH3) 2 (CH2) 2C (CH3) 2NHCSNH- then Fl is not FITC, eosin or erythosin ; or when L is monoamine menthane, Fl is not FITC (CA 131: 56155 (4) ) ; or when Lig is CGP12177 and L is a single bond, Fl is not NBD ; or when Lig is alprenolol i. e o-prop-2-enyl phenyl and L is-C (CH3) 2- or a single bond, Fl is not NBD.

Optionally additionally a) when Lig is XAC ie in Lig. a when each of R. al and R. a2 is propyl, R. a3 is H and R. a4 is-Ph-OCH2CONH (CH2) 2NH-, and L is a single bond Fl is not BODIPY TM 630/650 ; or b) when Lig is ABEA, ie m is 4 and L is a single bond Fl is not BODIPYTM 630/650.

Preferably a ligand or fluorescent ligand of the invention is an agonist which maintains its binding affinity and its functional activity or is an antagonist which maintains its binding affinity on linking or when linked to fluorescent moiety Fl.

Fluorescent ligands may have affinity such that they bind permanently, semi- permanently or transiently, ie may retain bound or may be washed away when unbound ligand is washed away.

Fluorescent ligands of the invention may be inherently optically active or may be functionalised, in known manner, to be optically active, and any such ligand may be present as a racemate or as one of its optically active isomers.

In a further aspect of the invention there is provided a novel reactive ligand of formula IV or IV'as hereinbefore defined or library thereof useful for linking to any suitable tag of formula V or V'as hereinbefore defined, with the proviso that <BR> <BR> when Lig is Lig. a and is 1, 3-dialkyl xanthine as hereinbefore defined wherein<BR> <BR> Xl and X2 are =O, R. a is H, R. al and R. a2 are both CH3 or both n-C3H7, then R. a4 is not 4-hydroxyphenol or PhOCH2CO2H ; or when R. al and R. a2 are both n-C3H7, then R. a4 is not PhOCH20CNHPhOH; PhOCH20CONsuccin, PhOCH2CONH2, PhOCH2CONH (CH2) 2NH2, PhOCH2cONH (CH2) 8NH2 PhOCH2COHNNH2, or PhOCH2CONH (CH2) 2N (CH2CH3. HOAc) CH2PhOH ; or when Lig is CGP12177 then L is not-C (CH3) 2 (CH2) 2C (CH3) 2NH2 (CA 121: 103486 ; or when Lig is aden, L is not -(CH2)2S(CH2)2NH2 (CA 125: 218348; or L is not (CH2) 6NH2 or CH2CONH (CH2) 6NH2 (CA 134: 2043); or L is not (CH2) 2NH2 or (CH2) 20 (CH2) 20 (CH2) 2NH2 (CA 135: 25706); or L is not (CH2) nNH2 where n is 2- 12 (CA 108 : 715) ; or when Lig is alprenolol L is not (CE2) 8NE2 or when Lig is propranolol L is not (CH2) 4NH2 (CA 124 : 8848) or when Lig is alprenolol L is not CH2C (CH3) 2NH2 (CA 108 : 215827) or when Lig is ICI 118551 L is not (CH2) 2NH2 or when Lig is propranolol L is not (CH2) 2NH2 (CA 98: 4564) Preferably a novel ligand-linker comprising a compound of formula IV wherein components are as hereinbefore defined and a reactive group YLjg is as hereinbefore defined, preferably of formula Lig L. I or Lig. LI'as hereinbefore defined.

In a further aspect of the invention there is provided a novel fluorophore linker of formula V or V'as hereinbefore defined or library thereof.

In a further aspect of the invention there is provided a kit comprising ligand precursors, linker precursors and tag precursors of formulae IV, IV', V, V'and/or VI as hereinbefore defined for preparing a library of compounds of formula I as hereinbefore defined.

In a further aspect of the invention there is provided the use of a fluorescent ligand of formula I or I'as hereinbefore defined or library thereof for visualising receptors or

receptor binding, assessing pharmacological properties of the fluorescent ligand, in high throughput screening of novel chemical entities that bind to the target receptor, in inhibiting an intracellular enzyme or inhibiting a drug transporter or a substrate of a drug transporter, in studying drug transport or drugs suitable for transport, in distinguishing healthy or diseased tissue and the like. Preferably the use comprises using any fluorescence detection technique more preferably confocal microscopy or fluorescence correlation spectroscopy. Preferably the use allows to calculate ligand affinity constants and concentration of sub-populations of a receptor type, intracellular enzyme or drug transporter as hereinbefore defined.

In a further aspect of the invention there is provided a method for receptor binding or inhibition, intracellular enzyme inhibition or drug transport or inhibition and visualisation comprising contacting a fluorescent ligand as hereinbefore defined with a sample in manner to facilitate binding or inhibition thereof or transport thereby, and detecting changes in fluorescence or location thereof.

A sample may comprise cell material, selected from cells, cell extracts, cell homogenates, purified or reconstituted proteins, recombinant proteins or synthesised proteins and the like, and includes a target for the compound of formula I. Samples comprising cell material may be derived from plants, animals, fungi, protists, bacteria, archae or cell lines derived from such organisms. Animal or plant cells used to prepare the sample may be healthy or disfunctional and are optionally used in the diagnosis of a disease such as leukaemia or cancer. In a preferred embodiment of the invention the sample comprises mammalian cells, extracts and homogenates thereof.

Preferably a sample comprises live cell material, more preferably including individual cells or sub cell compartments, most preferably comprising GPCRs, intracellular enzymes or drug transporters in living cells, membrane containing these proteins, solubilised receptors, enzymes or drug transporters or GPCR arrays. Cell material may be obtained in known manner by culturing cells or by expressing proteins in cells.

In a preferred embodiment the cell material is a cell expressing a GPCR, enzyme or drug transporter. GPCR's are possibly the single most important class of targets for current and prospective drug therapies.

More preferably the sample comprises GPCR receptors selected from adenosine Al- , A2A-, A2B-and A3-receptors, pi, ß2-and ps-adrenoceptors, or comprises inhibitors of intracellular enzymes such as cyclic nucleotide phosphodiesterases, most preferably CHO-cells expressing human adenosine Al-receptor or beta-adrenoceptor or an inhibitor of an intracellular enzyme such as an inhibitor of intracellular phosphodiesterases.

Cell material may be tagged prior to contact with the fluorescent ligand, for example by tagging with GFP, for example GFP tagged GPCR's, GFP tagged intracellular enzymes and GFP tagged drug transporters, or a native receptor, intracellular enzyme or a drug transporter to which a fluorescent antibody has been targetted, to allow

visualising of the cell receptors, enzymes or transporters, and overlay with the fluorescent ligands.

Receptors may be provided in membrane samples or in acutely dispersed cell samples, for example endogenous receptors such as Ai-AR in acutely dispersed cells.

The adenosine receptor binding site is located deep within the pocket of the receptor, whereby a fluorescent ligand with linker is a preferred fluorescent (ant) agonist.

Whilst there is considerable freedom in modifying the ligand and retaining antagonist binding activity, it is harder to retain agonist activating activity, ie activating the receptors functions on binding.

The method for drug transport of a substrate of a drug transporter would be to follow the uptake of the compound of formula I into the cell cytosol (if the transporter moves the drug into cells) OR after loading the cells with substrate to follow the dissappearance of the compound of formula I from the cells and its appearance in the extracellular medium (if the transporter moves the drug out of the cells-for example in the case that the transporter is an ATP-driven pump). Preferably the method comprises monitoring transport of a drug into a cell via an equilibrium transporter that moves the compound into the cell-then applying an inhibitor of this first equilibrium transporter, and monitoring the export of the drug from the cells via an ATP-driven pump transporter.

The method of inhibition of a drug transporter may be monitored by detecting binding to the transporter on the cell surface.

Preferably the method including detecting a change in fluorescence includes detecting a change in the intensity, excitation or emission wavelength distribution of fluorescence (single and multi photon), fluorescence lifetime, fluorescence polarisation or a combination thereof or the like. The optical response is detected by known means such as cameras, film, laser-scanning devices, fluorometers, photodiodes, quantum counters, microplate, microscopes, fluorescent microscopes such as epifluorescence or confocal, cytometers, readers and the like, preferably CSLM, confocal plate readers, fluorescence polarisation plate readers or FCS. Where the sample is examined using a flow cytometer, examination of the sample optionally includes sorting components of the sample according to their fluorescence response.

A method for binding or inhibition or detection according to the invention may be in vitro or in vivo.

In a particular advantage of the invention the novel fluorescent ligands are suitable for use in combination with FCS enabling the study of ligand-receptor binding at the single molecule level. Because of the nature of the events being monitored FCS is ideal for the study of thermodynamic and kinetic features of molecular interactions in solution. Another particular advantage of the invention is that the FCS approach can be adapted to monitor ligand-receptor binding at the single molecule level using photon counting fluorescence intensity measurements. This removes any requirement for the molecules to be moving within the confocal volume.

With ligands showing low background fluorescence it is not necessary to remove unbound ligand by washing before performing either confocal microscopy or FCS. It is therefore possible to measure fluorescence with time, in both time and concentration dependent manner.

Confocal microscopy (CSLM) allows visualisation of a section through a cell showing concentration of fluorophore at the cell edges indicating membrane receptor binding. Visualisation is of a particular plane of focus such that a"slice"through an individual cell may be observed, as known in the art. Different coloured channels may be selected to visualise different fluorophore types.

FCS is a non-invasive technique which analyses the diffusion characteristics of fluorescent species through a very small excitation volume (<10-151) by statistically analysing the pattern of their photon emissions. Thus fast-diffusing free ligand can be distinguished from slowly-diffusing receptor-bound ligand and quantified simultaneously when the volume is localised to the cell membrane. Preferably the method incorporating FCS comprises measuring fluctuations in fluorescence intensity in a confocal volume of <10-151. Statistical analysis of these fluctuations gives information about the speed of diffusion (i. e. mass) and concentration of the fluorescent molecules present. Thus free ligand (fast diffusing) and bound ligand (slow diffusing) can be quantified simultaneously on a single cell.

FCS (fluorescence correlation spectroscopy) correlates fluctuations in fluorescence emission of particles to parameters such as particle mass and concentration for the study of molecular interactions in solution. FCS essentially monitors spontaneous fluorescence intensity fluctuations of fluorescently tagged molecules in a microscopic detection volume (10-151) through analysis by a tightly focused laser beam.

These fluctuations provide information on the rate of diffusion or diffusion time of a particle which is directly dependent on the mass of the given molecule. When small and therefore rapidly diffusing molecules pass through the path of the laser they produce rapidly fluctuating fluorescence intensity patterns, whereas when larger molecules pass through the beam they produce bursts of fluorescence that are more sustained. Consequently the increase in the mass of a biomolecule, eg as a result of ligand binding, is detected as an increase in the diffusion time of the resultant biomolecule.

Fluorescence microscopy may be used to localise receptors at single cell or sub cellular level with sensitivity and speed. In this way high affinity tagged ligands could help to elucidate molecular characteristics of GPCR receptor subtypes, such as adenosine and the like receptors, their regional distribution and cellular localisation.

In a further aspect of the invention there is provided the use of a fluorescent target for the fluorescent ligand, for example, a Green Fluorescent Protein-tagged receptor, intracellular enzyme or drug transporter. In this case the spectral characteristics of the fluorescent ligand are chosen to allow separate detection of the location of both the fluorescent ligand and the fluorescent receptor, intracellular enzyme or drug

transporter. Cross-correlation fluorescence correlation spectroscopy or fluorescence intensity measurements will then, allow the quantitative analysis of ligand-receptor, ligand-enzyme, ligand-drug transporter or drug transport interactions in a single measurement. This is distinct from prior art methods involving GFP-protein translocation assays and assays involving fluorescence energy transfer (FRET).

Figure 1 exemplifies this approach.

In a further aspect of the invention there is provided a cell surface GPCR modified on its N-terminus or a naturally occurring domain to express a short epitope tag for a commercially available antibody (e. g. myc, haemaglutinin, FLAG). This is then expressed in CHO cells and a fluorescent antibody to the tag sequence is used in living cells to provide two-colour analysis of fluorescent ligand-receptor interactions as described for GFP-tagged proteins above.

In a further aspect of the invention there is provided CHO cells expressing a cell surface GPCR modified as claimed in Claim 37 for use with a fluorescent antibody to the tag sequence is used in living cells to provide two-colour analysis of fluorescent ligand-receptor interactions as described for GFP-tagged proteins above.

In a further aspect of the invention there is provided a kit comprising a compound of formula I or I'as hereinbefore defined and a target therefore provided as a cell line, membrane derived from such a cell line or protein solubilised from that cell line. The cell derived material may be provided in one of three forms : (1) from cells expressing a green fluorescent protein tagged receptor, intracellular enzyme or drug transporter ; (2) from cells expressing an epitope tag for a commercially available fluorescent antibody or (3) a wild-type protein for which, a specific fluorescent antibody is also provided.

In an alternative embodiment there is provided a kit comprising a compound of formula I or I'as hereinbefore defined and a fluorescent antibody to a native protein which can be used in native (non-recombinant) cells.

In each case, the spectral characteristics of the compound of formula I or I'and fluorescent antibody or green fluorescent protein are selected to allow optimum two- colour cross-correlation fluorescence correlation spectroscopy (single or multiphoton).

The invention is now illustrated in non-limiting manner with reference to the following figures and examples and accompanying synthesis schemes.

In the Figures: Fig 1 shows binding of BODIPY-XAC to CHO-K1 cells expressing the human Al- receptor with a Green fluorescent protein tag attached to the C terminus. (a) binding of red ligand ; (b) location of Al-GFP receptor and (c) co-localisation (yellow) of two signals obtained via confocal microscopy, more specifically Figure 1 shows images taken from confocal microscopy imaging of a) fluorescence derived from XAC BY- 630 binding to receptors on the surface of CHO cells observed at the red channel, b) fluorescence derived from green fluorescent protein fused to the C terminus of the

human adenosine Al-receptor, expressed by CHO cells indicating receptor locations observed via the green channel and c) overlaid images from a) and b) showing overlap of fluorescence and therefore confirming ligand binding is specific to receptors. Fig 2 shows binding of BODIPY-ABEA to CHO-K1 cells expressing the human Al-receptor with a Green fluorescent protein tag attached to the C terminus.

(a) binding of red ligand ; (b) location of A1-GFP receptor and (c) co-localisation (yellow) of two signals obtained via confocal microscopy.

In the Schemes: Scheme 1 shows synthesis routes for the synthesis of an adenosine receptor antagonist Lig-L-F1L Schemes 2 and 3 show synthesis routes for the synthesis of two adenosine receptor agonists Lig-L-F1L including the synthesis of ligand precursor Lig-L-ZL from linker precursor ZL'-L-ZL Scheme 4 shows synthesis routes for the synthesis of two beta adrenoceptor agonists Lig-L-F1L including the synthesis of ligand precursor Lig-L-ZL from linker precursor ZL'-L-ZL Examples A-C The following compounds are synthesised or modelled and binding affinity studied : Example A1/Bl/C 1 Adenosine receptors antagonists : XAC-BODIPY 630/650 (1) Example A2/B2 Adenosine receptor agonists : Adenosine-BODIPY 630/650 (2) NECA-BODIPY 630/650 (3) (ABEA-BODIPY 630) APEA-BODIPY 630/650 (3a) ABIPEA-BODIPY 630/650 (3b) Example A3/B3 Beta-adrenoreceptor agonists Salmeterol-BODIPY 630/650 (4) Clenbuterol-BODIPY 630/650 (9) Example A4/B4 Beta-adrenoreceptor antagonists CGP12177-BODIPY 630/650 (5) Propranolol-BODIPY 630/650 (6) ICI118551-BODIPY 630/650 (7) Alprenolol-BODIPY 630/650 (8) Example A5/B5 Inhibitors of cyclic nucleotide phosphodiesterases XAC-BODIPY 630/650 (1) Materials and Methods The H NMR spectra were acquired on a Bruker AM 250 (250 MHz) spectrometer, in CDC13 or DMSO-d6. Chemical shifts (8) are recorded in ppm with reference to the residual solvent signal/TMS. Coupling constants (J) are recorded in hertz, and signal multiplicities are described by s (singlet), d (doublet), dd (doublet of doublets), t (triplet), m (multiplet), br (broad). Where given, assignments are made based on homonuclear correlation spectroscopy (COSY-45) and, where available, are in full agreement with literature values (Jacobsen KA et al., R Med. Chem. (1985), 28, 1341-6).

(Analytical RP-HPLC was performed on a Waters Millenium LC system with 996 PDA eluent detection, using a Vydac c8 column (150mm x 4.6 mm) at a flow rate of 1.0 mL. min-1. The mobile phases used were: Solvent A, water, (degassed by helium bubble); Solvent B, acetonitrile, (degassed by sonication)).

A. SYNTHESIS Example Al-Synthesis of adenosine based fluorescent Ai-receptor antagonists 1. XAC-BY630 (1) Scheme 1 Reagents and conditions: (i) BODIPY 630/650-X-SE, DMF 2 h, RT, (72%).

XAC-BODIPY 630/650 was synthesised by reacting the primary alkyl amine group of XAC with BODIPY°-630/650-X-succinimidyl ester (Molecular Probes). XAC and BY630 were stirred in N, N-dimethylformamide for 2h at room temperature and the product purified by HPLC. XAC and analogues were synthesised by the method of Jacobsen et al J. Med. Chem 1985, 28, 1334-1340.

TOF ES+ found 974.3998 (C50Hs5BF2N907S requires 974.4006) Rt 12.5 min (35-100% v/v B, 30 min) 5H 0.87, 0.90 (6H, overlapping t, J 9. 3, Nl-, N3-CH2CH2CH3), 1.14-1. 25 (2H, m, 1. 36-1.62 (6H, m, C23H2, C25H2, N1/3-CH2CH2CH3), 1.68-1. 78 (2H, m, Nl/3- CH2CH2CH3), 2.04 (2H, t, J 7. 3, C22H2), 3.04-3. 19 (6H, m, Cl8H2, C19H2, C26H2), 3.86 (2H, t, J 7.4, N13-CH2CH2CH3), 4.01 (2H, t, J 7.1, N1/3-CH2CH2CH3), 4.52, 4.53 (4H, 2 x s, C15H2, C29H2), 6.95 (1H, d, J4. 2), 7.05-7. 10 (4H, m), 7.27-7. 30 (3H,

m), 7. 35-7.40 (2H, m), 7.41 (1H, br s), 7.54-7. 65 (3H, m), 7.70 (1H, s), 7.77 (1H, s), 7. 80-7. 92 (2H, s), 8. 01-8.23 (4H, m) (2 x CIIH, 2 x Cl2H, 2 x C32H, 2 x C33H, C35H, C36H, C38H, C39H, C41H, C43H, C44H, C47H, C48H, C49H, N9H, N17H, N20H, N27H) Example A2-synthesis of adenosine based fluorescent agonists at the human Ar-adenosine receptor (Al-AR) receptor based on 5'-N- ethvlcarboxamidoadenosine (NECA) with maintained functional activity Compounds 2,3, 3a and 3b were synthesised by reaction of suitably protected inosine derivatives, specifically with a chlorinating agent allowing introduction of a protected linker. Removal of protecting groups preceded conjugation of a fluorescent agent via the linking group.

Scheme 2 Reagents and conditions : (a) Ac20, pyridine, 40°C, 1 h, 97%. (b) POC13, N, N- dimethylaniline, reflux, 5 min, 85%. (c) (i) H2N (CH2) 4NHR, DIEA, EtOH, reflux, 18 h, (ii) sat. NH3/MeOH, 0°C, 2 h. 66%. (d) H2, Pd/C, MeOH: H20: AcOH (7: 2: 1), r. t., 2 h, 80% (e) BODIPY 630/650-SE, DMF, r. t. , 3 h, 63% 1. Adenosine-C4-BODIPY 630/650 (ABA-BY630) (2) ABA-BY630 was synthesised using the method and reagents and conditions described in Scheme 2 a-e in which R is COCH2Ph.

ES+ found 885. 4 (C43H48BF2N9O7S requires 885. 4) Rt 22.5 min (5-100% v/v B, 30 min) 2. NECA - C4 - BODIPY 630/650 (ABEA-BY630) (3).

N6-aminobutyl-5'-deoxy-5'-oxo-5'-ethylaminoadenosine (ABEA) was synthesised from commercially available reagents in 6 steps. The primary amine group of ABEA was acylated with the fluorophore BODIPY@630/650-X-succinimydyl ester (BY- 630, Molecular Probes) to afford BY630-ABEA, which was purified by RP-HPLC (Scheme 3).

The synthesis is shown in Scheme 3, with use of linker precursor of formula H2N (CH2) 4 HNCOOCH2Ph :

Scheme 3 Reagents and Conditions : (a) 2,2-Dimethoxypropane, TsOH, acetone, r. t., 18 h. (b) TEMPO, BAIS, MeCN : H20 (1: 1), r. t. , 4 h. (c) (i) SOC12, DMF, CHC13, reflux, 6 h.

(ii) EtNH2, CHC13, 5°C, 30 min. (d) H2N (CH2) 4NHZ, DIEA, ETCH, reflux, 18 h. (e) 0.1 M [HCl (aq), 50°C, 4 h. (f) H2, Pd/C, MeOH : H20: AcOH (9: 0.9 : 0.1), r. t. , 3 h. (g) BODIPY 630/650-X-SE, DMF, r. t. , 4 h.

Synthesis of linker modified ligand, compound of formula IV 2',3'-Isopropylideneinosine 1: Inosine (5.36 g, 0.02 mol) and tosic acid monohydrate (3. 80 g, 0.02 mol) were suspended in a mixture of 2,2- dimethoxypropane (50 cm3) and acetone (200 cm3) and stirred for 18 h. Sodium hydrogen carbonate (2.52 g, 0.02 mol) and water (40 cm3) were added and the suspension stirred for 15 min. The suspension was evaporated to constant volume and the crude product recrystallised from the residual water, yielding the acetonide 1 (3.71 g, 60%) as white needles; mp 266-268 °C (from H20) (lit. , 266 °C) ; [α]22D - 67.1 (c 0.59 in MeOH) (lit. , [α] 20D -66. 9 (c 0.8 in MeOH)); #H(250 MHz ; DMSO-d6) 1.31 (3 H, s, CH3), 1.53 (3 H, s, CH3), 3.53 (2 H, m, C5'H2), 4.22 (1 H, m, C4'H), 4.93 (1 H, dd, J6. 1 and 2.5, C3 H), 5.26 (1 H, dd, J6. 1 and 2.9, C2'H), 6.10 (1 H, d, J 2.9, C1'H), 8.10 (1 H, s, adenine CH), 8.31 (1 H, s, adenine CH); 8c (69.2 MHz ; DMSO-d6) 25.2, 27.0 (2 x acetonide), 61.4 (C5'), 81.3 (C4'), 83. 8 (C3'), 86.6 (C2'), 89. 6 (Cl), 113.1 (4°), 124.4 (4°), 138. 7 (CH), 146.1 (CH), 147.8 (4°), 156.5 (4°) ; m/z (ES+) 309 (MH+), 137 (M-ribose).

2', 3'-Isopropylidene-5'-oxoinosine 2 : Acetonide 1 (3.08 g, 10 mmol), TEMPO (313 mg, 2 mmol) and iodosobenzene diacetate (7.09 g, 22 mmol) were dissolved in MeCN : H20 (1: 1,50 cm3) and stirred, with the exclusion of light, for 4 h. The solvents were carefully evaporated from the resultant suspension and the reaction residue sequentially triturated with acetone and diethyl ether to yield the acid 2 (2.67

g, 83%) as a white powder; mp 224-229 °C (from diethyl ether) (lit., 274-276 °C) ; (found: C, 48.55 ; H, 4.3 ; N, 17.0. C13H14N4O6 requires C, 48.45 ; H, 4.4 ; N, 17.4%) ; AH (250 MHz; DMSO-d6) 1.33 (3 H, s, CH3), 1.51 (3 H, s, CH3), 4. 68 (1 H, d, J 1.6, C4 H), 5.36-5. 44 (2 H, m, C 2'H and C'H), 6.30 (1 H, s, C1'H), 8.02 (1 H, s, adenine CH), 8. 27 (1 H, s, adenine CH), 12.42 (1H, br s, NH; 8c (69. 2 MHz; DMSO-d6) 25.1, 26.7 (2 x acetonide), 83. 9,85. 8, 90.0 (4 x CH), 112.9 (4°), 124.4 (4°), 140.0 (CH), 145.8 (CH), 148.2 (4°), 156. 8 (4°), 171.8 (C=O) ; m/z (ES+) 323 (MH+), 137 (M- ribose).

6-Chloro-6-deoxy-5'-ethylamino-2', 3'-isopropylidene-5'-oxo-5'-deoxyinosine 3: (N. B. Rigorously dry reaction conditions and under an inert atmosphere) acid 2 (967 mg, 3 mmol), was suspended in CHC13 (15 cm3) to which was added N, N DMF (581 µL, 7.5 mmol) and SOC12 (1.09 cm3, 15 mmol). The suspension was placed in a hot oil-bath and maintained at reflux for 6 h. The resultant solution was evaporated and the yellow oil dissolved in THF (20 cm3) at 5 °C. Ethylamine (2.0 M solution in THF, 3.75 cm3, 7.5 mmol) was added drop wise, stirred at 5'C for 15 min and allowed to warm to room temperature. The solvent was evaporated, the residue dissolved in DCM (25 cm3) and washed with water (2 x 20 cm3) and saturated brine solution (2 x 20 cm3). The organic fraction was dried and evaporated to leave a yellow oil that was purified by column chromatography on silica (5 % MeOH-DCM) to give the title compound 3 (525 mg, 48%) as a yellow syrup ; [ (XI19D-12. 9 (c 0. 50 in CHC13) ; AH (250 MHz ; CDC13 ; Me4Si) 0.78 (3 H, t, J 7.3, CH2CH3), 1.41 (3 H, s, CH3), 1. 64 (3 H, s, CH3), 2.90-3. 11 (2 H, m, CH2CH3), 4. 74 (1 H, d, J 1. 9, C4'H), 5. 4. 6 (1 H, dd, J6. 2 and 2. 3, C2H), 5.54 (1 H, dd, J 6. 2 and 1. 9, C3'H), 6.24 (1 H, d, J 2.3, Cl H), 6.28 (1 H, br s, NH), 8. 35 (1 H, s, adenine CH), 8. 68 (1 H, s, adenine CH); 8c (69.2 MHz; Coda ; Me4Si) 14.2 (CH2CH3), 25.0, 26.9 (2 x acetonide), 33.9 (CH2CH3), 82. 9, 83. 4, 86. 7, 92. 0 (4 x CH), 114.6 (4°), 132.3 (4°), 144. 8 (CH), 150. 9 (4°), 151. 9 (4°), 152.2 (CH), 168. 1 (C=O) ; iiilz (ES-) 366 ((M-H)-), 153 (M-ribose).

N6- (4-Benzyloxycarbonylaminobutyl)-5'-ethylamino-2',3'-isopropy lidene-5'- oxo-5'-deoxyadenosine 4: Chloride 3 (337 mg, 0.92 mmol) was dissolved in EtOH (10 cm3) to which was added N-benzyloxycarbonylbutan-1, 4-diamine (305 mg, 1.37 mmol) and DIEA (159 p, L, 0.92 mmol). The solution was placed in a hot oil-bath and maintained at reflux for 18 h. The resultant solution was evaporated and the yellow oil purified by column chromatography on silica (2.5 % MeOH-DCM) to give the title compound 4 (445 mg, 88%) as a pale yellow gum; #H(250 MHz; CDC13 ; Me4Si) 0.99 (3 H, t, J 7. 1, CH2CH3), 1.43 (3 H, s, CH3), 1.55-1. 71 (7 H, m, CH3 and 2 x CH2), 3.20-3. 35 (2 H, m, CH2), 3.55-4. 01 (4 H, m, CH2CH3 and CH2), 4.81 (1 H, s, CH), 5.10 (3 H, m, benzyl CH2 and CH), 5.51 (1 H, d, J 5. 9, CH), 5.71 (1 H, d, J5. 9, CH), 6.10 (1 H, br s, NH), 6.16 (1 H, br s, NH), 7.30-7. 36 (5 H, m, aromatics), 7.86 (1 H, s, adenine CH), 8. 22 (1 H, s, adenine CH); 8c (69.2 MHz; CDC13 ; Me4Si) 13.7 (CH2CH3), 25.1, 26.6 (2 x acetonide), 26.8, 27.0, 40.0, 40.4 (4 x CH2), 61.5 (CH2CH3), 66.6 (benzyl CH2), 84.1, 84.7, 87.0, 91.6 (4 x CH), 113.7 (4°), 128.1 (C), 128.5 (CH), 136.7 (4°), 139.9 (CH), 152.8 (CH), 154.9 (4°), 156.5 (CH), 169.4 (C=O) ; m/z (ES+) 554 (MH+), 341 (M-ribose).

N6- (4-Benzyloxycarbonylaminobutyl)-5'-ethylamino-5'-oxo-5'-deox yadenosine 5: Adenosine derivative 4 (261 mg, 0.47 mmol) was dissolved in 1 M HCl (aq) : 1,4- dioxane (1: 1,4 cm3), placed in a 50 °C oil-bath and stirred for 4 h. The resultant solution was adjusted to-pH 8 (satd. NaHC03 (aq)), saturated with NaCl and extracted with EtOAc (3 x 5 cm3). The combined organic fractions were dried and evaporated and the crude product purified by preparative layer chromatography (10 % MeOH- DCM) to give the title compound 5 (160 mg, 66%) as a colourless oil; AH (250 MHz ; DMSO-d6) 1.08 (3 H, t, J 7. 2, CH2CH3), 1.45-1. 62 (4 H, m, C2H2 and C3H2), 2.98- 3.06 (2 H, m, C1H2), 3.17-3. 26 (2 H, m, CH2CH3), 3.37-3. 53 (2 H, m, C4H2), 4.12- 4.16 (1 H, m, C3'H), 4.31 (1 H, d, J 1.1, C4'H), 4.58-4. 65 (1 H, m, C2'H), 4.99 (2 H, s, benzyl CH2), 5.56 (1 H, d, J6.5, C2'-OH), 5.76 (1 H, d, J4.2, C3'OH), 5.96 (1 H, d, J7. 6, C'H), 7.25-7. 34 (6 H, m, aromatics and NH), 8. 01 (1 H, br s, carbamate NH), 8. 27 (1 H, s, adenine CH), 8.39 (1 H, s, adenine CH), 8.94 (1 H, t, J 5. 6, amide NH); 8c (69. 2 MHz ; DMSO-d6) 14.9 (CH2CH3), 26.6, 27.1, 33.4, 39. 5, 40.3 (5 x CH2), 65.3 (benzyl CH2), 72.2, 73.3, 84.9, 88.0 (4 x CH), 120.2 (4°), 127.9 (CH), 128.5 (CH), 137.5 (CH), 140.6 (4°), 152.6 (CH), 154.9 (4°), 156.3 (4°), 169.3 (4°) ; into (ES+) 514 (MH+).

N6- (4-Aminobutyl)-5'-ethylamino-5'-oxo-5'-deoxyadenosine (ABEA) 6: Adenosine derivative 5 (48 mg, 0.09 mmol) was dissolved in MeOH : H20 : AcOH (9: 0. 9 : 0. 1,5 cm3), to which was added 10 % Pd/C (10 mg). The flask was evacuated, filled with hydrogen (balloon) and stirred vigorously for 3 h. The reaction mixture was filtered through celite and the celite washed with MeOH. The combined organic filtrates were evaporated and the resultant oil evaporated again from MeCN (2 x 15 cm3) to give the title compound 6 (35 mg, quant. ) as a colourless oil; 8H (250 MHz; DMSO-d6) 1.08 (3 H, t, J 7. 2, CH2CH3), 1.46-1. 88 (6 H, m, 2 x CH2 and NH2), 2.63 (2 H, t, J6. 8, CH2), 3.16-3. 29 (2 H, m, CH2CH3), 3.40-3. 52 (2 H, m, CH2), 4.10-4. 15 (1 H, m, C3'H), 4.30 (1 H, d, J 1.3, C4'H), 4. 53-4.62 (1 H, m, C2'H), 5.96 (1 H, d, J 7.7, C1'H), 8.05 (1 H, br s, NH), 8. 27 (1 H, s, adenine CH), 8.39 (1 H, s, adenine CH), 8. 95 (1 H, t, J 5. 6, amide NH) ; into (ES+) 380 (MH+).

Synthesis of fluorescent ligand, compound of formula I ABEA-BY630 (3): ABEA 6 (5.74 mg, 15.1 µmmol) was dissolved in N,N-DMF (1 cm3) under an inert atmosphere and with the exclusion of light. A solution of Bodipy 630/650-X-succinimidyl ester (Molecular Probes) (5.0 mg, 7.55 µmmol, 1 cm3 N,N- DMF) was added and the reaction stirred for 4 h. The solution was evaporated and the crude product purified by preparative layer chromatography (10 % MeOH-DCM) to give the title compound 7 (3) (5.24 mg, 75%) as a purple powder ; m/z (ES+) found 947.37 (C45H51BF2N10O7SNa requires 947.36).

ABEA-BY630 3. NECA-CS-BODIPY 630/650 (APEA-BY630) (3a) This compound was synthesised using the method of Scheme 3 as described for Compound (3), with use of linker precursor of formula H2N(CH2)5NHCOOCH2Ph: APEA-BY630 was obtained having the formula :

Rt 8. 6 min (30-100% v/v B, 25 min) 4. NECA-PEG8-BODIPY 630/650 (ABIPEA-BY630) (3b) This compound was synthesised using the method of Scheme 3 as described for Compound (3), wherein intermediates 1 to 3 below are analogues of structures 4 to 7 respectively shown in Scheme 3, with use of linker precursor of formula H2N ((CH2)2O)2(CH2)2 NH COOCH2Ph: N6-(8-Benzyloxycarbonylamino-3,6-dioxaoctyl)-5'-ethylamino-2 ', 3'- isopropylidene-5'-oxo-5'-deoxyadenosine 1 : 8H (400 MHz, CDC13) 0. 88 (3H, t J 7.3, Et CH3), 1. 38 (3H, s, acetonide CH3). 1. 62 (3H, s, acetonide CH3), 3.03-3. 16 (2H, m, Et CH2), 3.40-3. 93 (14H, m, 6 x linker methylenes, C3'H, C4'H), 4.67 (1H, s, CfH), 5.11 (2H, s, benzyl CH2), 5.32 (1H, s, C1'H), 5. 80 (1H, br s, carbamate NH), 6.55 (1H, br s, C6-NH), 7.02 (1H, br s, amide NH), 7.28-7. 37 (5H, m, aromatic CH), 7.64 (1H, br s, adenine CH), 8. 29 (1H, s, adenine CH).

N6- (8-Benzyloxycarbonylamino-3, 6-dioxaoctyl)-5'-ethylamino-5'-oxo-5'- deoxyadenosine 2: #H(400 MHz; DMSO-d6) 1.08 (3H, tJ7. 2, Et CH3), 3.12-3. 17 (2H, m, linker CHa), 3.18-3. 25 (2H, m, Et CH2), 3.41 (2H, t J 6. 0, linker CH2), 3.49- 3.54 (4H, m, 2 x linker CH2), 3.57-3. 67 (4H, m, 2 x linker CHa), 4.14 (1H, br m, C3'H), 4.31 (1H, d J 1. 5, C4'H), 4.58-4. 63 (1H, m, C2'H), 5.00 (2H, s, benzyl CH2), 5.54 (1H, dJ6. 4, C2-OH), 5.74 (1H, d J 4.1, C3'-OH), 5.97 (1H, dJ7. 6, C'H), 7.25-7. 36 (6H, m, aromatic CH and carbamate NH), 7. 85 (1H, br s, C6-NH), 8.28 (1H, br s, adenine CH), 8.40 (1H, s, adenine CH), 8.87 (1H, t J 5. 6, amide NH).

N6- (8-Amino-3, 6-dioxaoctyl)-5'-ethylamino-5'-oxo-5'-deoxyadenosine 3: #H(400 MHz; DMSO-d6) 1.05 (3H, tJ7. 1, Et CH3), 1.86 (2H, br s,-NH2), 2.71-2. 80 (2H, m, linker CH2), 3.17-3. 26 (2H, m, Et CH2), 3.41-73 (10H, m, 5 x linker CH2), 4.15 (1H, br m, C3'H), 4.34 (1H, s, C4'H), 4.47-4. 54 (1H, m, C2'H), 5.95 (2H, br s, Cf-OH, C3- OH), 6.01 (1H, d J 7. 5, C1'H), 7.92 (1H, br s, C6-NH), 8.31 (1H, br s, adenine CH), 8.44 (1H, s, adenine CH), 8. 95 (1H, t J 5. 6, amide NH).

ABIPEA-BY630 was obtained having the formula : TOP ES+ found 985. 3993 (C47H56BF2N10O9S requires 985. 4013) Rt 8.3 min (35-100% v/v B, 25 min) Example A3-Synthesis of ß-Adrenoceptor agonists 1. Salmeterol-BODIPY 630/650 (4) and Derivative-Salmeterol-BODIPY 630/650 (4a) Salmeterol is linked to fluorophore via two different linking sites, in the following syntheses

In a first approach, a linker is substituted onto the salmeterol side-chain through which the fluorophore is subsequently attached. In the second approach the native alkyl side-chain of salmeterol is replaced with a linker and fluorophore. In this case, according to the invention, retention of binding, fluorescence and activity are uncertain and must therefore be verified and information provided with the fluorescent ligand, to provide a useful compound.

Scheme 4 Reagents and conditions: (i) (a) HCHO, HCl(aq), dioxane, 60°C (b) 2, 2- Dimethoxypropane, TsOH. (ii) Me3SI, NaH, THF. (iii) (a) BocNH (CH2) nNH2, EtOH, (b) HC1, Et2O. (iv) BODIPY 630/650-X-SE, DMF, RT. (v) (a) ZL'YL'-L-YLPL, EtOH, (b) HCl, Et20, ZL'YL'-L-YLPL iS and results in compound 4a

All of the following molecules rely upon the synthesis of the same two linker moieties as shown in Scheme 4 and described above, (where the hydrocarbon chain length can be easily varied, or altered chemically to e. g. an ethylene glycol structure to improve solubility).

2. Clenbuterol-BODIPY 630/650 (9)

Example A4-Synthesis of-Adrenoceptor antagonists All of the following molecules rely upon the synthesis of the same two linker moieties as shown in Scheme 4 and described in Example A3, (where the hydrocarbon chain length can be easily varied, or altered chemically to e. g. an ethylene glycol structure to improve solubility).

1. CGP 12177-BODIPY 630/650 (5) 2. Propranolol-BODIPY 630/650 (6) 3. ICI118551-BODIPY 630/650(7) 4. Alprenolol-BODIPY 630/650(8)

B. PHARMACOLOGY Example Binding of adenosine based fluorescent Al receptor antagonists 1. XAC-BY630 (1) The adenosine-A1 receptor (Al-AR) is a G-protein coupled receptor which is found in a variety of tissues including brain, heart, adipose tissue and muscle. By conjugating the Al-AR antagonist xanthine amine cogener (XAC) to the fluorophore BODIPY@-630/650 (BY630), we have synthesised a fluorescent Al-AR ligand, XAC-BY630, to allow visualisation of this receptor in living cells.

[3H] DPCPX binding alongside cyclic AMP and inositol phosphate accumulation assays were performed on CHO-A1 cells expressing the human Al-receptor. Images were acquired using a Zeiss LSM510 confocal microscope using CHO-A1 cells grown to 50% confluency on 8-well Labtek plates in Dulbecco's Modification of Eagle's Medium : Ham's F12 containing 5% foetal calf serum and 2mM glutamine. Cells were washed twice with HEPES-buffered saline prior to incubation at 22°C with compounds as indicated.

Spectroscopic analysis of XAC-BY630 and BY630 itself showed that their peak excitation (630, 632mn, respectively) and emission wavelengths (650, 653nm) were not substantially different. [3H] DPCPX binding studies on CHO-A1 cell membranes showed that XAC-BY630 had a lower affinity for the Al-AR than XAC (pKs=7. 79+0. 13 and 6. 820. 11, XAC and XAC-BY630, respectively, means. e. mean, n=4). XAC-BY630 also behaved as a competitive Al-AR antagonist at both 5'-N-ethylcarboxamidoadenosine-mediated inhibition of cAMP production (apparent pKB=6. 980. 15 vs. 8. 060. 24 for XAC, n=3) and stimulation of inositol phosphate production (apparent pKB=6. 260. 20 vs. 7. 460. 08 for XAC, n=4). Confocal imaging showed that XAC-BY630 bound to membrane-localised Al-ARs in a time-and concentration-dependent manner. Binding of XAC-BY630 (25-250nom) was detected after 5 min, and was predominantly located at the membrane after a 30 min. incubation. Membrane binding of XAC-BY630 was receptor-specific, since a 30 min pre-incubation with DPCPX (10-8-10-6M) caused a concentration-dependent inhibition of membrane binding (30 min, 50nM).

These studies indicate that XAC-BY630 is a functional Al-AR antagonist with moderate affinity which could be used to visualise the Al-AR in primary tissue and cell lines.

Fluorescence Correlation Speetroscopy (FCS).

FCS is a non-invasive technique which measures fluctuations in fluorescence intensity in a confocal volume of <10-151. Statistical analysis of these fluctuations gives information about the speed of diffusion (i. e. mass) and concentration of the fluorescent molecules present. Thus free ligand (fast diffusing) and bound ligand (slow diffusing) can be quantified simultaneously on a single cell. We have used FCS to measure binding of the fluorescent ligand, xanthine amine cogener- BODIPY"630/650 (XAC-BY630) to the human adenosine Al receptor (A1-AR).

CHO cells expressing either the human At-AR or an Al-AR-Topaz fusion were cultured on glass-bottomed 8-well plates and prepared for live cell measurement.

FCS measurements were made using a Zeiss Confocor 2, fitted with an Axiocam CCD camera for x-y positioning. Cells were incubated with ligands at 22°C for the times indicated and the confocal volume was positioned on the upper membrane.

Data were collected for 2x30s, following a 15s pre-bleach and analysed using a multi-parameter equation using Zeiss AIM software.

Initially, the diffusion characteristics of the Al-AR-Topaz fusion protein (Al-AR- Tpz) were detennined in CHO-AlTpz cells. Autocorrelation analysis showed the diffusion time (To) tor the A,-AR was 15. 00. 9ms (means. e. mean, n=84). A second component (#D=118~14µs) was also seen, probably caused by an optical event within the fluorophore ("blinking"). FCS analysis of XAC-BY630 in buffer showed a single component diffusion (D=60+211s, n=10). On the upper membrane of CHO-A1 cells incubated with XAC-BY630 (1-40nM, 10-60 min, n=71), two further slow-diffusing species were detected in addition to free ligand. The first component had a similar diffusion time (TDi=17. 4l. lms ; 69/71 cells) to that seen for Al-AR-Tpz, suggesting that it is receptor-bound ligand. The second was a very slow diffusing component (D2=345+41ms, 61/71 cells). Following preincubation with 8-cyclopentyl-1, 3-dipropyl xanthine (DPCPX) (1µM, 30 min), tD2 was present in 30/31 cells, suggesting this component is non-specific binding. However, the tDs component was present in only 17/31 cells. In addition, in cells exposed to 15nM XAC-BY630 for 30 min the amount of TDI component was reduced from 51. 814. 9 to 13. 65. 4 receptors/gM2 by DPCPX (n=8 and 4, respectively, Student's t-test, P<0. 05), further suggesting this component is Al-AR bound ligand.

We have used FCS to quantify binding to the Al-AR and measure receptor diffusion in single live cells. Further development allows quantitative receptor-ligand binding of the endogenous Ai-AR in acutely dispersed cells.

These studies indicate that XAC-BY630 is a functional Ai-AR antagonist with moderate affinity which could be used to visualise and measure binding to the Ai- AR, in primary tissue and cell lines.

Example 132-Binding of NECA based fluorescent A1-receptor agonists 2. BY630-ABEA (3) Functional studies were performed in CHO-K1 cells expressing both the human Al- AR and a c-fos-pGL3 reporter vector (CHO-Alfos cells). Cells were incubated for 24h in serum-free DMEM/F-12 media, then stimulated with agonist for 5h, in some cases following 30 min incubation with 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX). Luciferase expression was quantified using a Luclites kit according to manufacturer's instructions. Live cell confocal imaging was carried out on CHO-A1 cells or CHO cells expressing the A,-AR tagged on the C-terminus with a green fluorescent protein (CHO-AlTpz).

In CHO-Alfos cells, both BY630-ABEA and the Al-AR agonist N6-cyclopentyl adenosine (CPA) stimulated luciferase expression in a dose-dependent manner (pECso's of 7. 010. 04 (n=6) and 6. 760. 18 (n=5) for CPA and BY630-ABEA, respectively, means. e. mean). Stimulation was mediated by the Al-AR receptor, since the concentration response curves were shifted to the right in a competitive manner by 10nM DPCPX, yielding pKd values of 8. 720. 03 and 9. 050. 10 vs. CPA and BY630-ABEA, respectively (n=3). A higher dose of DPCPX (lOOnM), gave a pKd of 8. 620. 02 for CPA stimulation, but completely blocked the response to

BY630-ABEA (n=3). For receptor visualisation, CHO-Al cells were incubated with lOOnM BY630-ABEA for up to 60min. Binding of ligand to the membrane was detectable after 5 min, and was substantial after 30 min (n=3). Binding was to the Al-AR, since it was substantially reduced by preincubation with DPCPX (1pM, 30 min). In addition, experiments in CHO-AlTpz cells, showed co-localisation of ligand fluorescence at the membrane with that from the fluorescently tagged Al-AR.

Results are shown in Figure 1 which shows images taken from confocal microscopy imaging of a) fluorescence derived from ligand binding of a fluorescent ligand of the invention to CHO cells observed at the red channel, b) fluorescence derived from green fluorescent protein expressed by CHO cells indicating receptor locations observed via the green channel and c) overlaid images from a) and b) showing overlap of fluorescence and therefore confirming ligand binding is specific to receptors.

In conclusion, we have succeeded in synthesising a novel fluorescent agonist ligand for the human Al-AR. This ligand will be useful in monitoring the localisation of the endogenous Al-AR receptor in both acutely dispersed cells and cell lines.

C. LIGANDS ASSOCIATED WITH PHARMACOLOGICAL DATA Example C1 - Data sheets for library / catalogue compound comprising adenosine based fluorescent A'eeeptor antagonists 1. XAC-BY630 (1) Characterisation : Fluorescent adenosine Al-receptor antagonist.

Synthesis and analysis : see Al above.

Storage....-20°C (dark) Spectral Properties: Excitation Max 638nm Emission Max 655nm Fluorescence Lifetime 4.2 ns Emission quantum yield 0.33 Pharmacology : CHO-cells expressing human adenosine Al-receptor : Inhibition of 3H-DPCPX binding (membranes) pKB =-6. 82 + 0.11 Inhibition of 3H-DPCPX binding (whole cells) pKB =-6.9 Antagonism of NECA-stimulated cAMP accumulation pKI =-6. 98 + 0. 15 Antagonism of NECA-stimulated inositol phosphate accumulation pKI =-6. 26 + 0.20

Imaging : Picture of XAC-BY630/650 binding to CHO-A1 cells and CHO-A1-GFP cells Also pictures showing displacement of binding by non-fluorescent antagonist DPCPX.

Example D LIBRARY WITH DIFFERENT FLUORESCENTLY TAGGED LIGANDS Dl A library is assembled comprising 3 fluorescent ligands each ligand comprising ABIPEA fluorescently tagged with a fluorophore providing different fluorescence characteristics selected from BODIPY 630/650-X-SE, EvoBlue 30 SE, BODIPY FL ethylene diamine etc.

Fluorescently tagged ligands are obtained by the process of the invention as hereinbefore defined.

The library includes data sheets (C. above) for each ligand.

D2 An alternative library is assembled comprising 2 fluorescent ligands comprising adenosine and ABIPEA as herein before referred, each were divided into 3 samples and modified by incorporation of a linker of varying carbon chain length from C3-6, whereby the compounds of formula 1\1 comprised JL is amine, L is (CHs- 6 and YL is amine. The compounds were reacted with fluorophore providing different fluorescence characteristics selected from EvoBlue 30 SE and BODIPY 630/650 X- SE.

Fluorescently tagged ligands are obtained by the process of the invention as hereinbefore defined.

The library includes data sheets (C. above) for each ligand.

D3 An alternative library is assembled comprising 3 tagged ligands each ligand comprising ABIPEA tagged with a selection of tags as known in the art, including one tagged with a fluorophore.

The library includes data sheets (C. above) for each ligand.

The libraries are useful for conducting binding studies as known in the art for a desired fluorescent ligand having the desired fluorophore or for a selection of fluorescent ligands or for a selection of ligands one of which comprises a desired fluorophore.

A library was then selected for screening for binding at a desired receptor and a fluorescent ligand was selected which gave optimum pharmacology for the desired receptor. Choice of the library to be screened is facilitated by the rational design of the library which provides the required analogues to generate a positive selection.