JP2003529560A - New ligands and methods for preparing the same - Google Patents

Abstract

  (57) [Summary] Disclosed are processes for modifying a parent ligand by attaching a binding agent reactive with a portion of a target receptor to the parent ligand. The parent ligand binds to the binding agent so that a covalent bond can be formed between the binding agent and the receptor moiety. Also, compositions and probes containing the modified ligands, methods for detecting and / or quantifying receptors using the modified ligands of the invention, and methods for detecting and / or quantifying a target receptor using the modified ligands of the invention. Disclosed are methods of treating or preventing a condition.

Description

Detailed Description of the Invention

CROSS REFERENCE TO RELATED APPLICATIONS This application relates to US Provisional Patent Application No. 60/178, filed January 28, 2000.
, 756, the disclosures of which are incorporated herein by reference in their entireties.

TECHNICAL FIELD The present invention relates generally to ligand-receptor interactions. In particular, the invention relates to modified ligands that bind irreversibly to its cognate receptor, and methods for preparing such ligands. The novel ligands of the present invention are particularly useful for investigating protein function and useful as a drug for more efficiently inhibiting or stimulating cognate receptor function (in health care applications, agricultural applications and environmental applications). ) Has.

BACKGROUND ART Pharmacological receptors are intracellular or membrane-bound proteins that, after binding a specific ligand, produce a pharmacological effect. In this regard, the pharmacological receptor is (a) for detecting a ligand signal by forming a ligand-receptor complex, and (b) for conducting and transducing a signal that produces a pharmacological effect. Has a dual function.

Drugs can replace the endogenous physiological ligands that interact with the receptor.
A prerequisite for such drug-receptor interactions is the formation of drug-receptor complexes, as is the case for ligand-receptor interactions. In contrast to a physiological ligand that stimulates the effect after binding to the receptor (receptor-mediated effect), the drug is (a) an agonist or drug that stimulates the effect after binding to the receptor, and (b) a receptor-binding effect. It may be later classified as an agonist or drug that does not stimulate effects.

Several types of molecular interactions are possible for drug-receptor binding,
Includes ionic bonds, hydrogen bonds, and hydrophobic bonds due to van der Waals forces.
The majority of receptor interactions simultaneously involve several types of binding. Ionic bonds are important in the early stages of drug-receptor interactions because they have the best or longest range. After the first interaction, dipole-dipole coupling,
Fine tuning occurs, including hydrogen and hydrophobic bonds. All of these interactions also anchor the drug molecule in the active site of the receptor, but nevertheless the forces of interaction are so weak that their binding is reversible. Therefore, the pharmacological effect of any drug is often influenced by its own concentration in plasma, as decreasing plasma drug concentration increases dissociation of the drug molecule from its receptor.

[0006] Several agents are known that irreversibly or pseudo-irreversibly inhibit the enzyme, and the exact mechanism of inhibition results in slight differences in the inhibition profile and duration of inhibition.

Many inhibitors of acetylcholinesterase react covalently with this enzyme to form acyl enzymes that deacylate more slowly than acetyl enzymes formed with the natural substrate acetylcholine. This acetyl enzyme is rapidly formed by the attack of the active site serine on the substrate. Transfer of an acyl group to an enzyme
It occurs via a tetrahedral intermediate. This acetyl enzyme has a half-life of 10 μs,
It is rapidly hydrolyzed. These rapid acylation and deacylation steps result in a turnover rate of 10 ⁵ substrate molecules per enzyme molecule per second. Cholinesterase inhibitors such as physostigmine and neostigmine form the methylaminocarbamiol and dimethylaminocarbamoyl enzymes, which have half-lives for minutes of deacetylation. Therefore, by providing the enzyme with an alternative substrate, the catalysis of acetylcholine is prevented during the catalytic cycle for the carbamoylating agent. The rate constants of the respective acylation steps for the acetoxyester and carbamoxy ester substrates do not differ significantly, and therefore the longer residence time of the carbamoyl enzyme conjugate is an important factor in favorable inhibition.

Some other enzymes are inhibited by the covalent attachment of inhibitors, resulting in irreversibility. Hydrazine (phenelzine, an isoxalxazide metabolite) and acetylene agent (pargyline) are oxidized to reactive intermediates by monoamine oxidase. These intermediates attack the flavin cofactor bound on the enzyme. Such agents are called suicide substrates because their activation requires catalysis by the very enzyme they inactivate. Therefore, the inactivation process is mechanism-based. Currently, there are numerous examples of such substrates, and activation by these enzymes results in covalent modification of the enzyme or associated cofactors. Often this occurs by conjugation or association of the enzyme with its substrate, followed by attack of flanking groups. Some of the targets of suicide substrates have therapeutic significance. These include: penicillinase and alanine racemase during antibacterial design; GABA transaminase inhibitors of antiepileptic agents; lipoxygenase and cyclooxygenase inhibitors that control leukotriene and prostaglandin biosynthesis, respectively; formation of estrogenic hormones. -Blocking aromatase inhibitors; ornithine decarboxylase inhibitors as antiparasitic agents; and dopamine β-hydroxylase inhibitors for controlling catecholamine biosynthesis. Many suicide substrates serve as antimetabolites and are potential antineoplastic agents. The effect of these inhibitors, their relative dissociation constants, i.e. not only the K _m value as compared to the endogenous substrate but also on the rate competition between turnover and the inactivation events suicide substrate.

Omeprazole (PRILOSEC) is another well-known irreversible binding drug that has been published for clinical use. This drug inhibits gastric acid secretion by binding to H ⁺ , K ⁺ -ATPase present only in the apical membrane of parietal cells. Omeprazole is particularly useful in patients with hypergastrinemia and its peptic ulcer can be useful in patients not adequately controlled with H ₂ antagonists. Omeprazole contains a sulfinyl group in the bridge between the substituted benzimidazole and the pyridine ring. At neutral pH, the drug is a chemically stable, lipid-soluble, weak base lacking inhibitory activity. This neutral weak base reaches the parietal cells from the blood and diffuses into the secretory tubules where the drug is protonated and thereby trapped. The protonated drug rearranges to form sulfenic acid and sulfenamide. Sulfenamide covalently interacts with sulfhydryl groups at key sites in the extracellular (luminal) domain of transmembrane H ⁺ , K ⁺ -ATPase. Therefore, omeprazole must be considered as a prodrug that needs to be activated in order to be effective.

Despite the effectiveness of some drugs that irreversibly bind their targets, there is a lack of currently available methods to rationally design ligands for irreversibly binding target receptors. is doing.

DISCLOSURE OF THE INVENTION The present invention provides for the binding of a binding agent to a parent ligand that reversibly binds to the target receptor, where the binding agent is reactive with a portion of the target receptor and, as a result, , A covalent bond can be formed between the binding agent and its moiety), the modified ligand produced results, at least in part, from the unexpected discovery that it can irreversibly bind the target receptor.

Accordingly, in one aspect of the invention there is provided a process for modifying a parent ligand, wherein the process is such that a covalent bond can be formed between the binding agent and the moiety. Binding the parent ligand to a binding agent that is reactive with the portion of the target receptor to which the parent ligand binds.

[0013]   Suitably the binding agent is attached to the parent ligand via a spacer.

[0014]   Preferably the spacer is covalently attached to the parent ligand.

[0015]   Preferably the spacer is covalently attached to the binding agent.

The spacer includes an alkyl group, a heteroalkyl group, a cycloalkyl group, a heterocycloalkyl group, an oxoalkyl group, a heterooxoalkyl group, an alkenyl group, a heteroalkenyl group, an aralkyl group, a heteroaralkyl group, an aryl group and a hetero group. A group appropriately selected from the group consisting of aryl groups, or any other molecular structure that acts as a spacer. The length of this spacer arm is appropriately selected from the range between about 0Å and about 20Å.

Preferably, the spacer is a non-hydrolyzable group under physiological conditions.

Preferably, the binding agent is a sulfhydryl group specific binding agent, an amino group specific binding agent, a carboxyl group specific binding agent, a tyrosine specific binding agent, an arginine specific binding agent, a histidine specific binding agent, methionine. It is selected from the group consisting of specific binding agents, tryptophan specific binding agents, and serine specific binding agents.

Sulfhydryl group-specific binders include N-maleimide, N-maleimide derivatives and disulfide reagents (5′-dithiobis- (2-nitrobenzoic acid), 4,4 ′.
-Dithiodipyridine, methyl-3-nitro-2-pyridyldisulfide, and methyl-2-pyridyldisulfide).

Amino group specific binders include alkylating agents, including but not limited to α-haloacetyl compounds, aryl halides, aldehydes and ketones, isocyanates, isothiocyanates, imide esters, N-hydroxylsuccinimidyl esters, p- -A nitrophenyl ester, an acyl chloride, and an acylating agent, including, but not limited to, a sulfonyl chloride.

The carboxyl group-specific binder may be selected from the group consisting of carbodiimides and carboxyl group esterification reagents including, but not limited to, diazoacetate esters and diazoacetamide.

The tyrosine-specific binding agent may be selected from diazonium derivatives including, but not limited to, benzidine and bis-diazotized 3,3′-dimethylbenzidine.

Arginine-specific binders include glyoxal, phenylglyoxal, 2-
It may be selected from 1,2-dicarbonyl reagents including but not limited to 3-butanediol and 1,2-cyclohexanedione.

Histidine specific binders include alkylating agents including, but not limited to, α-haloacetyl compounds, aryl halides, aldehydes and ketones, and diethylpyrocarbonate, ethoxyformic anhydride, isocyanates, isothiocyanates, imide esters, Suitably selected from the group consisting of acylating agents including, but not limited to, N-hydroxyl succinimidyl ester, p-nitrophenyl ester, acyl chloride, and sulfonyl chloride.

The methionine-specific binding agent may be selected from the group consisting of alkylating agents including, but not limited to, α-haloacetyl compounds, aryl halides, aldehydes and ketones.

The tryptophan-specific binding agent may be selected from the group consisting of N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide and p-nitrophenylsulfenyl chloride.

The serine-specific binding agent may be selected from the group consisting of diisopropylfluorophosphoric acid and acrylsulfonyl fluorides including, but not limited to, phenylmethyl-sulfonyl fluoride.

The parent ligand can be any natural or non-natural ligand, but is preferably a biologically active ligand, including known drugs and naturally occurring or synthetic drug candidate compounds. .

In another aspect, the invention provides modified ligands produced by the processes broadly described above.

In yet another aspect of the invention, there is provided a modified ligand having the following general formula: LR ₁ -A (I), wherein L is a parent ligand; A is a binding agent that is reactive with the portion of the target receptor to which the parent ligand binds, such that a covalent bond can be formed between the binding agent and the moiety;
R ₁ is preferably an alkyl group, a heteroalkyl group, a cycloalkyl group, a heterocycloalkyl group, an oxoalkyl group, a heterooxoalkyl group, an alkenyl group, a heteroalkenyl group, an aralkyl group, a heteroaralkyl group, an aryl group and It is an optimal spacer containing a non-hydrolyzable group selected from the group consisting of heteroaryl groups.

In a preferred embodiment, the modified ligand is interactive with the es nucleoside transporter and / or the nucleoside / nucleotide / nucleobase sensitive protein, wherein the modified ligand is selected from the group consisting of: General formula:

[0032]

[Chemical 10] Where A is N-maleimide, 2-pyridyldithio, or halogen; X is NH, S, or O; Y is H, halogen, NH ₂ , or O. Z is H, halogen, or CH ₃ ; R _L is a spacer arm containing a group that is preferably non-hydrolyzable under physiological conditions; R ₂ is H, β-D
-Ribose, β-D-2-deoxyribose, or their 5'-monophosphate, 5'-diphosphate, and 5'-triphosphate.

Suitably, the modified ligand is the es nucleoside transporter and / or
Or a nucleoside / nucleotide / nucleobase-sensitive protein inhibitor and a general formula selected from the group consisting of:

[0034]

[Chemical 11] Here, R ₄ is 4- [N-methyl] cyclohexanecarboxylate, N-
[M-benzoate], 4- [p-phenyl] butyrate, N- [γ-butyrate], N- [α-acetate], or N- [ε-caproylate (capro).
ylate)];

[0035]

[Chemical 12] Here, R ₄ is 4- [N-methyl] cyclohexanecarboxylate, N-
[M-benzoate], 4- [p-phenyl] butyrate, N- [γ-butyrate], N- [α-acetate], or N- [ε-caproylate];

[0036]

[Chemical 13] Here, R ₅ is 4-carbonyl-α-methyl-α-toluene, 6- [α-methyl-α-tuloamido] -hexanoate (hexan).
oate), N- [3-propionate], or 6- [3′-propioamido] hexanoate; and

[0037]

[Chemical 14] Here, R ₅ is 4-carbonyl-α-methyl-α-toluene, 6- [α-methyl-α-turamide] -hexanoate, N- [3-propionate], or 6- [3′-propioamide]. It is hexanoate.

In another embodiment, modified ligands that bind to the serotonin receptor can be used.

In another aspect, the invention resides in a composition comprising a ligand modified as broadly described above with a pharmaceutically acceptable carrier.

In a further aspect of the invention there is provided a method of treating or preventing a condition that binds to a target receptor, which method comprises administering a therapeutically effective dose of a composition as broadly described above. Administering to a patient in need of treatment.

According to another aspect of the invention, there is provided a method of detecting the presence of a target receptor in a test sample, the method comprising the steps of: a modified ligand as broadly described above. Contacting the sample with the modified ligand binding to the target receptor; and detecting the presence of a complex comprising the modified ligand and the receptor in the contacted sample. Process.

In another aspect of the invention, there is provided a method of quantifying the presence of a target receptor in a test sample, the method comprising the steps of: a modified ligand as broadly described above. And contacting the sample with the modified ligand binding to the target receptor; measuring the concentration of a complex comprising the modified ligand and the receptor in the contacted sample. Associating the concentration of the measured complex with the concentration of the receptor in the sample.

In yet another aspect, the invention provides a method of detecting the presence of a target receptor on a cell or cell membrane, the method comprising the steps of: Modification as broadly described above. Contacting a modified ligand with a sample containing the cells or cell membranes, wherein the modified ligand binds to the target receptor; and the modified ligand and the contacted sample in the sample. A step of detecting the presence of a complex containing the cell or cell membrane.

In another aspect of the invention, there is provided a method of quantifying the presence of a target receptor on a cell or cell membrane, which method comprises the following steps: modified as broadly described above. Contacting a ligand with a sample containing the cell or cell membrane, wherein the modified ligand binds to the target receptor; the modified ligand and the cell or cell membrane in the contacted sample. Measuring the concentration of the complex comprising; and associating the measured concentration of the complex with the concentration of the receptor present on the cell or cell membrane.

In another aspect, the invention extends to a probe that covalently binds to a target receptor, the probe comprising a ligand modified as broadly described above having a receptor molecule associated therewith.

In one embodiment, the probe comprises a modified ligand that is interactive with the es nucleoside transporter and / or nucleoside / nucleotide / nucleobase-sensitive protein, as broadly described above.

In this aspect, the cells are preferably animal cells, more preferably mammalian cells, and more preferably human cells. Alternatively, the cells can be plant cells or microbial cells. Microbial cells include, but are not limited to, cells of bacterial, viral or fungal origin.

The present invention also includes the use of modified ligands and probes as broadly described above, particularly in the study, treatment and prevention of conditions associated with their corresponding target receptors.

In one embodiment, a process for modifying a parent ligand is provided,
This process involves attaching the parent ligand to a binding agent that is reactive with the portion of the target receptor to which the parent ligand binds, where when the parent ligand binds to the receptor, a covalent bond is added to the binding agent. It is formed between the above portion.

In one preferred embodiment, the binding agent is located on the ligand at a position that facilitates and / or allows the formation of covalent bonds with moieties of the target receptor. In another preferred embodiment, the receptor to which the ligand binds is the active site associated with biological activity. This receptor is, for example, a cell surface receptor. In one embodiment, binding of the modified ligand to the receptor is associated with an altered activity of the target receptor.

In another preferred embodiment, the parent ligand and / or receptor is naturally occurring. In another embodiment, the modified ligand is not a crosslinker. In another embodiment, the modified ligand optionally does not include a photoreactive group such as a photolabel. In another embodiment, the modified ligand does not include a label. In another embodiment, the receptor to which the ligand binds is RN
It is not a nucleic acid such as A or DNA. In another embodiment, the binding agent does not bind a portion of the nucleic acid and optionally binds to amino acid residues.

In one embodiment, a process for modifying a parent ligand is provided,
The process involves attaching the parent ligand to a binding agent that is reactive with the portion of the target receptor to which the parent ligand binds, wherein when the parent ligand binds to the receptor, a covalent bond is attached to the binding agent. Formed between the agent and the moiety, where the parent ligand specifically binds to the nucleoside transporter.

[0053] In another embodiment, a process for modifying a parent ligand is provided, the process comprising: a parent of a sulfhydryl group-specific binding agent that is reactive with a sulfhydryl group of a target receptor to which the parent ligand binds. Binding a ligand, wherein a covalent bond is formed between the binding agent and the sulfhydryl group when the parent ligand binds to the receptor, and wherein the parent ligand is a serotonin receptor Binds specifically with.

In one embodiment, a modified ligand having the general formula: L—R ₁ -A (I) is provided, wherein L specifically binds to a target receptor comprising a nucleoside transporter. Is a parent ligand; where A is the portion of the target receptor to which the parent ligand binds such that a covalent bond is formed between the binding agent and the moiety when the parent ligand binds to the receptor. Is a binder that is reactive; and R ₁ is an optional spacer.

In another embodiment, a modified ligand having the general formula: LR ₁ -A (I) is provided, wherein L is a parent ligand that specifically binds to the target serotonin receptor. Where A is the sulfhydryl of the target receptor to which the parent ligand binds such that a covalent bond is formed between the binder and the sulfhydryl group of the receptor when the parent ligand binds to the receptor. A binder that is reactive with a group; and R ₁ is an optional spacer.

The disclosures of all patents, patent applications, publications and published patent applications referred to herein are hereby incorporated by reference in their entireties.

Detailed Description 1. Definitions Unless defined otherwise, all technical and scientific terms used herein are commonly understood by one of ordinary skill in the art to which this invention belongs. Has the same meaning as one. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials have been described. For purposes of the present invention, the following terms are defined below.

The article “one (“ a ”and“ an ”)” is used herein to refer to one or more than one (ie, at least one) of the grammatical subject matter of the article. Used in. By way of example, "an element" means one element or more than one element.

By “attached”, a direct or indirect binding of a binding agent to a ligand in a manner that resists the separation of the binding agent from the ligand under normal physiological conditions. Connection is meant.
Thus, the term "bound", as used herein, is used between a binding agent and a ligand or to interfere with the separation of the binding agent from this ligand under normal physiological conditions. Another ionic bond, a hydrogen bond, a Van der Waals force, a covalent bond or a combination thereof formed between the spacer and the binder and the ligand are included within this range.

Throughout this specification, unless the context requires otherwise, the word “including (“ com
"prise", "comprises," and "comprising") "
Is meant to include the stated step or element or group of steps or group of elements, but not to exclude any other step or element or group of steps or group of elements. By "means a portion of the modified ligand that is reactive with the portion of the receptor that binds to the derived parent ligand of the modified ligand, where a covalent bond is formed between the binding agent and the receptor portion. It is possible. In this binding, the binding agent may be sufficient to promote covalent binding to the receptor moiety, either individually or in the presence of an auxiliary binding agent. The auxiliary binding agent can be an enzyme that catalyzes the formation of a covalent bond between the binding agent and the receptor moiety, or it can be an activator that can cause this formation.

By “isolated” is meant material that is substantially free or essentially free of components normally associated with its native state.

As used herein, the term “ligand” refers to an agent that binds, interacts with, or otherwise associates with a target receptor. The factor may bind to the target receptor if the target receptor is in its native conformation, or if it is partially or totally unfolded or denatured. According to the present invention, the ligand is not limited to factors that bind to the recognized functional region of the target receptor (eg, the hormone binding site of the receptor) and the like. In the practice of the present invention, a ligand can also be a factor that binds to any surface or internal sequence or conformational domain of the target receptor. Preferably, the ligand is a molecule (including drugs) that affects physiological function.
The ligand can also be an endogenous ligand.

By “obtained from” a sample (eg, an extract containing a receptor)
Is isolated or derived from a particular source, such as a suitable cell or tissue source, including humans, animals, plants, or microorganisms.

The term “patient”, as used herein, refers to any organism for which treatment or prevention of a condition associated with the receptor is desired using the methods of the invention. However, it is understood that "patient" does not imply that the condition is present. Thus, the patient may include microorganisms, plants or animals. Preferably, the patient is a human or other mammal, and includes any individual who is desired to be tested or treated using the methods of the invention. Suitable mammals within the scope of the present invention include primates, domestic animals (e.g. sheep, cows, horses, donkeys, pigs), clinical test animals (e.g. rabbits, mice, rats, guinea pigs, hamsters), Examples include, but are not limited to, companion animals (eg, cats, dogs) and captive wild animals (eg, foxes, deer, dingos).

By “pharmaceutically acceptable carrier” is meant a solid or liquid filler, diluent or encapsulating substance that may be safely used in topical or systemic administration.

The term “pharmaceutically acceptable salt” as used herein refers to the non-toxic salts of the modified ligands of the invention, which salts are generally suitable with the free base. Prepared by reaction with organic or inorganic acids. Representative salts include the following: acetate, benzenesulfonate, benzoate, hydrogen carbonate, hydrogen sulfate, hydrogen tartrate, borate, bromide, calcium edetate, cansylate, carbonate. , Chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate,
Esylate, fumarate, gluceptate
ate), gluconate, glutamate, glucoryl arsanilate, hexyl resorcinate, hydrabamin (hy)
drabamine), hydrobromide, hydrochloride, hydronaphthalenecarboxylate, iodide, isothionate, lactate, lactobionate.
ate), laurate, malate, maleate, mandelate, mesylate, methyl bromide, methyl nitrate, methyl sulfate, mucinate, napsylate (n
apsylate), nitrate, oleate, oxalate, pamoate (pam)
aote), palmitate, pantothenate, phosphate / diphosphate, polygalacturonate, salicylate, stearate, basic acetate, succinate,
Tannic acid salts, tartrate salts, teoclate, tosylate, triethiodide, valerate.

The term “receptor” as used herein refers to a structure comprising a molecule or cluster of molecules specific for one or more ligands, where the ligand-receptor binding, interaction. , Otherwise the association results in, alters or abolishes the function of this receptor. This receptor is preferably (but not exclusively) a protein. Representative receptors include, but are not limited to, insulin receptor, epidermal growth factor receptor, γ-aminobutyric acid receptor, nicotinic acetylcholine receptor, serotonin receptor, α- and β-adrenoceptor, dopamine. Receptors, histamine receptors, prostanoid receptors, adenosine receptors, cyclic nucleotide receptors, glutamine receptors, cytokine receptors, atrial natriuretic peptide receptors, and prostaglandin receptors. This receptor is a transporter (eg glucose, amino acid transporter, sodium-proton exchanger, chloride-bicarbonate exchanger, sodium pump, calcium pump, proton pump); channel (eg sodium channel, potassium channel, calcium channel) , And chloride channels); enzymes, such as oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases. But,
It is understood that the receptors need not be proteins, and that they may contain nucleic acids, for example amino groups found on adenine, guanine and cytosine, which can be targeted by the binding agents according to the invention. Alternatively, the receptor has one or more carboxyl groups and / or 1 that can be targeted by the binding agent.
It can be a carbohydrate with more than one amino group (eg, an amino-containing carbohydrate such as aminophenylglycoside) or a lipid (eg, present in the phosphate head group of some phospholipids).

As used herein, a “reporter molecule” provides, by its chemical nature, an analytically identifiable signal that allows the detection of a complex comprising a ligand and its cognate receptor. A molecule that does is meant. The term "reporter molecule" also extends to applications of cell aggregation or inhibition of aggregation, such as red blood cells on latex beads.

The term “sample”, as used herein, refers to any suitable sample that may contain a target receptor according to the present invention. The sample can be extracted, untreated, processed, diluted or concentrated from any suitable source and can contain one or more cells and / or cell membranes. The sample may include whole cells, degenerated cells, cell membranes or parts thereof. Alternatively, the sample may contain the isolated receptor. Suitably the sample may contain cells obtained from a tissue biopsy.
Alternatively, the sample can include cells or cell lines cultured in vitro.

The term “spacer” as used herein refers to chemical linkers, polymers, peptides, etc. that spatially separate a ligand and a binder. Preferably, the spacer is chosen so that it does not interfere with the binding of the modified ligand to the receptor.

By “therapeutically effective amount” is meant in the context of treatment of conditions associated with the receptor,
By administration, either as a single dose or as part of a series of doses, to a patient in need of such treatment is meant an amount effective to treat this condition. This effective amount will vary depending on the health and physical condition of the individual being treated, the taxon of the individual being treated, the formulation of the composition, the diagnosis of the medical condition, and other relevant factors. This amount, which can be determined through routine trials, is expected to be used in a relatively wide range.

2. Modified Ligands The present invention resides, at least in part, in the surprising discovery that a binding agent can bind to a parent ligand to form a modified ligand. The ligand binds irreversibly to the target receptor to which the parent ligand binds reversibly. The irreversible binding of the modified ligand to the receptor is due to the binding agent and the moiety present on the receptor (preferably one or more functional amino acid side groups) (often
Referred to herein as a "functional group"). This covalent bond is formed by the association of the modified ligand with the receptor, followed by attack of the adjacent reactive functional group by the binding agent. This irreversible interaction of the modified ligand with the receptor results in either a persistent inhibition (as an antagonist) or stimulation (as an agonist) of receptor function. The normal function or activity of this receptor resumes only after the new receptor is synthesized.

Parental ligands that can be modified include ligands that interact with the es nucleoside transporter and / or nucleoside / nucleotide / nucleobase-sensitive proteins. The two forms of nucleoside transporters are classified based on their sensitivity to inhibition by NBMPR (nitrobenzylthioinosine). Griffith et al., Biochim. Biophys. Acta, vol
． 1286, pp. 153-181 (1996). The group of NBMPR-sensitive transporters is designated as es (equilibrium sensitive) and the insensitive group is designated as ei (equilibrium insensitive). 1
In one embodiment, compounds are provided that have differential binding to the es receptor for the ei receptor. Compounds that selectively and irreversibly bind to the es receptor are provided. Expression of the es receptor is positively associated with the carcinogenic state of cells.

Among the many reactive functional amino acid side chains on the es nucleoside transporter protein that can be used for covalent attachment, the sulfhydryl group of cysteine residues is probably the most pharmacologically and biologically is important. Early studies on the effects of sulfhydryl reagents on nucleoside transport in mammalian cells include:
It was shown to cause significant inhibition of uridine uptake in various cultured cells (
Pagemann & Richey (1974), Biochim. Bioph
s. Acta, vol. 344, pp. 263-305; Pagemann et al. (1978), J. Am. Cell. Physiol. , Vol. 97, pp. 49-
72; Pagemann & Wohlhueter (1980), Curr. T
op. Membr. Transp. , Vol. 14, pp. 225-230; B
elt (1983), Biochem. Biophys. Res. Commun
． , Vol. 110, pp. 417-423; Belt (1983), Mol.
Pharmacol. , Vol. 24, pp. 479-484). However, most of the early work was directed at total nucleoside uptake, and e
Few attempts have been made to discern differences in susceptibility to sulfhydryl reagents by the s and ei transport systems. Furthermore, the sulfhydryl reagents used are predominantly organomercury compounds, which have been demonstrated to perturb plasma structure at concentrations as low as 200 μM (Belt & Noel (1985), Bioche.
m. J. , Vol. 232, pp. 681-688).

The ligand is also serotonin (5-hydroxytryptamine, ie 5-
It can be modified into a ligand that binds to the HT) receptor. The 5-HT receptor includes various receptor subtypes (Peroutka, S.J.
． , CNS Drugs, (1995), vol. 4 (Appendix 1), pp. 18-
28). Ion channel 5-HT ₃ receptor (Derkash, V et al.,
Nature, (1989), vol. 339, pp. Except for 706-709), other 5-HT receptors belong to a large family of 7-transmembrane G-protein coupled receptors. The clinical significance of the effects of 5-HT is, for example, neurological disorders, CNS disorders, psychiatric disorders and emotional disorders.
r) (migraine, anxiety, depression, schizophrenia, obsessive-compulsive disorder, psychosis, aggression, hostility, eating disorders, gastrointestinal disorders, hypertension, maintenance of circadian rhythm in sleep-wake cycle, sexual Activity, compulsive behavior, temperature, vomiting, and maintenance of cardiovascular and motor function). Therefore, drugs that target the 5-HT receptor have broad clinical use. Ligands that bind to the 5-HT receptor can have effects by binding to, for example, the cardiovascular system, platelets, gastrointestinal tract, and brain (Erspamer, V. Ed., "5-Hydroxytr.
yptamine and related indolealklylami
nes ", Handbuch der Expermentellen Pha
rmakologie, Vol. 19. Springer-Verlag, Be
rlin, (1996), pp. 132-181).

Thus, for example, the ligand 5-HT can be modified as disclosed herein. Other ligands that can be modified include 5-HT precursors and agonists and antagonists of the 5-HT receptor known in the art.
Examples include: precursor 5-hydroxytryptophan used as antidepressant; 5-HT _1A -agonist used as tranquilizer and antihypertensive; sumatriptan used for migraine. (Selective 5HT ₁ receptor agonists); 5-HT ₂ -antagonists such as methysergide used in migraine prophylaxis and carcinoid syndrome; 5-HT such as ketaserine used to lower blood pressure in hypertensive patients ₂ -antagonist;
And selective 5-HT ₃ that is used to treat cytostatic vomiting and radiation induced emesis -. Antagonists (Beasley, C.M., et al., Psych
opharmacology, (1992), vol. 107, pp. 1-10
Bolden-Watson, C .; And Richelson, E .; , Lif
e Sciences, (1993), vol. 52, pp. 1023-102
9; Koe, B .; K, J. Clin. Psychiatry, (1990), v
ol. 51, pp. 13-17).

Serotonin-binding ligands that can be modified include ligands developed for CNS disorders such as anxiety (eg, benzodiazepines, or 5-HT _1A agonists (eg, buspirone, gepirone, ipsapirone, and flesinoxan). In the case of anxiolytics, they have advantages over benzodiazepines because they do not have the sedative and drug dependent disorders of benzodiazepines (Barrett, JE., And Vanover, KE.
, Psychopharmacology, (1993), vol. 112, p
p. 1-12. ). Other serotonin binding ligands include ligands developed for the treatment of depression such as SSRIs (eg fluoxetine). SSRIs have also been shown to be effective in treating bulimia and obsessive-compulsive disorder, and also specific cognitive aspects of obesity, panic disorder, premenstrual syndrome, diabetic neuropathy, chronic pain, and Alzheimer's disease. May be useful in the treatment of Other useful antidepressants that are relatively potent antagonists of the 5-HT ₂ receptor include tricyclic antidepressants such as trazadone and nefazodone (C
owen, P.M. J. , And Anderson. I. M. , "5-Hydrox
ytryptamine in Psychiatry "(Sandler, M
, Copen, A .; , & Harnett, S .; Chapter), Oxford Univ
ersity Press, New York, (1991)). The ligand is
Compounds developed for the treatment of schizoprenia (
Antipsychotics including clozapine, risperidone and olanzapine) (Weil-Malhe)
rbe, H .; , Serotonin and schizophrenia. "
The Central Nervous System. Vol. 3, Ser
Otonin in Health and Disease ", Essman
, W. B. Ed., Spectrum Publications, Inc. , Ne
w York, (1978), pp. 231-291). Ligands associated with the treatment of eating disorders such as SSRI and fenfluramine can be used. Fenfluramine may be useful in the treatment of other diseases such as autism, premenstrual syndrome, seasonal affective disorders, and attention deficit disorder. Ligands associated with alleviation of gastrointestinal disorders, such as ondansetron and granisetron, can be used to treat radiation-induced vomiting associated with cancer chemotherapy, as well as amelioration of nausea and recovery from general anesthesia. Used in the treatment of resulting vomiting. Other ligands include metoclopramide and cisapride. A ligand used in the control of the sleep-wake cycle (eg L-tryptophan),
Alternatively, a non-selective 5-HT agonist or 5-HT antagonist (for example,
Ritanserin) can be used (for review, see Wauquier, A., and Dugovic, C., Ann NY Acad. Sci., (1990).
), Vol. 600, pp. 447-459). Other ligands include etanserin, which is used as an antihypertensive agent, 5-
HT ₂ receptor antagonists.

2.1. Discussion on Reactivity of Protein Structure Peptides and proteins are composed of amino acids polymerized together through the formation of peptide (amide) bonds. The peptide-bonded polymer forms the backbone of the polypeptide structure. There are 20 common amino acids found throughout nature, each of which is an identifying side chain of a particular chemical structure.
side chain), charge, hydrogen bonding ability, hydrophilicity (or hydrophobicity), and reactivity. This side chain is not involved in peptide formation and therefore does not interfere or react with its environment.

Amino acids can be classified by type depending on the characteristics of their side chains. The most significant amino acids for covalent purposes are those with accessible ionizable side chains (eg, aspartic acid, glutamic acid, lysine, arginine, histidine, cysteine, and tyrosine). Methionine and tryptophan also contain ionizable side chains, which are not readily accessible. This is because they are usually buried deep in the molecular structure of the receptor due to their hydrophobic nature.

Both aspartic acid and glutamic acid contain carboxylic acid groups with ionizing properties. Carboxylic acid groups in proteins can be derivatized through the use of amide bond-forming agents or through active esters or reactive carbonyl intermediates.

Lysine, arginine, and histidine have ionizable amine-containing side chains. These amine-containing side chains are typically exposed on the surface of proteins,
And can be easily derivatized. The most important reaction that can occur with these residues is
Alkylation and acylation.

Cysteine is the only amino acid containing a sulfhydryl group. The most important reaction of cysteine groups in proteins is the formation of disulfide bridges with another cysteine molecule. Cysteine sulfhydryls and cystine disulfides (called cystine residues) undergo a variety of reactions, including alkylation to form stable thioether derivatives, acylation to form relatively labile thioesters, and many oxidation and reduction processes. Can cause Cysteine and cystine groups are relatively hydrophobic and are commonly found within protein cores. Deformer or "
Accessing the sulfhydryl groups of large proteins without the presence of a "driver" is often difficult. Nevertheless, such steric hindrance is due to the sulfhydryl group, leading edge in selectivity.
ge) is given. Therefore, without the use of a modifier, the physiological ligand or drug may be incorporated within the internal structure of the receptor to access functionally reactive sulfhydryl groups located deep within the ligand binding site. It is used to direct binders as described therein. Irreversible binding agents targeted with sulfhydryl groups may have probably lower levels of non-specific binding (in clinical terminology "less side effects") than any other functional group.

Tyrosine contains a phenolic side chain. Although this amino acid only solubilizes slightly in water, the ionizable nature of the phenolic group sometimes causes it to appear in the hydrophilic regions of proteins, usually at or near the surface. Therefore, unlike cysteine residues, tyrosine derivatization proceeds with less need for deformers to further open the protein structure. Tyrosine can be specifically targeted for modification via its phenolate anion by acylation.

In summary, a protein molecule may contain up to 9 amino acids whose side chains are easily derivatized. These 9 residues have sufficient reactivity for modification reactions 8
Including a functional group of principle. These are guanidinyl group of arginine, γ-carboxyl group and β-carboxyl group of glutamic acid and aspartic acid, sulfhydryl group of cysteine, imidazolyl group of histidine, ε of lysine.
An amino acid, the thioether part of methionine, the indolyl group of tryptophan and the phenolic hydroxyl group of tyrosine. Since methionine and tryptophan are generally buried inside the protein and thus protected from the conjugate dissolved in the solvent, they show only some selected reactivity in intact proteins. Other ionizing groups are usually either exposed on the protein surface or can be accessed with the aid of deformers or "drivers." Therefore, they are easy targets for the conjugate. However, among amino acid side chains with multiple reactive functional groups on proteins that can be used for labeling, the sulfhydryl group of cysteine residues is most preferably both pharmaceutical and biologically important. It has been reported that in most macromolecules there is at least one copy of the reactive sulfhydryl group located at or near the ligand binding site of the target macromolecule. Destruction of this reactive sulfhydryl functional group by a sulphydryl reducing agent has been shown to affect the function of many macromolecules.

(2.2. Receptor moiety capable of conjugation) A sulfhydryl moiety having a thiolate ion as an active species is the most reactive functional group in a protein. Reactive thiol with about 8.6 pK _a is
The pK towards _a and it is expected to increase with pH which gradually increases beyond the pK _a.

In the process of modifying the parent ligand, it is advantageous to take advantage of the presence of this highly reactive sulfhydryl group, which is typically located near or at the ligand binding site of the receptor. To do. The drug or physiological ligand may be chemically modified to include a binder that reacts faster with the thiol group than with any other reactive functional group. Upon association of the modified drug with its receptor, the sulfhydryl-group-directed conjugate attacks any sulfhydryl group located within its accessible proximal; which binds the drug to its receptor. Creates a covalent bond.

In addition to sulfhydryl groups, there are also other highly reactive functional groups present on the amino acid side chains, which can be chemically modified. Conjugates that are reactive with these functional groups are discussed below.

(2.3. Binder) (2.3.1. Sulfhydryl group-specific binder) (N-maleimide derivative) Maleimide is used particularly at a pH of less than 7 (at this time, other nucleophilic groups are protons). It is believed to be fairly specific for the sulfhydryl group. In acidic and near neutral solutions, the reaction rate with simple thiols is about 100 times faster than the corresponding simple amines. Although this rate increases with pH, the reaction with amino groups also becomes significant at high pH. The other major competitive reaction is the hydrolysis of maleimide to maleic acid. However, at pH 7, the apparent rate of hydrolysis was only 3.2 × 10 ⁻⁴ min ⁻¹ in 0.1 M sodium phosphate buffer at 20 ° C., which was too slow to react with sulfhydryl groups. Do not block. Degradation rates only dominate at pH above neutral. The thiol undergoes a Michael reaction with the maleimide, producing an adduct exclusively on the double bond. The resulting thioether bond is very stable and cannot be cleaved under physiological conditions.

(Disulfide Reagent) Disulfide exchange occurs when a sulfhydryl group reacts with a disulfide. Some of the most commonly used disulfide reagents are 5,5'-dithiobis- (2-nitrobenzoic acid), 4,4'-dithiodipyridine, methyl-3-nitro-2-pyridyldisulfide. , And methyl-
2-pyridyl disulfide. The protein disulfide formed is 2-
It is easily reversed in the presence of free mercaptans such as mercaptoethanol or dithiothreitol. The reduction of protein disulfide to its body sulfhydryl group allows the protein to restore its function. Thus, this class of irreversible binding drugs provides an additional safety mechanism to prevent various therapeutic complications (eg, overdose, overreaction, etc.).

(2.3.2. Amino Group-Specific Binder) This amino group is another strong nucleophilic group in proteins. However, its abundance and ubiquitous in the protein, as well as due to the relatively high pK _a of the ammonium ion, most of the reagents react with amino groups can also react with other functional groups. Thus, amino groups may not be ideal targets for modified ligands according to the present invention unless a significant cysteine residue is present at the drug binding site. Nevertheless, many stable acylation products are still formed with this amino group and only with this amino group. Most common reactions of amines are alkylation and acylation reactions. Some of the important alkylating factors that can be incorporated into the ligand structure are: α-haloacetyl compounds (eg haloacetate,
Haloacetamide), N-maleimide derivatives, aryl halides (eg dinitrofluorobenzene, trinitrobenzene sulphonate), aldehydes (eg glutaraldehyde, formaldehyde) and ketones (eg pyridoxal phosphate). Acylating agents include, but are not limited to, isocyanates, isothiocyanates, imide esters, N-hydroxylsuccinimidyl esters, p-nitrophenyl esters, acyl chlorides, and sulfonyl chlorides.

(2.3.3. Carboxyl Group-Specific Binder) The most important chemical modification reaction of the carboxyl group utilizes a carbodiimide-mediated process. For proteins, the optimum pH for the reaction is about 5, which is difficult to achieve in most physiological conditions. Other reagents such as diazoacetate esters and diazoacetamide can also be used to esterify carboxyl groups. Similar to carbodiimides, these reagents react with high specificity to protein carboxyl groups under mild acid conditions.

2.3.4. Tyrosine-Specific Binder Factor Tyrosine, histidine, and other aromatic residue proteins are electron-rich.
These residues cause electrophilic substitution reactions on the aromatic ring. Useful electrophiles for reaction with tyrosine and histidine in proteins are diazonium compounds. Other protein compounds such as lysine, tryptophan, cysteine, and arginine residues react very slowly, so that diazonium reagents can be considered tyrosine-selective. Diazonium ions are generally labile even at neutral pH and maximal reaction with proteins is typically achieved at alkaline pH. The tyrosine phenolate ion also reacts with acylating agents in a manner similar to the amino group. However, tyrosine groups are generally considered to have lower reactivity. This is not because the phenolate ion has a lower nucleophilicity, but because the tyrosine residue is usually buried in the protein and is therefore generally less reactive due to its hydrophobicity.

(2.3.5. Arginine-Specific Binder) The predominant reaction of the guanidinyl portion of the arginine residue is the reaction with the 1,2-dicarbonyl reagent. Commonly used vicinal diketones include glyoxal, ferglyoxal, 2-3-butanedione and 1,2-cyclohexanedione.

2.3.6. Histidine-Specific Binders Many alkylating agents that react with the imidazolyl moiety of histidine are referred to earlier, but their kinetics are generally Inferior to the nucleophilic group of. Even with α-haloacetates, N-carboxymethylation is generally slow compared to sulfhydryl groups. However, such reactive .alpha.-halocarboxyl groups may have affinity labels (e.g., p-toluenesulfonylphenylamine chloromethylketone) (p-toluenesulfophenylphenylene chloro).
Specific reactions can be achieved when incorporated into romethylketone), p-toluenesulfonyl lysine chloromethyl ketone). In addition to α-haloacetyl groups, other alkylating agents are not reactive towards histidine. In the acylating reagent, histidine forms an acylation product that is generally unstable and can cause spontaneous hydrolysis. The most important acylating agents commonly used for the modification of histidine are diethylpyrocarbonate or ethoxyformic anhydride. The acylated imidazole is reversed at alkaline pH resulting in histidine recovery. Deacylation is accomplished very quickly at hydroxylamine and neutral pH.

2.3.7 Methionine-Specific Binder The primary chemical modification of methionine is alkylation. Methionine is often
Being located within the hydrophobic interior of the protein, methionine tends to provide a high degree of selectivity. Only alkylating reagents that bind to the ligand are accessible to these buried methionines.

2.3.8 Tryptophan-Specific Binders Due to their hydrophobicity, tryptophan residues are generally buried inside the protein. Tryptophan residues are N-bromosuccinimide and 2-
It can be modified with hydroxy-5-nitrobenzyl bromide. A separate reagent, p
-Nitrophenylsulfenyl chloride has been used to modify the indolyl moiety. This reaction is selective for tryptophan and cysteine residues.

2.3.9 Serine-Specific Binders Many reactive reagents such as diisopropylfluorophosphate, phenylmethyl-sulfonyl fluoride and other acrylsulfonyl fluorides react with the active site serine. Has been found. Care should be trained in the use of such reagents as the competitive hydrolysis reactions are strong.

2.4 Design of the Modified Ligands of the Invention Covalent bond formation between the modified ligand and the receptor can be catalyzed by an enzyme, triggered by an activator, or its own binding agent. Promoted by. This covalent bond is preferably formed with a functional group located at or near the ligand binding site of the receptor. This strategy is
It is advantageous as it ensures a high degree of specificity.

The design of irreversible binding ligands depends on the chemical, biological and molecular properties of both the ligand and the receptor. In each instance, the binder to be introduced on the chemical structure of the ligand may be different and may require a particular arrangement. In general, the ligand is preferably a functional group that allows binding of the binding agent without affecting the activity of the ligand to bind its receptor and induce the biological activity of the ligand, or such a functional group. It includes functional groups that can be modified to contain groups. In this respect, the modified ligand need not have the same biological activity as the parent ligand (eg, need not be activated in vivo by some metabolic or catabolic step).

Some of the conditions and requirements to be considered for binder selection and placement are as follows: Determination of reaction specificity for specific functional groups of the receptor (eg, amino, sulfhydryl, carboxyl, guanidyl, imidazolyl, and other amino acid side chains) required for binder selection. The choice depends on the availability of any functional groups on the receptor to which the drug molecule is linked. The non-bonding binding agent of the modified ligand must be specific for this functional group.

2. Hydrophobicity and hydrophilicity of the binder. Receptors in a hydrophobic environment may require a hydrophobic ligand to reach the receptor. For example, a modified ligand with a hydrophilic binder cannot access the corresponding functional group located deep inside the hydrophobic core of the receptor.

3. Cleavability of the binder. In some cases, it may be desirable to separate the modified ligand bound to the receptor. For example,
When toxic drugs (corresponding to the parent ligand) are modified, a safe mechanism must be introduced to preemptively prevent situations such as overdose. In this case, the use of cleavable conjugation factors can restore conjugation if complications occur. A number of cleavable bonds can be used for this purpose. These include disulfide bonds, amidine bonds, mercury group bonds, vicinal glycol bonds,
Examples include an azo bond, a sulfone bond, an ester bond and a thioester bond.
In this regard, the conjugate agent itself may be cleavable, or if the binding agent breaks through the spacer and is attached to the modified ligand, the spacer may be cleavable. In this respect the spacer may be selected from the group consisting of: (
N-succinimidyl 3- (2-pyridyldithio) propionate, succinimidyloxycarbonyl-α-methyl-α- (2-pyridyldithio) toluene,
3- (2-pyridyldithio) propionylhydrazine, disuccinimidyl tartrate, N- [4- (p-azidophenylazo) -benzoyl] -3-aminohexyl-N'-oxysuccinimide ester, 4,4'- Difluoro-3,3'-
Dinitrophenyl-sulfone, 3- (4-azido-2-nitrobenzoylseleno) propionic acid, 2-methylmaleic anhydride.

4. Binder size and geometry. The presence of a binding agent on the modified ligand must allow binding of that ligand to the receptor. Upon binding of the modified ligand to the receptor, the binding agent must be in close proximity to the receptor moiety to effect conjugation. Spacers as defined herein may require the binding agent to be in close proximity to the receptor moiety. In such cases, the length of the spacer depends on the distance required for the binding agent to reach the effective site of conjugation adjacent to the receptor moiety. Structural analysis of receptors using X-ray crystallography, nuclear magnetic resonance, etc., in combination with molecular modeling, in the identification of receptor moieties to be conjugated and in the selection and placement of binders on the chemical structure of the ligand. Can help. The receptor portion can be at the ligand binding site of the receptor. Preferably, the receptor portion is outside the ligand binding site of the receptor. Without being limited to any theory,
It is possible that this latter approach may be advantageous. This is because it is more likely to prevent the destruction of the receptor function upon binding with the modified ligand. On this basis, the parent agonist can be modified to cross-link cognate receptors, thereby causing a continuous stimulation of receptor function ("turn-on of the receptor"). Alternatively, the parent antagonist is
It can be modified to crosslink its cognate receptor, thereby causing a continuous inhibition of receptor function ("turn-off" of the receptor).

3. Compositions The present invention also includes compositions that include a modified ligand as described herein with a pharmaceutically acceptable carrier.

The invention also features a method of treating or preventing a condition associated with a target receptor, which method, as broadly described above, provides a therapeutically effective amount to a patient in need of such treatment. The step of administering the composition is included.

Depending on the particular route of administration, various pharmaceutically acceptable carriers well known in the art may be used. These carriers include sugar, starch, cellulose, and their derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline, and fever. It can be selected from substance-free water.

Any suitable route of administration may be employed for providing the compositions of the invention to the patient. For example, oral administration, rectal administration, parenteral administration, sublingual administration, buccal administration, intravenous administration, intraarterial injection, intramuscular injection, intradermal administration, subcutaneous administration, inhalation administration, intraocular administration, intraperitoneal administration, brain Indoor administration, transdermal administration and the like can be used.

Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, suppositories, aerosols, transdermal patches and the like. These dosage forms may also include injecting or implanting a controlled release device specifically designed for this purpose or other form of implant modified to act in addition to this modality. Controlled release of modified ligands can be achieved, for example, with hydrophobic polymers (including acrylics, waxes, relatively high aliphatic alcohols, polylactic acid and polyglycolic acid), and certain cellulose derivatives (eg hydroxypropylmethylcellulose). It can be provided by coating the ligand. In addition, controlled release can be provided by using other polymeric matrices, liposomes and / or microspheres.

Compounds suitable for oral or parenteral administration are discrete units such as capsules, sachets or tablets, each containing a predetermined amount of one or more immunogenic agents of the invention. As well as powders or granules, or solutions or suspensions in aqueous liquids, solutions or suspensions in non-aqueous liquids, water-in-oil emulsions or oil-in-water liquid emulsions. Such compositions may be prepared by any of the methods of pharmacy but all methods result in the association of one or more modified ligands as described above with the carrier which constitutes one or more necessary ingredients. Includes. In general, the composition uniformly and thoroughly mixes the modified ligand of the invention with a liquid carrier or a finely divided solid carrier or both, and then, if desired, the desired product. Prepared by molding into a presentation.

The modified ligand can be in the form of a pharmaceutically acceptable salt as is known in the art.

The compositions may be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically useful. In this regard, the dose of modified ligand administered to a patient should be sufficient to effect a beneficial response (eg, amelioration of the condition being treated) in the patient over time. The amount of modified ligand administered will depend on the age of the subject being treated,
It may depend on the subject being treated, including gender, weight and general health.
In this respect, the precise amount of modified ligand for administration will depend on the judgment of the practitioner. In determining an effective amount of the modified ligand to administer in the treatment or prevention of a condition associated with the target receptor, the physician can assess the progression of the condition.

In any case, one of ordinary skill in the art can readily determine an appropriate dosage of the modified ligand of the present invention. Such a dosage may be on the order of nanograms to milligrams of a modified ligand of the invention.

4. Detection of Target Receptors and Cells or Cell Membranes Comprising These The invention also features a method of determining the presence of a target receptor in a test sample. This method comprises the steps of contacting a sample with a modified ligand as described in Section 2, wherein the modified ligand binds to a target receptor, and the modified ligand in the contacted sample and the modified ligand. Detecting the presence of a complex containing the receptor.

The invention also includes a method of quantifying the presence of a target receptor in a test sample. The method comprises contacting a sample with a modified ligand as broadly described above, wherein the modified ligand binds to a target receptor, and the modified ligand and the receptor in the contacted sample. And measuring the concentration of the complex comprising, and correlating the measured concentration of the complex with the concentration of the receptor in the sample.

The invention also provides a method of detecting the presence of a target receptor on a cell or cell membrane. This method comprises the step of contacting a sample containing cells or cell membranes with a modified ligand as described broadly above, wherein the modified ligand binds to a target receptor, and Detecting the presence of a complex comprising the modified ligand and the cell or cell membrane.

Also included by the present invention is a method of quantifying the presence of target receptors on cells or cell membranes. The method involves contacting a sample containing cells or cell membranes with a modified ligand as described broadly above, wherein the modified ligand binds to a target receptor. The concentration of the complex containing the modified ligand and the cell or cell membrane is then measured in the contacted sample, and the measured concentration of the complex is the concentration of the receptor presented on the cell or cell membrane. Associated.

This modified ligand can be used as a means to identify the actual drug pocket or drug binding region on the receptor molecule. Since the binding of these modified ligands to their target is irreversible, the actual site of drug interaction is MassS.
Various techniques such as peptide fingerprinting with pec can be used to identify on the receptor. This information can be used to identify molecules that bind to a particular drug pocket.

Any suitable technique can be used to determine the formation of this complex. For example, a modified ligand having a reporter molecule (sometimes referred to herein as a "probe") that binds a modified ligand according to the present invention may be used in the art to determine and / or quantify ligand-receptor interactions. In any suitable assay known in. For example, scintillation counting, autoradiography, fluorography, flow cytometry, UV spectroscopy, fluorescence spectroscopy, chemiluminescence imaging, fluorescence microscopy, confocal microscopy, electron microscopy etc. may be used in this regard.

The reporter molecule may be attached to any suitable portion of any modified ligand, including binders and spacers, if any. Preferably, the binding of the reporter molecule to the modified ligand is chosen so that the reporter molecule does not interfere with the binding of the modified ligand to the receptor.

It is understood that the reporter molecule that binds the antigen binding molecule can include: direct binding of the reporter molecule to the modified ligand; indirect binding of the reporter molecule to the modified ligand; Binding of the reporter molecule to another assay reagent that subsequently binds; and binding of the modified ligand to the subsequent reaction product.

Reporter molecules include chromogens, chromophores, catalysts, enzymes, fluorescent dyes, chemiluminescent molecules,
It may be selected from the group comprising lanthanide ions (eg europium (Eu ³⁴ )), radioisotopes, spin labels, and direct visual labels.

In the case of direct visual labels, those used include colloidal metallic or non-metallic particles, dye particles, enzymes or substrates, organic polymers, latex particles, liposomes, or other containing signal producing substances. It can be made from vesicles and the like.

A number of enzymes suitable for use as reporter molecules are available in the US patent specifications, US Pat. Nos. 4,366,241, 4,843,000, and 4,849.
, 338. Suitable enzymes useful in the present invention include alkaline phosphatase, horseradish peroxidase, luciferase, β-galactosidase, glucose oxidase, lysozyme, malate dehydrogenase and the like. This enzyme may be used alone or in combination with a second enzyme present in solution.

Suitable fluorescent dyes include fluorescein, isothiocyanate (FITC)
, Tetramethylrhodamine isothiocyanate (TRITC), R-phycoerythrin (RPE), and Texas Red (Texas Red). Other exemplary fluorescent dyes include Dower et al. (Int
Fluorescent dyes discussed by International Publication WO 93/06121). See also US Pat. No. 5,573.
, 909 (singer et al.) And 5,326,692 (Blinkley).
Et al.) For the fluorescent dyes described in Alternatively, reference is made to US Pat.
227,487, 5,274,113, 5,405,975, 5,433,896, 5,442,045, 5,451,663.
No. 5,453,517, No. 5,459,276, No. 5,516.
No. 864, No. 5,648,270, and No. 5,723,218.

Preferably, the reporter molecule is ³ H, ¹²⁵ I, ¹⁴ C, ³² P, ³³ P,
And radioisotopes such as ³⁵ S.

5. Applications (Treatment) The modified ligands of the present invention, which preferably exhibit irreversible binding agents, have substantial advantages over conventional reversible binding agents. First, dose-related inhibition or stimulation may be present for a long time after the drug has cleared plasma. In other words, the effect of the drug appears to be longer lasting than expected from the plasma elimination half-life of the drug. Second, due to the longer lasting drug effect, these irreversible binders can be administered less frequently and at lower doses. This is,
Minimize adverse side effects and prevent cumulative toxicity induced by the drug itself or by its metabolites themselves. Third, the binding of the irreversible antagonist to the receptor is permanent, so blocking the receptor response is no longer a competitive inhibitory mechanism. This irreversible antagonism, at any concentration, prevents the antagonist from producing maximal effects at a given receptor. Furthermore, if the modified ligand is made highly radioactive, it can be used as a therapeutic agent to specifically kill cells bearing cognate receptors, or it can be used for imaging.

(Diagnostics) Another application of this ligand modification technique is to further elucidate different receptor subpopulations that can be targeted to rescue dysfunction in various complex physiological processes. More importantly, this modified drug allows the inventors to "react" at specific receptors without undergoing redundant, tedious, and complex manipulations of genetic material.
It allows to develop / identify animal models that are “defective”. Thus, the complex physiological mechanism and function of various receptors in a "real-life" context can be studied and analyzed. further,
One of ordinary skill in the art can also study how a variety of different receptors are linked and affected by each other's function. The effectiveness of such animal models also
By simultaneously blocking multiple receptor populations of interest, researchers
It makes it possible to predict and elucidate the therapeutic outcome of various drugs. Thus, the present invention can be used to profile cells as well as the different receptors present in tissues, organs or systems. Such receptor profiling
To discover new drug targets, to predict possible side effects of drugs, and how different cells communicate with each other, the health of their cells, and their It can be advantageously used to determine whether to respond to a particular extracellular stimulus (eg, a drug).

[0128]   The invention is now described with reference to the following non-limiting examples.

Examples Example 1: Equilibrative nucleoside transporter in mouse melanoma SP2 / 0-Ag14 cells.
Effect of N-ethylmaleimide on transporter) Representative N-maleimide derivatives were used as sulfhydryl reagents (advantageously specific for sulfhydryl groups). Sulfhydryl reagents react only with specific accessible sulfhydryl groups on proteins, allowing for specific inhibition and good penetration into cells due to the uncharged nature of the compound. N-ethylmaleimide (NEM) is the smallest maleimide reagent capable of forming stable thioesters with the reactive sulfhydryl groups of proteins. The sensitivity of the es and ei nucleoside transport systems towards NEM was demonstrated. We show that the sensitivity of the nucleoside transport system towards NEM using a variety of different cell lines is a general phenomenon and is not restricted to a particular cell or tissue type.

Previous studies of the es nucleoside transport system (Patterson et al. (1997), M.
ol. Pharmacol. , Vol. 13, p. 114; Gati et al. (1983).
, Mol. Pharmacol. , 23, pp. 146-1520) have suggested that the sugar component of the nucleoside is important for the binding of the nucleoside to the es nucleoside transport site. Thus, in the present invention, the linker moiety binds to the pyrimidine / purine ring of this nucleoside, avoiding disruption of effective inhibition of the es nucleoside transporter, providing a novel probe for its transporter regulatory site.

We have found that a novel group of nucleosides, cytidyl 4- [N-maleimidomethyl] cyclohexane-1-carboxylic acid (CrMCC), and their derivatives and analogs, are found in human cell es nucleoside transporter proteins. It has been found to have the ability to irreversibly inhibit CrMcc or 1-[[4-[(4-amino-1-β-D-ribofuranosyl-2 (1H) -pyrimidione) carbonyl] cyclohexyl] methyl] -1H-pyrrole-2,5-
The direct and rapid synthetic route used to synthesize the dione is shown in FIG.

To demonstrate the sensitivity of the es and ei nucleoside transport systems to NEM,
Various mammalian cell lines were loaded with different concentrations of NEM (
0-30 mM). FIG. 1 shows that equilibrium transport of ³ H-uridine (final concentration 50 μM) in mouse myeloma SP2 / 0-Ag14 cells (measured by 5-second interval uptake) was inhibited by NEM in a clear biphasic manner. Indicates. About 60
Transport activity of ˜70% was inhibited in NEM with an IC ₅₀ value of 0.15 mM. The remaining 30-40% transport activity was gradually eliminated by NEM at a concentration of approximately 3 mM. Similar biphasic inhibition by NEM also revealed human HL-60 and human MCF.
Observed in -7 cells, which carry both es and ei transport systems (L
ee et al. (1995) Biochem. Biophys. Acta. 1268, 200-208). Monophasic ³ H-uridine transport curves in NEM dose response experiments were observed for cells with es only (mouse EL-4 cells) or ei only (rat Morris 7777) transport system. IC ₅₀ value is EL-4
And Morris 7777 about 0.1 mM and 1.5, respectively.
It was mM. These results suggested that the monophasic curves of total ³ H-uridine transport observed in NEM dose response experiments were a reflection of the existence of two different equilibrium nucleoside transport systems in these cells. NEM sensitive components are
The es transport system, and the NEM insensitive component is the ei transport system. But,
The ei transporter is less sensitive to NEM inhibition and the higher concentrations of N
It can be inhibited with EM or by prolonged exposure to NEM. This observation is consistent with the general finding that the sulfhydryl groups of the enzyme show considerable variation in their reactivity. This reactivity, over several stages of inactivity,
It ranges from non-reactive to no immediate reaction and to immediate reaction.

To further demonstrate that the reduction of uridine influx by the es transport system was due to NEM-induced chemical modification of carrier proteins, which subsequently affected transport affinity, and the es nucleoside transport activity was ³ H-. To confirm the finding that it can be demonstrated by NBMPR binding capacity, mouse myeloma SP2 / 0-Ag14 cells pre-treated or untreated with 0.3 mM NEM for 1 min have high affinity on the cell membrane. Assayed for derivative composition of the ³ H-NBMPR binding site. FIG. 2 shows that K _d values for ³ H-NBMPR binding (corrected for non-specific binding determined in the presence of 20 μM NBTGR) were significantly changed in response to NEM treatment. Immediately 1 minute after NEM exposure, 0 for untreated cells
． The apparent Kd value was 1.9 ± 0.4 nM compared to 16 ± 0.02 nM. However, 40,000 ± for NEM treated and untreated cells, respectively.
B _max values for 2,600 and 41,000 ± 1,000 sites / cell were not significantly different. These results indicate that important disulfide bonds are close to or close to the nucleoside transport / binding site of the es transport system, where disulfide bond formation in NEM affects carrier transport affinity. Provide further proof that they have been deployed. The suggestion that the key sulfhydryl group on the es transporter protein is probably located on or near, but not on, the nucleotide transport / binding site has been suggested by NBMP.
Derived from the observation that R, dilazep, dipyridamole (at 30 mM) and uridine (at 10 mM) are unable to protect this sulfhydryl group from NEM modification (Lee et al. (1995), Biochem. Biophys.
． Acta. 1268, 200-208). NEM is effective as an inhibitor of the es transporter protein, but it is toxic and must be modified for therapeutic purposes.

Example 2 (Synthesis and Characterization of CrMCC) The strategy for selectively and irreversibly inhibiting the es transporter protein is a reactive group-specific covalent binder for the driver (driver). Attaching (maleimide), which can deliver the covalent agent to the desired target
Is. The most suitable driver is the physiological ligand itself (nucleoside). Cytidine (a pyrimidine nucleoside) is selected among other physiological nucleotides due to its "functional inactivity" and thus minimizes "non-specific" drug binding. For the spacer arm that connects the maleimide to cytidine, cyclohexanecarboxylic acid is chosen for the pilot study. This configuration is NB
It mimics the chemical structure of MPR (Fig. 5b). In addition, other advantages such as hydrophobicity (provided by chlorohexane) and stability (provided by carboxylic acid) are also considered.

CrMcc or 1-[[4-[(4-amino-1-β-D-ribofuranosyl-2 (1H) -pyrimidione) carbonyl] cyclohexyl] methyl] -1H-
The direct and rapid synthetic route used to synthesize the pyrrole-2,5-diones is shown in FIG.

N-succinimidyl 4- [N-maleimidomethyl] cyclohexane-1-carboxylate (SMCC) and cytidine were separately dissolved in anhydrous dimethyl sulfoxide (DMSO) before the reaction. The reaction was initiated when the two reagents were mixed at room temperature and shielded from light. The molar concentration ratio of SMCC cytidine in this mixture was 0.10M: 0.15M, and the pH was 7 in the reaction system.
． It was 5 to 8.0. Within 4 hours CrMCC was found in the reaction mixture,
Then, using an absorption wavelength of 300 nm, HPLC (AKTA Purifier,
C ₁₈ reverse phase column (Resource R) scanned on Pharmacia)
It can be separated from cytidine and SMCC by PC, Pharmacia).
Cytidine was eluted with 100% water and CrMCC and SMCC were eluted with water containing 15% acetonitrile at a flow rate of 1.5 ml / min. CrMC
The hydrophobicity of C was greater than that of cytidine, but less than that of SMCC. The formation of CrMCC reached near maximum after 48 hours of reaction between SMCC and cytidine at 22-24 ° C (Fig. 4). Computer program UNI
The area under the peak was calculated using CORN (version 2.0).

The HPLC-purified CrMCC was freeze-dried by freeze-drying (freeze-drying). The activity of CrMCC is as follows when it is stored at -20 ° C in a desiccator.
It was stable for at least 3 months. The molecular weight of CrMCC was measured by LC-MS (Wat
ers). In short, CrMCC is 20% at a flow rate of 3 ml / min.
_C18 reverse phase column (Resource RPC
, Pharmacia). The capillary voltage for micromass mass spectrometry was set to 3.0-3.5V and the cone voltage was set to 20V.
V2 gas flow was 700 L / hr and electrospray was negative. Mass spectra were collected under full scan operation (scanning range 1-1000 m / z). The molecular weight of CrMCC was determined by monitoring protonated molecular ions, and this was similar to the predicted value of 462.5. The CrMCCs synthesized were consistently larger than 95%.

The light absorption spectrum of CrMCC was measured by uv-vis spectrophotometer. FIG. 5A shows that there are two λ _max in the optical absorption spectrum of CrMCC. One of these is 254 nm (similar to λ _max for cytidine),
And the other is 300 nm (similar to λ _max of SMCC). The absorption profile of CrMCC with two λ _max is very close to that of NBMPR (FIG. 5B). This indicated that CrMCC consisted of both pyrimidine and cyclohexane in its chemical structure. Subsequent structural studies using NMR confirmed this finding.

Example 3 Effect of CrMCCs on ³ H-NBMPR Binding in Human HL-60 Promyelocytic Leukocyte Plasma Membrane To synthesize ³ H-CrMCC, a radioreactive cytidine ( ³ H -Cytidine)
Was used. Since high concentrations of substrate increase the yield of CrMCC, non-radioactive cytidine was pre-mixed with radioactive ³ H-cytidine in a 100: 1 concentration ratio prior to reaction with SMCC (see Example 2). That).

Purified HL-60 plasma membrane (0.13M NaCl, suspended in reaction solution)
0.02 M NaHCO ₃ , pH 7.0), cytidine, SMCC, and C
Prepare stepwise concentrations of rMCC for 5 minutes, then expose to ³ H-NBMPR (5 nM) for an additional 30 minutes. This reaction is referred to as a membrane filtration method (Lee and Jarvis.
(1988), Biochem. J. , 249, 557-564). The data shown in FIG. 6 are corrected for nonspecific binding determined in the presence of 20 μM NBTGR. FIG. 6 shows that the specificity of ³ H-NBMPR for HL-60 unsealed plasma membrane was inhibited by CrMCC with an IC ₅₀ value of 10 μM. In contrast, S in inhibition of ³ H-NBMPR binding
IC ₅₀ values for MCC and cytidine are 100 μM and> respectively.
It was 1 mM. The K _i value for CrMCC was calculated to be 1 μM.

To analyze the effect of CrMCCs on the binding kinetics of ³ H-NBMPR, purified HL-60 plasma membranes were used with 0, 10, and 50 μM CrMCCs.
Pretreatment for 30 minutes, followed by a further 30 minutes of graded concentrations of ³ H-NBMPR (0.2
~ 8 nM). The double reciprocal plot of this result is shown in FIG. The line of this plot was bisected on the horizontal axis. This indicates an altered B _max value, but an unchanged K _d value in the presence of CrMCC. For the data shown, the apparent K _d values for ³ H-NBMPR binding were 0.36 ± 0.04, 0.31.
± 0.03 and 0.39 ± 0.05 nM, 1.56 ± 0.05, 0.59 ± for membranes treated with 0, 10 and 50 μM CrMCC, respectively.
B _max values for 0.01 and 0.30 ± 0.01 pmol / mg protein. The data were corrected for non-specific binding determined in the presence of 20 μM nitrobenzylthioguanosine (NBTGR), a non-radioactive competing ligand. These results suggested a non-competitive ³ H-NBMPR binding by CrMCC. This is a characteristic feature of irreversible antagonism (antagonism).

Any clinically useful drug should have little or no cytotoxicity in its therapeutic dosage range. Exponentially growing HL-60 cells at a starting cell density of 5 × 10 ⁴ cells / ml in RPMI medium containing 10% FBS were added to graded concentrations of CrMCC, cytidine and SMCC (0-100 μM) at 3
Exposed for days. Electronic particle analyzer (electronic particle analyzer)
Cell densities were counted using a Le analyzer (Sysmex). Figure 8 shows that both CrMCCs and cimetidine were 100μ after 3 days of exposure.
A concentration as high as M has little effect on the growth of HL-60 cells,
Or indicate none at all. In contrast, SMCC (one of the parent compounds) is HL-
It was highly toxic to 60 cells with an IC ₅₀ value of less than 0.5 μM for cell proliferation. The toxicity of SMCC is due to non-specific interactions with any accessible sulfhydryl group of cells. Cimetidine is expected to have little or no inhibition on cell proliferation. Because this nucleoside is fairly "inert" and does not induce nucleotide imbalances in the concentration range tested.

Example 4 Binding of ³ H-CrMCC to Human HL60 Promyelocytic Leukocyte Plasma Membrane Due to availability of radioactive CrMCC ( ³ H-CrMCC), biochemistry of CrMCC to es nucleoside transporter. It becomes possible to study physical characteristics. This experiment was performed to examine the binding rate of ³ H-CrMCC to the unsealed plasma membrane of HL-60 cells. FIG. 9 shows that binding of ³ H-CrMCC (final concentration 30 μM) to purified HL-60 plasma membrane was significantly slowed. A minimum of 5 minutes is required to achieve a maximum binding value of 12 nmol / mg HL-60 plasma membrane protein. This differs from the binding of reversible antagonists such as NBMPR, dipyridamole and dilazep. The binding of these reversible antagonists was known to be rapid and almost complete within the first minute of incubation. The binding reaction of Figure 9 was stopped by the membrane filtration method and the data was corrected for the filter blank.

It is important to confirm that the binding of ³ H-CrMCC to the unsealed HL-60 plasma membrane is actually irreversible. Purified unsealed HL-60 plasma membrane was incubated with 30 μM ³ H-CrMCC for 10 minutes. Then, this mixture was diluted 20 times, and the diluted mixture was
Dissociation occurred at different time intervals at room temperature. FIG. 10 shows ³ H-CrMCC.
Shows little or no dissociation from the binding site of at least 60 minutes after 20-fold dilution. Even 1 mM cytidine present in the diluted medium failed to displace ³ H-CrMCC from its binding site. In contrast, HL-6
The binding of 30 μM ³ H-cimetidine to 0 plasma membrane was low, and upon dilution was rapidly and completely dissociated (insert in FIG. 10). Taken together with this finding, non-competitive inhibition of ³ H-NBMPR binding by CrMCC (FIG. 7) was obtained.
It has been suggested that the interaction of the with the binding site is indeed irreversible. Figure 1
The dissociation reaction shown at 0 was stopped by membrane filtration and the data was corrected for filter blanks.

To determine the concentration dependence of binding of ³ H-CrMCC to its binding site, purified unsealed HL-60 plasma membranes were spiked with graded concentrations of ³ H-CrMC.
The reaction was terminated by membrane filtration by incubation with C (3-800 μM) for 10 minutes (FIG. 11). This data was decomposed into at least two components, a high affinity component (Kd = 23.8 ± 2.2 μm) and a low affinity component (inserted in FIG. 11). The kinetic constants for low affinity components are ³ H-CrMC
It could not be evaluated in the existing concentration range of C. Low affinity components are non-specific ³ H-C
It is also possible that it is the rMCC binding site. The data shown in FIG. 11 was corrected for the filter blank.

It is important to determine the stability of the ³ H-CrMCC-receptor complex at different pH conditions. Purified unsealed HL-60 plasma membrane was
Incubated with μM ³ H-CrMCC for 5 minutes. The reaction mixture was then diluted 20-fold with reaction buffers of different pH values (pH 0.85-7.5). This dissociation reaction was stopped by the membrane filtration method. The data was corrected for filter blanks. FIG. 12 shows that the ³ H-CrMCC receptor complex has a physiological p
It is very stable at H. However, the ligand-receptor complex
． At a pH below 5, it started to disintegrate.

Earlier studies with the Sfluhydryl Reagent NEM indicated that cysteine residues were probably e
It was suggested to be placed very close, but not above, the nucleoside transport / binding site of the s nucleoside transporter protein. Therefore,
If ³ H-CrMCC binds to the same site that NEM does, then N
Substrates that cannot inhibit EM action (Lee et al. (1995) Biochim. Biop.
hys. Acta, Vol. 1268, pp. 200-208), as well as ³ H-CrM.
Has little or no effect on CC binding. FIG. 13 shows 1 mM physiological nucleosides (eg cytidine, uridine, adenosine and inosine) and 20 μM es nucleoside transport inhibitors (eg NBMPR).
And dipyridamole) are actually ³ H-CrMC on the HL-60 plasma membrane.
It shows that the binding of C (final concentration 30 μM) could not be inhibited. In contrast, 1m
M NEM inhibits binding of ³ H-CrMCC at 0.5 mM C
It was as effective as rMCC. This finding was found by NEM and CrMCC
It was suggested that it reacted with the same sulfhydryl group on the s nucleoside transporter protein.

Example 5 Effect of Other Sulfhydryl-Reactive Covalent Arms on the Binding of ³ H-Cytidine Molecules to Human HL-60 Promyelocytic Leukocyte Plasma Membrane Cytidine via a cyclohexanecarboxylic acid spacer arm. It has been successfully demonstrated that a maleimide (sulfhydryl group-reacting agent) bound to E. coli is effective in irreversibly inhibiting the binding of ³ H-NBMPR to the es transporter protein. In this experiment, the ability of the ³ H-cytidine molecule to bind to the HL-60 plasma membrane was tested for its irreversible es nucleoside transporter inhibitor (after binding to the same or different sulfhydryl-based reagents via different spacer arms). Comparisons are provided to provide an indirect indication of the suitability of the spacer arm used to make Therefore, ³ H-cimetidine was chemically modified to incorporate various sulfhydryl-reactive lateral arms at the 6-amino position of the pyrimidine ring. Compounds used to modify ³ H-cytidine include m-maleimidobenzoyl-N-hydroxysuccinimide (MBS), N-
Succinimidyl 4- [p-maleimidophenyl] butyrate (SMPB),
N- [γ-maleimidobutyryloxy] succinimide, 4-succinimidyloxycarbonyl-α-methyl-α- [2-pyridylthio] toluene (SMPT
), N-succinimidyl 3- [2-pyridyldithio] propionate (SP
DP), N-succinimidyl maleimide acetate (AMAS), and N-
[Ε-maleimidocaproyloxy] succinimide (EMCS), and N
-Succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC). Purified HL-60 plasma membrane was loaded with 10
Incubated with 0 μM of these chemically modified ³ H-cytidine analogs for 5 minutes. The reaction was stopped by the membrane filtration method. FIG. 14 shows N-α-
The maleimidoacetoxylic acid (AMA) covalent arm has a HL-60 plasma membrane,
It is subsequently shown to promote the highest irreversible binding of ³ H-cytidine to 3- [2-pyridylthio] propionic acid (PDP). However, N-ε-maleimidocaproic acid (EMC), m-maleimidobenzoic acid (MB), N-γ-maleimidobutyric acid (GMB) and 4- [p-maleimidophenyl] butyric acid (MPB) are -Compared to maleimidomethylcyclohexanecarboxylic acid (MCC), it was as effective as the covalent arm. In contrast, α-methyl-α- [2-pyridyldithio] toluenecarboxylic acid (MPT) was the least effective. The symbol X in FIG. 14 represents cytidine.

(Example 6) (Identification of ³ H-CrMCC binding protein in human HL-60 promyelocytic leukemia cell membrane) An attempt was made to identify a cell membrane protein labeled with ³ H-CrMCC. ³ H-CrMCC (20 μM) -labeled HL-60 cell membrane protein dissolved in SDS was applied to a C ₁₈ reverse phase column (Resource RPC, Pharmacia).
) And FPLC (ATKA FPLC, Pharmacia) under the following conditions:
): Column volume (CV, 3 ml), starting buffer A (0.05%
Water containing TFA), eluent B (acetonitrile containing 0.065% TFA),
Flow rate (2 ml / min), detection (280 nm), elution (3 CV with 0% B, 0-5)
1 CV containing% B, 5 CV containing 5% B, 15 CV containing 5 to 100% B, 10
Rinse with 0%). FIG. 15 shows a typical UV (λ = 280 nm) absorbance profile of HL-60 cell membranes after separation by FPLC using a C ₁₈ reverse phase column. The arrow in the chromatogram indicates the peak of the protein labeled with ³ H-CrMCC (see FIG. 16). The eluate was collected at 1 ml / min and the radioactivity was determined by liquid scintillation counter. Figure 1
6 is the chromatogram fraction numbers 43 to 44, 49 to 50 and 5
It is shown that there are at least three major radioactivity peaks at 7-59. The sharp radioactivity peak at the fraction number 6 is the decomposition product of ³ H-CrMCC (
That ^is, due to non-specific binding of ³ H-CrMCC to 3 H- cytidine) and membrane lipids.

Example 7 Development of Irreversible Binding Drugs of Adenosine Origin In light of the successful production of an irreversible inhibitor of the es transporter protein using cytidine as a lead compound, the present invention is characterized by the addition of covalent arms. Can be reproduced by attaching to a physiological nucleoside (eg, adenosine). Based on the general procedure shown in Figure 3 and using the chemistries listed in Example 5, < ³ > H-adenosine was chemically modified to provide various sulfhydryl reactivity at the 6-amino position of the purine ring. Incorporated covalently bound arms. HL-60 cell membranes were incubated for 5 minutes with 100 μM of these chemically modified ³ H-adenosine analogs. The reaction was stopped by the membrane filtration method. Figure 1
7 is 4- [p-maleimidophenyl] butyric acid (MPB) (4- [p-malei
It is shown that the midophenyl] butyryl acid) covalent arm (followed by N-α-maleimidoacetic acid (AMA)) most strongly promotes irreversible binding of ³ H-adenosine (100 μM) to HL-60 cell membranes. However, 3- [2-pyridyldithio] -propionic acid (PDP), N- [epsilon] -maleimidocaproic acid (EMC) (N- [epsilon] -maleimidecaproiclica)
cid), m-maleimidobenzoic acid (MB), and N-maleimidomethylcyclohexanecarboxylic acid (MCC) were equally less effective as covalent arms. In contrast, N-γ-maleimidobutyric acid (GMB) and α-methyl-
α- [2-Pyridyldithio] toluene carbonic acid (MPT) was the least effective.

(Example 8) (Inhibitory effect of irreversible reaction of 5-HT analog on binding of ³ H-5-HT in mouse brain membrane) (A. Synthesis of compound) Figures 19a, 20a, 21a and 22a. The indicated compound (LBT3001 (
1- [2- (5-hydroxy-1H-indol-3-yl] -ethyl) -pyrrole-2,5-dione) and LBT3002 (4- (2,5-dioxo-2,5-dihydro-, respectively). Pyrrol-1-yl) -N- [2- (5-hydroxy-1)
H-indol-3-yl) -ethyl] -butyramide
), LBT3004 (3- (2,5-dioxo-2,5-dihydro-pyrrole-
1-yl) -N- [2- (5-hydroxy-1H-indol-3-yl) -ethyl] -propionamide), and LBT3005 (4- (2,5-dioxo-2,5-dihydro-pyro) -L-1-ylmethyl) -cyclohexanecarboxylic acid [2- (5-hydroxy-1H-indol-3-yl) -ethyl] -amide)
The chemical structure of) demonstrates its effectiveness as an irreversible binding compound and provides examples of modified ligands that are modified 5-HT receptor binding ligands containing a binding agent.

The reaction scheme for the synthesis of LBT3001 (FIG. 19b) is as follows: 5-
Hydroxytryptamine (212.7 mg, 10 mmol) was dissolved in saturated sodium bicarbonate solution (25 ml) at 0 ° C. N-Ethoxycarbonyl imide (177.6 mg, 1.
05 mmol) was added with stirring. Thirty minutes later, the ice water tank was changed to a warm water tank (30 ° C ~
40 ° C.). The reaction solution was then stirred in a warm water bath for about 1 hour. The solution was then extracted with ethyl acetate (3 x 50 ml). The ethyl acetate layer was then washed with deionized water (2 x 20 ml) to bring the pH to near neutral and then with brine (20 ml). The organic layer was then dried with anhydrous MgSO ₄ . The solvent was removed under reduced pressure to give a crude product as a yellow oil. Purification via flash chromatography (30% ethyl acetate in hexanes) gave a yellow to orange crystalline product (187.2 mg, 73%).

The reaction scheme for the synthesis of LBT3002 (FIG. 20b) is as follows: 5-Hydroxytryptamine hydrochloride (42.5 mg, 0.2
mmol) and 3-maleimidobutyric acid (40.3 mg, 0.22 mmol),
Suspended in 1 ml 2-methoxyethyl ether (DME). In this solution, N
-Methylmorpholine (NMM, 25 μl, 0.22 mmol) and 1,3-dicyclohexylcarbodiimide (DCC, 45.4 mg, 0.22 mmol) were added. The solution was stirred at room temperature for 3 hours. The product was added to the gradient solvent eluent (0
Purify directly by flash chromatography using methylene chloride with 3% methanol to give a brown oil (40.7 mg, 60%).

The reaction scheme for the synthesis of LBT3004 (FIG. 21b) is as follows: 5-
Hydroxytryptamine hydrochloride (42.5 mg, 0.2 mmol) and 3-maleimidopropionic acid (37.2 mg, 0.22 mmol) were added to 1 m.
Suspended in 1 of 2-methoxyethyl ether (DME). To this solution was added N-methylmorpholine (NMM, 25 μl, 0.22 mmol) and 1,3-dicyclohexylcarbodiimide (DCC, 45.4 mg, 0.22 mmol). The solution was stirred at room temperature for 3 hours. The product was eluted with a gradient solvent (0% to 1
． Direct purification by flash chromatography using 5% to 3% methanol in methylene chloride) gave an orange oil (46.0 mg, 70%).

The reaction scheme for the synthesis of LBT3005 (FIG. 22b) is as follows: 4-aminomethyl-cyclohexanecarboxylic acid ((ml) saturated NaHCO ₃ (80 ml).
(2.83 g, 18.0 mmol) was vigorously stirred at 0 ° C. In this solution, N
-Ethoxycarbonyl imide (finely ground, 3.05 g, 18.0 mmol)
Was added in small portions. Once the addition is complete (about 10 minutes), deionized water (80 ml)
) Was added and the mixture was stirred at room temperature for 40 minutes. The resulting mixture is 1
The pH was adjusted to 1-2 with M HCl and extracted with ethyl acetate (3 × 80 ml). The organic layer was washed with deionized water (50 ml), then brine (20 ml).
ml). The organic phase was dried with magnesium sulphate and evaporated under reduced pressure to give the crude product. Purification by flash chromatography (hexane: ethyl acetate: acetic acid = 60: 39: 1) gave the product in the solid state (2.69 g, 63%). 5-hydroxytryptamine hydrochloride (
42.5 mg, 0.2 mmol) and maleimidomethyl-cyclohexanecarboxylic acid (52.2 mg, 0.22 mmol) were suspended in 1 ml methoxyethyl ether (DME). To this solution, N-methylmorpholine (NMM, 25
μl, 0.22 mmol) and 1,3-dicyclohexylcarbodiimide (D
CC, 45.4 mg, 0.22 mmol) was added. The solution was stirred at room temperature for 3 hours. The product was directly purified by flash chromatography using a gradient solvent eluent (ethyl acetate: hexane (1: 1) to ethyl acetate: hexane (3: 1)) to give a light brown oil (28. 3 mg, 35.8%).

Other 5-HT receptor binding compounds as shown in FIG.
One of the reaction schemes shown in 2b can be used to similarly modify for irreversible binding to their target sites.

B. Assay FIG. 18 shows the inhibitory effect of various 5-HT analogs on the high affinity binding of ³ H-5-HT to mouse brain membranes. Reaction buffer (0.13M NaCl, 0.
Purified mouse brain membranes suspended in 02M NaHCO ₃ , pH 7.0 were added to graded concentrations of LBT3001 (■), LBT3002 (□), LBT3004 (○).
), And LBT3005 (●) for 5 minutes, and then ³ H-5-HT (
It was exposed to a final concentration of 5 nM) for another 30 minutes. The reaction was stopped by the membrane vacuum filtration method. The data shown were collected for non-specific binding determined in the presence of 1 mM non-radioactive 5-HT. The results were plotted against control binding determined in the absence of inhibitor.

FIG. 18 shows that binding of ³ H-5-HT to mouse brain membranes is inhibited by all analogs of 5-HT in an apparently biphasic manner.

LBT3001 (1- [2- (5-hydroxy-1H-indol-3-yl] -ethyl) -pyrrole-2,5, an analog that does not contain a spacer arm between 5-HT and the maleimide molecule. -Dione) (FIG. 19a) effectively binds ³ H-5-HT to its high and low affinity binding sites (IC ₅₀ values of about 0.001 μM and about 50 μM, respectively). Inhibited. LBT3002 (FIG. 20a), an analog containing three carbon molecules in the spacer arm, has high and low affinity binding sites (IC ₅₀ values of approximately 0.000 each).
³ H-5-HT binding to ³ μM and about 200 μM). An analog containing two carbon molecules in the spacer arm, LBT3004 (FIG. 21a), has high and low affinity binding sites (IC ₅₀ values of about 0 for each).
． ³ H-5-HT binding to 2 μM and approximately 50 μM) was inhibited. LBT3005 which is an analog containing cyclohexane carboxyl molecule in the spacer arm
(FIG. 22a) inhibited ³ H-5-HT binding to its high and low affinity binding sites (IC ₅₀ values of about 0.5 μM and about 300 μM, respectively). These results clearly show that irreversible binding drugs can be designed using the described techniques. Moreover, this technique is applicable to many small molecules and can be applied to further improve the effects of existing molecules.

The entire disclosures of all publications, patents and patent applications referred to herein are incorporated by reference.

Throughout this specification the aim is to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or collection of specific features.
Those skilled in the art will immediately appreciate that various modifications and changes can be made in the particular embodiments illustrated without departing from the scope of the present invention in view of the present disclosure. All modifications and variations are intended to be included within the scope of the appended claims.

The invention is illustrated by the preferred embodiments and is described with reference to the following figures.

[Brief description of drawings]

FIG. 1 shows NEM inhibition of balanced ³ H-uridine transport in mouse myeloma SP2 / 0-Ag14 cells.

FIG. 2 ³ H-NBMP binding in mouse myeloma SP2 / 0-Ag14 cells.
Figure 3 shows the effect of NEM on R equilibrium kinetics.

[Figure 3]   Figure 3 shows a general reaction scheme for the synthesis of CrMCC.

[Figure 4]   FIG. 4 shows the rate of CrMCC formation.

[Figure 5]   FIG. 5 shows the optical absorption profile of CrMCC and NBMPR.

FIG. 6 shows inhibition of ³ H-NBMPR binding by CrMCC, cytidine, and SMCC in human HL-60 promyelocytic leukemia plasma membrane.

FIG. 7: ³ H-NBMP binding to human HL-60 promyelocytic leukemia plasma membrane.
3 shows the effect of CrMCC on R kinetics.

FIG. 8: CrMCC, Cytidine and SMCC on HL-60 cell proliferation.
Shows the effect of.

FIG. 9: ³ H-CrMCC against human HL-60 promyelocytic leukemia plasma membrane.
Indicates the time course of the join.

FIG. 10 shows ³ H- from the binding site of human HL-60 promyelocytic leukemia plasma membrane.
³ shows the dissociation of CrMCC and ³ H-cytidine.

FIG. 11: ³ H-CrMC against human HL-60 promyelocytic leukemia plasma membrane.
The concentration dependence of C binding is shown.

FIG. 12 shows the effect of pH on the dissociation of ³ H-CrMCC from its binding site on the human HL-60 promyelocytic leukemia plasma membrane.

FIG. 13 shows inhibition of ³ H-CrMCC binding to human HL-60 promyelocytic leukemia plasma membrane.

FIG. 14 shows covalent binding of sulfhydryl-reactive ³ H-cytidine analogs to human HL-60 promyelocytic leukemia plasma membrane.

FIG. 15 shows the UV absorbance profile of reverse phase chromatography of human HL-60 promyelocytic leukemia plasma membrane protein.

FIG. 16 shows the reverse phase chromatographic radioactivity profile of human HL-60 promyelocytic leukemia plasma membrane protein.

FIG. 17 shows covalent binding of sulfhydryl-reactive ³ H-adenosine analogs to human HL-60 promyelocytic leukemia plasma membrane.

FIG. 18 shows inhibition of ³ H-5-HT binding to mouse brain membranes by various 5-HT analogs.

FIG. 19a shows the chemical structure of LBT3001 (1- [2- (5-hydroxy-1H-indol-3-yl) -ethyl] -pyrrole-2,5-dione).

FIG. 19b   Figure 19b shows a reaction scheme for the synthesis of LBT3001.

FIG. 20a shows LBT3002 (4- (2,5-dioxo-2,5-dihydro-
Pyrrol-1-yl) -N- [2- (5-hydroxyl-1H-indole-3
-Yl) -ethyl] -butyramide).

FIG. 20b   Figure 20b shows a reaction scheme for the synthesis of LBT3002.

FIG. 21a shows LBT3004 (3- (2,5-dioxo-2,5-dihydro-
Pyrrol-1-yl) -N- [2- (5-hydroxyl-1H-indole-3
-Yl) -ethyl] -proprionamide).

FIG. 21b   Figure 21b shows a reaction scheme for the synthesis of LBT3004.

FIG. 22a shows LBT3005 (4- (2,5-dioxo-2,5-dihydro-
1 shows the chemical structure of pyrrol-1-yl-methyl) -cyclohexanecarboxylic acid [2- (5-hydroxy-1H-indol-3-yl) -ethyl] -amide).

FIG. 22b   Figure 22b shows a reaction scheme for the synthesis of LBT3005.

FIG. 23   FIG. 23 shows the structures of exemplary ligands that can be modified to include a binder.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 1/08 A61P 1/08 3/00 3/00 3/04 3/04 9/12 9/12 15 / 08 15/08 25/00 25/00 25/04 25/04 25/06 25/06 25/18 25/18 25/22 25/22 25/24 25/24 25/28 25/28 35/00 35/00 35/02 35/02 43/00 105 43/00 105 111 111 111 114 114 C07D 403/12 C07D 403/12 C07H 19/067 C07H 19/067 19/167 19/167 G01N 33/50 G01N 33 / 50 Z 33/566 33/566 // G01N 33/15 33/15 Z (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE, TR), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, E, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD , RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK , DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL , TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW Term (Reference) 2G045 BB14 BB50 CB01 FA29 FB02 FB06 FB07 FB08 JA01 JA02 4C057 BB02 DD01 LL17 LL18 LL31 LL41 4C063 AA01 BB03 BB09 CC06 DD04 EE01 4C086 AA01 AA02 AA03 AA04 BC13 EA17 EA18 GA07 MA01 MA04 NA05 NA13 NA14 ZA01 ZA08 ZA12 ZA16 ZA18 ZA42 ZA66 ZA70 ZA71 ZA81 ZB21 ZB26 ZB27 ZC35 ZC42

Claims (70)

[Claims] 1. A process for modifying a parent ligand, the process comprising the step of attaching a binding agent to the parent ligand that is reactive with a portion of a target receptor, wherein the receptor is bound to the parent ligand. Bind, so that a covalent bond can be formed between the binding agent and the moiety. 2. The process of claim 1, wherein the binding agent is attached to the parent ligand via a spacer. 3. The spacer is covalently attached to the parent ligand.
The process of claim 2. 4. The process of claim 2, wherein the spacer is covalently attached to the binding agent. 5. The process of claim 2, wherein the spacer comprises a group selected from the group consisting of: alkyl group, heteroalkyl group, cycloalkyl group, heterocycloalkyl group, Oxoalkyl group, heterooxoalkyl group, alkenyl group, heteroalkenyl group, aralkyl group, heteroaralkyl group, aryl group, and heteroaryl group. 6. The binding agent is a sulfhydryl group-specific binding agent.
The process of claim 4. 7. The process of claim 4, wherein the binder is selected from the group consisting of a binder specific for amino groups and a binder specific for carboxyl groups. 8. The process of claim 1, wherein the binding agent is selected from the group consisting of: process: sulfhydryl group specific binding agent, amino group specific binding agent, Carboxyl group specific binding agent, tyrosine specific binding agent, arginine specific binding agent, histidine specific binding agent, methionine specific binding agent, tryptophan specific binding agent, and A binding agent specific for serine. 9. The process of claim 8, wherein the sulfhydryl group-specific binder is selected from the group consisting of N-maleimide and N-maleimide derivatives. 10. The process according to claim 9, wherein the N-maleimide derivative is 5′-dithiobis- (2-nitrobenzoic acid), 4,4′-dithiodipyridine, methyl-3-nitro. -2-pyridyl disulfide, and methyl-2-
A process selected from the group consisting of pyridyl disulfides. 11. The process of claim 8, wherein the amino group-specific binder is selected from the group consisting of alkylating agents and acylating agents. 12. The process according to claim 11, wherein the alkylating agent is selected from the group consisting of α-haloacetyl compounds, aryl halides, aldehydes, and ketones. 13. The process according to claim 11, wherein the acylating agent is selected from the group consisting of: isocyanate, isothiocyanate, imide ester, N-hydroxylsuccinimidyl ester, p. -Nitrophenyl ester, acyl chloride, and sulfonyl chloride. 14. The process of claim 8, wherein the carboxyl group-specific binder is selected from the group consisting of carbodiimides and carboxyl group esterifying agents. 15. The process according to claim 14, wherein the carboxyl group esterifying agent is selected from the group consisting of diazoacetate esters and diazoacetamide. 16. The process of claim 8 wherein the tyrosine specific binder is a diazonium derivative. 17. The process of claim 16, wherein the diazonium derivative is selected from the group consisting of benzidine and bis-diazotized 3,3′-dimethylbenzidine. 18. The process of claim 8 wherein the arginine specific binding agent is selected from 1,2-dicarbonyl agents. 19. The process according to claim 18, wherein the 1,2-dicarbonyl agent is selected from the group consisting of glyoxal, phenylglyoxal, 2-3-butanedione, and 1,2-cyclohexanedione. Process. 20. The process of claim 8, wherein the histidine-specific binder is selected from the group consisting of alkylating agents and acylating agents. 21. The process of claim 20, wherein the alkylating agent is selected from the group consisting of α-haloacetyl compounds, aryl halides, aldehydes, and ketones. 22. The process according to claim 20, wherein said acylating agent is selected from the group consisting of: diethylpyrocarbonate, ethoxyformaldehyde, isocyanate, isothiocyanate, imide ester, N. -Hydroxyl succinimidyl ester, p-nitrophenyl ester,
Acyl chloride, and sulfonyl chloride. 23. The process of claim 8, wherein the methionine-specific binding agent is an alkylating agent. 24. The process according to claim 23, wherein the alkylating agent is selected from the group consisting of α-haloacetyl compounds, aryl halides, aldehydes, and ketones. 25. The process of claim 8, wherein the tryptophan specific binder comprises N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide and p-nitrophenylsulfenyl chloride. A process selected from the group. 26. The process according to claim 8, wherein the serine-specific binder is selected from the group consisting of diisopropyl fluorophosphate and acrylsulfonyl fluoride. 27. The process of claim 26, wherein the serine-specific binding agent is phenylmethyl-sulfonyl fluoride. 28. A modified ligand produced by the process of claim 1. 29. A modified ligand having the general formula: LR ₁ -A (I), wherein L is a parent ligand; A is reactive with a portion of the target receptor. A binding agent capable of binding the parent ligand to the receptor such that a covalent bond is formed between the binding agent and the moiety; and R ₁ is any spacer , Modified ligands. 30. The modified ligand according to claim 29, wherein the spacer comprises a perhydrolytic group selected from the group consisting of: an alkyl group, a heteroalkyl group, a cycloalkyl group, a heterocyclo. Alkyl group, oxoalkyl group,
Heterooxoalkyl group, alkenyl group, heteroalkenyl group, aralkyl group,
Heteroaralkyl, aryl, and heteroaryl groups. 31. A composition comprising the modified ligand of claim 28 or claim 29 in combination with a pharmaceutically acceptable carrier. 32. A method of treating or preventing a condition associated with a target receptor, the method comprising the modified ligand of claim 28 or claim 29 in combination with a pharmaceutically acceptable carrier. Administering a therapeutically effective dose of a product to a patient in need of such treatment. 33. A method of detecting the presence of a target receptor in a test sample, the method comprising the step of: contacting the sample with a modified ligand according to claim 28 or claim 29, wherein Binding the target receptor when the modified ligand is present in the test sample; and the presence of a complex comprising the modified ligand and the receptor in the contacted sample. A step of detecting. 34. A method of quantifying the presence of a target receptor in a test sample, the method comprising: contacting the sample with a modified ligand according to claim 28 or claim 29, wherein: Binding the target receptor if the modified ligand is present in the test sample; measuring the concentration of the complex comprising the modified ligand and the receptor in the contacted sample And; relating the concentration of the measured complex to the concentration of the receptor in the sample. 35. A method of detecting the presence of a target receptor on a cell or cell membrane, the method comprising the step of: contacting a modified ligand according to claim 28 or claim 29 with a sample containing the cell or cell membrane. Wherein the modified ligand binds to the target receptor when present in the cell or cell membrane;
And detecting the presence of the modified ligand and the complex comprising the cell or cell membrane in the contacted sample. 36. A method for quantifying the presence of a target receptor on a cell or cell membrane, comprising the step of: contacting the modified ligand of claim 18 or 19 with a sample containing the cell or cell membrane. Wherein the modified ligand binds to the target receptor if the modified ligand is present on the cell or cell membrane; the modified ligand in the contacted sample, and the cell or Measuring the concentration of a complex comprising a cell membrane; and correlating the measured concentration of the complex with the concentration of the receptor present on the cell or cell membrane. 37. A probe covalently bound to a target receptor, said probe comprising a modified ligand according to claim 28 or claim 29, said ligand having a reporter molecule associated with said ligand. ,probe. 38. The method of claim 3 comprising the modified ligand of claim 30.
7. The probe according to 7. 39. Use of a modified ligand according to claim 28 or claim 29 or a probe according to claim 37 in the study, treatment and prevention of conditions associated with target receptors. 40. Use of a modified ligand according to claim 28 or claim 29 or a probe according to claim 37 in the study of target receptor function. 41. A process for modifying a parent ligand, the process comprising: binding a binding agent to the parent ligand, the binding agent being reactive with a portion of a target receptor to which the parent ligand binds, Wherein when the parent ligand binds to the receptor, a covalent bond is formed between the binding agent and the moiety, and the parent ligand specifically binds to the nucleotide transporter. 42. The process of claim 41, wherein the binding agent binds to the parent ligand via a spacer. 43. The process according to claim 42, wherein the spacer is selected from the group consisting of: an alkyl group, a heteroalkyl group,
A cycloalkyl group, a heterocycloalkyl group, an oxoalkyl group, a heterooxoalkyl group, an alkenyl group, a heteroalkenyl group, an aralkyl group, a heteroaralkyl group, an aryl group, and a heteroallyl group. 44. The process of claim 41, wherein the binding agent is selected from the group consisting of: a binding agent specific for sulfhydryl groups,
Amino group specific binding agent, carboxyl group specific binding agent, tyrosine specific binding agent, arginine specific binding agent, histidine specific binding agent, methionine specific binding agent, Tryptophan specific binding agents, and serine specific binding agents. 45. The process of claim 41, wherein the binder is N
A process, which is a binder specific for a sulfhydryl group selected from the group consisting of maleimide and N-maleimide derivatives. 46. The process according to claim 45, wherein the N-maleimide derivative is 5′-dithiobis- (2-nitrobenzoic acid), 4,4′-dithiodipyridine, methyl-3-nitro. -2-pyridyl disulfide, and methyl-2
A process selected from the group consisting of pyridyl disulfide. 47. The process of claim 41, wherein the binder is selected from the group consisting of alkylating agents and acylating agents. 48. A process for modifying a parent ligand, the process comprising attaching to the parent ligand a binding agent specific for a sulfhydryl group that is reactive with a sulfhydryl group of a target receptor to which the parent ligand binds. Coupling, wherein when the parent ligand binds to the receptor, a covalent bond is formed between the binding agent and the sulfhydryl group, and the parent ligand specifically binds to the serotonin receptor. To process 49. A modified ligand produced by the process of claim 41. 50. A modified ligand produced by the process of claim 48. 51. A modified ligand having the general formula: L—R ₁ -A (I), wherein L is a parent ligand that specifically binds to a target receptor containing a nucleotide transporter. A is a binding agent that is reactive with a portion of a target receptor, wherein the parent ligand binds to the receptor, such that when the parent ligand binds to the receptor, the binding agent and the portion A modified ligand, wherein the covalent bond is formed between the binder; and R ₁ is an optional spacer. 52. The modified ligand according to claim 51, wherein the spacer comprises a non-perhydrolytic group selected from the group consisting of: an alkyl group, a heteroalkyl group, a cycloalkyl group, a hetero group. A cycloalkyl group, an oxoalkyl group, a heterooxoalkyl group, an alkenyl group, a heteroalkenyl group, an aralkyl group, a heteroaralkyl group, an aryl group, and a heteroallyl group. 53. The binder is a sulfhydryl group-specific binder.
52. The modified ligand of claim 51. 54. The modified ligand of claim 52, wherein the sulfhydryl group-specific binder is selected from the group consisting of N-maleimide and N-maleimide derivatives. . 55. The modified ligand of claim 51, wherein the parent ligand specifically binds a nucleotide transporter. 56. The modified ligand of claim 51, wherein the modified ligand is reactive with nucleotide transporters and / or nucleoside / nucleotide / nucleobase sensitive proteins, wherein The modified ligand according to claim 1, wherein the modified ligand has a general formula selected from the group consisting of: Where A is N-maleimide, 2-pyridyldithio, or halogen; X is NH, S or O; Y is H, halogen, NH ₂ , or O; Z is H , Halogen, or CH ₃ ; R ₁ is a spacer arm, which optionally comprises a non-perhydrolytic group, preferably under physiological conditions; and R ₂ is H, β-D-ribose, β-D-deoxyribose, or their 5'-mono-phosphates, 5'di-phosphates, and 5'tri-phosphates. 57. A modified ligand according to claim 51, wherein the modified ligand has the general formula selected from the group consisting of: Here, R ₄ is 4- [N-methyl] cyclohexanecarboxylate, N-
[M-benzoate], 4- [p-phenyl] butyrate, N- [γ-butyrate], N- [α-acetate], or N- [ε-caproylate]; Here, R ₄ is 4- [N-methyl] cyclohexanecarboxylate, N-
[M-benzoate], 4- [p-phenyl] butyrate, N- [γ-butyrate], N- [α-acetate], or N- [ε-caproylate]; Here, R ₅ is 4-carbonyl-α-methyl-α-toluene, 6- [α-methyl-α-turamide] -hexanoate, N- [3-propionate], or 6- [3′-propioamide]. Hexanoate; and Here, R ₅ is 4-carbonyl-α-methyl-α-toluene, 6- [α-methyl-α-turamide] -hexanoate, N- [3-propionate], or 6- [3′-propioamide]. Hexanoate. 58. general formula: A modified ligand having a _{L-R 1 -A (I)} , where, L is located in the parent ligand that specifically binds to a target serotonin receptors; A is A binding agent that is reactive with a sulfhydryl group of a target receptor, wherein the target ligand is bound by the parent ligand, such that the parent ligand binds to the receptor, and the parent ligand binds to the receptor A modified ligand, wherein a covalent bond is formed between the binder and the sulfhydryl group; and R ₁ is an optional spacer. 59. The modified ligand of claim 58, wherein the parent ligand comprises serotonin or precursors or analogs thereof. 60. The modified ligand of claim 58, wherein the modified ligand has a structure selected from the group consisting of: LBT3001 (1- [2- (5-hydroxy-1H-indol-3-yl]
-Ethyl) -pyrrole-2,5-dione) LBT3002 (4- (2,5-dioxo-2,5-dihydro-pyrrol-1-
Yl) -N- [2- (5-Hydroxy-1H-indol-3-yl) -ethyl] -butyramide) LBT3004 (3- (2,5-dioxo-2,5-dihydro-pyrrol-1-
Yl) -N- [2- (5-Hydroxy-1H-indol-3-yl) -ethyl] -propionamide); and LBT3005 (4- (2,5-dioxo-2,5-dihydro-pyrrol-1-
Ylmethyl) -cyclohexanecarboxylic acid [2- (5-hydroxy-1H-indol-3-yl) -ethyl] -amide). 61. A composition comprising the modified ligand of claim 51 and a pharmaceutically acceptable carrier. 62. A composition comprising the modified ligand of claim 58 and a pharmaceutically acceptable carrier. 63. A method of detecting the presence of a target receptor in a test sample, comprising the step of: contacting the sample with a modified ligand of claim 51, wherein the modified A said ligand is present in said test sample, said binding to said target receptor; and detecting the presence of a complex comprising said modified ligand and said receptor in said contacted sample. Including the method. 64. A method of quantifying the presence of a target receptor in a test sample, the method comprising: contacting the sample with a modified ligand according to claim 51, wherein the modified ligand is A ligand is present in the test sample, which binds to the target receptor; measuring the concentration of the complex comprising the modified ligand and the receptor in the contacted sample; and Correlating the concentration of the measured complex with the concentration of the receptor in the sample. 65. A method of detecting the presence of a target receptor in a test sample, the method comprising: contacting the sample with a modified ligand of claim 58, wherein the modified A said ligand is present in said test sample, said binding to said target receptor; and detecting the presence of a complex comprising said modified ligand and said receptor in said contacted sample. Including the method. 66. A method of quantifying the presence of a target receptor in a test sample, the method comprising: contacting the sample with a modified ligand of claim 58, wherein the modified ligand is A ligand is present in the test sample, which binds to the target receptor; measuring the concentration of the complex comprising the modified ligand and the receptor in the contacted sample; and Correlating the concentration of the measured complex with the concentration of the receptor in the sample. 67. A probe that covalently binds to a target receptor, said probe comprising the modified ligand of claim 51, said ligand having a reporter molecule associated with said ligand. 68. Use of a modified ligand according to claim 51 or a probe according to claim 67 in the study of target receptor function. 69. A probe that covalently binds to a target receptor, said probe comprising the modified ligand of claim 58, said ligand having a reporter molecule associated with said ligand. 70. Use of the modified ligand according to claim 58 or the probe according to claim 69 in the study of target receptor function.