WO2008076916A2 - Thallium-sensitive agents and methods of using the same - Google Patents

Abstract

The present application provides novel thallium-sensitive agents and assays using those agents, e.g., for identifying modulators of ion channels, channel-linked receptors or ion transporters.

Description

Thallium-Sensitive Agents and Methods of Using the Same

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority from U.S. Provisional Patent Application No. 60/875,105, filed on December 15, 2006, the contents of which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The field of the invention relates to ion channel assays.

BACKGROUND

Ion channels are important targets for a large number of therapeutic indications as well as for safety profiling of new drugs. Typically, ion channels, e.g., potassium channels, are assayed using patch clamp techniques or fluorescent membrane potential dyes. The former is typically a medium to low-throughput technique. The latter allows for high throughput, but often lacks specificity. The ability to use a high-throughput functional assay for the detection and characterization of small molecule modulators of potassium channels is highly desirable.

SUMMARY

Accordingly, the present application describes, e.g., novel thallium-sensitive agents and their use in screening assays, e.g., high-throughput screening assays. Such assays can be used, for example, to identify modulators of ligand- and voltage-gated channels, e.g., potassium channels, in an automated homogenous assay format, for example, using FLIPR™ or FLIPRTetra™ devices (Molecular Devices Corporation, Sunnyvale, CA).

In one aspect, the application provides compounds represented by Structure I, wherein Ri and R₂ are each independently a moiety that comprises up to 45 carbon atoms and, optionally, comprising one or more N, O, S, F, Cl, Br or I atoms (e.g., 1, 2, 3, 4, 5, 6, or 7 such atoms); R₃, R₄ and R₅ are each independently H, F, Cl, Br, I or a moiety that comprises up to 45 carbon atoms and, optionally, comprising one or more N, O, S or F atoms (e.g., 1, 2, 3, 4, 5, 6, or 7 of such atoms); R₆ is H, F, Cl, Br, I, CN, NO₂, NH₂, alkylamino or dialkylamino; R₇, Rs and R9 are each independently F, Cl, Br, I, NO₂, NH₂, or CN, or a moiety comprising up to 45 carbon atoms and, optionally, comprising one or more N, O, S or F atoms; any two of R₇, Rs or R₉ may together define a ring (e.g., 1, 2, 3, 4, or even 5 such rings); A is O, S, Se or N; and n is 0 or 1. Such compounds can be, e.g., compounds as represented by Structures (1), (2), (3), (4), (5), (6) or (7). Each Ri and R₂ can be independently neutral or charged, e.g, having one or more positive or negative charges. The heterocyclic ring can be connected to the coumain fragment in by any connectivity, e.g., at the 2, 3, 4 or 5 position.

The coumarin fragment can be connected at the 2 position, such compounds being represented by Structure II. A can be S, as represented by Structure IV. Re can be H, such compounds being represented by Structure VII.

In any of the above compounds, R3 can be represented by, e.g.,

wherein Ri₀ and Rn are each independently H or a moiety that includes up to 8 carbon atoms and, optionally, one or more N, O, S or F atoms; and wherein a and b are each independently a integer between 1 and 6, inclusive. In such compounds, Rio and Rn can each be, e.g.,

In any of the above compounds, Ri and R₂ can each independently be, e.g.,

wherein R₄₃ and R₄₄ are each independently H or a moiety that includes up to 8 carbon atoms and, optionally one or more N, O, S or F atoms; and wherein a and c are each independently an integer between 1 and 6, inclusive. In such compounds, R₄3 and R₄₄ can each be, e.g.,

In another aspect, the application provides compounds represented by Structure XX, wherein R₄5, Ri6, Rn and Ris are each independently H or a moiety that includes up to 12 carbon atoms and, optionally, one or more N, O, S or F atoms; G is CHO or a moiety that includes up to 16 carbon atoms and, optionally, one or more N, O, S or F atoms; and e and f are each independently a integer between 1 and 6, inclusive. For example, the compound can be represented by Structures (10), (11), (12) or (13). The compound can be represented by Structure XXI, where G is CHO and e and f are each 1. In the compounds, R₁5, Ri6 and Ris can each independently be, e.g.,

In the compounds, Ris can be, e.g.,

The compound can be represented by Structure XXII, wherein G is G'. G' can be, e.g.,

In another aspect, the invention provides compositions comprising one or more of any of the compounds described herein. For example, the composition can include compounds (3), (10), (11), (12) and (13). Compounds (3), (10), (11), (12) and (13) can be present in the composition from, e.g., about 25 to about 40 percent by weight, from about 9 to about 30 percent by weight, from about 9 to about 30 percent by weight, from about 15 to about 50 percent by weight and from about 15 to about 50 percent by weight, respectively. Compounds (3), (10) + (11), (12) + (13) can be present in the composition from about 31 to about 40 percent by weight, from about 20 to about 30 percent and from about 30 to about 50 percent by weight, respectively. The ratio of compound (1 l):compound (10) can be about 1:3 and/or the ratio of compound (13):compound (12) can be from about 1 :3.

In still another aspect, the application provides a kit comprising one or more of any of the compounds described herein. The kit can include one or more masking compounds. The kit can include a surfactant (such as polyethylene glycol), a buffer, and/or a thallium salt, such as thallium sulfate.

In yet another aspect, the application provides a method for detecting the activity of a target transport structure, the method comprising: (a) contacting a cell expressing a target transport structure with the thallium sensitive agent described herein; (b) contacting the cell with a candidate modulator; (c) contacting the cell with an assay buffer containing a thallium salt solution; and (d) detecting a signal generated by the thallium sensitive agent to determine the effect of the candidate modulator on the activity of the target transport structure.

In another aspect, the application provides a method for identifying a modulator of a target transport structure, the method comprising: (a) contacting a cell expressing a target transport structure with the thallium sensitive agent described herein; (b) contacting the cell with a candidate modulator; (c) contacting the cell with an assay buffer comprising a thallium salt solution; and (d) detecting a signal generated by the thallium sensitive agent to determine the effect of the candidate modulator on the activity of target transport structure.

The cell can expresses an ion channel that is a cation channel permeable to thallium ions. The cation channel can be selected from the group consisting of potassium ion channel, sodium ion channel, and calcium ion channel. The potassium ion channel can be a calcium-activated and voltage-gated channel. The potassium ion channel can be an SK, Maxi-K, HERG and KCNQ channel. The cation channel can be a ligand-gated VRl channel.

The cation channel can be a non-selective ion channel. The non-selective ion channel can be an acetylcholine receptor, glutamate receptor, kainate, NMDA receptor, 5-hydroxytryptamine-gated receptor-channel, ATP-gated (P2X) receptor- channel, nicotinic acetylcholine-gated receptor-channel, vanilloid receptor, ryanodine receptor-channel, IP3 receptor-channel, cation channel activated in situ by intracellular cAMP, and cation channel activated in situ by intracellular cGMP. The thallium salt solution can include a water soluble thallium salt, e.g., selected from the group consisting OfTl₂SO₄, Tl₂CO₃, TlCl, TlOH, TlNO₃ and TlOAc.

The assay buffer can be ClTree, and may further include sodium gluconate; potassium gluconate; calcium gluconate; magnesium gluconate; HEPES and glucose.

Cells may be grown in a low Cl - cell growth medium, e.g., containing no more than 2 mM Cl^". The low Cl - cell growth medium can include sodium gluconate; potassium gluconate; MgSO₄JH₂O; NaHCO₃; calcium gluconate; NaH₂PO₄; HEPES; Glucose; lOOxVitamins; 5Ox amino acids; and glutamine.

The thallium sensitive agent may be a thallium sensitive fluorescent agent or thallium sensitive non-fluorescent agent. The thallium sensitive agent can be, e.g., ThalKal, e.g., made by a process described herein.

The cell can express a channel-linked receptor selected from the group consisting of GPCR, metabotropic glutamate receptor, muscarinic acetylcholine receptor, dopamine receptor, and serotonin receptor. The cell can express an ion transporter selected from the group consisting of dopamine ion transporter, glutamate ion transporter, seratonin ion transporter, sodium-potassium ATPase, proton- potassium ATPase, sodium/calcium exchanger, and potassium-chloride ion co- transporter. The thallium sensitive agent can be a fluorescent agent, and the method can further include contacting the cell with an extracellular masking agent (quenching compound) after contacting the cell with the thallium sensitive agent.

The modulating compound (e.g., a candidate compound) can activate or inhibit the ion channel, channel-linked receptor, or ion transporter.

The method can further include adding a stimulus solution to the thallium salt solution. The stimulus solution can include an agent selected from the group consisting of ionophore, KCl, nicotine, acetylcholine, muscarin, and carbamylcholine.

The method can further include measuring the signal after step (b). Detecting a signal may include detecting a decrease in the signal. A decrease in signal can indicate that the candidate agent is an activator of the ion channel, channel-linked receptor, or ion transporter. Detecting a signal can include detecting an increase in the signal. An increase in signal may indicate that the candidate agent is an activator of the ion channel, channel-linked receptor, or ion transporter. Detecting a signal can include detecting no change in the signal. No change in the signal can indicate that the candidate agent is an inhibitor of the ion channel, channel- linked receptor, or ion transporter.

In another aspect, the application provides compounds represented by Structure I' (of FIG. 8E), wherein Ri and R₂ are each independently a moiety that comprises up to 45 carbon atoms and, optionally, comprising one or more N, O, S, F, Cl, Br or I atoms (e.g., 1, 2, 3, 4, 5, or even 6 such atoms); R3, R₄ and R5 are each independently H, F, Cl, Br, I or a moiety that comprises up to 45 carbon atoms and, optionally, comprising one or more N, O, S or F atoms (e.g., 1, 2, 3, 4, 5, 6, or 7 of such atoms); Re is H, F, Cl, Br, I, amino, alkylamino, dialylamino, NO₂, or CN; and β is a moiety that comprises up to 45 carbon atoms and that includes a heterocyclic 5- or 6-membered ring system, e.g., 1, 2, 3, or even 4 such rings. In some embodiments, the heterocyclic ring is directly bonded to the coumarin fragment.

In some embodiments, the moiety further comprises one or more N, O, S or F atoms in addition to the one or more heteroatoms of the one or more rings. For example, the heterocyclic 5- or 6-membered ring system can be an aromatic ring system. For example, the heterocyclic ring system can be a 5- membered ring system that includes 2, 3, 4, or even 5 heteroatoms, such as O, S, Se, or N. In particular embodiments, the 5-membered ring system is a thiadiazole ring system, such as a 1,3,4-thiadiazole ring system. For example, such a system can have one of the structures shown directly below.

For example, the heterocyclic ring system can be a 6-membered ring system that includes 1 or more heteroatoms, e.g., 2, 3, 4, or even 5. In some embodiments, the 6-membered ring system includes 1 heteroatom and the heteroatom is N. For example, such a ring system can be a pyridine ring system. In particular embodiments, such a system can have one of the structures shown directly below. While the 3 -isomer is shown below, connectivity can be other than through carbon 3, e.g., it can be through 1 or 2.

As used in this application, the following words or phrases have the meanings specified.

A "transport structure" is any cellular structure involved in transporting ions across a cellular membrane. Exemplary transporter structures include ion channels, ion channel-linked receptors, and ion transporters.

An "ion channel" is any protein or proteins that forms an opening or a pore in a cellular membrane where the pore or opening is capable of permitting ions to flow therethrough. A "channel-linked receptor" is any protein or proteins that are linked to ion channels, where the protein activity affects the activity of an ion channel. An "ion transporter" is any protein or proteins that transports ions across a cellular membrane. A "modulator" is any compound or agent that can alter the activity of an ion channel, i.e., alter the movement or transport of ions through an ion channel. The modulator can be an organic molecule or chemical compound (naturally occurring or non-naturally occurring), such as a biological macromolecule (e.g., nucleic acid, protein, non-peptide, or organic molecule), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues, protein or protein fragment. Modulators are evaluated for the potential to act as inhibitors or activators of a biological process or processes, e.g., to act as agonist, antagonist, partial agonist, partial antagonist, antineoplastic agents, cytotoxic agents, inhibitors of neoplastic transformation or cell proliferation, and cell proliferation- promoting agents. The activity of a modulator may be known, unknown or partially known.

A "channel blocker" is a compound that inhibits, directly or indirectly, the movement of ions through an ion channel. The compound may exert its effect by directly occluding the pore, by binding and preventing opening of the pore, or by affecting the time and frequency of the opening of the ion channel.

A "channel opener" is a compound that activates the movement of ions through an ion channel. The compound may regulate ion channels by affecting the duration and/or frequency of the opening of the ion channel. Alternatively, the compound may change the voltage dependence of voltage-gated ion channels, such that the ion channel is open.

An "agonist" is a molecule that is able to activate the ion channel, channel- linked receptor or ion transporter.

An "antagonist" is a molecule that affects the agonist action or which inhibits the activity of the ion channel, channel-linked receptor or ion transporter. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and equipment or software similar or equivalent to those described herein can be used in the practice of the present invention, suitable methods, equipment, and software are described below. All publications and other references mentioned herein are incorporated by reference in their entirety for all that they contain. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features and advantages of the invention will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. IA and IB are schematic representations of generalized structures of some fluorescent thallium sensitive compounds.

FIGS. 2A-2D are schematic representations of generalized structures of fluorescent thallium sensitive dyes having O, S, Se, or NH in a heterocyclic ring attached to a coumarin fragment.

FIGS. 3A-3C show generalized structures for fluorescent thallium sensitive dyes having S in the heterocyclic ring attached to the coumarin fragment and also having various Rβ substitution.

FIGS. 4A-4C are generalized structures for fluorescent thallium sensitive dyes having S in a heterocyclic ring attached to a coumarin fragment and also having various R₆-R₉ substitution.

FIGS. 5A-5F are generalized structures of fluorescent thallium sensitive dyes having S in a heterocyclic ring attached to a coumarin fragment and also having various R3-R6 substitution. FIG. 6 are generalized structures for fluorescent thallium sensitive dyes for a specific class of Ri-R₃ substitution.

FIG. 7 are structures of some exemplary fluorescent thallium sensitive dyes.

FIG. 8 are structures of some other exemplary fluorescent thallium sensitive dyes. FIGS. 8A-8C are structures of some other exemplary fluorescent thallium sensitive dyes.

FIGS. 8D is a schematic representation of generalized structures of some other fluorescent thallium sensitive compounds. FIG. 8E are structures of still other exemplary fluorescent thallium sensitive dyes.

FIG. 9 is a generalized structure of synergists.

FIGS. 10A- 1OB are generalized structures for other synergists. FIG. 11 is some exemplary functionality for synergists.

FIG. 12 are structures for some exemplary synergists.

FIG. 13 are structures for some other exemplary synergists.

FIGS. 14-16 together illustrate a synthetic scheme, illustrating one synthetic method for producing compound (1). FIGS. 17-19 together illustrate a synthetic scheme, illustrating one synthetic method for producing compound (2).

FIGS. 20-22 together illustrate a synthetic scheme, illustrating one synthetic method for producing compound (3).

FIG. 23 is a synthetic scheme, illustrating the formation of compound (11) from compound (10).

FIGS. 24A-24B is a synthetic scheme, illustrating the formation of compounds (12) and (13).

FIG. 25 are possible structures useful for producing the fluorescent thallium sensitive compounds of FIG. 8. FIG. 26 is an illustration that provides a schematic overview of an exemplary assay described in the present application.

FIG. 27 is a graph illustrating that signal increases observed in experiments using ThalKal in voltage-gated channels is potassium-dose dependent.

FIG. 28 is a graph illustrating that a no-wash potassium channel assay, described herein, provides favorable results as compared to a potassium channel wash assay.

FIG. 29 is a graph illustrating that signal changes observed in experiments using ThalKal are dependent upon the thallium concentration used in the experiments.

FIGS. 30A and 30B are a set of graphs illustrating the results of assays where cisapride and dofetilide were used as modulators.

FIGS. 31A-3 IB are a set of graphs that illustrate the response of ThalKal to thallium as compared to the response of known fluorescent probe BTC. FIGS. 32A-32C are a set of graphs that illustrate the effect of l,2-bis(2- aminophenoxy)ethane-N,N,N^l,N'-tetraacetic acid (BAPTA) and BAPTA derivatives on enhancing the outcome of assays using purified compound (3).

FIGS. 33A-33B are a set of graphs that illustrate that an assay performed using ThalKal (Fig. 33A) prepared in accordance with the presently described methods yields results similar to an assay performed using highly purified compounds (10) and (1 1) (Fig. 33B).

FIG. 34 is a graph that provides the results of assays performed using ThalKal (blue circles) and highly purified compound (3) along with compounds (10) and (1 1) (red squares) when dofetilide was used as an inhibitor of hERG channels.

DETAILED DESCRIPTION

The present application describes novel thallium-sensitive agents and assay methods for detecting and identifying compounds that modulate (e.g., activate, increase, inhibit, or reduce) the activity of ion channels, e.g., potassium, calcium and/or sodium channels. Such modulators may be useful, e.g., for treating a variety of conditions such as cation channel-associated diseases, diseases associated with channel-linked receptors, and bacterial, fungal, inflammatory or immunological disorders.

A. THALLIUM SENSITIVE AGENTS

The present application provides novel thallium sensitive agents. In the methods described herein, thallium sensitive agents are employed as an indicator of flux of thallium across membranes. The thallium sensitive agents are sufficiently sensitive so as to produce detectable changes in fluorescence or optical intensity in response to changes in thallium ion concentration in the cell cytoplasm. Types of thallium sensitive agents that can produce a detectable signal include, e.g., fluorescent compounds, optionally in combination with synergists and/or non-fluorescent compounds.

IA. THALLIUM SENSITIVE FLUORESCENT AGENTS THAT INCLUDE A COUMARIN

FRAGMENT BONDED TO SYSTEM THAT INCLUDES A 5-MEMBERED HETEROCYCLIC RING SYSTEM THAT INCLUDES A SINGLE HETEROATOM IN THE RING Referring to FIG. IA, generally, some of the thallium sensitive fluorescent compounds can be represented by Structure I, which includes a coumarin dye core bonded to a system that includes a heterocyclic 5-membered ring system, e.g., an aromatic heterocyclic 5-membered ring system, that includes a single heteroatom in the ring system. In such compounds, Ri and R₂ are each independently a moiety that includes up to 45 carbon atoms. The moieties that include up to 45 carbon atoms, optionally, but often desirably, include one or more N, O, S, F, Cl, Br or I atoms, which can act as chelating atoms when they possess lone pairs of electrons. R₃, R₄ and R₅ are each independently H, F, Cl, Br, I or a moiety that includes up to 45 carbon atoms and, optionally, but often desirably, one or more N, O, S or F atoms. Re is H, F, Cl, Br, I or CN and R₇, R₈ and R₉ are each independently F, Cl, Br, I, NO₂, NH₂ or CN, or a moiety including up to 45 carbon atoms and, optionally, but often desirably, one or more N, O, S or F atoms. In the compounds represented by Structure I, any two Of R₇, Rs or R₉ may together define a ring. In addition, in compounds represented by Structure I, A is O, S, Se or N. When A is O, S or Se, n is O (no hydrogen) and when A is N, n is 1, meaning the nitrogen has a hydrogen atom bonded thereto. Any compound described herein can be in salt form, e.g., when A is nitrogen, the nitrogen can has two hydrogen atoms such that the nitrogen atom has a positive charge.

In some implementations, the moieties that include up to 45 carbon atoms have fewer carbon atoms, e.g., up to 40, up to 35, up to 25 or up to 20 carbon atoms. In embodiments in which the moieties that include up to 45 carbon atoms include one or more N, O, S, F, Cl, Br or I, or one or more N, O, S or F, the compounds can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or even 12 heteroatoms, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or even 12 O or S atoms. Any one or more of Ri, R₂, R₃, R₄, R₅, R₇, Rs or R₉ can be, e.g., an alkyl group, such a straight chain, branched, mono- or polycyclic alkyl group. Examples of straight chain and branched alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec -butyl, t-butyl, amyl, isoamyl, sec-amyl, 1,2-dimethylpropyl, 1, 1- dimethylpropyl, pentyl, hexyl, 4-methylpentyl, 1-methylpentyl, 2-methylpentyl, 3- methylpentyl, 1, 1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,2- dimethylbutyl, 1,3-dimethylbutyl, 1,2,2-trimethylpropyl, 1, 1,2-trimethylpropyl, heptyl, 5-methylhexyl, 1-methylhexyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 4,4- dimethylpentyl, 1 ,2-dimethylpentyl, 1,3-dimethylpentyl, 1,4-dimethylpentyl, 1,2,3- trimethylbutyl, 1 , 1 ,2-trimethylbutyl, 1,1,3-trimethylbutyl, octyl, 6-methylheptyl, 1- methylheptyl, 1,1,3,3-tetramethylbutyl and nonyl. Examples of cyclic alkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, and cyclodecyl. Any one or more of R3, R₄, R5, R7, Rs or R9 can be alkoxy, e.g., such as those derived from primary, secondary or tertiary alcohols. In some implementations, the alkoxy group includes between one carbon atom and about 12 carbon atoms. Other examples of alkoxy groups include those derived from aromatic hydroxy compounds, such as those that include up to 20 carbon atoms (e.g., those derived from phenols and naphthols).

When any two of R₇, Rs or R₉ together define a ring, the ring can be carbocyclic or heterocyclic. The ring can also be aromatic or non-aromatic. For example the ring can be a 5-, 6- or 7-membered ring. When one of R7, Rs or R9 together define a ring, the ring can be optionally substituted with one or more alkyl groups or alkoxy groups described herein (or others). Also, the ring can be optionally substituted with one or more F, Cl, Br or I.

Referring to FIG. IB, in desirable implementations, the thallium sensitive fluorescent compounds are represented by Structure II in which the coumarin fragment is connected at the 2 position of the 5-membered heterocyclic compound. Often these are desirable since 2-substituted derivatives are generally more available. Nevertheless, the 3-, A-, or even 5-substituted isomer can be utilized.

As exemplified in FIGS. 2A-2D, A can be O, S, Se or NH, corresponding to Structures III, IV, V and VI, respectively. In desirable implementations A is S.

Referring to FIGS. 3A-3C, while R₆ can be H (FIG. 3A, Structure VII), F, Cl, Br, I (FIG. 3 C, Structure IX) or CN (FIG. 3 B, Structure VIII), in desirable embodiments, R₆ is H. Changing the electronegativity of R₆ can tune the fluorescence of the compound.

Referring to FIG. 4A, in some embodiments, A is S, R₆ is H and Rs is a 2- methoxy ethane group (Structure X). Referring to FIG. 4B, in other embodiments, A is S, R₆ is CN and Rs is a methyl group (Structure XI). Referring to FIG. 4C, in still other embodiments, A is S, R₆ is I and Rs is a 2-furan group (Structure XII).

Referring to FIG. 5A, in some embodiments, A is S, R₆ is H and R3 is a methyl group (Structure XIII). Referring to FIG. 5B, in some embodiments, A is S, Re is H and R₅ is a methoxy group (Structure XIV). Referring to FIG. 5C, in some instances, A is S, R₆ is H and R₄ is Cl (Structure XV). Referring to FIG. 5D, in still other embodiments, A is S, R₆ is H and R₅ is F (Structure XVI). Referring to FIG. 5E, in still other embodiments, A is S, Re is H and R₅ is a t-butyl group (Structure XVII). Referring now to FIG. 5F, in desirable embodiments, A is S and each of R₃, R₄, R₅ and R₆ is H.

Referring now to FIG. 6, in desirable embodiments, R₃ is represented by fragment Fl . In such desirable embodiments, Rio and Rn are each independently H (acid form) or a moiety that includes up to 8 carbon atoms and, optionally, but often desirably, one or more N, O, S or F atoms. In such embodiments, a and b are each independently an integer between 1 and 6, inclusive. In desirable embodiments, a and b are both 1. In particular implementations, Rio and Rn are each an acetoxymethyl group, which is represented by fragment F2. In desirable implementations, Ri and R₂ are represented by fragment F3 and fragment F4, respectively. In such implementations, c and d are each independently an integer from 1-6, inclusive. For example, Rio and/or Rn can be H or acetoxymethyl.

Referring to FIG. 7, in particular implementations, the thallium sensitive fluorescent compounds are represented by compounds (1), (2) or (3). While compounds (1), (2) and (3) of FIG. 7 have acetoxymethyl groups, the compounds can be provided, e.g., in acid form (or its conjugate base form, e.g., its Li, Na, K, Ca or Mg salt form). In still other embodiments, some of the groups are in acid form and some are in ester form, e.g., in mixed form.

Referring to FIG. 8, in still other particular implementations, the thallium sensitive fluorescent compounds are represented by compounds (4), (5), (6) or (7). While the compounds (4), (5), (6) and (7) of FIG. 8 are shown as methyl esters, the compounds can be, e.g., provided in acid form (or its conjugate base form, e.g., its Li, Na, K, Ca or Mg salt form), or they can be provided as another ester, e.g., an acetoxymethyl ester or ethyl ester. In still other embodiments, mixed groups are provided. In still other particular embodiments, the thallium sensitive fluorescent compounds are represented by those structures (40), (42), (44), (46), (47), (48), (49), (50), (52), (54), (56), (58) and (60) of FIGS. 8A-8C. While compounds shown in FIGS. 8A-8C have acetoxymethyl groups, the compounds can be provided, e.g., in corresponding acid form (or its conjugate base form, e.g., its Li, Na, K, Ca or Mg salt form). In still other embodiments, some of the groups are in acid form and some are in ester form, e.g., in mixed form.

With respect to the Schiff base derivatives, e.g., compound (52) of FIG. 8B, Ar can be any aromatic ring system, such as a carbocyclic or heterocyclic aromatic ring system, optionally substituted with a moiety that include up to 45 carbon atoms, and that, optionally includes one or more N, O, S, F, Cl, Br or I (in addition to the atoms of the ring). For example, the aromatic ring system can be an imidazole, benzimidazole, purine, pyrazole, indazole, oxazole, benzoxazole, isoxazole, benzis oxazole, thiazole or benzothiazole, pyridine, quinoline, isoquinoline, pyrazine, quinooxaline, acridine, pyrimidine or quinazoline ring system. For example, the ring system can include 1, 2, 3, or even 4 such rings.

IB. THALLIUM SENSITIVE FLUORESCENT AGENTS THAT INCLUDE A COUMARTN FRAGMENT BONDED A SYSTEM THAT INCLUDES A 5- or 6- MEMBERED HETEROCYCLIC RING SYSTEM THAT INCLUDES ONE OR MORE HETEROATOMS IN THE RING

Referring now to FIG. 8D, generally, some other thallium sensitive fluorescent compounds can be represented by Structure I', which includes a coumarin dye core bonded to a system (β) that includes a heterocyclic 5- or 6-membered ring system, e.g., an aromatic heterocyclic 5-membered ring system, that includes one or more heteroatoms, e.g., 2 or 3 heteroatoms, that are each the same or that are each different. For example, each heteroatom can be independently O, S, Se or N. The system (β), can include one or more F, Cl, Br, I, NO₂, NH₂ or CN, or one or more moieties that include up to 45 carbon atoms and, optionally, but often desirably, one or more N, O, S or F atoms.

In such compounds, Ri and R₂, R3, R₄, R5 and Re can be any of those moieties described in reference to FIG. IA.

For example, β in the compounds of Structure I' can be or can include one or more imidazole, benzimidazole, purine, pyrazole, indazole, oxazole, benzoxazole, isoxazole, benzisoxazole, thiazole or benzothiazole, pyridine, quinoline, isoquinoline, pyrazine, quinooxaline, acridine, pyrimidine or quinazoline ring systems. For example, β can include 1, 2, 3, or even 4 such rings systems. The heterocylic ring can be bonded directly to the coumarin fragment.

FIG. 8E shows structures for specific examples of this type of compound. While compounds (62), (63), (64), (65) and (68) shown in FIGS. 8E have 5 acetoxymethyl groups, the compounds can be provided, e.g., in corresponding acid form (or its conjugate base form, e.g., its Li, Na, K, Ca or Mg salt form). In still other embodiments, some of the groups are in acid form and some are in ester form, e.g., in mixed form.

With respect to the Schiff base derivatives, e.g., compound (65) of FIG. 8E, o Ar can be any aromatic ring system, such as a carbocyclic or heterocyclic aromatic ring system, optionally substituted with a moiety that include up to 45 carbon atoms, and that, optionally includes one or more N, O, S, F, Cl, Br or I (in addition to the atoms of the ring). For example, the aromatic ring system can include an imidazole, benzimidazole, purine, pyrazole, indazole, oxazole, benzoxazole, isoxazole, 5 benzis oxazole, thiazole or benzothiazole, pyridine, quinoline, isoquinoline, pyrazine, quinooxaline, acridine, pyrimidine or quinazoline ring system. For example, the ring system can include 1, 2, 3, or even 4 such rings.

2. SYNERGISTS 0 Certain agents can work in conjunction with the thallium sensitive fluorescent agents to provide an effect that is greater than obtained using fluorescent agents alone. These agents are referred to herein as synergists. Without intending to be bound by theory, synergists are believed to bind calcium in the cell such that less free calcium is available to bind fluorescent thallium sensitive agent and may have a higher affinity5 for calcium than other metals, e.g., thallium and/or potassium.

Referring to FIG. 9, generally, useful non-fluorescent synergists are represented by Structure XX. In such compounds, R₄₅, R₁₆, Rn and Ris are each independently H or a moiety that includes up to 12 carbon atoms and, optionally, yet desirably, one or more N, O, S or F atoms. G is CHO or a moiety that includes up to0 16 carbon atoms and, optionally, yet often desirably, one or more N, O, S or F atoms. In compounds of Structure XX, e and f are each independently an integer between 1 and 6, inclusive. In certain implementations, e and f are both 1. The moiety that includes up to 12 carbon atoms and/or the moiety that includes up to 16 carbon atoms can be, e.g., any of the alkyl groups discussed above.

Referring to FIG. 1OA, in some non- fluorescent synergist compounds G is CHO and e and f are each 1 (Structure XXI). Referring to FIG. 1OB, in other embodiments, G is G' in which G' is a moiety that includes up to 16 carbon atoms and, optionally, yet often desirably, one or more N, O, S or F atoms (Structure XXII).

Referring to FIG. 11, in desirable embodiments, G' (of Structure XXII) is a benzothiazole F6 or a substituted benzothiazole group.

While still referring to FIG. 11, in Structures XX, XXI or XXII, Ri₅, Ri₆ and Ri₈ can each independently be H (acid form), acetoxymethyl F7 or (O=C)CH₃ F8. In certain implementations, Rn is fragment F9. While F9 shows the acetoxymethyl ester derivative, another ester or the acid form can be provided. In addition, some of the groups can be provided in acid form and others in ester form on a single molecule.

Referring now to FIG. 12, two synergists are shown (compounds 10 and 11). While acetoxymethyl ester derivatives are shown, another ester or the acid form can be provided. In addition, some of the groups can be provided in acid form and some in ester form. Other esters include methyl, ethyl, isopropyl, butyl and n-butyl.

Referring now to FIG. 13, two other synergists are shown (compounds 12 and 13). While acetoxymethyl ester derivatives are shown, another ester or the acid form can be provided. In addition, some of the groups can be provided in acid form and some in ester form. Other esters include methyl, ethyl, isopropyl, butyl and n-butyl.

3. COMPOSITIONS INCLUDING THALLIUM AGENTS

Compositions including thallium sensitive agents, e.g., fluorescent and synergists, can be made by combining one or more of the fluorescent agents and/or one or more of the synergists.

Referring back now to FIGS. 7 and 8, in some embodiments, the composition includes one or more of compounds (1), (2), (3), (4), (5), (6) and (7). The compounds can be provided as the acid form, the acetoxymethyl ester or another ester. Referring back now to FIGS. 12 and 13, in some embodiments, in addition to one or more of the compounds (1), (2), (3), (4), (5), (6) and (7), the composition can also include one or more of compounds (10), (11), (12) and (13). Compounds (10), (11), (12) and (13) can be provided in the acid form, the acetoxymethyl ester form or in another ester form.

In desirable embodiments, the composition of includes compound (3) and compounds (10), (11), (12) and (13). In some embodiments, compounds (3), (10), (11), (12) and (13) are present in the composition from about 25 to about 40 percent by weight, from about 9 to about 30 percent by weight, from about 9 to about 30 percent by weight, from about 15 to about 50 percent by weight and from about 15 to about 50 percent by weight, respectively.

In some embodiments, compounds (3), (10), (11), (12) and (13) are in the composition and compound (3), combined compounds (10) and (11) and combined compounds (12) and (13) are present in the composition from about 31 to about 40 percent by weight, from about 20 to about 30 percent and from about 30 to about 50 percent by weight, respectively.

In some embodiments, compounds (3), (10), (11), (12) and (13) are in the composition and a ratio of compound (1 l):compound (10) is about 1:3 and/or the ratio of compound (13):compound (12) is from about 1 :3.

4. KITS INCLUDING THALLIUM AGENTS

Kits including thallium sensitive agents, e.g., fluorescent and non-fluorescent agents, can be made by combining one or more of the fluorescent agents and/or one or more of the non-fluorescent agents and packaging the compounds and/or compositions as a kit.

In desirable embodiments, a kit is prepared by combining compound (3) and compounds (10), (11), (12) and (13). In addition to any of the compounds or compositions disclosed herein, the kit can also include one or more masking compounds, one or more surfactants, such as polyethylene glycol or polypropylene glycol, one or more buffers and/or a thallium salt, such as thallium sulfate.

Any kit may include instructions for use (see attached Appendix I).

5. METHODS OF MAKING THALLIUM SENSITIVE AGENTS Referring to FIGS. 14-16, compound (1) can be prepared by reacting

CHO(OH)-BAPTA derivative compound (14) with compound (15) in the presence of basic piperidine. Piperidine deprotonates one of the acid hydrogens α to the ethyl ester group. The resulting enolate (not shown) adds to the CHO group, followed by intramolecular cyclization and then dehydration to give compound (16). Generally, the reaction shown in FIG. 14 is carried out at a temperature of from about 50 ⁰C to about 150 ⁰C, e.g., from 60 ⁰C to about 100 ⁰C, and for a time from about 30 minutes to about 500 minutes, e.g., from about 60 minutes to about 250 minutes. Reaction of compound (16) with LiI in acetonitrile produces the shown lithium carboxylate (17). Generally, the reaction shown in FIG. 15 is carried out at a temperature of from about 50 ⁰C to about 150 ⁰C, e.g., from 60 ⁰C to about 100 ⁰C, and for a time from about 1 day to about 7 days, e.g., from about 2 days to about 4 days. Treatment of compound (17) with acid water yields the acid form (17'). The lithium carboxylate (17) can be reacted with bromomethylacetate in dimethyl formamide (DMF) and diisopropylethyl amine (DIEA) to give compound (1). Generally, the reaction shown in FIG. 16 is carried out at a temperature of from about 25 ⁰C to about 100 ⁰C, e.g., from 35 ⁰C to about 75 ⁰C, and for a time from about 30 minutes to about 500 minutes, e.g., from about 60 minutes to about 250 minutes.

Referring to FIGS. 17-19, compound (2) can be prepared by reacting CHO(OH)-BAPTA derivative compound (14) with compound (20) in the presence of basic piperidine. Piperidine deprotonates one of the acidic hydrogens α to the ethyl ester group. The resulting enolate (not shown) adds to the CHO group, followed by intramolecular cyclization and then dehydration to give compound (21). Reaction of compound (21) with LiI in acetonitrile produces the shown lithium carboxylate (22). Treatment of compound (22) with acidic water yields the acid form (22'). The lithium carboxylate (22) can be reacted with bromomethylacetate in dimethyl formamide (DMF) and diisopropylethyl amine (DIEA) to give compound (2). The reaction conditions used in the preparation of compound (2) are generally those used in the preparation of compound (1).

While (15) is the 2-substituted isomer, the 3 or 4-substituted isomer can be utilized to make the corresponding compound as well. Referring to FIGS. 20-22, compound (3) can be prepared by reacting

CHO(OH)-BAPTA derivative compound (14) with compound (25) in the presence of basic piperidine. Piperidine deprotonates one of the acidic hydrogens α to the ethyl ester group. The resulting enolate (not shown) adds to the CHO group, followed by intramolecular cyclization and then dehydration to give compound (26). Reaction of compound (26) with LiI in acetonitrile produces the shown lithium carboxylate (27).

Treatment of compound (27) with acidic water yields the acid form (27'). The lithium carboxylate (27) can be reacted with bromomethylacetate in dimethyl formamide (DMF) and diisopropylethyl amine (DIEA) to give compound (3). The reaction conditions used in the preparation of compound (3) are generally those used in the preparation of compound (1).

While the above synthetic scheme has been shown as complete reactions, in some embodiments, the reactions are not complete, and so there are competing reactions. For example, often when compound (3) is made, compounds (10), (11),

(12) and (13) are often generated as co-products. This can be desirable because in a single synthesis, fluorescent thallium sensitive compounds and synergist compounds can be prepared.

Referring to FIGS. 20 and 21, if cyclization is not complete, during lithiation, compound 10' also is formed. The next step of the reaction scheme (shown in FIG.

22) also then yields compound 10 of FIG. 23. Compound (11) is also often present, which is made by conversion of the acetoxymethyl ester to the acetate.

Referring now to FIGS. 24A and 24 B, compounds (12) and (13) also result from side-reactions. In particular, FIG. 24A shows that during the synthesis of compound (25), disulfide compound (35) is also generated. FIG. 24B shows that disulfide (35) can react with the aldehyde of compound (14) to form a Schiff base, which then cyclizes and then is oxidized. Treatment of the resulting cyclized and oxidized intermediate with LiI and then bromomethyl acetate gives compound (12).

Compound (12) then often converts form the acetoxymethyl ester to the acetate, to give compound (13).

Referring now to FIGS. 8 and 25, compounds (4), (5), (6) and (7) can be made by utilizing compounds (4'), (5 '), (6') and (7') (or equivalents thereof) in the synthetic scheme described above.

Referring back now to FIG. 8E, the pyridine derivative can be made using the methods described herein, along with a corresponding 1, 2 or 3 -substituted pyridine ester, such as ethyl isonicotinate, instead of the substituted thiophene derivative. B. METHODS OF USING THALLIUM SENSITIVE AGENTS

The present application provides useful assays that employ the thallium sensitive agents described above. The assays can be used, e.g., to determine whether a test compound is an ion channel modulator, e.g., a potassium, calcium, or sodium channel modulator. The methods described herein can also be used, for example, to observe the activity of an ion channel, ion channel-linked receptor, and/or ion transporter, directly or indirectly. The methods involve detecting the flux of thallium ions across membranes. The transport of thallium sensitive agents and thallium ions into or out of cells is followed by an increase or decrease in detectable signal. For example, when using a fluorescent thallium sensitive agent as described herein, flux of thallium ions across a membrane results in a change in intensity of fluorescence inside the cell, resulting in recordable signals. Activation of a cation channel enhances the rate of influx of thallium ions (resulting in, e.g., a change in fluorescence of a thallium sensitive fluorescent compound), and inhibition decreases the rate of influx of thallium ions (resulting in, e.g., no or little change in the fluorescence of a thallium sensitive fluorescent agent). Generally, fluorescence of a thallium sensitive agent remains unchanged if no thallium ion is bound to it. Thus, if a cell is loaded with a thallium sensitive agent, and an ion channel in the cell's membrane is blocked by a candidate channel modulator, thereby inhibiting thallium influx, little or no change in fluorescence in the cell will be detected. Simple and convenient optical methods can be employed to detect ion flux (influx or efflux), e.g., flux of thallium ions in, e.g., a live cell system. Useful methods are described, for example, in U.S. Patent Publication No. 2002/0168625 (U.S. Serial Number 09/975,891), which is incorporated herein by reference in its entirety. Alternatively or in addition, certain assays can be performed as non- fluorescence-based assays. For example, a thallium sensitive agent may react with thallium ions to form a product that forms a precipitate or a colored product, and thus causes detectable changes in optical density of a test mixture. In such cases, absorbance can be recorded, e.g., by a spectrophotometer, before, during, and after addition of thallium ions, and addition of channel modulators. Cells expressing ion channels and/or receptors can be loaded with thallium sensitive non-fluorescent agent and washed with, e.g., a buffered chloride-free solution. The transport of thallium into cells causes an increase or decrease in the optical density signal. Thallium ions pass through open channels and change the optical density inside the cell, resulting in recordable signals. Activation of an ion channel enhances the rate of influx of thallium ions (resulting in an increased formation of precipitant or colored product) and inhibition decreases the rate of influx of thallium ions (resulting in no or little change in precipitation or colored product formation). Generally, the optical density remains the same if no thallium ions react with the thallium sensitive compound compound. Thus, if an ion channel is blocked and thallium ion influx is inhibited, little or no change in optical density is detected.

General Influx Methods

Influx assays to identify potential modulators of target transporter structures are provided herein. Typically, such assays involve incubating a test mixture that includes: a) cells expressing the target ion channels (e.g. potassium, sodium, or calcium ion channel), ion channels that are linked to receptors (e.g. GIRK), channel- linked receptors (e.g. GPCR), and/or ion transporters (e.g. glutamate transporter); b) a detectable (i.e., signal generating) thallium sensitive agent; c) thallium ions; and d) a candidate modulator, i.e., a potential ion channel, channel-linked receptor or ion transporter activity modulator. Typically, the optical signal of the thallium sensitive agent is measured before the modulator is added and the assay is performed under conditions suitable for ion channel, channel-linked receptor or ion transporter activity to occur. The optical signal of the thallium sensitive agent is measured after the agent is added and a change in the optical signal indicates movement of thallium ions through the channel or transporter.

The methods can be performed using whole cells that express ion channels. In general, such methods might include: a) culturing the cells under suitable conditions; b) contacting or loading the cells with a thallium sensitive agent (e.g., a cell permeable thallium sensitive agent); c) treating the cells (e.g., washing or adding a masking agent) to remove or mask excess agent from the environment outside of the cells; d) measuring the detectable signal to obtain a baseline measurement; e) contacting the cells with a solution that includes thallium ions and an appropriate stimulus solution (i.e., a solution that activates the ion channel, channel-linked receptor, or ion transporter), contacting the cells with a candidate modulator; and f) detecting any signal change. Alternatively or in addition, whole cells expressing ion channels and channel- linked receptors can be used. Such methods might include: a) culturing cells expressing an ion channel and channel-linked receptor; b) contacting or loading the cells with a thallium sensitive agent; c) treating the cells (e.g., washing or adding masking agent) to remove or mask excess thallium sensitive agent; d) measuring the detectable signal to obtain a baseline measurement; e) contacting the cells with a candidate modulator; f) measuring the detectable signal; g) contacting the cells with a solution which includes thallium ions and an appropriate stimulus solution for the channel-linked receptor; and h) measuring the detectable signal. Alternatively or in addition, the methods can be performed using whole cells expressing ion transporters. Such methods may include: a) culturing cells expressing an ion transporter; b) contacting or loading the cells with a thallium sensitive agent; c) treating the cells to remove or mask excess thallium sensitive agent; d) measuring the detectable signal to obtain a baseline measurement; e) contacting the cells with a candidate modulator; 6) measuring the detectable signal; f) contacting the cells with a solution which includes thallium ions and an appropriate stimulus solution for the ion transporter; and g) measuring the detectable signal.

Skilled practitioners will appreciate that control experiments can be performed to facilitate analysis of the effects of a candidate modulator. Control experiments can be performed using, e.g., (1) native, untransfected cells under identical conditions of the methods of the invention; (2) the addition of thallium ions to the test mixture in the absence of stimulus solution; (3) cells under identical conditions of the methods of the invention, but without the candidate modulator of the target transport structure added to the test mixture; and/or (4) cells under identical conditions to the methods of the invention, but using known modulators of the target transport structure.

Changes in optical signal can be observed using any method known to those of skill in the art. An exemplary device that is particularly useful for observing such changes is the Fluorometric Image Plate Reader (FLIPR™; Molecular Devices Corporation, Sunnyvale, CA).

General Efflux Methods

Efflux assays typically employ the same types of cells used in the influx assays, which are loaded with a thallium sensitive agent described herein. Cells are contacted with a thallium composition (e.g., for about 5, 10, 15, or 20, or more than 20, minutes) to load the cells, such that thallium ions and thallium sensitive agent both reside in the cell at the start of the assay. The cells can then be washed to remove excess thallium ions and assayed, e.g., using a device such as a FLIPR™, to detect changes in signal. In lieu of washing cells, a masking agent can be added. Channels can be stimulated to open by the addition of any one of a number of ligands or by changing the membrane potential of the cell, such as by changing potassium concentrations, to permit efflux of ions through the ion channels. In this system, efflux can be detected, e.g., by detecting a decrease in the detectable signal (e.g., a decrease in fluorescence). Typically, in efflux methods, similar equipment and conditions are used as for influx methods.

Stimulus Solutions

A stimulus solution is a solution capable of activating a target transport structure (e.g., an agonist). Some ion channels/transporters are known to be constitutively active and thus do not require a "stimulus" in addition to the thallium ion tracer. For channels that do require a stimulus, that stimulus may be a ligand, e.g., a molecule that binds to the channel or channel linked receptor and turns it on (e.g., an agonist). Alternatively or in addition, a stimulus can be a change in membrane potential for voltage-gated channels. Typically, voltage-gated channels are activated by either direct electrical stimulation with electrodes or using a stimulus solution that contains an ionic composition capable of causing depolarization (such as high external potassium). Thallium or potassium ions can also act as a stimulus for voltage-gated channels. In such cases, thallium ions can act as both a tracer and a depolarizing stimulus. In influx assays, thallium ions can be added just before, during, or after the addition of a stimulus.

Stimulus solutions can be selected based on the type of transport structure used in a given assay. Selecting an appropriate stimulus solution is within the skill of the art. In some instances, a stimulus solution includes a buffer that does not include activating reagents, such that the target transport structure remains substantially at rest. In those instances, the stimulus solution includes reagents that do not activate the transport structure of interest, but facilitate activation when a modulating agent is added to the cells to initiate the assay.

The stimulus solution selected for use with voltage-dependent ion channels (e.g., the N-type calcium channel or KCNQ2 channel) often depends upon the sensitivity of the ion channel to the resting potential of the cell membrane. For methods using these voltage-dependent ion channels, the stimulating solution may include activating reagents that serve to depolarize the membrane (e.g., ionophores, valinomycin, etc.).

A stimulus solution selected for use with some voltage-dependent ion channels for activation by depolarization of the cell membrane includes potassium salt at a concentration such that the final concentration of potassium ions in the cell-containing well is in the range of about 10-150 mM (e.g., 50 mM KCl, or in thallium-based assays, 25 mM K₂SO₄, since chloride may cause precipitation of thallium ion). Alternatively or in addition, voltage-dependent ion channels can be stimulated by an electrical stimulus.

A stimulus solution selected for use with channel-linked receptors and ligand- gated ion channels depends upon ligands known to activate such receptors. For example, nicotinic acetylcholine receptors can be activated by nicotine or acetylcholine; similarly, muscarinic acetyl choline receptors may be activated by addition of muscarine or carbamylcholine. A stimulating solution for use with these systems may include, e.g., nicotine, acetylcholine, muscarine or carbamylcholine.

Cells and Cell-Free Systems

Methods described herein may employ cells expressing, e.g., one or more of 1) ion channels permeable to thallium; 2) ion channels and channel-linked receptors that are permeable to thallium ions; and/or 3) ion transporters permeable to thallium ions. Cells used for the methods described herein can be naturally occurring. Alternatively or in addition, it may be useful to employ cells transformed or transfected with heterologous nucleic acids encoding the transport structures, i.e., recombinant cells. Such cells can be particularly useful in that they might predominantly express a single type of structure of interest. It is well within the skill of artisans in the field to determine which types of cells should be used in a given assay.

Many cells that can be genetically engineered to express a heterologous cell surface protein are known. Useful cells include, e.g., bacterial cells, yeast cells and mammalian cells. Exemplary cells include, e.g., human embryonic kidney (HEK) cells, HEK 293 cells (U.S. Pat. No. 5,024,939; Stillman et al. 1985, MoI. Cell Biol. 5, 2051-2060), Chinese hamster ovary (CHO) cells (ATCC Nos. CRL9618, CCL61, 5 CRL9096), Xenopus laevis oocyte, (XLO) cells, baby hamster kidney (BHK) cells (ATCC No. CCLlO), mouse L cells (ATCC No. CCLI.3), Jurkats (ATCC No. TIB 152) and 153 DG44 cells (Chasin (1986) Cell. Molec. Genet. 12: 555) human embryonic kidney (HEK) cells (ATCC No. CRL1573), PC12 cells (ATCC No. CRL17.21) and COS-7 cells (ATCC No. CRL1651). o Cells can be cultured, e.g., in solution or on a solid support, and can be adherent or non-adherent. Useful solid supports include, e.g., glass or plastic culture dishes, beads, or multi-well plates. Although any number of cells capable of eliciting a detectable fluorescence signal in an assay may be used in a multi-well plate, skilled practitioners will appreciate that the number of cells seeded into each well may be5 chosen so that the cells are at or near confluence, but not overgrown, when the assays are conducted, so that the signal-to-background ratio of the signal is increased.

Alternatively or in addition, the methods can be performed using membranes (e.g., membrane vesicles) that include a transport structure. In some instances, the system may be essentially cell-free. The use of membrane vesicles are known to0 those of skill in the art.

The methods described herein are useful with potassium channels. The methods may also be useful with, e.g., other ion channels, channel-linked receptors, such as a receptor (e.g. GPCR), signal transduction pathways that are linked to or able to modulate the activity of an ion channel and proteins that are linked to ion channels,5 bacterial porins, and ion transporters.

Ion Channels

Ion channels include, e.g., ligand- or voltage-gated, stretch-activated cation channels, selective or non-selective cation channels. 0 Types of ligand-gated non-selective cation channels include, e.g., acetylcholine receptors, glutamate receptors such as AMPA, kainate, and NMDA receptors, 5-hydroxytryptamine-gated receptor-channels, ATP-gated (P2X) receptor- channels, nicotinic acetylcholine-gated receptor-channels, vanilloid receptors, ryanodine receptor-channels, IP3 receptor-channels, cation channels activated in situ by intracellular cAMP, and cation channels activated in situ by intracellular cGMP.

Types of ion channels, e.g., voltage-gated ion channels, include Ca²⁺, K⁺, and Na⁺ ion channels. Channels can be expressed exogenously or endogenously. Channels can be stably or transiently expressed in native and/or engineered cell lines.

Types Of K⁺ channels include, e.g., KCNQl (KvLOTl), KCNQ2, KCNQ3, KCNQ4, KCNQ5, HERG, KCNEl(IeK, MinK), Kvl.5, Kir 3.1, Kir 3.2, Kir 3.3, Kir 3.4, Kir 6.2, SUR2A, ROMKl, Kv2.1, KvI.4, Kv9.9, Kir6, SUR2B, KCNQ2, KCNQ3, GIRKl, GIRK2, GIRK3, GIRK4, hlKl, KCNAl, SURl, Kvl.3, HERG (Conley, E. C. and Brammer, W. J.; 1999, The Ion Channel, Factsbook IV. Voltage- Gated Channels, Academic Press, London, UK), intracellular calcium-activated K⁺ channels, rat brain (BK2) (McKinnon, D. (1989) J. Biol Chem. 264, pp. 9230-8236); mouse brain (BKl) (Tempel, et al. (1988) Nature 332, pp. 837-839), and the like. Types of Na⁺ channels include, e.g., rat brain I and II (Noda, et al. 1986, Nature 320, pp. 188-192); rat brain III (Kayano, et al. 1988, FEBS Lett. 228, pp. 187- 194); human II (ATCC No. 59742, 59743 and Genomics 1989,5:204-208), and the like.

Types Of Ca²⁺ channels include, e.g., human calcium channel α_ls α₂, β and/or γ subunits (U.S. application Ser. Nos. 07/745,206 and 07/868,354), the ryanodine receptor (RyR) and the inositol 1,4,5 -triphosphate receptor (IP3_R) (T. Jayaraman et al., J. Biol. Chem., 267, pp. 9474-77 (1992); A. M. Cameron et al., Proc. Natl. Acad. Sci. USA, 92, pp. 1784-44 (1995)), rabbit skeletal muscle O₁ subunit (Tanabe, et al. (1987) Nature 328, pp. 313-E318); rabbit skeletal muscle α₂ subunit (Ellis, et al. (1988) Science 241, pp. 1661-1664); rabbit skeletal muscle p subunit (Ruth, et al. (1989) Science 245, pp. 1115-1118); rabbit skeletal muscle gamma subunit (Jay, et al. (1990) Science 248, pp. 490-492), and the like.

Channel-Linked Receptors

The methods can be applied to indirectly measure the activity of channel- linked receptors and signal transduction systems. For example, the activity of channel-linked receptors can be determined, where the activation of the receptor initiates subsequent intracellular events that lead to the modulation of ion channel activity. Such modulation may result from interactions between receptor subunits with ion channels (e.g., GPCR βγ subunits and GPCR-linked K⁺ channels (e.g., GIRKs)) or by changes in the concentrations of messenger molecules such as calcium, lipid metabolites, or cyclic nucleotides, which modulate ion channel activity.

Exemplary useful G-protein-coupled receptors include, e.g., muscarinic acetylcholine receptors (mAChR), adrenergic receptors, serotonin receptors, dopamine receptors, angiotensin receptors, adenosine receptors, bradykinin receptors, metabotropic excitatory amino acid receptors, and the like.

Another type of indirect assay involves determining the activity of receptors that when activated result in a change in the level of intracellular cyclic nucleotides, e.g., cAMP, cGMP. For example, activation of some dopamine, serotonin, metabotropic glutamate receptors and muscarinic acetylcholine receptors results in an increase or decrease in the cAMP or cGMP levels in the cytoplasm. Furthermore, there are cyclic nucleotide-gated ion channels, e.g., rod photoreceptor cell channels and olfactory neuron channels (Altenhofen, W. et al. (1991) Proc. Natl. Acad. Sci U.S.A. 88:9868-9872 and Dhallan et al. (1990) Nature 347: 184-187), that are permeable to cations upon activation by binding of cAMP or cGMP. Thus, in accordance with the methods described herein, a change in cytoplasmic ion levels, caused by a change in the amount of cyclic nucleotide activation of photo-receptor or olfactory neuron channels, can be used to determine function of receptors that cause a change in cAMP or cGMP levels when activated. In cases where activation of the receptor results in a decrease in cyclic nucleotide levels, it may be useful to expose the cells to reagents that increase intracellular cyclic nucleotide levels, e.g., forskolin, prior to adding a receptor-activating compound to the cells in the assay.

Cells used for this type of assay can be generated by co-transfecting a host cell with DNA encoding an ion channel (such as GIRK) and DNA encoding a channel- linked receptor (e.g., certain metabotropic glutamate receptors, muscarinic acetylcholine receptors, dopamine receptors, or serotonin receptors) which, when activated, cause a change in cyclic nucleotide levels in the cytoplasm.

Any cells expressing a receptor protein capable upon activation of causing a change in the activity of an ion channel may be useful in the methods. For example, cells expressing a receptor protein capable, upon activation, of directly increasing the intracellular concentration of calcium (e.g., G-protein-coupled receptors), e.g., by opening gated calcium channels, or indirectly affecting the concentration of intracellular calcium by causing initiation of a reaction that utilizes Ca ⁺ as a second messenger, may be useful. Cells that endogenously express such channel-linked receptors or ion channels, and cells which may be transfected with a suitable vector encoding one or more of such cell surface proteins, are known to those of skill in the art, or may be identified by those of skill in the art.

Receptors include, e.g., muscarinic receptors, e.g., human M2 (GenBank accession #M16404); rat M3 (GenBank accession #M16407); human M4 (GenBank accession 4M16405); human M5 (Bonner, et al, (1988) Neuron 1, pp. 403-410); and the like; neuronal nicotinic acetylcholine receptors, e.g., the human α₂, human 013, and human β₂, subtypes disclosed in U.S. Ser. No. 504,455 (filed Apr. 3, 1990, which is hereby expressly incorporated by reference herein in its entirety); the human 015, subtype (Chini et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89: 1572-1576), the rat α₂ subunit (Wada, et al. (1988) Science 240, pp. 330-334); the rat α₃ subunit (Boulter, et al. (1986) Nature 319, pp. 368-374); the rat α₄ subunit (Goldman, et al. (1987) Cell 48, pp. 965-973); the rat α₅ subunit (Boulter, et al. (1990) J. Biol. Chem. 265, pp.

4472-4482); the chicken α₇ subunit (Couturier et al. (1990) Neuron 5:847-856); the rat β₂ subunit (Deneris, et al. (1988) Neuron 1, pp. 45-54) the rat β₃ subunit (Deneris, et al. (1989) J. Biol. Chem. 264, pp. 6268-6272); the rat β₄ subunit (Duvoisin, et al. (1989) Neuron 3, pp. 487-496); combinations of the rat α subunits, rat NMDARl receptor (Moriyoshi et al. (1991) Nature 354:31-37 and Sugihara et al. (1992)

Biochem. Biophys. Res. Comm. 185:826-832); mouse NMDA el receptor (Meguro et al. (1992) Nature 357:70-74); rat NMDAR2A, NMDAR2B and NMDAR2C receptors (Monyer et al. (1992) Science256: 1217-1221); rat metabotropic mGluRl receptor (Houamed et al. (1991) Science 252: 1318-1321); rat metabotropic mGluR2, mGluR3 and mGluR4 receptors (Tanabe et al. (1992) Neuron 8: 169-179); rat metabotropic mGluR5 receptor (Abe et al. (1992) J. Biol. Chem. 267: 13361-13368) and the like; adrenergic receptors, e.g., human beta 1 (Frielle, et al. (1987) Proc. Natl. Acad. Sci. 84, pp. 7920-7924); human alpha 2 (Kobilka, et al. (1987) Science 238, pp. 650-656); hamster beta 2 (Dixon, et al. (1986) Nature 321, pp. 75-79) and the like; dopamine receptors, e.g., human D2 (Stormann, et al. (1990) Molec. Pharm. 37, pp. 1-6); mammalian dopamine D2 receptor (U.S. Pat. No. 5,128,254); rat (Bunzow, et al. (1988) Nature 336, pp. 783-787) and the like; serotonin receptors, e.g., human 5HTIa (Kobilka, et al. (1987) Nature 329, pp. 75-79); serotonin 5HT1C receptor (U.S. Pat. No. 4,985,352); human 5HT.sub. lD (U.S. Pat. No. 5,155,218); rat 5HT2 (Julius, et al. (1990) PNAS 87, pp.928-932); rat 5HTIc (Julius, et al. (1988) Science 241, pp. 558- 564) and the like.

Ion Transporters

Ion transporters include, e.g., neurotransmitter ion transporters (e.g. dopamine ion transporter, glutamate ion transporter or seratonin ion transporter) (Gadea, A. and Lopez-Colome, A. M., J. Neurosci. Res., 2001, 63, 453-460) sodium-potassium ATPase, proton-potassium ATPase (Silver, R. B. and Soleimani, M., Am. J. Physiol, 1999, 276, F799-F81 1), sodium/calcium exchanger, and potassium-chloride ion co- transporter (Gillen, C. M. et al., J. Biol. Chem., 1996, 271, 16237-16244).

Buffers and Growth Media

Any buffer with buffering capacity of about pH 5.5 to 9.0, such as HEPES and TRIS, can be used in the methods described herein. Buffers are well known in the art and can be readily obtained from the literature, e.g., Molecular Cloning; A Laboratory Manual (2nd edition, Sambrook, Fritch, and Maniatis 1989, Cold Spring Harbor Press) or Short Protocols in Molecular Biology (Ausubel, F. M., et al., 1989, John Wiley & Sons). Although all assays described herein can be performed in known physiological Cl^" containing buffers, Cl -free buffer conditions and low Cl^" cell growth media often produce particularly robust and consistent results (Cl^" may cause precipitation of thallium).

Any growth medium known in the art may be used in the methods described herein. Cell growth media can include, e.g., of one or more of the following: sodium gluconate; potassium gluconate; MgSO₄OJH₂O; NaHCOs; calcium gluconate;

NaH₂PO₄; glucose; vitamins; amino acids; glutamine and buffer (e.g., HEPES). An exemplary useful cell growth media composition includes sodium gluconate (109 mM); potassium gluconate (5.4 mM); MgSO₄O.7H₂O (0.8 mM); NaHCO₃ (26.2 mM); calcium gluconate (3.6 mM); NaH₂PO₄ (1.2 mM); HEPES, pH 7.3 w/NaOH (25 mM); Glucose (5.6 mM); lOOxVitamins (10 ml/1); 5Ox amino acids (20 ml/1); and glutamine (2 mM). Useful buffers and growth media are described, e.g., in U.S. Patent Publication No. 2002/0168625 (US Serial Number 09/975,891), which is incorporated herein by reference in its entirety.

Thallium Compositions

In the methods described herein, thallium ion flux across membranes is measured using thallium sensitive agents. Solutions of thallium salts provide the thallium ions. Useful thallium salts include those that are water soluble, e.g., TI₂SO₄, Tl₂CO₃, TlCl, TlOH, TlOAc, TlNO₃ salts, and the like.

Extracellular Masking Agents

Where fluorescent thallium sensitive agents are used, excess fluorescent compound can be removed using an extracellular masking agent (i.e., a quencher). Using a masking agent may obviate the need to wash unloaded thallium sensitive fluorescent agent from the cells. Masking agents are typically non-cell permeant and non-toxic, and can be light absorbing compounds having an absorbance spectrum that overlaps with the excitation or emission spectrum of the thallium sensitive agent. The masking agent can significantly absorb the emission of the thallium sensitive agent. Masking agents can be of a chemical composition that prevents their passage into cells and can be charged or very large compounds. The concentration range for masking agents will range from micromolar to millimolar, depending on their light absorbing properties. Useful masking agents include, e.g., tartrazine, amaranth, and Acid Red 112, or a mixture of any such quenchers. Masking agents are known in the art for their useful properties as stains and dyes in other applications and are described, e.g., in the Sigma-Aldrich Handbook of Dyes, Stains, and Indicators (Floyd G. Green, 1990, St. Louis, Mo.).

Candidate Modulators

Essentially any chemical compound can be used as a potential modulator in the assays described herein. Any compound, such as a small organic or inorganic molecule, amino acid, polypeptide, nucleic acid, peptide nucleic acid, carbohydrate, or polysaccharide can be used. Candidate modulators can be synthetic, naturally occurring, or a combination of synthetic and natural components. If desired, the test compound can be a member of a library of test compounds (e.g., a combinatorial chemical library)

Useful compounds include, e.g., those that can be dissolved in aqueous or organic (especially DMSO-based) solutions. Skilled practitioners will appreciate that chemical compounds are widely available, e.g., from Sigma Chemical Co. (St. Louis, Mo.), Aldrich Chemical Co. (St. Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika (Buchs, Switzerland), and the like.

High-Throughput Screening The methods can be adapted for high-throughput screening. High-throughput screening assays are known and employ the used of microtiter plates or pico-, nano-, or micro-liter arrays.

High-throughput methods can be performed using whole cells expressing target transport structures. For example, a method can be performed by: 1) culturing cells; 2) optionally adhering the cells onto a solid support; 3) loading the cells with a cell permeant thallium sensitive agent that produces a detectable signal; 4) treating the cells (e.g., washing or adding extracellular masking agents) to remove excess thallium sensitive agent; 5) measuring the detectable signal; 6) adding a solution containing thallium ions and appropriate stimulus solution; 7) adding a candidate modulatory compound; 8) measuring detectable signal; and 9) recording changes in the detectable signal (i.e., before and after the addition of thallium ions, stimulus solution and modulatory compound). A change in the detectable signal indicates the effect of the channel modulators.

The assays permit high throughput screening of large chemical libraries, e.g., by automating assay steps and providing candidate modulatory compounds from any convenient source to assay. Assays run in parallel on a solid support (e.g., microtiter formats on microtiter plates in robotic assays) are well known. Automated systems and methods for detecting and measuring changes in optical detection (or signal) are known (U.S. Pat. No. 6, 171,780; 5,985,214; 6,057, 1 14). High throughput screening methods can include screening a combinatorial library containing a large number of potential therapeutic modulating compounds (Borman, S, C. & E. News, 1999, 70(10), 33-48). Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art. A combinatorial chemical library is a collection of diverse chemical compounds generated using either chemical synthesis or biological synthesis, to combine a number of chemical building blocks, such as reagents. For example, a linear combinatorial chemical library, such as a polypeptide library, is typically formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks. Exemplary combinatorial chemical libraries include, e.g., peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka, Int. J Pept. Prot. Res., 1991, 37:487-493 and Houghton, et al, Nature, 1991, 354, 84-88). Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, e.g., peptoids (PCT Publication No. WO 91/19735); encoded peptides (PCT Publication WO 93/20242); random bio-oligomers (PCT Publication No. WO 92/00091); benzodiazepines (U.S. Pat. No. 5,288,514); diversomers, such as hydantoins, benzodiazepines and dipeptides (Hobbs, et al., Proc. Nat. Acad. Sci. USA, 1993, 90, 6909-6913); vinylogous polypeptides (Hagihara, et al., J. Amer. Chem. Soc. 1992, 114, 6568); nonpeptidal peptidomimetics with beta-D-glucose scaffolding (Hirschmann, et al., J. Amer. Chem. Soc, 1992, 114, 9217-9218); analogous organic syntheses of small compound libraries (Chen, et al., J. Amer. Chem. Soc, 1994, 116, 2661; Armstrong, et al. Ace Chem. Res., 1996, 29, 123-131); or small organic molecule libraries (see, e.g., benzodiazepines, Baum C&E News, Jan. 18,1993, page 33,); oligocarbamates (Cho, et al., Science, 1993, 261, 1303); and/or peptidyl phosphonates (Campbell, et al., J. Org. Chem. 1994, 59, 658); nucleic acid libraries (see, Seliger, H et al., Nucleosides & Nucleotides, 1997, 16, 703-710); peptide nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083); antibody libraries (see, e.g.,

Vaughn, et al., Nature Biotechnology, 1996, 14(3), 309-314 and PCT/US96/10287); carbohydrate libraries (see, e.g., Liang, et al., Science, 1996, 274, 1520-1522 and U.S. Pat. No. 5,593,853, Nilsson, U J, et al., Combinatorial Chemistry & High Throughput Screening, 1999 2, 335-352; Schweizer, F; Hindsgaul, O. Current Opinion In Chemical Biology, 1999 3, 291-298); isoprenoids (U.S. Pat. No. 5,569,588); thiazolidinones and metathiazanones (U.S. Pat. No. 5,549,974); pyrrolidines (U.S. Pat. Nos. 5,525,735 and 5,519,134); morpholino compounds (U.S. Pat. No. 5,506,337); benzodiazepines (U.S. Pat. No. 5,288,514) and other similar art. Devices for preparing combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem. Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar, Ltd., Moscow, RU, 3D Pharmaceuticals, Exton, Pa., Martek Bio sciences, Columbia, Md., etc.).

Combinatorial chemical libraries are screened in one or more assays, as described herein, to identify library members (particular chemical species or subclasses) that display modulating ability (Borman, S., supra; Dagani, R. C. & E. News, 1999, 70(10), 51-60). Modulating compounds thus identified can serve as conventional lead compounds or can themselves be used as potential or actual therapeutics.

It is sometimes useful to run positive controls to ensure that the components of the assays are working properly. In an example of a positive control, a known ion channel opener compound is contacted with the sample mixture of the assay, and the resulting increase in ion channel activity is determined according to the methods herein. In another example, for cells expressing ion channels, a known ion channel blocker compound can be added, and the resulting decrease in ion channel activity is similarly detected. It will be appreciated that candidate modulators can be combined with compounds having known effects on transport structures. For example, known ion channel openers or blockers can be used to find modulators, which further effect the ion channel activation or suppression that is otherwise caused by the presence of the known ion channel modulator. It may be possible to screen up to several thousand different candidate modulators in a single day. Each well of a microtiter plate can be used to run a separate assay against a selected potential modulator, or if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator. Thus, a single standard microtiter plate can assay about 100 (96) modulators. If 1536 well plates are used, then a single plate can easily assay from about 100 to about 1500 different compounds. It is possible to assay many different plates per day. Assay screens for up to about 6,000-20,000, and even up to about 100,000-1,000,000 different candidate modulator compounds are possible using the methods described herein.

The following Examples are presented to demonstrate the methods and compounds of the present invention and to assist one of ordinary skill in making and using the same. The Examples are not intended in any way to otherwise limit the scope of the disclosure of the protection granted by Letters Patent granted hereon.

EXAMPLES

The disclosure is further described in the following examples, which do not limit its scope.

Example 1. Synthesis of Crude Compound (1):

Referring again now to FIGS. 14-16, to a mixture of piperidine (10 μL) and methanol (1.0 mL) was added 5.9 mg of compound (14), which was purchased from Genolite Biotek and used as received, and 3.0 mg of compound (15), which was purchased from Aldrich and used as received. The mixture was heated at about 7O⁰C with stirring for about 1.5 hours. After such time, solvent and excess compound (15) were removed on a rotary evaporator. The residue was reconstituted with 3.5 mL of acetonitrile, and then the reconstituted material was treated with 80.0 mg of LiI. The mixture was heated under reflux for 4 days. The lithium salts were collected by centrifuge.

To a mixture of 1.5 mL of dimethylformamide (DMF) and diisopropylethylamine (DIEA, 40 μL) were added 4.5 mg of the above-collected lithium salts. To this mixture was added 40 μL of bromomethylacetate, which was obtained from Aldrich and used as received. This mixture was allowed to set at room temperature for about 4 hours, and then the solvent was removed. The resulting residue was treated with ethyl acetate and the remaining solids were filtered off. The ethylacetate was removed giving crude compound (1).

Example 2. Synthesis of ThalKal:

Referring again now to FIGS. 20-25, to a mixture of piperidine (1 drop) and methanol (2.0 mL) was added 5.0 mg of compound (14) and 10.0 mg of crude compound (25), which was produced according to the synthetic procedure shown in FIG. 24 (and contained compound 35). The mixture was heated at about 7O⁰C with stirring for about 1.5 hours. After such time, solvent and excess compound (25) were removed on a rotary evaporator. The residue was reconstituted with 3.5 mL of acetonitrile, and then the reconstituted material was treated with 80.0 mg of LiI. The mixture was heated under reflux for 4 days. The desired lithium salts were collected by centrifuge.

To a mixture of 1.0 mL of dimethylformamide (DMF) and diisopropylethylamine (DIEA, 40 μl) were added 2.0 mg of the above-collected lithium salts. To this mixture was added 40 μL of bromomethylacetate. This mixture was allowed to set at room temperature for about 4 hours, and then the solvent was removed. The resulting residue was treated with ethyl acetate and the remaining solids were filtered off. The ethylacetate was removed giving the crude ThalKal product as a thick oil, which was purified using preparative thin layer chromatography (TLC). HPLC-MS showed that the purified ThalKal product includes compounds (3), (10) + (11) and (12) + (13), and that they are present in the composition from about 31 to about 40 percent by weight, from about 20 to about 30 percent by weight and from about 30 to about 50 percent by weight, respectively. In addition, HPLC-MS showed that the ratio of compound (1 l):compound (10) is about 1 :3 and that the ratio of compound (13):compound (12) is from about 1 :3.

Example 3. Exemplary Fluorescence-Based FLIPR™ Assays for Potassium Channels Using a Thallium Sensitive Agent

Specific potassium channel assays that employ thallium-sensitive probes and their use for high throughput screening are described in the art. This Example provides data generated in such assays using a novel thallium-sensitive agent described in the present application. The experiments described below employ a thallium sensitive agent referred to as ThalKal, which is a composition that includes compounds (3), (10) plus (11) and (12) plus (13) of FIGS. 7, 12 and 13, respectively. Methods of synthesizing ThalKal are described herein. The assays can be used, e.g., for detecting modulators of ligand- and voltage-gated potassium and sodium channels, expressed in mammalian cells. The methods can be carried out in an automation friendly homogenous assay format, e.g., on FLIPR™ or FLIPRTetra™. The following is a general description of methods used to generate data described in the present Example. The instrument used for the assays was a FLIPR3. 384-well plates were used. Cells were plated (10,000/well for CHO and 17,500/well for HEK) and incubated overnight. Media was removed and dye was loaded at 50 uL/well. Plates were incubated for 60 minutes at 37°C with 5% CO2.

Inhibitors/activators were diluted in Cl - free assay buffer and incubated for 15 minutes at RT. The assay was performed on a FLIPR™ using filter 2 (545-590 nm) and initiated by adding Cl^'-free assay buffer containing 1.25 mM TI₂SO₄ and 5 mM K₂SO₄. Specific instructions for an exemplary assay are provided in Appendix I. FIG. 26 provides a schematic overview of an exemplary assay. This particular assay exploits the permeability of thallium(I) (Tl⁺) for potassium(I)(K⁺) channels. The novel Tl⁺-sensitive fluoresent indicator (ThalKal), which includes AM esters is loaded into the cells in the presence of a masking dye. The AM esters enter the cells through passive diffusion and are enzymatically cleaved, releasing Tl⁺ reactive agent. The cells are stimulated with either a mixture of K⁺ and Tl⁺ or a ligand in the presence Of Tl⁺. The increase of fluorescence in a FLIPR™ or FlexStation^® assay implies influx of Tl⁺ into the cell specifically through the potassium channel and is thus a measure of channel activity. The masking dye blocks extracellular background fluorescence from media, compounds, and excess ThalKal. FIG. 27 is a graph illustrating a comparison of a potassium titration performed with and without thallium in a CHO-hERG line. The data shows that the signal observed is due only to the interaction of ThalKal with thallium. An excess amount of potassium ion (above 25 mM) results in a decrease of signal due to competitive inhibition of thallium. Thus, the graph shows that the signal increase observed for voltage gated channels is K -dose dependent. K⁺ changes the membrane potential of the cell, which opens up the voltage-gated channel. At high K⁺ concentrations, a reduction of the signal can be observed due to the competition of K⁺ and Tl⁺ to enter the cell. The optimal K⁺ concentration was high enough to result in a robust response and low enough not to compete with the Tl⁺ influx through the channel. For ligand-gated cell lines, the appropriate concentration of ligand was determined in the presence of thallium. A potassium channel assay was performed in HEK-hERG cells as a wash assay as described by Weaver et al. (Weaver et al. 2005 J. Biomol. Screening 9(8) 671-777) and as a non-wash assay using masking dye technology as described in the general protocol, above. FIG. 28 is a graph that compares the results of the two protocols. In a high throughput setting, a mix and read set-up for an assay is useful. In addition to saving time, it also avoids the need to wash cells and with that increases the precision in the assay.

FIG. 29 is a graph illustrating data generated in a thallium ion titration in a CHO-hERG cell line. The assay was performed using 10 mM potassium ion to trigger opening of the channel. The signal observed was dependent on the thallium concentration used. The optimal concentration resulted in a robust response while at the same time kept the thallium concentration in the assay at a minimum. 2.5 mM Tl⁺ yielded a near maximum signal and was chosen for this assay system. The assay was run in chloride-free buffer to avoid TlCl precipitation..

FIGS. 30A and 30B are graphs illustrating the results of assays where cisapride (a peristaltic stimulant) and dofetilide, (an antiarrhythmic drug), both known inhibitors of the hERG channel, were used as modulators. The reported IC50 for cisapride using Patchclamp technology ranges from 6.5-44 nM, which is close to the value of 64 nM obtained using the present assays. The IC50 values determined by this FLIPR™ potassium channel assay for dofetilide are 23 nM for HEK-hERG and 6.2 nM for CHO-hERG, which are well within the reported and expected range of 10-110 nm.

FIGS. 31A-3 IB are a set of graphs that illustrate the response of a composition comprising ThalKal (which composition was prepared according to the methods described herein) to thallium as compared to the response of a known thallium sensitive agent, i.e., an AM ester of known fluorescent probe BTC (BTC-AM). As can be observed in FIGS. 31A-31B, assays performed using ThalKal display results similar to those using BTC-AM, but have lower intensity, tighter %CV, and improved Z-value.

Compound (3), in highly purified form, yielded reduced fluorescence in the potassium assay experiments described immediately below. Compositions including ThalKal, prepared according to the methods described herein, yielded excellent results. FIGS. 32A-32C illustrate the effect of l,2-bis(2-aminophenoxy)ethane- NjNjN'jN'-tetraacetic acid (BAPTA) and BAPTA derivatives on enhancing the outcome of assays using purified compound (3). FIG. 32A illustrates that highly purified compound (3) showed decreased fluorescence in the absence of BAPTA of any type. FIG. 32B illustrates that highly purified compound (3) also showed decreased fluorescence in the presence of an AM ester of BAPTA (BAPTA-AM), (obtained from Invitrogen, Inc.). However, adding compounds (10) and (1 1) restores the desired response, as is shown in FIG. 32C. Thus, it appears that ThalKal prepared in accordance with the present application includes at least one BAPTA derivative that enhances the performance of the assay. Without intending to be bound by theory, it is believed that BAPTA-CHO-OH localizes differently than BAPTA-AM in the cell, possibly in the vicinity of the membrane, where it can neutralize calcium influx through calcium channels more efficiently than does BAPTA.

High-pressure liquid chromatography/mass spectroscopy revealed that compounds (10) and (11) were components of the ThalKal composition (data not shown). FIGS. 33A and 33B illustrate, in a side-by-side manner, that an assay performed using ThalKal prepared in accordance with the presently described methods (FIG. 33A) yields results similar to an assay performed using highly purified compounds (3), (10) and (11) (FIG. 33B). FIG. 34 is a graph that provides the results of assays performed using ThalKal, and highly purified compound (3) along with compounds (10) and (11) when dofetilide was used as an inhibitor of hERG channels. As can be observed in FIG. 34, similar results were obtained, providing further evidence that compounds (3), (10) and (1 1) are components of the ThalKal composition. OTHER EMBODIMENTS

A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention.

For example, while thallium compounds have been utilized herein, other compounds can be used in the assays. For example, compounds that include or that generate cations having a similar ionic radius as thallium (I) can be also utilized. For example, Rb (I) or Au (I) compounds can be utilized.

Still other embodiments are within the scope of the following claims.

Claims

WHAT WE CLAIM IS:

1. Compounds represented by Structure I

I wherein

Ri and R₂ are each independently a moiety that comprises up to 45 carbon atoms and, optionally, comprising one or more N, O, S, F, Cl, Br or I atoms;

R₃, R₄ and R₅ are each independently H, F, Cl, Br, I or a moiety that comprises up to 45 carbon atoms and, optionally, comprising one or more N, O, S or F atoms;

R₆ is H, F, Cl, Br, I or CN;

R₇, Rs and R9 are each independently F, Cl, Br, I, NO₂, NH₂ or CN, or a moiety comprising up to 45 carbon atoms and, optionally, comprising one or more N, O, S or F atoms; any two Of R₇, Rs or R₉ may together define a ring;

A is O, S, Se or N; and n is O or 1.

2. Compounds of claim 1, wherein the coumarin fragment is connected at the 2 position, the compounds being represented by Structure II

3. Compounds of claim 1, wherein A is S, the compounds being represented by Structure IV

IV

4. Compounds of claim 1, wherein R₆ is H, the compounds being represented by Structure VII

VII

5. Compounds of any one of claims 1-4, wherein R3 is represented by

wherein Ri₀ and Rn are each independently H or a moiety that includes up to 8 carbon atoms and, optionally, one or more N, O, S or F atoms; and wherein a and b are each independently a integer between 1 and 6, inclusive.

6. Compounds of claim 5, wherein Rio and Rn are each

7. Compounds of any one of claims 1-6, wherein Ri and R₂ are each independently

wherein Ri₃ and R₁₄ are each independently H or a moiety that includes up to 8 carbon atoms and, optionally one or more N, O, S or F atoms; and wherein a and c are each independently an integer between 1 and 6, inclusive.

8. Compounds of claim 7, wherein Ri₃ and R₁₄ are each

9. Compounds of claim 1 represented by Structures (1), (2), (3), (4), (5), (6) or (7),

0), (46) or (47)

10. Compounds represented by Structure XX

XX wherein

Ri5, Ri6, Rn and Ris are each independently H or a moiety that includes up to 12 carbon atoms and, optionally, one or more N, O, S or F atoms;

G is CHO or a moiety that includes up to 16 carbon atoms and, optionally, one or more N, O, S or F atoms; and e and f are each independently a integer between 1 and 6, inclusive.

11. Compounds of claim 10, wherein G is CHO and e and f are each 1, the compounds being represented by Structure XXI

XXI

12. Compounds of claim 10 or 11, wherein Ri₅, Ri₆ and Ris are each independently

13. Compounds of any one of claims 10-12, wherein Ri₈ is

4. Compounds of claim 10 represented by Structures (10) or (11)

1 1

15. Compounds of claim 10, wherein G is G', the compounds being represented by Structure XXII

XXII

16. Compounds of claim 15, wherein G' is

17. Compounds of claim 10 represented by Structures (12) or (13)

18. A composition comprising one or more compounds of any one of claims-17.

19. A composition comprising one or more compounds of any one of claims-9 and one or more compounds of any one of claim 10-17.

20. The composition of claim 19, wherein the one or more compounds of any one of claims 1-9 are selected from the group consisting of

and wherein the one or more compounds of any one of claims 10-17 are selected from the group consisting of

21. The composition of claim 20, wherein the composition comprises compounds (3), (10), (11), (12) and (13).

22. The composition of claim 21, wherein compounds (3), (10), (11), (12) and (13) are present in the composition from about 25 to about 40 percent by weight, from about 9 to about 30 percent by weight, from about 9 to about 30 percent by weight, from about 15 to about 50 percent by weight and from about 15 to about 50 percent by weight, respectively.

23. The composition of claim 21, wherein compounds (3), (10) + (11), (12) + (13) are present in the composition from about 31 to about 40 percent by weight, from about 20 to about 30 percent and from about 30 to about 50 percent by weight, respectively.

24. The composition any one of claims 21-23, wherein the ratio of compound (1 l):compound (10) is about 1:3 and/or the ratio of compound (13):compound (12) is from about 1 :3.

25. A kit comprising a compound of any one of claims 1-17 or a composition of any one of claims 18-21.

26. The kit of claim 25, further comprising one or more masking compounds.

27. The kit of claim 25 or 26, further comprising a surfactant, such as polyethylene glycol.

28. The kit of any one of claims 25-27 ^', further comprising a buffer.

29. The kit of any one of claims 25-28, further comprising a thallium salt, such as thallium sulfate.

30. A method for detecting the activity of a target transport structure, the method comprising: (a) contacting a cell expressing a target transport structure with the thallium sensitive agent of any one of claims 1 to 24;

(b) contacting the cell with a candidate modulator;

(c) contacting the cell with an assay buffer containing a thallium salt solution; and

(d) detecting a signal generated by the thallium sensitive agent to determine the effect of the candidate modulator on the activity of the target transport structure.

31. A method for identifying a modulator of a target transport structure, the method comprising:

(a) contacting a cell expressing a target transport structure with the thallium sensitive agent of any one of claims 1 to 24;

(b) contacting the cell with a candidate modulator;

(c) contacting the cell with an assay buffer comprising a thallium salt solution; and

(d) detecting a signal generated by the thallium sensitive agent to determine the effect of the candidate modulator on the activity of target transport structure.

32. The method of claim 30 or 31, wherein the cell expresses an ion channel that is a cation channel permeable to thallium ions.

33. The method of claim 31, wherein the cation channel is selected from the group consisting of potassium ion channel, sodium ion channel, and calcium ion channel.

34. The method of claim 31, wherein the cation channel is a potassium ion channel.

35. The method of claim 34, wherein the potassium ion channel is a calcium- activated and voltage-gated channel.

36. The method of claim 34, wherein the potassium ion channel is selected from the group consisting SK, Maxi-K, HERG and KCNQ channels.

37. The method of claim 31, wherein the cation channel is a ligand-gated VRl channel.

38. The method of claim 31, wherein the cation channel is a non-selective ion channel.

39. The method of claim 38, wherein the non-selective ion channel is selected from the group consisting of acetylcholine receptor, glutamate receptor, kainate, NMDA receptor, 5-hydroxytryptamine-gated receptor-channel, ATP-gated (P2X) receptor-channel, nicotinic acetylcholine-gated receptor-channel, vanilloid receptor, ryanodine receptor-channel, IP3 receptor-channel, cation channel activated in situ by intracellular cAMP, and cation channel activated in situ by intracellular cGMP.

40. The method of claim 30 or 31, wherein the thallium salt solution comprises a water soluble thallium salt.

41. The method of claim 40, wherein the thallium salt is selected from the group consisting OfTl₂SO₄, Tl₂CO₃, TlCl, TlOH, TlNO₃ and TlOAc.

42. The method of claim 41, wherein the thallium salt is Tl₂SO₄.

43. The method of claim 30 or 31, wherein the assay buffer is Cl^'-free.

44. The method of claim 43, wherein the the assay buffer further comprises sodium gluconate; potassium gluconate; calcium gluconate; magnesium gluconate; HEPES and glucose.

45. The method of claim 30 or 31, wherein the cells are grown in a low Cl - cell growth medium, containing no more than 2 mM Cl^".

46. The method of claim 45, wherein the low Cl - cell growth medium comprises sodium gluconate; potassium gluconate; MgSO₄.7H₂O; NaHCO₃; calcium gluconate; NaH₂PO₄; HEPES; Glucose; lOOxVitamins; 5Ox amino acids; and glutamine.

47. The method of claim 30 or 31, wherein the thallium sensitive agent is a thallium sensitive fluorescent agent or thallium sensitive non-fluorescent agent.

48. The method of claim 30 or 31, wherein the cell expresses a channel-linked receptor selected from the group consisting of GPCR, metabotropic glutamate receptor, muscarinic acetylcholine receptor, dopamine receptor, and serotonin receptor.

49. The method of claim 30 or 31, wherein the cell expresses an ion transporter selected from the group consisting of dopamine ion transporter, glutamate ion transporter, seratonin ion transporter, sodium-potassium ATPase, proton- potassium ATPase, sodium/calcium exchanger, and potassium-chloride ion co- transporter.

50. The method of claim 30 or 31, wherein the thallium sensitive agent is a fluorescent agent, and wherein the method further comprises contacting the cell with an extracellular fluorescent quenching compound after contacting the cell with the thallium sensitive agent.

51. The method of claim 30 or 31, wherein the candidate modulating compound activates or inhibits the ion channel, channel-linked receptor, or ion transporter.

52. The method of claim 30 or 31, further comprising adding a stimulus solution to the thallium salt solution.

53. The method of claim 52, wherein the stimulus solution contains an agent selected from the group consisting of ionophore, KCl, nicotine, acetylcholine, muscarin, and carbamylcholine.

54. The method of claim 31, further comprising measuring the signal after step (b).

55. The method of claim 31, wherein said modulator activates or inhibits the ion channel, channel-linked receptor, or ion transporter.

56. The method of claim 31, wherein detecting a signal comprises detecting a decrease in the signal.

57. The method of claim 56, wherein a decrease in signal indicates that the candidate agent is an activator of the ion channel, channel-linked receptor, or ion transporter.

58. The method of claim 31, wherein detecting a signal comprises detecting an increase in the signal.

59. The method of claim 58, wherein an increase in signal indicates that the candidate agent is an activator of the ion channel, channel-linked receptor, or ion transporter.

60. The method of claim 31, wherein detecting a signal comprises detecting no change in the signal.

61. The method of claim 60, wherein no change in the signal indicates that the candidate agent is an inhibitor of the ion channel, channel-linked receptor, or ion transporter.

62. A method of making a thallium sensitive agent, comprising (a) combining a compound of Structure XXV

XXV wherein R₂0 is C1-C20 straight chain, branched or cyclic alkyl, and R₇, Rs and R₉ are each independently F, Cl, Br, I, NO₂, NH₂ or CN, or a moiety comprising up to 45 carbon atoms and, optionally, comprising one or more N, O, S or F atoms; any two Of R₇, Rs or R₉ may together define a ring;

with a compound of Structure XXVI

XXVI wherein R₂i, R₂₂ and R₂3 are each independently a moiety that comprises up to 45 carbon atoms and, optionally, comprising one or more N, O, S, F, Cl, Br or I atoms, together with a material comprising a base, such an amine, to provide a compound having a coumarin fragment bonded to moiety that comprises a 5- membered ring heterocycle.

63. The method of claim 62, further comprising forming a metal salt from the compound having the coumarin fragment.

64. The method for claim 63, further comprising forming an ester from the formed metal salt.

65. The method of claim 62, wherein the compound of Structure XXV comprises compound (25), and wherein the compound of Structure XXVI comprises compound (14).

66. The method of claim 62 in which compound (35) is also combined during the combining of the compound of Structure XXV and the compound of Structure XXVI.

67. Compounds made by any one of the methods of claims 62-66.

68. A method for detecting the activity of a target transport structure, the method comprising:

(a) contacting a cell expressing a target transport structure with the thallium sensitive agent made by any one of the methods of claims 62-66.

(b) contacting the cell with a candidate modulator;

(c) contacting the cell with an assay buffer containing a thallium salt solution; and

(d) detecting a signal generated by the thallium sensitive agent to determine the effect of the candidate modulator on the activity of the target transport structure.

69. A method for identifying a modulator of a target transport structure, the method comprising:

(a) contacting a cell expressing a target transport structure with the thallium sensitive agent made by any one of the methods of claims 62-66;

(b) contacting the cell with a candidate modulator;

(c) contacting the cell with an assay buffer comprising a thallium salt solution; and

(d) detecting a signal generated by the thallium sensitive agent to determine the effect of the candidate modulator on the activity of target transport structure.

70. Compounds represented by Structure I'

coumarin fragment

wherein

Ri and R₂ are each independently a moiety that comprises up to 45 carbon atoms and, optionally, comprising one or more N, O, S, F, Cl, Br or I atoms;

R₃, R₄ and R₅ are each independently H, F, Cl, Br, I or a moiety that comprises up to 45 carbon atoms and, optionally, comprising one or more N, O, S or F atoms;

R₆ is H, F, Cl, Br, I or CN; and β is a moiety that comprises up to 45 carbon atoms and includes a heterocyclic 5- or 6-membered ring system.

71. The compounds of claim 70, wherein the moiety further comprises one or more N, O, S or F atoms in addition to the one or more heteroatoms of the ring.

72. The compounds of claim 70 or 71, wherein the heterocyclic 5- or 6- membered ring system is an aromatic ring system.

73. The compounds any one of claims 70-72, wherein the heterocyclic ring system is a 5-membered ring system that includes 3 heteroatoms.

74. The compounds of claim 73, wherein the 5-membered ring system is a thiadiazole ring system.

75. The compounds of claim 74, wherein the 5-membered ring system is a 1,3,4-thiadiazole ring system.

76. The compounds any one of claims 70-72, wherein the heterocyclic ring system is a 6-membered ring system that includes 1 heteroatom.

77. The compounds of claim 76, wherein the heteroatom is N.

78. The compounds of claim 77, wherein the ring system is a pyridine ring system.

79. The compounds of claim 77, wherein the ring system is a pyridine-oxide ring system.

80. The compounds of claim 70, represented by structures (62) or (63)

81. The compounds of claim 70, represented by structures (64) or (68). H₃