JP2006503853A - Cystic fibrosis membrane conductance regulator protein inhibitor and method of use thereof - Google Patents

Abstract

The present invention provides compositions, pharmaceutical preparations and methods for the inhibition of cystic fibrosis membrane conductance regulator protein (CFTR) that are useful in the study and treatment of CFTR-mediated diseases and conditions. The compositions and pharmaceutical preparations of the present invention may comprise one or more thiazolidinone compounds and may further comprise one or more pharmaceutically acceptable carriers, excipients and / or adjuvants. The methods of the invention, in certain embodiments, comprise administering an effective amount of a thiazolidinone compound to a patient suffering from a CFTR-mediated disease or condition. In another aspect, the present invention provides a method of inhibiting CFTR, comprising contacting a subject's cells with an effective amount of a thiazolidinone compound. Furthermore, the invention features a non-human animal model of CFTR-mediated disease caused by administering a thiazolidinone compound to a non-human animal in an amount sufficient to inhibit CFTR.

Description

BACKGROUND OF THE INVENTION Cystic fibrosis transmembrane conductance regulator (CFTR) protein is a cAMP-activated chloride (Cl ⁻ ) channel expressed in mammalian airway, intestine, pancreas and testicular epithelial cells. CFTR is cAMP- mediated Cl ^- is a chloride channel that participates in the secretion. Hormones, cAMP is increased for example β- adrenergic agonist, or toxins such as cholera toxin, cAMP-dependent protein kinase is activated, CFTR Cl ^- channel is phosphorylated, thereby the channel is opened. As cellular Ca ²⁺ increases, different apical membrane channels are also activated. Phosphorylation by protein kinase C opens or closes the apical membrane Cl ^- channel. CFTR is located primarily in the epithelium where it provides a pathway for the movement of Cl ^- ions across the apical membrane and provides key points to regulate transdermal salt and water transport rates. CFTR chloride channel function is associated with a wide range of diseases, such as cystic fibrosis (CF), and some forms of male infertility, polycystic kidney disease and secretory diarrhea.

  Cystic fibrosis (CF), a hereditary fatal disease, is caused by mutations in CFTR. Observations of human cystic fibrosis (CF) patients and CF mouse models show the importance of intestinal and pancreatic fluid transport and the function of CFTR in male infertility (Grubb et al., 1999, Physiol. Rev. 79: S193 -S214; Wong, PY, 1997, Mol. Hum. Reprod. 4: 107-110). However, the mechanism by which defective CFTR causes airway disease remains unclear and is the main cause of CF morbidity and mortality (Pilewski et al., 1999, Physiol. Rev. 79: S215-S255). The main issues in understanding CF airway disease are insufficient CF mouse models (mouse models show little or no airway disease), lack of large animal models of CF, and chronic infection and inflammation Limiting the availability of uninjured human CF airways. High affinity CFTR-selective inhibitors were not available to study airway disease mechanisms in CF or to create CF phenotypes in large animal models.

High affinity CFTR inhibitors also have clinical uses in the treatment of secretory diarrhea and cystic kidney disease and in the inhibition of male infertility. The compounds diphenylamine-2-carboxylate (DPC) and 5-nitro-2- (3-phenylpropyl-amino) -benzoate (NPPB) inhibit CFTR at high concentrations, but the inhibitory effect is nonspecific ( Cabantchik et al., 1992, Am. J. Physiol. 262: C803-C827; McDonough et al., 1994, Neuron 13: 623-634; Schultz et al., 1999, Physiol. Rev. 79: S109-S144) . The best CFTR inhibitor available for electrophysiological and other cell-based studies, glibenclamide, is used at concentrations> 100 μM (Sheppard et al., 1992, J. Gen. Physiol. 100: 573 -Hongre et al., 1994, Pflugers Arch. 426: 284-287). However, in this concentration, glibenclamide also other Cl ^- inhibiting transporter and K ⁺ channels (Edwards et al, 1993, Br J. Pharmacol 110:.... 1280-1281; Rabe et al, 1995, Pflugers Arch. 429: 659-662; Yamazaki et al., 1997, Circ. Res. 81: 101-109). Although effective small molecule inhibitors of other ion transport proteins are well known, small molecules with specific CFTR inhibitory capacity suitable for the treatment of secretory diseases are not available.

  Accordingly, there is a need for the development of CTFR inhibitor compounds and animal models useful for the study and treatment of CF and methods of using such compounds for the treatment and adjustment of secretory diseases. The present invention seeks to solve these and other needs and overcomes the deficiencies found in the prior art.

SUMMARY OF THE INVENTION The present invention provides compositions, pharmaceutical preparations and methods for the inhibition of cystic fibrosis membrane conductance regulator protein (CFTR) useful for the study and treatment of CFTR-mediated diseases and conditions. The compositions and pharmaceutical preparations of the present invention may comprise one or more thiazolidinone compounds or derivatives and may further comprise one or more pharmaceutically acceptable carriers, excipients and / or adjuvants. The methods of the invention, in certain embodiments, comprise administering an effective amount of a thiazolidinone compound or derivative to a patient suffering from a CFTR-mediated disease or condition. In another aspect, the present invention provides a method of inhibiting CFTR, comprising contacting a subject's cells with an effective amount of a thiazolidinone compound or derivative. Furthermore, the invention features a non-human animal model of CFTR-mediated disease caused by administering to a non-human animal an amount of a thiazolidinone compound or derivative sufficient to inhibit CFTR.

  These and other objects and advantages of the present invention will become apparent from the following detailed description.

  The present invention will be more fully understood with reference to the accompanying drawings. The drawings are for illustrative purposes only.

  Before describing the present invention, it is to be understood that the present invention is not limited to the particular embodiments described and, as such, may naturally vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, and that the scope of the present invention is limited only by the appended claims. It is.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although 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 are now described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials associated with the methods and / or materials to which those publications mentioned.

  As used in this specification and the appended claims, the singular forms “a (an)” and “the”, unless the context clearly dictates more than one Note that the subject is included. Thus, for example, “an inhibitor” includes a plurality of such inhibitors, and “the cell” includes one or more cells and equivalents well known to those skilled in the art. , Etc.

  The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application and are incorporated herein by reference. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such publications based on the previous invention. In addition, the publication date provided may be different from the actual publication date. The actual publication may need to be confirmed separately.

  The definitions used herein are provided for clarity and should not be considered limiting. Technical and scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which this specification belongs.

DETAILED DESCRIPTION OF THE INVENTION The present invention is based on the discovery of thiazolidinone compounds and derivatives that are high affinity CFTR inhibitors. The structures and formulations and methods of use of the compounds and derivatives of the present invention are described in more detail below.

Definitions “Cystic fibrosis transmembrane conductance regulator protein-mediated condition or indication” or “CFTR-mediated condition or indication” is the activity of cystic fibrosis transmembrane conductance regulator protein (CFTR), eg, the activity of CFTR in ion transport Any condition, disorder or disease caused by or a sign of such a condition, disorder or disease. Such a condition, disorder or disease, or a symptom of such a condition, disorder or disease can be treated by inhibition of CFTR activity, eg, inhibition of CFTR ion transport. CFTR activity is associated, for example, with intestinal secretion in response to various agonists such as cholera toxin (e.g., Snyder et al. 1982 Bull. World Heath Organ. 60: 605-613; Chao et al. 1994 EMBO J. 13: 1065-1072; see Kimberg et al. 1971 J. Clin. Invest. 50: 1218-1230).

  As used herein, a “CFTR inhibitor” is a compound that decreases the efficiency of ion transport by CFTR, particularly with respect to transport of chloride ions by CFTR. Preferably, the CFTR inhibitors of the present invention are specific CFTR inhibitors, i.e. CFTR activity without significantly affecting or adversely affecting the activity of other ion transporters such as other chlorine transporters, potassium transporters, etc. Is a compound that inhibits Preferably, the CFTR inhibitor is a high affinity CFTR inhibitor, for example, having a CFTR affinity of at least about 1 μM, usually about 1 to about 5 μM.

  As used herein, “treating, treatment” includes treatment of a disease, condition, disorder or indication in a subject, where the disease, condition, disorder or indication is mediated by the activity of CFTR. This treatment (1) prevents a disease, condition or disorder, i.e., a subject who has been exposed to or prone to the disease, condition or disorder but has not yet experienced or shown the signs Preventing the development of clinical signs of disease in (2) preventing the disease, condition or disorder, i.e. preventing or reducing the development of the disease, condition or disorder, or clinical signs thereof, or (3) Reducing a disease, condition, or disorder, that is, causing a regression of the disease, condition, or disorder, or clinical signs thereof.

  A “therapeutically effective amount” or “effective amount” is a treatment as described above for a disease, condition, disorder, or indication mediated by the activity of CFTR when administered to a mammal or other subject in need of treatment. Means an amount of a compound of the invention sufficient to carry out. The amount of a compound of the invention that constitutes a “therapeutically effective amount” will vary depending on the compound, the disease and its severity, and the age, weight, etc. of the subject to be treated, but those skilled in the art will be able to It can be determined routinely by knowledge and this disclosure.

  The terms “subject” and “patient” mean one or more members of any mammal or non-mammal species that may be in need of the pharmaceutical methods, compositions and treatments described herein. Thus, subjects and patients include primates (including humans), dogs, cats, ungulates (e.g., horses, cows, swine (e.g., pigs), birds, and other Examples include, but are not limited to, subjects of particular interest, which are commercially important humans and non-human animals (eg, domestic animals and domesticated animals).

  “Mammal” means one or more members of any mammalian species, including, for example, dogs, cats, horses, cows, sheep, rodents, and the like, and primates, particularly humans. Animal models other than humans, particularly mammals such as primates, mice, rabbits, etc. may be used for experimental investigations.

  As used herein, the term “unit dosage form” refers to physically discrete units suitable as unit dosages for human and animal subjects, each unit being a pharmaceutically acceptable diluent. A predetermined amount of a compound of the invention, calculated in an amount sufficient to produce the desired effect, in association with the carrier, vehicle, or vehicle. The specifications for the novel unit dosage forms of the present invention depend on the particular compound used and the effect to be achieved, as well as the pharmacodynamics associated with each compound in the host.

  The term “physiological conditions” is meant to include conditions that are compatible with living cells, eg, primarily aqueous conditions such as temperature, pH, salinity, etc. that are compatible with living cells.

  “Pharmaceutically acceptable excipient” means an excipient that is generally useful in preparing a pharmaceutical composition that is safe, non-toxic, biological, and otherwise undesirable. And excipients suitable for veterinary use as well as human pharmaceutical use. As used in the specification and claims, a “pharmaceutically acceptable excipient” includes both one or more such excipients.

  As used herein, “pharmaceutically acceptable derivatives” of the compounds of the present invention include their salts, esters, enol ethers, enol esters, acetals, ketals, orthoesters, hemiacetals, hemiketals. , Acids, bases, solvates, hydrates or prodrugs. Such derivatives can be readily prepared by those skilled in the art using well-known methods for such derivatization. The resulting compound may be administered to an animal or human if there is no substantial toxic effect, and such a compound is pharmaceutically active or a prodrug.

  “Pharmaceutically acceptable salt” of a compound of the invention means a salt that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. Such salts include (1) formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc .; organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentane Propionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3- (4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfone Acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methyl Bicyclo [2.2.2] oct-2-ene-1-carboxylic acid, glucoheptonic acid, 4,4'-methylenebis- (3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropion , Trimethylacetic acid, tert-butylacetic acid, laurylsulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, etc., or (2) acidic protons present in the parent compound Is replaced by a metal ion, such as an alkali metal ion, alkaline earth ion, or aluminum ion; or is coordinated with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, etc. Mention may be made of the salts formed.

  “Pharmaceutically acceptable ester” of a compound of the invention means an ester that is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound, eg, an acidic group such as carboxylic acid, phosphoric acid, phosphine. Examples include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl, and heterocyclyl esters of acids, sulfonic acids, sulfinic acids, and boronic acids.

  “Pharmaceutically acceptable enol ether” of a compound of the invention means an enol ether that is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound, for example, the chemical formula C═C (OR) (wherein , R is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl or heterocyclyl), but is not limited to these.

  “Pharmaceutically acceptable enol ester” of a compound of the invention means an enol ester that is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound, eg, the formula C═C (OC (O) R ) Wherein R is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl or heterocyclyl, but is not limited to these.

  “Pharmaceutically acceptable solvate or hydrate” of a compound of the invention means a solvated or hydrated complex that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. Examples include, but are not limited to, one or more solvents or water molecules of the compounds of the invention, or complexes with 1 to about 100, or 1 to about 10, or 1 to about 2, 3 or 4 solvents or water molecules. Not.

"Prodrug" means any compound that releases an active parent compound of formula (I) in vivo when administered to a mammalian subject. Prodrugs of compounds of formula (I) contain functional groups that are hydrolyzed under standard physiological conditions to the corresponding carboxy, hydroxy, amino or sulfhydryl groups. Examples of such functional groups include esters of hydroxy groups in compounds of formula (I) (eg acetic acid, formic acid and benzoic acid derivatives) and carbamates (eg N, N-dimethylaminocarbonyl) and the like. However, it is not limited to these. Another example is a dipeptide or tripeptide ester of a hydroxy or carboxy group in the compound of formula (I). The preparation of such functional groups is well known in the art. For example, a compound of formula (I) to which a hydroxy group is attached (e.g., when X ₁ , X ₂ , X ₃ , Y ₁ or Y ₂ is hydroxy) is associated with a carboxylic acid or a dipeptide having a free carboxy terminus. Treatment under esterification conditions well known in the art may provide the desired ester functionality. Similarly, a compound of formula (I) to which a free carboxy group is attached can be combined with an alcohol or a serine residue (e.g., --N (H) -C (H) (CH ₂ OH) -C (O)-) Treatment with tripeptides containing groups under esterification conditions well known in the art may yield the desired ester functionality. Further, the compound of formula (I) to which the carboxylic ester group is bonded is treated with a different carboxylic acid ester under standard transesterification reaction conditions to obtain the compound of formula (I) to which the desired ester functional group is bonded. It may be generated. All such functional groups are considered to be within the scope of the present invention.

As used herein, “organic group” and “organic radical” mean any carbon-containing functional group, such as aliphatic groups, cyclic groups, aromatic groups, functionalized derivatives thereof and / or Or the hydrocarbon group classified as various combinations thereof is mentioned. “Aliphatic group” means a saturated or unsaturated linear or branched hydrocarbon group and includes, for example, alkyl, alkenyl, and alkynyl groups. The term “alkyl group” means a substituted or unsubstituted, saturated linear or branched hydrocarbon group or chain (eg, C ₁ -C ₈ ), for example, methyl, ethyl, isopropyl, tert-butyl, heptyl , N-octyl, dodecyl, octadecyl, amyl, 2-ethylhexyl and the like. Suitable substituents include carboxy, protected carboxy, amino, protected amino, halo, hydroxy, protected hydroxy, mercapto, lower alkylthio, nitro, cyano, monosubstituted amino, protected monosubstituted amino, disubstituted amino, C ₁ -C ₇ alkoxy, C ₁ -C ₇ acyl, and the like C ₁ -C ₇ acyloxy. The term “substituted alkyl” refers to hydroxy, protected hydroxy, amino, protected amino, cyano, halo, trifluoromethyl, monosubstituted amino, disubstituted amino, lower alkoxy, mercapto, lower alkylthio, carboxy, protected carboxy, amino and / or Or the said alkyl group substituted 1-3 times by the hydroxy salt is meant. As used in connection with substituents on heteroaryl rings, “substituted (cycloalkyl) alkyl” and “substituted cycloalkyl” are those listed for “substituted alkyl” groups, as defined below. Is substituted with the same functional group. The term “alkenyl group” means an unsaturated, straight chain or branched hydrocarbon group having one or more carbon-carbon double bonds, such as a vinyl group. The term “alkynyl group” means an unsaturated, straight chain or branched hydrocarbon group having one or more carbon-carbon triple bonds. The term “cyclic functional group” means a closed ring hydrocarbon group that is classified as an alicyclic group, aromatic group, or heterocyclic group. The term “alicyclic group” means a cyclic hydrocarbon group having properties similar to an aliphatic group. The term “aromatic group” or “aryl group” means a monocyclic or polycyclic hydrocarbon group, which may contain one or more heteroatoms, and is further defined below. “Heterocyclic group” means a closed ring hydrocarbon in which one or more atoms in the ring is an element other than carbon (eg, nitrogen, oxygen, sulfur, etc.) and is further defined below.

  An “organic group” may be functionalized or include additional functional groups associated with organic groups such as carboxyl, amino, hydroxyl, etc. (which may or may not be protected) But you can. For example, the phrase “alkyl group” includes not only pure open chain saturated hydrocarbon alkyl substituents such as methyl, ethyl, propyl, t-butyl, etc., but also other substituents well known in the art, hydroxy, Also included are alkyl substituents having alkoxy, mercapto, alkylthio, alkylsulfonyl, halo, cyano, nitro, amino, carboxyl, and the like. Thus, “alkyl groups” include ethers, esters, haloalkyls, nitroalkyls, carboxyalkyls, hydroxyalkyls, sulfoalkyls, and the like.

  The terms “halo group” or “halogen” are used interchangeably herein to indicate a fluoro, chloro, bromo or iodo group. Preferred halogens are chloro and fluoro.

  The term “haloalkyl” refers to an alkyl group as defined above substituted with one or more halogen groups. The halogen atoms may be the same or different. “Dihaloalkyl” refers to the above alkyl group substituted by two halo groups, which may be the same or different. The term “trihaloalkyl” refers to the above alkyl group substituted by 3 halo groups, which may be the same or different. “Perhaloalkyl” refers to a haloalkyl group as defined above wherein each hydrogen atom in the alkyl group is replaced by a halogen atom. The term “perfluoroalkyl” denotes a haloalkyl group as defined above wherein each hydrogen atom in the alkyl group is replaced by a fluoro group.

  The term “cycloalkyl” means a mono-, bi- or tricyclic saturated ring that is fully saturated or partially unsaturated. Examples of such functional groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, cyclooctyl, cis- or trans-decalin, bicyclo [2.2.1] hept-2-ene, cyclohex- 1-enyl, cyclopent-1-enyl, 1,4-cyclooctadienyl, and the like.

  The term “(cycloalkyl) alkyl” refers to an alkyl group, as defined above, that is replaced with one of the above cycloalkyl rings. Examples of such functional groups include (cyclohexyl) methyl, 3- (cyclopropyl) -n-propyl, 5- (cyclopentyl) hexyl, 6- (adamantyl) hexyl, and the like.

The term “substituted phenyl” refers to halogen, hydroxy, protected hydroxy, cyano, nitro, mercapto, alkylthio, trifluoromethyl, C ₁ -C ₇ alkyl, C ₁ -C ₇ alkoxy, C ₁ -C ₇ acyl, C ₁ -C ₇ acyloxy, carboxy, oxy carboxy, protected carboxy, carboxymethyl, protected carboxymethyl, hydroxymethyl, protected hydroxymethyl, amino, protected amino, (monosubstituted) amino, protected (monosubstituted) amino, (disubstituted) amino , carboxamide, protected carboxamide, N- (C ₁ ~C ₆ alkyl) carboxamide, protected N- (C ₁ ~C ₆ alkyl) carboxamide, N, N-di (C ₁ -C ₆ alkyl) carboxamide, trifluoromethyl, N - ((C ₁ ~C ₆ alkyl) sulfonyl) amino, N- (phenylsulfonyl) amino or a substituted or unsubstituted phenyl, for example bi Is selected from the group consisting of Eniru or naphthyl group, in one or more portions, in some cases, it specifies a phenyl group substituted with moiety of 2 or 3.

  Examples of the term “substituted phenyl” include mono- or di (halo) phenyl groups such as 2-, 3- or 4-chlorophenyl, 2,6-dichlorophenyl, 2,5-dichlorophenyl, 3,4-dichlorophenyl. 2-, 3- or 4-bromophenyl, 3,4-dibromophenyl, 3-chloro-4-fluorophenyl, 2-, 3- or 4-fluorophenyl; mono- or di (hydroxy) phenyl groups such as , 2, 3, or 4-hydroxyphenyl, 2,4-dihydroxyphenyl, their protected hydroxy derivatives, etc .; nitrophenyl groups, such as 2-, 3- or 4-nitrophenyl; cyanophenyl groups, such as 2- , 3- or 4-cyanophenyl; mono- or di (alkyl) phenyl groups such as 2-, 3- or 4-methylphenyl, 2,4-dimethylphenyl, 2-, 3- or 4- (iso- Propyl) phenyl, 2-, 3- or 4-ethyl Phenyl, 2-, 3- or 4- (n-propyl) phenyl; mono- or di (alkoxy) phenyl groups such as 2,6-dimethoxyphenyl, 2-, 3- or 4- (isopropoxy) phenyl, 2-, 3- or 4- (t-butoxy) phenyl, 3-ethoxy-4-methoxyphenyl, etc .; 2-, 3- or 4-trifluoromethylphenyl; mono- or di-carboxyphenyl or (protected carboxy) Phenyl groups such as 2-, 3- or 4-carboxyphenyl or 2,4-di (protected carboxy) phenyl; mono- or di- (hydroxymethyl) phenyl or (protected hydroxymethyl) phenyl, such as 2-, 3- or 4- (protected hydroxymethyl) phenyl or 3,4-di (hydroxymethyl) phenyl; mono- or di- (aminomethyl) phenyl or (protected aminomethyl) phenyl; or mono- or di- (N -(Methyl Sulfonylamino)) phenyl, for example 2-, 3- or 4- (N- (methylsulfonylamino)) phenyl. The term “substituted phenyl” refers to disubstituted phenyl groups having different substituents, such as 3-methyl-4-hydroxyphenyl, 3-chloro-4-hydroxyphenyl, 2-methoxy-4-bromophenyl, 4-ethyl-2-hydroxyphenyl, 3-hydroxy-4-nitrophenyl, 2-hydroxy-4-chlorophenyl and the like.

  The term “(substituted phenyl) alkyl” means one of the above substituted phenyl groups attached to one of the above alkyl groups. Examples of such functional groups include 2-phenyl-1-chloroethyl, 2- (4′-methoxyphenyl) ethyl, 4- (2 ′, 6′-dihydroxyphenyl) -n-hexyl, 2- (5 '-Cyano-3'-methoxyphenyl) -n-pentyl, 3- (2', 6'-dimethylphenyl) propyl, 4-chloro-3-aminobenzyl, 6- (4'-methoxyphenyl) -3- Carboxyhexyl, 5- (4′-aminomethylphenyl) -3- (aminomethyl) pentyl, 5-phenyl-3-oxopent-1-yl, (4-hydroxynaphth-2-yl) methyl and the like can be mentioned.

  As noted above, “aromatic” or “aryl” refers to 5- and 6-membered carbocycles. Also, as noted above, “heteroaryl” is optionally substituted with 1 to 4 heteroatoms, such as oxygen, sulfur, and / or nitrogen atoms, particularly nitrogen atoms alone or with sulfur or oxygen ring atoms. A 5- or 6-membered ring which may be These 5- and 6-membered rings may be completely unsaturated.

  Furthermore, the optionally substituted 5-membered or 6-membered ring may be optionally condensed to form an aromatic 5-membered or 6-membered ring structure. For example, the ring may be optionally fused to form an aromatic 5- or 6-membered ring structure, such as a pyridine or triazole structure, preferably a benzene ring.

  The following ring structures are examples of heterocyclic (whether substituted or unsubstituted) radicals indicated by the term “heteroaryl”: thienyl, furyl, pyrrolyl, pyrrolidinyl, imidazolyl, isoxazolyl, triazolyl, thiadiazolyl, oxadiazolyl , Tetrazolyl, thiatriazolyl, oxatriazolyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, oxazinyl, triazinyl, thiadiazinyltetrazolo, 1,5- [b] pyridazinyl and purinyl, and benzofused derivatives such as benzoxazolyl, benzthiazolyl, Benzimidazolyl and indolyl.

Substituents for the optionally substituted heteroaryl ring are 1-3 halo, trihalomethyl, amino, protected amino, amino salt, monosubstituted amino, disubstituted amino, carboxy, protected carboxy, carboxylate , Hydroxy, protected hydroxy, hydroxy group salt, lower alkoxy, mercapto, lower alkylthio, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, (cycloalkyl) alkyl, substituted (cycloalkyl) alkyl, phenyl, substituted phenyl, phenyl Alkyl, and (substituted phenyl) alkyl. The substituent for the heteroaryl group is as defined above, and in the case of trihalomethyl, it can be trifluoromethyl, trichloromethyl, tribromomethyl or triiodomethyl. As used in connection with the above substituents for heteroaryl rings, the terms "lower alkoxy" means a C ₁ -C ₄ alkoxy group, similarly, the term "lower alkylthio", C ₁ ~ C ₄ means an alkylthio group.

The term "(monosubstituted) amino", phenyl, substituted phenyl, alkyl, substituted alkyl, C ₁ -C ₄ acyl, C ₂ -C ₇ alkenyl, C ₂ -C ₇ substituted alkenyl, C ₂ -C ₇ alkynyl, shows a C ₇ -C ₁₆ alkylaryl, amino group having one substituent selected from the group consisting of C ₇ -C ₁₆ substituted alkylaryl and heteroaryl group. The (monosubstituted) amino can further have an amino protecting group, as encompassed by the term “protected (monosubstituted) amino”. The term "(disubstituted) amino", phenyl, substituted phenyl, alkyl, substituted alkyl, C ₁ -C ₇ acyl, C ₂ -C ₇ alkenyl, C ₂ -C ₇ alkynyl, C ₇ -C ₁₆ alkylaryl, an amino group having two substituents selected from C ₇ -C ₁₆ substituted alkylaryl and heteroaryl. The two substituents may be the same or different.

  The term “heteroaryl (alkyl)” refers to an alkyl group, as defined above, substituted at any position by a heteroaryl group, as defined above.

  “Optional” or “optionally” means that the event, situation, feature or element described below may occur but is not required, and the description is Or when the situation occurs and when it doesn't happen. For example, “a heterocyclo group optionally mono- or disubstituted with an alkyl group” means that an alkyl group may be present, but that is not necessary, the description is that the heterocyclo group is mono or It is meant to include di-substituted situations and situations where the heterocyclo group is not substituted by an alkyl group.

The term “electron-withdrawing group” means that a functional group on a molecule can attract electrons more strongly toward the functional group than a hydrogen atom, compared to when hydrogen occupies the same position in the molecule. Means. Examples of electron withdrawing groups include, for example, halogen groups, —C (O) R groups (wherein R is alkyl); carboxylic acid and ester groups; —NR ₃ ⁺ groups (wherein R is alkyl) Or nitro; —OR and —SR groups where R is hydrogen or alkyl; and organic groups containing such electron withdrawing groups (as defined herein); ), Such as, but not limited to, haloalkyl groups (including perhaloalkyl groups).

  Compounds that have the same molecular formula but differ in the nature or sequence of bonding of atoms or the spatial arrangement of atoms are termed “isomers”. Isomers that differ in the arrangement of their atoms in space are termed "stereoisomers". Stereoisomers that are not mirror images of one another are termed “diastereomers”, and stereoisomers that are non-superimposable mirror images are termed “enantiomers”. A pair of enantiomers is possible when a compound has an asymmetric center, for example when the asymmetric center is bound to four different functional groups. Enantiomers can be characterized by the absolute configuration of their asymmetric centers, described by Cahn and Prelog's R- and S-rank rules, or molecules can rotate the plane of polarized light and be dextrorotatory or levorotatory (i.e., respectively) (+)-Or (-)-isomer). A chiral compound can exist as either individual enantiomer or as a mixture thereof. A mixture containing equal proportions of enantiomers is called a “racemic mixture”.

  The compounds of the present invention may have one or more asymmetric centers; therefore, such compounds can be generated as individual (R)-or (S) -stereoisomers or mixtures thereof. . Unless otherwise stated, the description or nomenclature for a particular compound in the specification and claims is intended to include both the individual enantiomers and mixtures thereof, racemic or not. Methods for determination of stereochemistry and separation of stereoisomers are well known in the art (eg “Advanced Organic Chemistry”, 4th edition, J. March, John Wiley and Sons, New York, 1992, 4th. (See chapter discussion).

Overview The present invention relates to thiazolidinone compositions, thiazolidinone derivative compositions and methods for their use in high affinity inhibition of cystic fibrosis conductance regulator protein (CFTR), and their use for the study and treatment of CFTR-mediated diseases and conditions Provide a method. The discovery of the subject thiazolidinone compounds and derivatives thereof is based on the screening of many potential candidate compounds using assays designed to identify CFTR inhibitors that interact directly with CFTR. The inhibitory compounds of the present invention are probably CFTR because multiple CFTR activators that act on different activation pathways were included in the study leading to the identification of the target compound without sticking to any particular theory or mode of operation Cl ^- transport pathways, or inhibiting by acting in vicinity. Screening 50,000 different compounds identified several 2-thioxo-4-thiazolidinone compounds and derivatives as effective CFTR inhibitors. These compounds and derivatives are not chemically and structurally related to conventional well-known CFTR activators or conventional well-known CFTR inhibitors DPC, NPPB or glibenclamide. The most potent CFTR inhibitors identified from screening, Cl in human airway cells ^- had a K _I of ~300nM to inhibition of current. Inhibition was rapid, reversible and CFTR-specific.

  The compositions and methods of the present invention are described in more detail below.

(式中、
X₁、X₂およびX₃はそれぞれ、水素、有機基、ハロ基、ニトロ基、アゾ基、ヒドロキシル基およびメルカプト基から選択され；Y₁、Y₂およびY₃はそれぞれ、水素、有機基、ハロ基、ニトロ基、アゾ基、ヒドロキシル基およびメルカプト基から選択され；A₁およびA₂はそれぞれ、酸素および硫黄から選択され、A₃は硫黄およびセレンから選択され；A₄は1または複数の炭素原子またはヘテロ原子を有し、存在してもしなくてもよい）。A₄が存在しない場合、中央複素環は5員環である。 Thiazolidinone compounds and derivatives The thiazolidinone compounds and derivatives used in the compositions and methods of the present invention have a heterocycle of 5 or more atoms, an aryl-substituted nitrogen, at least one sulfur, oxygen, or selenium heteroatom, and a heterocycle Containing one or more carbonyl or thiocarbonyl groups attached to More specifically, the subject thiazolidinone compounds and derivatives may have the following chemical formula (I), or pharmaceutically acceptable derivatives thereof, as individual stereoisomers or mixtures thereof:

(Where
X ₁ , X ₂ and X ₃ are each selected from hydrogen, organic group, halo group, nitro group, azo group, hydroxyl group and mercapto group; Y ₁ , Y ₂ and Y ₃ are each hydrogen, organic group, Selected from halo, nitro, azo, hydroxyl and mercapto groups; A ₁ and A ₂ are each selected from oxygen and sulfur, A ₃ is selected from sulfur and selenium; A ₄ is one or more Having carbon atoms or heteroatoms, which may or may not be present). When A ₄ is not present, the central heterocycle is a 5-membered ring.

(式中、
X₁、X₂およびX₃はそれぞれ、水素、有機基、ハロ基、ニトロ基、アゾ基、ヒドロキシル基およびメルカプト基から選択され；Y₁、Y₂およびY₃はそれぞれ、水素、有機基、ハロ基、ニトロ基、アゾ基、ヒドロキシル基およびメルカプト基から選択され；A₁およびA₂はそれぞれ、酸素および硫黄から選択される)。特定の態様では、X₁は電子吸引性基であってもよく、ハロアルキル基、ジハロアルキル基、トリハロアルキル基(例えば、トリフルオロアルキル基)またはフルオロ基を含んでもよい。Y₂は、独立してアルキル、ヒドロキシル、カルボキシル、ニトロ、カーボネート、カルバメート、アルコキシ、アルキルカルボニル、およびハロ基からなる群から選択され、Y₁は独立して、ヒドロキシルおよびブロモ基から選択され、Y₃は独立して水素および窒素基から選択される。 In certain embodiments, the thiazolidinone compounds and derivatives of formula (I) above have the formula (Ia):

(Where
X ₁ , X ₂ and X ₃ are each selected from hydrogen, organic group, halo group, nitro group, azo group, hydroxyl group and mercapto group; Y ₁ , Y ₂ and Y ₃ are each hydrogen, organic group, Selected from halo, nitro, azo, hydroxyl and mercapto groups; A ₁ and A ₂ are each selected from oxygen and sulfur). In certain embodiments, X ₁ may be an electron withdrawing group and may include a haloalkyl group, a dihaloalkyl group, a trihaloalkyl group (eg, a trifluoroalkyl group) or a fluoro group. Y ₂ is independently selected from the group consisting of alkyl, hydroxyl, carboxyl, nitro, carbonate, carbamate, alkoxy, alkylcarbonyl, and halo groups, Y ₁ is independently selected from hydroxyl and bromo groups, Y ₃ is independently selected from hydrogen and nitrogen groups.

(式中、
X₁、X₂およびX₃の少なくとも1つは電子吸引性基であり；Y₁、Y₂およびY₃はそれぞれ、水素、アルキル、ヒドロキシル、カルボキシル、ニトロ、カーボネート、カルバメート、アルコキシ、アルキルカルボニル、およびハロ基から選択される)。1つの態様では、X₁は2、3、または4から選択される1つの位置にあり；Y₂は、2、3、または4から選択される1つの位置にあり；Y₁およびY₃は水素であってもよい。 In many embodiments, the subject thiazolidinone compounds and derivatives of formula (I) may include 3-aryl-5-arylmethylene-2-thioxo-4-thiazolidinones of formula (Ib):

(Where
At least one of X ₁ , X ₂ and X ₃ is an electron withdrawing group; Y ₁ , Y ₂ and Y ₃ are each hydrogen, alkyl, hydroxyl, carboxyl, nitro, carbonate, carbamate, alkoxy, alkylcarbonyl, And a halo group). In one embodiment, X ₁ is at one position selected from 2, 3, or 4; Y ₂ is at one position selected from ₂ , 3, or 4; Y ₁ and Y ₃ are It may be hydrogen.

(式中、
Y₁〜Y₃は上記の通りである)。1つの態様では、トリフルオロメチル基は2、3、または4から選択される1つの位置にあり；Y₂は、2、3、または4から選択される1つの位置にあり；Y₁およびY₃はこの態様では水素であってもよい。 3-Aryl-5-arylmethylene-2-thioxo-4-thiazolidinone more particularly has the chemical formula (Ic):

(Where
Y _{1 to} Y ₃ are as described above). In one embodiment, the trifluoromethyl group is at one position selected from 2, 3, or 4; Y ₂ is at one position selected from ₂ , 3, or 4; Y ₁ and Y ₃ may be hydrogen in this embodiment.

すなわち、3-[(3-トリフルオロメチル)フェニル]-5-[(4-ニトロフェニル)メチレン]-2-チオキソ-4-チアゾリジノン；

すなわち、3-[(3-トリフルオロメチル)フェニル]-5-[(4-オキシカルボキシフェニル)メチレン]-2-チオキソ-4-チアゾリジノン；

すなわち、3-[(3-トリフルオロメチル)フェニル]-5-[(4-カルボキシフェニル)メチレン]-2-チオキソ-4-チアゾリジノン；

すなわち、3-[(3-トリフルオロメチル)フェニル]-5-[(3,4-ジヒドロキシフェニル)メチレン]-2-チオキソ-4-チアゾリジノン；

すなわち、3-[(3-トリフルオロメチル)フェニル]-5-[(3,5-ジブロモ-4-ヒドロキシフェニル)メチレン]-2-チオキソ-4-チアゾリジノン；および

すなわち、3-[(3-トリフルオロメチル)フェニル]-5-[(3-ブロモ-4-ヒドロキシ-5-ニトロフェニル)メチレン]-2-チオキソ-4-チアゾリジノン。また、上記列挙した任意の化合物のトリフルオロメチル基はフェニル環の2位または4位としてもよい。 In some embodiments of the present invention, the thiazolidinone compounds of the present invention may include:

3-[(3-trifluoromethyl) phenyl] -5-[(4-nitrophenyl) methylene] -2-thioxo-4-thiazolidinone;

3-[(3-trifluoromethyl) phenyl] -5-[(4-oxycarboxyphenyl) methylene] -2-thioxo-4-thiazolidinone;

3-[(3-trifluoromethyl) phenyl] -5-[(4-carboxyphenyl) methylene] -2-thioxo-4-thiazolidinone;

3-[(3-trifluoromethyl) phenyl] -5-[(3,4-dihydroxyphenyl) methylene] -2-thioxo-4-thiazolidinone;

3-[(3-trifluoromethyl) phenyl] -5-[(3,5-dibromo-4-hydroxyphenyl) methylene] -2-thioxo-4-thiazolidinone; and

That is, 3-[(3-trifluoromethyl) phenyl] -5-[(3-bromo-4-hydroxy-5-nitrophenyl) methylene] -2-thioxo-4-thiazolidinone. In addition, the trifluoromethyl group of any of the above listed compounds may be in the 2-position or 4-position of the phenyl ring.

Pharmaceutical Preparations The present invention also provides pharmaceutical preparations of the above subject thiazolidinone compounds. The subject compounds can be incorporated into various formulations for therapeutic administration by various routes. More particularly, the compounds of the invention can be formulated into pharmaceutical compositions by combining with suitable, pharmaceutically acceptable carriers, diluents, excipients and / or adjuvants, or Formulations may be in solid, semi-solid, liquid or gaseous form preparations such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injectable solutions, inhalants and aerosols. Preferably, the formulation does not contain detectable DMSO (dimethyl sulfoxide). DMSO is not a pharmaceutically acceptable carrier, diluent, excipient or adjuvant for non-topical parenteral or enteral administration. The formulation is designed to be administered to a subject or patient in need of administration via many different routes such as oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intratracheal administration, etc. May be.

  In one embodiment, topical administration (eg, by transdermal administration) is the subject. Topical formulations may take the form of transdermal patches, ointments, pastes, lotions, creams, gels and the like. Topical formulations may include one or more of penetrants, thickeners, diluents, emulsifiers, dispersion aids, or binders. Where the compound is formulated for transdermal delivery, the compound may be formulated for use with or with a penetration enhancer. Penetration enhancers include chemical penetration enhancers and physical penetration enhancers, which facilitate delivery of the compound through the skin and may also be referred to as “permeation enhancers” in the same sense. Physical penetration enhancers include, for example, electrophoresis techniques such as iontophoresis, the use of ultrasound (or “phonophoresis”), and the like. A chemical penetration enhancer is an agent that is administered before, immediately after, or with compound administration, thereby increasing the permeability of the skin, particularly the stratum corneum, and enhancing the penetration of the drug through the skin.

Compounds used to enhance skin permeability include sulfoxide, dimethyl sulfoxide (DMSO) and decylmethyl sulfoxide (C ₁₀ MSD); ethers such as diethylene glycol monoethyl ether, decaoxyethylene-oleyl ether, and diethylene glycol Monomethyl ethers; surfactants such as sodium laurate, sodium ryyl sulfate, cetyltrimethylammonium bromide, benzalkonium chloride, Poloxamer (231, 182, 184), Tween (20, 40, 60, 80) and lecithin; 1-substituted azacycloheptan-2-one, especially 1-n-dodecylcyclazacycloheptan-2-one; alcohols such as ethanol, propanol, octanol, benzyl alcohol, etc .; petrolatum such as Vaseline (petroleum jel ly) (petrolatum), mineral oil (liquid petrolatum), etc .; fatty acids such as C ₈ -C ₂₂ and other fatty acids (eg isostearic acid, octanoic acid, oleic acid, lauric acid, valeric acid); C ₈ -C ₂₂ Fatty alcohols (eg, oleyl alcohol, lauryl alcohol); lower alkyl esters of C _{8 to} C ₂₂ fatty acids and other fatty acids (eg, ethyl oleate, isopropyl myristate, butyl stearate, methyl laurate, isopropyl myristate, palmitate) Isopropyl, methyl propionate, ethyl oleate); monoglycerides of C _{8 to} C ₂₂ fatty acids (eg glyceryl monolaurate); tetrahydrofurfuryl alcohol polyethylene glycol ether; 2- (2-ethoxyethoxy) ethanol; diethylene glycol monomethyl ether; Polyethylene oxy Alkylaryl ethers of de; polyethylene oxide monomethyl ether; polyethylene oxide dimethyl ether; C ₆ -C ₈ diacid - lower alkyl esters (e.g., diisopropyl adipate); ethyl acetate; acetoacetate; polyols and esters thereof, for example , Propylene glycol, ethylene glycol, glycerol, butanediol, polyethylene glycol, and polyethylene glycol monolaurate; amides and other nitrogen-containing compounds such as urea, dimethylacetamide (DMA), dimethylformamide (DMF), 2-pyrrolidone, N -Alkyl pyrrolidones such as 1-methyl-2-pyrrolidone; ethanolamine, diethanolamine and triethanolamine; terpenes; alkanones and organic acids, in particular salicyl Examples include acids and salicylates, citric acid and succinic acid. Other chemical and physical penetration enhancers are described, for example, by Transdermal Delivery of Drugs, AFKydonieus (ED) 1987 CRL Press; Percutaneous Penetration Enhancers, eds. Smith et al. (CRC Press, 1995); Lenneruas et al., J Pharm Pharmacol 2002; 54 (4): 499-508; Karande et al., Pharm Res 2002; 19 (5): 655-60; Vaddi et al., J Pharm Sci 2002 July; 91 (7): 1639-51; Ventura et al., J Drug Target 2001; 9 (5): 379-93; Shokri et al., Int J Pharm 2001; 228 (1-2): 99-107; Suzuki et al., Biol Pharm Bull 2001; 24 (6): 698-700; Alberti et al., J Control Release 2001; 71 (3): 319-27; Goldstein et al., Urology 2001; 57 (2): 301-5; Kiijavainen et al., Eur J Pharm Sci 2000; 10 (2): 97-102; and Tenjarla et al., Int J Pharm 1999; 192 (2): 147-58.

  When a compound is formulated with a chemical penetration enhancer, the penetration enhancer is selected for compatibility with the compound, so that the compound is easily delivered through the subject's skin, for example, so that the compound is delivered to the systemic circulation. Present in a sufficient amount. In one embodiment, the compound is formulated with a penetration enhancer other than DMSO.

  In one embodiment, the compound is provided in a drug delivery patch, such as a transmucosal or transdermal patch, and can be formulated with a penetration enhancer. Patches are generally a backing layer (impermeable to the compound and other formulation ingredients), a matrix in contact with one side of the backing layer (this matrix provides sustained release of the compound, which is controlled release) And an adhesive layer (present on the same side as the matrix of the backing layer). The matrix can be selected as appropriate for the route of administration, and can be, for example, a polymer or hydroxygel matrix.

  In pharmaceutical dosage forms, the subject compounds of the invention may be administered in the form of a pharmaceutically acceptable derivative, such as a salt, and are suitably associated alone or with other pharmaceutically active compounds. And combinations thereof may be used. The following methods and excipients are merely exemplary and are in no way limiting.

  For oral administration, the subject compounds can be used alone or in combination with suitable additives to produce tablets, powders, granules or capsules, for example, with: conventional additives such as Lactose, mannitol, corn starch or potato starch; binders such as crystalline cellulose, cellulose derivatives, acacia gum, corn starch or gelatin; disintegrants such as corn starch, potato starch or sodium carboxymethylcellulose; lubricants such as talc or stearic acid Magnesium; and, if desired, diluents, buffers, wetting agents, preservatives and fragrances. Of particular interest is the formulation of the subject thiazolidinone compound and buffer, which protects the compound from the low pH of the stomach environment. It is also preferable to provide an enteric coating to avoid precipitation of the compound during passage through the stomach.

  The subject compounds of the present invention can be obtained by dissolving, suspending or emulsifying the compounds in an aqueous or non-aqueous solvent such as vegetable oil or other similar oils, synthetic fatty acid glycerides, higher fatty acids or esters of propylene glycol; If desired, it can be formulated into injectable preparations with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizing agents and preservatives. Particularly interesting solubilizers include vitamin E TPGS (d-α-tocopheryl polyethylene glycol 1000 succinate), cyclodextrin, and the like.

  The compounds of the invention can be used in aerosol formulations to be administered by inhalation. The compounds of the present invention can be formulated in pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.

  Furthermore, the subject compounds can be made into suppositories by mixing with various bases such as emulsifying bases or water-soluble bases. The compounds of the present invention can be administered rectally via a suppository. Suppositories can include vehicles such as cocoa butter, carbowax, and polyethylene glycol, which dissolve at body temperature but solidify at room temperature.

  Unit dosage forms for oral or rectal administration such as syrups, elixirs and suspensions may be provided, in which case each dosage unit such as 1 teaspoon, 1 tablespoon, tablet or suppository is 1 Or a predetermined amount of the composition comprising a plurality of inhibitors. Similarly, unit dosage forms for injection or intravenous administration may contain the inhibitor in a composition as a solution dissolved in sterile water, saline or another pharmaceutically acceptable carrier.

  Depending on the subject and condition being treated, and the route of administration, the subject compound may be administered at a dose of, for example, 0.1 μg to 10 mg / kg body weight / day. The range is wide. In general, the effectiveness of the therapeutic effect on different mammals varies widely, and doses are typically 20, 30 or 40 times lower (per unit body weight) in humans than in rats. Similarly, the mode of administration can greatly affect the dose. We found that intestinal fluid secretion induced by cholera toxin in mice was effectively blocked by a single intraperitoneal dose of about 10-20 μg, and about 10 times higher doses were effective in rats. . Thus, for example, an oral dose may be about 10 times the injected dose. Higher doses may be used for local delivery routes.

  A typical dose is a solution suitable for intravenous administration; one sustained release capsule containing tablets taken 2-6 times daily, or a proportionally higher amount of active ingredient taken once daily It may be a tablet. Sustained release effects can be obtained by capsule materials that dissolve at different pH values, by capsules that release gradually by osmotic pressure, or by any other known controlled release means.

  For use in the subject methods, the subject compounds may be formulated with other pharmaceutically active agents, such as other CFTR-inhibitors.

  Pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents that can be used with the present invention are readily available to the general public. In addition, pharmaceutically acceptable auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like are readily available to the general public.

  Persons of ordinary skill in the art may vary the dosage level as a function of the particular compound, the severity of the symptoms, and the subject's susceptibility to side effects. The preferred dose for a given compound can be readily determined by one skilled in the art by various means.

  Kits are provided comprising a unit dose of the subject compound, usually in oral or injectable doses. In such a kit, in addition to the container containing the unit dose, there is an information package insert that describes the use of the drug in the treatment of the subject's pathological condition and the resulting benefits. Preferred compounds and unit doses are those described herein above.

Conditions Suitable for Treatment Using the CFTR Inhibitors of the Invention The CFTR inhibitors disclosed herein are CFTR-mediated conditions, e.g., any condition mediated by CFTR in the activity of CFTR in ion transport, i.e. any Useful for the treatment of a condition, disorder or disease, or a sign of such a condition, disorder or disease. Such conditions, disorders, diseases, or symptoms thereof are suitable for treatment by inhibiting CFTR activity, eg, inhibiting CFTR ion transport.

  In one embodiment, the CFTR inhibitors of the present invention are used for the treatment of conditions associated with abnormally increased intestinal secretion, particularly acute abnormally increased intestinal secretion. CFTR activity is associated with intestinal secretion in response to various agonists such as cholera toxin (eg, Snyder et al. 1982 Bull. World Heath Organ. 60: 605-613; Chao et al. 1994 EMBO J. 13: 1065 -1072; see Kimberg et al. 1971 J. Clin. Invest. 50: 1218-1230). Thus, the CFTR inhibitor of the present invention can be administered in an amount effective to inhibit CFTR ion transport and thereby reduce intestinal fluid secretion.

  Thus, CFTR inhibitors can be used to treat intestinal inflammatory diseases and diarrhea, particularly secretory diarrhea. Secretory diarrhea is the leading cause of infant death in developing countries, where approximately 5 million people die each year (Gabriel at el., 1994 Science 266: 107-109). Several studies, including studies using CF mice, show that CFTR is the final common pathway for intestinal chloride ion (and hence intestinal fluid) secretion in response to various agonists (Synder et al. 1982, Bull. World Heath Organ. 60: 605-613; Chao et al., 1994 EMBO. J. 13: 1065-1072; and Kimberg et al., 1971, J. Clin. Invest. 50: 1218-1230 ). The mouse model of intestinal secretion used herein demonstrates that CFTR inhibition by systemic administration of the inhibitor at a non-toxic dose effectively blocks cholera toxin-induced intestinal secretion. See example).

  Diarrhea that may be suitable for treatment using the CFTR inhibitors of the present invention may include exposure to various pathogens or pathogens, such as cholera toxin (cholera), E. coli (especially enterotoxigenicity (ETEC)). Shigella, Salmonella, Campylobacter, Clostridium difficile, parasites (e.g. Giardia, Shigella amoeba), Cryptosporidiosis, Cyclospora), diarrhea viruses (e.g. rota Virus), food poisoning, or toxin exposure that increases CFTR-mediated intestinal secretion (but not limited to).

Other diarrhea includes diarrhea associated with AIDS (eg, AIDS-related diarrhea) and inflammatory gastrointestinal diseases such as ulcerative colitis, inflammatory colitis (IBD), Crohn's disease, and the like. Is adjusted three expression of the main mediators of inflammation by intestinal salt transport intestinal transepithelial Cl ^- by both the inhibition of growth and epithelial NaCl absorption of secretion may contribute to diarrhea in ulcerative colitis (See, for example, Lohi et al., 2002, Am. J. Physiol. Gastrointest. Liver Physiol. 283 (3): G567-75).

  The CFTR inhibitors of the present invention can also be used in the treatment of conditions such as multiple cystic kidneys, and have also been found to be used as male infertility agents by inhibiting CFTR activity in the testis.

  The CFTR inhibitors of the present invention can be screened in larger animal models (eg, the rabbit model described in Spira et al., 1981, Infect. Immun. 32: 739-747). Furthermore, a fecal analysis test using live Vibrio cholerae can be performed, and further, the CFTR inhibitor of the present invention can be characterized.

CFTR-deficient non-human animal models and human tissue models The CFTR inhibitors of the present invention are also used to create non-human animal models of diseases associated with reduced CFTR function (eg, reduced ion transport). be able to. Increased evidence for decreased mucociliary clearance and bacterial clearance in CFTR-deficient subjects, particularly those affected by cystic fibrosis (CF), due to defects in fluid and macromolecular secretion by the airway submucosal glands Increasing; however, functional studies in human airway glands are limited to airways with severe disease obtained during lung transplantation (Jayaraman et al. 2001 Proc. Natl. Acad. Sci. USA 98: 8119- 8123). Acute CFTR inhibition can determine the role of CFTR in water, salt and macromolecular secretion by submucosal glands. With high affinity CFTR inhibitors, non-human animal models that mimic CFTR-deficiency in humans, eg, mimic the human CF phenotype, can be created pharmacologically. In particular, large animal models of CFTR deficiency (e.g. CF) assess the pathophysiology of the onset and progression of airway disease in CF, and evaluate the efficacy of CF treatment, e.g. candidates for treatment of CFTR-deficiency or its indications Especially used for screening drugs.

  Inhibition of CFTR ion transport may appear in airway and pancreatic diseases, and male infertility. For example, inhibition of CFTR channels in the lungs and airways affects airway surface fluid, leading to mucus accumulation, which blocks the airways and collects large amounts of mucus on the lung wall, making it the best environment for infection to occur Can lead to chronic lung disease. This same phenomenon occurs in the pancreas, and the accumulated mucus interferes with the exocrine function of the pancreas, preventing essential food processing enzymes from reaching the intestines.

  Such non-human animal models can be created by administering a certain amount of a CFTR inhibitor effective to reduce CFTR activity in ion transport. Of particular interest is the use of the CFTR inhibitors of the present invention to induce the cystic fibrosis (CF) phenotype in non-human animals. For example, administering a certain amount of a CFTR inhibitor effective to inhibit the CFTR receptor in the lung effectively mimics the CFTR defect found in CF. Delivery routes for CFTR inhibitors are described in detail above. Depending on the non-human animal used, the subject compound may be administered intraperitoneally, subcutaneously, or by other routes, for example, 1 to 3 times daily at a dose of 50-500 μg / kg body weight. May be created. Oral administration may be up to about 10 times intraperitoneal or subcutaneous administration.

  A non-human animal model of CFTR-related disease can be used as a model for any suitable condition associated with decreased CFTR activity. Such conditions include those associated with CFTR mutations that cause abnormalities in epithelial ion and water transport. These abnormalities are associated with mucociliary clearance of the airways, and other mucosal and ductal epithelial disorders. Conditions that can be pharmacologically modeled by inducing a CFTR-deficient phenotype in non-human animals include cystic fibrosis (including atypical CF), idiopathic chronic pancreatitis, vas deferens, Examples include, but are not limited to, mild lung disease and asthma. For a review of diseases associated with decreased CFTR function, see, for example, Noone et al. Respir Res 2 328-332 (2001). Non-human animal models made with CFTR inhibitors can also function as models for microbial infection (eg, bacterial, viral, or fungal infections, particularly respiratory infections) in CFTR-deficient subjects. In one particularly interesting embodiment, the CFTR inhibitors of the present invention are used to pharmacologically induce the cystic fibrosis (CF) phenotype.

  Animals suitable for use in creating the animal model of the present invention include any animal, particularly mammals such as non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, Mice, gerbils, hamsters, ferrets, etc.), rabbits, pigs (eg, pigs, minipigs), horses, dogs, cats and the like. Large animals are particularly interesting.

  CFTR inhibitors can be contacted with isolated human tissue to create an ex vivo model of disease. Such tissue is contacted with an amount of a CFTR inhibitor effective to reduce CFTR activity in the tissue. Contact may be as short as 15 minutes, or as long as 2 hours or longer. Target human tissues include, but are not limited to, lungs (including trachea and airways), liver, pancreas, testis and the like. Physiological, biochemical, genomic or other studies can be performed on tissues treated with inhibitors to identify new therapeutic target molecules important in the pathophysiology of the disease. For example, tissues isolated from humans who do not have CF can be exposed to sufficient inhibitors to induce the CF phenotype, and such studies have been conducted and are important new in the pathophysiology of CF A therapeutic target molecule can be identified.

Synthesis of Compounds of the Invention Compounds of the invention may be synthesized by methods well known to those skilled in the art or disclosed in US Pat. Nos. 5,326,770 and 6,380,186, all of which are hereby incorporated by reference in their entirety. It may be prepared by similar methods or by methods similar to those described below.

  In the following description, it is understood that combinations of substituents and / or variables of the formulas shown are only allowed if such combinations yield stable compounds.

  One skilled in the art will also recognize that in the methods described below, the functional group of the intermediate compound may need to be protected by a suitable protecting group. Such functional groups include hydroxy, amino, mercapto and carboxylic acid. Suitable protecting groups for hydroxy include trialkylsyl or diarylsilyl (eg t-butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl and the like. Suitable protecting groups for amino, amidino and guanidino include t-butoxycarbonyl, benzyloxycarbonyl and the like. Suitable protecting groups for mercapto include —C (O) —R where R is alkyl, aryl or aralkyl, p-methoxybenzyl, trityl and the like. Suitable protecting groups for carboxylic acid include alkyl, aryl or aralkyl esters.

  Protecting groups are well known to those skilled in the art and may be added or removed by standard techniques as described herein.

  The use of protecting groups is described in detail in Theodora W. Greene, Peter G. M. Wuts, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley-Interscience. The protecting group may also be a polymer resin such as a Wang resin or a 2-chlorotrityl chloride resin.

  One skilled in the art will recognize that such protected derivatives of compounds of formula (I) may not have pharmacological activity per se, as described above (eg, in the overview and in thiazolidinone compounds and derivatives). It will also be appreciated that pharmacologically active compounds of the invention can be formed when administered to a mammal and subsequently metabolized in the body. As such, such derivatives may be described as “prodrugs”. All prodrugs of compounds of formula (I) are included within the scope of the present invention.

The following reaction scheme shows a method for producing the compound of the present invention. One skilled in the art will appreciate that the compounds of the invention can be prepared by similar methods or by methods well known to those skilled in the art. In general, the starting components may be obtained from sources such as Aldrich or synthesized according to sources well known to those skilled in the art (eg, Smith and March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition). (See Wiley Interscience, New York). In addition, various substituted functional groups (e.g., X ₁ , X ₂ , X ₃ , Y ₁ , Y _2, Y _3, etc.) of the compounds of the present invention can be converted into starting components, intermediates by methods well known to those skilled in the art. It may be bound to body components and / or the final product.

  In the following reaction scheme, R represents an alkyl or aralkyl group, and W represents a halogen atom such as Cl, Br, or I.

Following Reaction Scheme I, relates to the preparation of compounds of formula (1), these compounds are as described above (e.g., an overview and thiazolidinone compounds and the derivatives thereof) is a compound of the present invention, wherein A ₄ is absent , A ₁ , A ₂ , A ₃ , X ₁ , X ₂ , X ₃ , Y ₁ , Y ₂ and Y ₃ are as described above (eg in overview and in thiazolidinone compounds and derivatives).
Reaction scheme I

  In general, compounds of formula (I) are prepared by first treating a compound of formula (a) with 1 equivalent of a base, such as NaOH, at ambient temperature. Thereafter, the compound of formula (b) dissolved in a suitable solvent such as THF is added to the reaction mixture. The resulting reaction mixture is then stirred for a period of between about 1 hour and about 24 hours. An acid such as HCl is then added to the reaction mixture. The resulting reaction mixture is then stirred for a period of between about 1 hour and about 24 hours. The compound of formula (c) is then isolated from the reaction mixture by standard isolation and purification techniques. Subsequent treatment of the compound of formula (c) with the compound of formula (d) under standard Knoevenagel condensation conditions provides the desired product of formula (I).

Further, the compound of the chemical formula (I) can be prepared according to the following reaction scheme 2. Where A ₁ , A ₂ , A ₃ , Y ₁ , Y ₂ and Y ₃ are as described above (eg in the overview and in the thiazolidinone compounds and derivatives) and W is halo:
Reaction scheme 2

In general, a compound of formula (I) is prepared by first converting a compound of formula (e) with a compound of formula (f) and a catalytic amount of piperidine in standard Knoevenagel condensation conditions, for example, in glacial acetic acid, alcohol or another suitable solvent. For example by refluxing in the presence of. The compound of formula (g) is then isolated from the reaction mixture by standard isolation and purification techniques. Subsequent treatment of the compound of formula (g) with the compound of formula (h) under standard Ullmann condensation conditions, for example, in the presence of Cu or Cu ₂ O or CuO, results in the desired product of formula (I). can get.

Further, the compound of the chemical formula (I) can be prepared according to the following reaction scheme 3. Where A ₁ , A ₂ , A ₃ , Y ₁ , Y ₂ and Y ₃ are as described above (eg in the overview and in the thiazolidinone compounds and derivatives) and W is halo:
Reaction scheme 3

  In this reaction scheme, the first step is a Ullmann condensation between a compound of formula (e) and a compound of formula (h) to produce a compound of formula (c) and then a compound of formula (d) The desired product of formula (1) is obtained after Knoevenagel condensation with

  Starting compounds of formula (e) can be purchased from different chemical suppliers or by methods well known to those skilled in the art or by FCBrown et al., J. Am. Chem. Soc., 78, 384- 388 (1956); RE Strube, Organic Synthesis, CV 4, 6; KSMarkley and EEReid, J. Am. Chem. Soc., 52, 2137-2141 (all of which are hereby incorporated by reference in their entirety. Can be synthesized by methods similar to those disclosed in (incorporated).

In a manner similar to that described above, 3-[(3-trifluoromethyl) phenyl] -5-[(4-carboxyphenyl) methylene] -2-thioxo-4-thiazolidinone (herein CFTR _inh -172 and (See FIG.1C) and the synthesis of analogs with trifluoromethyl and carboxy substituents at different positions (see, e.g., FIG.1D) were synthesized using 2-thioxo-3- [a-trifluoro Achieved by Knoevenagel condensation of methyl-4-phenyl] -4-thiazolidinone (a = 2, 3 or 4) with b-carboxybenzaldehyde (b = 2, 3 or 4) in the presence of piperidine. The precipitate was filtered, washed with ethanol, dried, and recrystallized 2-3 times from ethanol to give bright yellow crystals (yield 70-85%). The structure was confirmed by ¹ H-NMR. The purity was> 99% as judged by thin layer chromatography and HPLC.

EXAMPLES The following examples are presented in order to fully disclose and explain how to make and use the present invention to those skilled in the art and to the extent that the inventors consider the invention to be. It is not intended to limit the above, and does not indicate that the following experiment is all, but is merely an experiment conducted. Efforts have been made to ensure accuracy with respect to numbers used (eg amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

  Although the synthesis | combination of the compound of this invention is illustrated using the following Example, it is not limited to these.

Synthesis example
Synthesis of 3-[(3-trifluoromethyl) phenyl] -5-[(4-carboxyphenyl) methylene] -2-thioxo-4-thiazolidinone
A. To a stirred solution of 3-trifluoromethylaniline (1.6 g, 10 mmol) and triethylamine (1 g, 10 mmol) dissolved in ethyl acetate (10 mL) was added carbon disulfide (0.8 g, 10 mmol) over a period of 30 minutes. It was dripped. When the addition was almost half complete, a mild exothermic reaction began, which could be easily controlled by using an ice bath intermittently. After stirring overnight, the thick yellow slurry was filtered and the precipitate was washed with 50 mL diethyl ether and air dried to give 3 g of a pale yellow dithiocarbamate solid (89%). mp92-95 ° C (dec.).

B. Stir sodium chloroacetate (0.6 mL of NaHCO ₃ solution containing chloroacetic acid (0.064 g, 0.46 mmol), prepared from pH 8-9), cool to 5-10 ° C. and dithiocarbamate (0.3 g, 0.9 mmol ) Was added over 10 minutes. While continuing to stir, the flask was warmed to ambient temperature. After stirring for 2 hours, the solution was cooled to 10 ° C., acidified with concentrated hydrochloric acid, and the reaction mixture was heated to 90-95 ° C. for 30 minutes. The resulting precipitate was filtered, washed with water and recrystallized from ethanol to give 0.103 g of 2-thioxo-3- (3-trifluoromethylphenyl) -4-thiazolidinone as shiny crystals, 83% The yield was obtained. mp 177-178 ° C.

C. 2-Thioxo-3- (3-trifluoromethylphenyl) -4-thiazolidinone (0.1 g, 0.36 mmol) and 4-carboxyl obtained above dissolved in absolute alcohol (1 mL) and piperidine (1 drop) A mixture of benzaldehyde (0.054 g, 0.36 mmol) was stirred and refluxed for 30 minutes. The yellow precipitate was filtered, washed with ethanol, dried and recrystallized from ethanol to give 0.108 g (73%) of the title compound as a yellow crystalline solid. mp: 180-182 ° C.

D. The following compounds were prepared in a similar manner as described above:
3-[(3-trifluoromethyl) phenyl] -5-[(3-carboxy-4-hydroxyphenyl) methylene] -2-thioxo-4-thiazolidinone;
3-[(3-trifluoromethyl) phenyl] -5-[(3,4,5-trihydroxyphenyl) methylene] -2-thioxo-4-thiazolidinone;
3-[(3-trifluoromethyl) phenyl] -5-[(2,3,4-trihydroxyphenyl) methylene] -2-thioxo-4-thiazolidinone;
3-[(3-trifluoromethyl-4-fluoro) phenyl] -5-[(3-carboxy-4-hydroxyphenyl) methylene] -2-thioxo-4-thiazolidinone; and
3-[(4-Fluoro-3-trifluoromethyl) phenyl] -5-[(4-carboxyphenyl) methylene] -2-thioxo-4-thiazolidinone.

  The following materials and methods were used in the examples below.

Cell lines, mice and compounds Fischer rat thyroid (FRT) cells co-expressing human wild-type CFTR and the halogenated indicator YFP-H148Q were generated as previously described (Galietta et al. 2001 J. Biol. Chem. 276: 19723-19728). Coon cells in 96-well black wall microplates (Corning Costar) supplemented with 5% fetal bovine serum, 2 mM L-glutamine, 100 U / mL penicillin, and 100 μg / mL streptomycin at a density of 20,000 cells per well Seeded in modified F12 medium. The assay was performed 48 hours after plating when cells were confluent (~ 40,000 cells / well).

  Initial cleaning was performed using various collections of 50,000 drug-like compounds from ChemBridge (San Diego, Calif.), Obtained as 10 mM stock solutions dissolved in DMSO and diluted to 100 mM in 96-well microplates. Structure-activity analysis was performed on analogs purchased from ChemBridge and ChemDiv (San Diego, Calif.).

  Wild type and cystic fibrosis (ΔF508 homozygous mutation) mice were bred at the CF Animal Core facility at the University of California (San Francisco, UCSF). The animal protocol was approved by the UCSF Committee on Animal Research.

T84 and Caco-2 cells were obtained from the UCSF cell culture facility. T84 cells were cultured in a 1: 1 mixture of DHEM and Hams F12 supplemented with 5% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, plated on Snapwell inserts (Corning Costar) and humidified (5% Grow at 37 ° C. in an O ₂ /95% CO ₂ ) atmosphere. Cells 10-14 days after plating were used. Caco-2 cells were cultured in DMEM containing 10% fetal bovine serum, 1% non-essential amino acids, 100 U / mL penicillin, and 100 μg / mL streptomycin and cultured on Snapwell inserts. Cells 21-24 days after plating were used. Wild-type mice with CD1 genetic background were bred as described above. Male Wistar rats (200-250 g) were purchased from the Jackson Laboratory. The animal protocol was approved by the UCSF Committee on Animal Research. Human colon fragments were newly obtained at the time of resection surgery, transported in ice-cold saline, and used within 1 hour after resection.

Forskolin, 8-bromo cGMP, amiloride, cholera toxin and STa toxin were purchased from Sigma Chemical Co. (St. Louis, MO). CFTR _act -16 was purchased from ChemBridge (San Diego, CA).

Screening procedure
3 meter robot arm, CO ₂ incubator, plate washer, liquid processing workstation, barcode reader, delidding station, and two FluoStar fluorescent plate readers (BMG Labtechnologies, Offenburg, Germany), 2 each The assay was performed using a custom screening system (Beckman) consisting of two syringe pumps and an HQ500 / 20X (500 ± 10 nm) excitation and HQ535 / 30M (535 ± 15 nm) emission filter (Chroma). The robotic system was integrated using modified SAMI version 3.3 software (Beckman) for the two plate readers. The custom software is written in VBA (Visual Basic for Application) to calculate the normalized fluorescence gradient (obtained from the halide inflow rate) minus the baseline from the stored data file. It was.

The assay consists of placing a 40-60 96-well plate containing FRT cells in an incubator (37 ° C., 90% humidity, 5% CO ₂ ), and a 96- containing a test compound and a disposable plastic pipette tip in the carousel. Constructed by placing a well plate. To initiate the assay, each well of a 96-well plate was washed 3 times in PBS (300 μl / wash), leaving 50 μl PBS. Add 10 μL CFTR-activation cocktail (5 μM forskolin, 100 μM IBMX, PBS containing 25 μM apigenin), and after 5 minutes, add one test compound (0.5 μL of 1 mM DMSO solution) to each well, The final concentration was 10 μM. After 10 minutes, the 96-well plate was transferred to a plate reader and a fluorescence assay was performed. Fluorescence was added to each well for 2 s (baseline), followed by a rapid addition (<0.5 s) of 160 μl equimolar PBS (in which 137 mM Cl ⁻ was replaced by I ⁻ ) for 12 s by recording continuously the (200 ms / point) fluorescence, CFTR-mediated I ^- assayed for transportation.

Intracellular [cAMP] and toxicity assays
[cAMP] and phosphatase assays were performed as previously reported (Galietta et al. 2001 J. Biol. Chem. 276: 19723-19728). Cytotoxicity was assessed by the dihydrorhodamine method 24 hours after cell incubation with 0-1000 μM inhibitor. Animal toxicity was assessed by measuring serum chemicals and hematology in mice (UCSF Clinical Laboratory) 5 days after daily intraperitoneal injections of 0-100 μg / kg inhibitor.

MDR-1 activity By measuring ³ H-vincristin accumulation in an immortalized human tracheal cell line, 9HTEo- / Dx, where endogenous expression of MDR-1 is upregulated by selection in increasing doxorubicin concentrations MDR-1 activity was assessed (Rasola et al. 1994 J. Biol. Chem. 269: 1432-1436). Cells were seeded in 24-well microplates (200,000 cells / well). After 48 hours, the cells were washed with a solution containing 130 NaCl, 2 KCl, 1 KH ₂ PO ₄ , 2 CaCl ₂ , 10 Na-Hepes (pH 7.3) and 10 glucose (expressed in mM) for 1 hour 37 Incubated at 200 ° C. with 200 μl of the same solution containing ³ H-vincristine (0.7 μM; 1 μCi / mL). The cells were then washed 3 times with ice-cold solution and lysed in 0.25M NaOH. The amount of vincristine was determined by scintillation counting.

Short-circuit current test using CFTR-expressing FRT cells
Snapwell inserts containing CFTR-expressing FRT cells or human bronchial epithelial cells were placed in a Ussing chamber system. In FRT cells, the half-chamber was filled with 5 mL of 75 mM NaCl and 75 mM sodium gluconate (top) and 150 mM NaCl (basal) (pH 7.3), and the basolateral membrane was permeabilized with 250 μg / mL amphotericin B (Galietta et al. al. 2001 J. Biol. Chem. 276: 19723-19728). For bronchial epithelial cells and T84 cells, both half chambers contained Krebs bicarbonate solution. Half chambers were continuously bubbled with air (FRT cells) or with 5% CO ₂ (bronchi and T84 cells) and maintained at 37 ° C. Short circuit currents were recorded continuously using a DVC-1000 voltage clamp (World Precision Instruments, Sarasota, FL) using an Ag / AgCl electrode and a 1M KCl agar bridge.

Cl ^- was a patch clamp analysis film current channel activity was measured in whole cell structure. Cl ^- in the recording channel, the extracellular (bath (bath bath)) solution contained the following (expressed in mM): 150 NaCl, 1 CaCl 2, 1 MgCl 2, 10 glucose, 10 mannitol, 10 TES (pH 7.4 ), The intracellular (pipette) solution contained: 120 CsCl, 1 MgCl ₂ , 10 TEA-Cl, 0.5 EGTA, 1 Mg-ATP, 10 Hepes (pH 7.3). CFTR was activated by extracellular solution containing forskolin (5 μM). The time course of membrane conductance was monitored in response to -100 and +800 mV AC voltage pulses. At a fixed time, the protocol was interrupted and a current-voltage relationship was created (from -100 to +100 mV, voltage pulse in 20 mV increments). Was activated by; volume - ^- sensitive Cl channels hypotonic solution (250 mOsm / kg extracellular NaCl which was reduced to 120NaCl). Calcium - sensitive Cl ^- channel in human bronchial epithelial cells, and the 100 [mu] M UTP was activated by the addition to the extracellular solution.

ATP- sensitive K ⁺ channel patch clamp analysis extracellular (bath) solution (expressed in mM) 130 NaCl, 2 KCl, 1 KH 2 PO 4, 2 CaCl 2, 2 MgCl 2, 10 Na-Hepes (pH 7.3 ) And membrane potential in pancreatic β-cell line INS-1 containing 10 glucose. Pipettes contained the following (expressed in mM): 140 KCl, 1 CaCl ₂ , 2 mM MgCl ₂ , 10 EGTA, 0.5 MgATP, 10 K-Hepes (pH 7.3). After achieving the whole cell structure, the amplifier was switched to current-clamp mode.

Intestinal fluid secretion and short circuit current
At the beginning of the three assays, fluid retention in the ileal loop was measured (Oi et al. 2002 Proc. Natl. Acad. Sci. USA 99: 3042-3046; Gorbach et al. 1971 J. Clin. Invest. 50: 881 -889). Mice with CD1 genetic background (8-10 weeks old, 25-35 g body weight) (or △ F508 homozygous mice) were starved for 24 hours and anesthetized with intraperitoneal ketamine (40 mg / kg) and xylazine (8 mg / kg) did. Body temperature was maintained at 36-38 ° C. during surgery using a heating pad. The abdominal cavity was excised small, exposing the small intestine, and a closed ileal loop (length 20-30 mm) close to the cecum was isolated by suture. The loops were injected with 100 μl PBS monotoxic or with PBS containing cholera toxin (1 μg). In some experiments, the inhibitor (150 μg / kg) was administered by intraperitoneal injection. The abdominal resection was closed with suture and the mice were awakened from anesthesia. The mice were anesthetized at 6 hours, the intestinal loop was externalized, the mesentery and connective tissue were removed, and the loop length and weight were measured.

In a closed adult mouse model of secretory diarrhea, mice were bred with 0.1 mL of 7% bicarbonate buffer (or buffer only) containing cholera toxin (10 μg) using an orogastric needle. (Richardson et al. 1986 Infect. Immun. 54: 522-528; Gabriel et al. 1999 Am J. Physiol 276: G58-G63). The four experimental groups were as follows: control (buffer only), cholera treatment, cholera-treatment + inhibitor (150 μg / kg ip 2 minutes before gavage), and inhibitor only. After 6 hours, the mice were euthanized, the small intestine (from the pylorus to the cecum) was externalized, and the associated mesentery and connective tissue were removed. The intestine was weighed and then opened vertically, the lumen fluid was removed (by blotting) and weighed again. Fluid retention was calculated from the ratio of intestinal weight before and after lumen fluid removal. For short-circuit current measurements, rat colon strips were isolated, the muscle layer removed by blunt dissection, placed in a Ussing chamber (area 0.7 cm ² ), and oxygenated bicarbonate containing 10 μM indomethacin. Immerse in salt Ringer solution. Short-circuit current was measured after inhibiting Na ⁺ current with amyloid (10 μM) followed by stimulation with forskolin (20 μM) followed by addition of inhibitor.

Synthesis of ¹⁴ C-labeled CFTR _inh -172 (Figure 6)
Carbon disulfide (0.8 g, 10 mM) was added dropwise over 30 minutes to a stirred solution of 3-trifluoromethylaniline (1.6 g, 10 mM) and triethylamine (1 g, 10 mM) dissolved in ethyl acetate (10 mL). The product 2-thioxo-3- (3-trifluoromethylphenyl) -4-thiazolidinone was synthesized. An ice bath was used to prevent overheating during the reaction. After stirring overnight, the thick yellow slurry was filtered and the precipitate was washed with 50 mL ether and air dried to yield 3 g (89% yield) of pale yellow dithiocarbamate solid (mp 92-95 ° C.). Obtained. NaBr- ¹⁴ C- acetate was stirred (Br @ - ¹⁴ C-acetate (Amersham), 55mCi / mmol, 64mg, 0.6mL of water 0.46 mM, prepared from pH8~9 with NaHCO _3), cooled to 5 to 10 ° C. Dithiocarbamate (0.3 g, 0.9 mM) was added over 10 minutes. Stirring was continued until the flask was warmed to room temperature. After 2 hours, the solution was cooled to 10 ° C., acidified with concentrated HCl, and heated to 90-95 ° C. for 30 minutes. The resulting precipitate was filtered, washed with water and recrystallized from ethanol to give 103 mg of the desired product as shiny crystals (83% yield). mp 177-178 ° C .; specific activity ( ¹⁴ C) 55 mCi / mmol.

In the synthesis of 2-thioxo-3- (3-trifluoromethylphenyl) -5- [4-carboxyphenylmethylene] -4-thiazolidinone ( ¹⁴ C-5) ( ¹⁴ C-CFTR _inh -172), the absolute alcohol ( 2-thioxo-3- (3-trifluoromethylphenyl) -4-thiazolidinone ( ¹⁴ C-5) (100 mg, 0.36 mM) and 4-carboxybenzaldehyde (54 mg, 0.36 mM) dissolved in piperidine (1 drop) mM) was refluxed for 30 minutes. The yellow precipitate was filtered, washed with ethanol, dried and recrystallized from ethanol to give 108 mg (73% yield) of yellow crystals. mp 180-182 ° C .; specific activity ( ¹⁴ C) 54 mCi / mmol.

Pharmacokinetic study
A bolus of ¹⁴ C-CFTR _inh -172 (50 μCi) in PBS containing 3% DMSO (titrated to pH 7.4 using NaOH) was administered intravenously to the rat's vein for 1 minute (male). Sprague-Dawley rats, 360-420g). Blood was collected from the catheter at specific times. ¹⁴ C-radioactivity was determined in plasma (isolated by centrifugation of whole blood at 14,000 g for 10 min) using a LS-6500 Multi-Purpose Scintillation Counter (Beckman) by scintillation counting (Scintiverse SE, Fisher, CA). Were determined. Pharmacokinetic analysis was performed using WinNonlin software (Pharsight). After the final blood / tissue sample was collected, the rats were sacrificed by pentabarbital overdose. All animal procedures were approved by the UCSF Committee on Animal Research.

Tissue distribution and emission studies
A bolus of ¹⁴ C-CFTR _inh -172 (2 μCi) was administered intravenously via the tail vein to mice (male CD1 mice, 30-35 g) over 1 minute. Mice were sacrificed at 5, 30, 120 and 240 minutes. Organs were removed, weighed and homogenized in distilled water (10-50 vol%). Radioactivity was determined by scintillation counting of homogenate (25-50 μL) and expressed as total ¹⁴ C-radioactivity per organ (or per gram tissue for skeletal muscle). At the same time, blood, urine and bile (from gallbladder or duodenum) were collected and ¹⁴ C-radioactivity was measured and expressed as / mL fluid. Excretion studies were performed with urine and stool collections over the first 24 hours after administration of ¹⁴ C-CFTR _inh -172. Tissue distribution studies were also performed on rats prepared as for pharmacokinetic studies.

Analysis of inhibitor metabolism Spotted on silica plates with aliquots of body fluid (plasma, urine, bile) and liver homogenate, and by thin layer chromatography using ethyl acetate: hexane: methanol (1: 1: 0.1) solvent system separated. For the original inhibitor, rf˜0.5 was obtained. Autoradiography was performed with a Transcreen LE amplification system (Kodak) using Hyperfilm (Amersham). ¹⁴ C-labeled CFTR _inh -172 standard was included on all plates.

Short-circuit current measurement (Example 7)
For cell studies, Snapwell inserts containing T84 cell monolayers were placed in a Ussing chamber system (Navicyte, Harvard Apparatus, Holliston, Mass.). Half chamber is filled with Krebs-bicarbonate solution (expressed in mM), NaCl 120, NaHCO ₃ 25, KH ₂ PO ₄ 3.3, K ₂ HPO ₄ 0.8, MgCl ₂ 1.2, CaCl ₂ 1.2, glucose 10. And then continuously bubbled with 5% CO ₂ /95% O ₂ . High K ⁺ buffer (expressed in mM) NaCl 65, KCl 67.5, KH 2 PO 4 1.5, CaCl 2 1, MgCl 2 0.5, HEPES 10, containing glucose 10. Low Cl ^- buffer contains sodium gluconate 120 (expressed in mM), KH ₂ PO ₄ 3.3, K ₂ HPO ₄ 0.8, MgCl ₂ 1.2, CaCl ₂ 1.2, HEPES 10, glucose 10 (maintained at 37 ° C). Contained and continuously bubbled with air. Mice were anesthetized with intraperitoneal ketamine (40 mg / kg) and xylazine (8 mg / kg) for measurement in the mouse colon. The ileum was removed, washed with ice-cold Krebs buffer, opened along the border of the intestinal membrane, and placed in a μ-Ussing chamber (area 0.7 cm ² , World Precision Instruments, Sarasota, FL). For measurement in the human intestine, the colon segment was bluntly dissected and the muscle layer removed and placed as described above. The half chamber was filled with oxygenated Ringer bicarbonate solution containing 10 μM indomethacin. Short circuit currents were recorded using a DVC-1000 voltage clamp (World Precision Instruments) with an Ag / AgCl electrode and 1M KCl agar bridge. Agonists / inhibitors were added to the half-chamber as follows.

In vivo intestinal fluid secretion in mouse and rat models (Examples 5 and 7)
Mice with a CD1 genetic background (8-10 weeks old, 25-35 g body weight) were allowed access to water for 24 hours but no food. Mice were anesthetized as described above and body temperature was maintained at 36-38 ° C. using a heating pad during surgery. A small abdominal incision was made, the small intestine was exposed, and the closed ileal loop (length 20-30 mm) near the cecum was isolated by suture. Loops were injected with 100 μL of PBS alone or PBS containing cholera toxin (1 μg). In some experiments, CFTR _inh -172 (0-200 μg) was administered by intraperitoneal injection at specific times before and after cholera toxin injection. The abdominal incision was closed by suturing and the mouse was awakened from anesthesia. The mice were anesthetized at 6 hours, the intestinal loop was externalized, the mesentery and connective tissue were removed, and the loop length and weight were measured.

To measure enterotoxin-induced fluid secretion in a rat closed loop model, male Wistar rats (body weight 200-250 g) were anesthetized with sodium pentobarbital (45 mg / kg). Loops (40-60 mm) were isolated and injected with 300 μl of PBS alone or PBS containing cholera toxin (10 μg) or STa toxin (0.1 μg). In some experiments, CFTR _inh -172 (200 μg) was ingested by intraperitoneal injection after administration of cholera toxin or STa toxin. Loop length and weight were measured at 3 hr (STa) or 6 hr (cholera toxin).

In the study of orally administered CFTR _inh -172, an open loop mouse model was used. In this model, mice receive 7% bicarbonate buffer or cholera toxin (1 μg in 7% bicarbonate buffer) alone, and CFTR _inh -172 (200 μg in vitamin E TPGS, see below). Forced feeding was done using a mouth-stomach feeding needle. After 6 hours, the small intestine (from the pylorus to the cecum) was externalized and the associated mesentery and connective tissue were removed. The intestine was weighed and opened longitudinally, the lumen fluid was removed and weighed again to quantify fluid retention.

Caco-2 permeability assay
Caco-2 cells were cultured on porous inserts and a monolayer resistance of 400-600 Ωcm ^-1 was obtained. For transport studies, the medium was replaced with an equal volume of Hank buffered salt solution (HBSS) containing 15 mM glucose and 25 mM HEPES (pH 7.3). After 1 hr, CFTR _inh -172 (25 μM) was added to the upper chamber and the plate was gently rocked at 37 ° C. At specified times, 50 μL of solution was removed from the lower (receiver) chamber and the CFTR _inh -172 concentration was measured by UV absorbance (385 nm). Apparent permeability (Papp) was calculated from the following formula:
Papp = dC / dT X (Vr / AC0)
(Where dC / dT indicates the rate of increase of the CFTR _inh -172 concentration in the receiver chamber, Vr is the volume of the receiver chamber, A is the monolayer surface area, and CO is the CFTR _inh -172 in the donor chamber. Initial concentration).

Pharmacokinetics and oral bioavailability studies Mice were temporarily anesthetized with halothane and ¹⁴ C-CFTR _inh -172 (12 μCi) (d-α-tocophenyl polyethylene glycol 1000 succinate, solubilized with vitamin E TPGS, in water 0.5% w / v CFTR _inh -172) in a dissolved 10% w / v TPGS suspension was orally gavaged. For comparison, other mice received ¹⁴ C-CFTR _inh -172 (2 μCi) intravenously via tail vein infusion. Blood was collected from the tail vein at specific times and plasma ¹⁴ C radioactivity was measured. At 6 hours, the mice were sacrificed by pentobarbital overdose, the organs were removed and the radioactivity in the homogenate was measured.

Biological Example 1
The main screening techniques used to identify compounds Screening invention CFTR inhibitors by direct CFTR- inhibitor interaction CFTR Cl ^- were designed to identify conductance inhibitor. As outlined in FIG. 1A, CFTR was pre-stimulated in CFTR-expressing FRT cells by activating a cocktail containing forskolin, IBMX and apigenin. Multiple mechanisms (cAMP increase, phosphodiesterase inhibition, and direct CFTR binding) by activation of CFTR by directly rather than more proximal step in the signal transduction pathway CFTR Cl ^- inhibitor that blocks transport path could be identified. FRT cells co-expressed a yellow fluorescent protein-based Cl ⁻ / I ⁻ sensor that provided a quantitative fluorescence reading of inhibitory potency (eg Jayaraman et al., 2000, J. Biol. Chem. 275: 6047-6050; Galietta et al., 2001, Am, J. Physiol. 281: C1734-C1742). After pre-stimulating CFTR and adding the compound, the cells undergo an inward I ^- gradient, leading to an influx of I- ^and a decrease in fluorescence. Each assay to record baseline fluorescence for 2 seconds, followed, I ^- was constructed from a continuous record of fluorescence of 12 seconds after the rapid addition of a solution containing. Compounds were tested separately at a 10 μM concentration in a 96-well format using a fully automated high throughput screening device (see Example 2 below).

FIG. 1B is a graph showing a representative curve as relative YFP fluorescence versus time from a primary screen of 50,000 compounds using the assay of FIG. 1A. The 49,993 compound had no significant effect on the kinetics of I ^- influx, as quantified from the slope of fluorescence decreasing after I ^- addition (<10% reduction in slope). Seven compounds produced a small decrease in negative slope (10-52%), almost all of which were 2-thioxo-4-thiazolidinone heterocycles with substituted phenylmethylene and phenyl moieties (Figure 1C) Having a similar core structure. Over 250 analogs with a thiazolidinone core structure were subsequently screened to identify the most potent CFTR inhibitors.

FIG. 1D shows that the most effective thiazolidinone CFTR inhibitor identified in the screening is 3-[(3-trifluoromethyl) phenyl] -5-[(4-carboxyphenyl) methylene] -2-thioxo-4-thiazolidinone (Referred to herein as CFTR _inh -172), as well as five analogs with significant inhibitory potency. Thus, the following compounds were identified as CFTR inhibitors: 3-[(3-trifluoromethyl) phenyl] -5-[(4-carboxyphenyl) methylene] -2-thioxo-4-thiazolidinone (CFTR _inh − 172); 3-[(3-trifluoromethyl) phenyl] -5-[(4-nitrophenyl) methylene] -2-thioxo-4-thiazolidinone (CFTR _inh -020); 3-[(3-trifluoro Methyl) phenyl] -5-[(4-oxycarboxyphenyl) methylene] -2-thioxo-4-thiazolidinone (CFTR _inh -029); 3-[(3-trifluoromethyl) phenyl] -5-[(3 , 4-Dihydroxyphenyl) methylene] -2-thioxo-4-thiazolidinone (CFTR _inh -185); 3-[(3-trifluoromethyl) phenyl] -5-[(3,5-dibromo-4-hydroxyphenyl ) Methylene] -2-thioxo-4-thiazolidinone (CFTR _inh -214); and 3-[(3-trifluoromethyl) phenyl] -5-[(3-bromo-4-hydroxy-5-nitrophenyl) methylene ] -2-Thioki 4-thiazolidinone (CFTR _inh -236). The most effective CFTR inhibitors have one or more electron withdrawing groups, such as 3-trifluoromethyl groups on ring 1, as described above, and electron withdrawing groups or polar substituents on ring 2. Had. CFTR _inh -172 was selected for further analysis. The relative potencies were as follows: 0.2 (CFTR _inh -020), 0.3 (CFTR _inh --029), 1.0 (CFTR _inh -172), 0.2 (CFTR _inh -185), 0.1 (CFTR _inh -214), And 0.1 (CFTR _inh -236).

To investigate the effect of the ring position of trifluoromethyl and carboxyl substituents, 8 analogs of CFTR _inh -172 were synthesized. In these analogs, the substituents moved to each unique position on rings 1 (trifluoromethyl) and 2 (carboxy). Relative inhibition of 3-[(a-trifluoromethyl) phenyl] -5-[(b-carboxyphenyl) methylene] -2-thioxo-4-thiazolidinone analogues compared to CFTR _inh -172 (efficacy 1.0) The efficacy was as follows: 0.69 (a = 2, b = 2), 0.70 (2, 3), 0.66 (2, 4), 0.74 (3, 2), 0.90 (3, 3), 0.67 ( 4, 2), 0.64 (4, 3) and 0.56 (4, 4).

Biological Example 2
Characterization of CFTR _inh -172 The level of CFTR inhibition of a particular dose of the subject thiazolidinone compound was determined using the fluorescence assay shown in FIG. 1A and described above. FIG. 2A shows dose-inhibition data for CFTR _inh -172 as relative YFP fluorescence versus time. Significant CFTR inhibition was seen with this thiazolidinone compound at a concentration of 0.3-0.6 μM. Figure 2B, CFTR _inh -172 (shown in the graph as the relative transport rate versus time of addition or after washout) inhibition by the completed ~10min (t _1/2 = 4min), reversed after washout and t _{1 / 2 to 5} min (inset). The relative transport rates shown in Figure 2C show that CFTR _inh -172 effectively inhibits CFTR activation by multiple types of agonists not included in the activation cocktail used for initial screening. Indicates. These agonists include genistein, CPT-cAMP, CPX, 8-MPO and the potent benzoflavone CFTR activator UCCF-029 (2- (4-pyridinium) benzo [h] 4H-chromen-4-one bisul And benzimidazolone CFTR activator UCCF-853 (Galietta, et al., 2001, J. Biol. Chem. 276: 19723-19728).

Electrophysiological experiments were also performed to establish the inhibitory potency and specificity of CFTR _inh -172. FIG. 3A shows rapid, dose-dependent inhibition of short circuit currents in CFTR-expressing FRT cells with CFTR _inh -172 added to a solution soaking the apical cell surface. FIG. 3B shows the mean dose-inhibition relationship of CFTR _inh -172 (K _d ˜300 nM, Hill count ˜1) and glibenclamide (K _d ˜300 μM) tested under the same conditions.

This similar inhibitory potency against thiazolidinones is found in cells that naturally express wild-type CFTR, such as primary cultures of T84 cells and human bronchial epithelial cells, and transfected FRT cells that express G551D-CTFR and ΔF508-CFTR ( Found after cold calibration). In studies in bronchial cells, Na ⁺ channels were blocked with amiloride, so the baseline current was largely CFTR-dependent. When CFTR _inh -172 was applied from the apical side after maximal CFTR activation with CPT-cAMP, the short circuit current was strongly inhibited (FIG. 3C, left). CFTR _inh -172 also inhibited short circuit current when added from the basolateral side (FIG. 3C, right).

Whole cell membrane current was measured in CFTR expressing FRT cells as illustrated in FIG. 3D. Stimulation with 5 μM forskolin produced a membrane current of 381 ± 47 pA / pF (n = 4) at +100 mV (total membrane capacity 21 ± 3 pF). The current-voltage relationship was linear as expected for pure CFTR current (FIG. 3F). Extracellular perfusion with 2 μM CFTR _inh -172 resulted in a rapid decrease in current at all membrane potentials, suggesting voltage-independent CFTR inhibition. Using low concentrations of CFTR _inh -172 (0.2 μM) and confirming the lack of voltage dependence of channel blockade resulted in ˜50% inhibition (FIG. 3F).

The specificity of CFTR _inh -172 for inhibition of CFTR was also investigated. Two non-CFTR-Cl ^- were studied channel. CFTR _inh -172 at a concentration of 5 μM did not inhibit Ca ²⁺ -activated Cl ⁻ secretion generated by adding UTP (100 μM) to the apical soaking solution in polarized human bronchial epithelial cells (FIG. 4A). Maximum UTP- dependent short-circuit current, in the absence of _{CFTR inh -172 9.9 ± 0.5μA / cm} 2, was the existing ^{10.0 ± 0.2μA / cm 2 (SE} , n = 4). 5 μM CFTR _inh -172 also did not block the volume activated Cl ⁻ current induced in FRT cells by extracellular perfusion with 250 mosM / kg hypotonic solution (FIG. 4B).

The activity of the CFTR homolog, ATP-binding cassette transporter MDR-1 (multiple drug resistance protein-1) was measured with 9HTE _0- / Dx overexpressing MDR-1 (Rosola et al. 1994 J. Biol. Chem 269: 1432-1436). Vincristine accumulation, which is inversely proportional to active drug release by MDR-1, was greatly increased by the MDR-1 inhibitor verapamil (100 μM) (FIG. 4C). CFTR _inh -172 (5 μM) did not affect vincristine accumulation, and thus MDR-1 was not inhibited.

Another homolog of CFTR is the sulfonylurea receptor (SUR) that regulates the activity of ATP-sensitive K ⁺ channels (K-ATP channels) (Aguilar-Bryan and Bryan 1999 Endocr. Rev. 20: 101-135). SUR1 is expressed in pancreatic β cells, where membrane potential and insulin release are controlled. Like glibenclamide, sulfonylureas cause insulin release (and a hypoglycemic response) by blocking K-ATP channels and membrane depolarization. To determine whether CFTR _inh -172 also blocks K-ATP channels, the membrane potential in a rat pancreatic β cell line, INS-1, was measured (FIGS. 4D, 4E). In contrast to the large membrane depolarization caused by glibenclamide, CFTR _inh -172 (2 and 5 μM) did not depolarize the membrane potential. 5 μM CFTR _inh -172 caused much less hyperpolarization than that caused by the K-ATP channel activator diazoxide (100 μM). With the addition of studies, 5 [mu] M of CFTR _inh -172 water channel (AQP1), urea transporter ^{(UT-B), Na +} / H + exchanger (NHE3) and Cl ^- / HCO ₃ ^- exchanger body (AE1) It was shown not to block.

Another analysis showed that 5 μM CFTR _inh -172 did not affect cellular cAMP production or phosphatase activity. In FRT cells, the basal cAMP level is 225 ± 22 fmol / well, which is 1290 ± 190 fmol / well (no inhibitor) and 1140 ± 50 (+ CFTR _inh -172) 30 minutes after stimulation with 20 μM forskolin. increased to n = 3). Judging using the dihydrorhodamine assay, CFTR _inh -172 was non-toxic to FRT cells after 24 hours at concentrations up to 100 μM. In mice, intraperitoneal injection of 1000 μg / kg CFTR _inh -172 daily for 7 days did not cause obvious toxicity. Food and water intake did not decrease. Serum electrolyte concentration, glucose, liver function index, serum creatinine, amylase and hematocrit did not change. Furthermore, very large doses of CFTR _inh -172 single systemic dose (10 mg / kg) did not cause obvious toxicity.

Biological Example 3
In vivo efficacy
The efficacy of CFTR _inh -172 was tested in vivo using two assays of intestinal fluid secretion induced by cholera toxin and by short-circuit analysis in isolated intestines. In the first assay, a series of small intestine closed loops were created in vivo and small volumes of saline or saline containing cholera toxin were injected into the lumens of alternating loops. Lumen fluid retention was determined after 6 hours. As shown in FIG. 5A, there was significant fluid retention and swelling in the cholera toxin treatment loop, but the adjacent control (saline) loop remained empty. A single dose of CFTR _inh -172 (150 μg / kg ip) prior to cholera toxin infusion effectively prevented fluid retention in the toxin-treated intestinal loop.

Data from a series of these experiments are summarized in the graph of FIG. 5B. CFTR _inh -172 significantly reduced fluid secretion to fluid secretion in the saline control loop, and inactive thiazolidinone analogs did not block fluid secretion. As suggested by previous data (Gabriel et al. 1994 Science 266: 107-109), the cholera toxin-treated intestinal loop from homozygous △ F508-CFTR mice remains empty and CFTR is involved in intestinal secretion Was shown to do. In the second assay, intestinal fluid secretion was induced by oral administration of cholera toxin (10 μg) and CFTR _inh -172 was administered systemically. After 6 hours, there was significant fluid accumulation as measured by weighing the entire small intestine. CFTR _inh -172 administration significantly reduced intestinal fluid retention as visually confirmed and quantified by the ratio of intestinal weight before and after lumen fluid removal (FIG. 5C).

FIG. 5D shows CFTR _inh -172 inhibition of short circuit current across intact rat colonic mucosa. After inhibition of Na ⁺ current by amiloride, forskolin caused a rapid increase in short circuit current. CFTR _inh -172 added to the mucosal solution inhibited the short circuit current with greater efficiency than when added to the capsular solution. This may be associated with impaired access to colonic epithelial cells via residual submucosa. Addition of 5 μM CFTR _inh -172 to mucosal solution alone reduced the short circuit current by> 80%. These results, CFTR Cl by CFTR _inh -172 in the intestine ^- electrophysiological evidence for channel inhibition is provided.

Biological Example 4
Pharmacokinetic analysis Pharmacokinetic analysis in rats was performed by sequentially measuring serum ¹⁴ C radioactivity after a single intravenous bolus injection of ¹⁴ C-labeled CFTR _inh -172. The total amount of inhibitor injected (400 μg, ˜1 mg / kg) was effective as an antidiarrheal drug in rats. FIG. 7 shows that the kinetics of serum ¹⁴ C radioactivity fits well with the 2-compartment model, the distribution volume is 1.2 L, and the AUC (area under the curve) is 3.8 μg · hr / mL. The half-time was 0.14 hr (redistribution) and 10.3 hr (discharge). ¹⁴ C-labeled CFTR _inh -172 was not detected in plasma 72 hours after administration, or in liver or kidney homogenate 14 days after administration.

The tissue distribution of ¹⁴ C-labeled CFTR _inh -172 was determined from organ homogenates and body fluid radioactivity after a single intravenous bolus injection. FIG. 8, Panel A summarizes the ¹⁴ C distribution in the main organ at the specified time after CFTR _inh -172 injection in mice. ¹⁴ C radioactivity was observed mainly within the liver and kidney within 5 minutes and decreased with time. Little radioactivity was found in the brain, heart, skeletal muscle or testis. At later times (30-240 minutes), ¹⁴ C radioactivity accumulated in the intestine. Figure 3, panel B shows a similar organ distribution of ¹⁴ C radioactivity in rats measured 60 minutes after iv bolus injection. Little radioactivity was found in the brain, heart and skeletal muscle. In some experiments, rats were sacrificed 10 days after injection of ¹⁴ C-labeled CFTR _inh -172 (50 μCi).

To determine the mechanism of CFTR _inh -172 accumulation in kidney, liver and intestine, ¹⁴ C radioactivity was measured in serum, urine and bile. Mean urinary radioactivity was 4.2 ± 1.2 × 10 ⁵ cpm / mL in the first 2 hours after injection. The ratio of ¹⁴ C radioactivity in urine to blood ranges from 5 to 7: 1, comparable to a urine to serum osmotic ratio of -5: 1 (1550 mOsm to 310 mOsm), with CFTR _inh -172 being a renal tubule Suggested to be removed from the kidney by glomerular filtration without absorption or secretion. The renal clearance mechanism of CFTR _inh -172 clearance was supported by roughly similar kinetics (data not shown) with decreased ¹⁴ C radioactivity in serum, urine and kidney tissue. The possibility of CFTR _inh -172 accumulation in bile was investigated based on observation of rapid accumulation of ¹⁴ C radioactivity in the liver and slow accumulation in the intestine. ¹⁴ C radioactivity was concentrated ˜9-fold in bile versus blood after administration to mice. To determine if bile CFTR _inh -172 was excreted in the stool or returned to circulation, urine and stool collections were performed on mice for the first 24 hours after radiolabel inhibitor injection. 93 ± 3% excretory radioactivity was found in urine, supporting the main renal excretion mechanism by enterohepatic circulation.

To determine if ¹⁴ C radioactivity measured in organs and fluids corresponds to intact or chemically modified CFTR _inh -172, thin-layer chromatography and autoradiography were used to analyze urine, serum and bile samples, and It performed on the supernatant of the liver homogenate prepared by centrifugation. FIG. 9 shows a single spot of rf˜0.5 for the original CFTR _inh -172 introduced in the bolus injection. Autoradiography of body fluids and organ homogenates showed a single spot at the same rf, indicating that no chemical modification of CFTR _inh -172 occurred.

CFTR _inh -172 is a weak acid with a pKa of 5.5 as determined by spectrophotometric pH titration. At physiological pH, ˜1% CFTR _inh -172 exists as a non-ionized acid with low polarity and high membrane permeability. The rapid uptake of CFTR _inh -172 in the above cell model suggests the feasibility of an orally bioavailable preparation with a warning that protection from low gastric pH may be necessary to prevent precipitation Is done. The results of these pharmacokinetic studies indicate that CFTR _inh -172 is gradually removed in rodents without being chemically modified by renal clearance, and that CFTR _inh -172 is concentrated in the bile and accumulates in the intestine. It is. CFTR _inh -172 did not significantly cross the blood-brain barrier, and there was little accumulation of CFTR _inh -172 in other important organs including heart, lung, skeletal muscle, and testis. Slow renal clearance of CFTR _inh -172, accumulation in the intestine, and little penetration into the blood brain barrier are advantageous for diarrhea prevention applications.

Biological Example 5
Dose response and duration of inhibitory effect of CFTR _inh -172 The purpose of this example is to determine that a single intraperitoneal administration of CFTR _inh -172 inhibits fluid secretion stimulated by cholera toxin in a mouse intestinal loop model It was to extend the above observations on what can be done. In particular, the purpose of this example was to measure the dose response relationship of CFTR _inh -172 inhibitory effects and the apparent half-life for duration.

Initially, intestinal loop fluid absorption and secretion kinetics were determined and mouse models were characterized. To study absorption, loop body fluid volume was measured at specific times after injecting 200 μL of PBS into individual loops. FIG. 10, panel A showed rapid fluid absorption, with 50% fluid remaining in ˜25 minutes. Intraperitoneal administration of CFTR _inh -172 at a dose that strongly inhibits intestinal fluid secretion induced by cholera toxin (20 μg) did not change the fluid absorption rate (measured at 30 minutes) compared to the control (Figure 10, Panel A, inset). To study secretion, cholera toxin (1 μg dissolved in 0.1 mL PBS) was injected into the intestinal loop. Figure 10, Panel B shows a slow onset of fluid secretion over 6 hours, consistent with previous studies in rodent models (Gorbach et al. J. Clin. Invest. 1971 50-881-889: Oi et al. Proc. Natl. Acad. Sci. USA 2002 99: 3042-3046). The rapid absorption of fluid in the intestine under normal conditions suggests that fluid accumulated in the intestinal lumen after active secretion is absorbed as soon as the secretion is blocked, and CFTR inhibition is not limited to cholera inhibition. Administration after the toxin is expected to be effective in preventing fluid retention.

FIG. 11, panel A summarizes the results of a CFTR _inh -172 dose response study in mice that received a single dose of inhibitor by intraperitoneal injection immediately after cholera toxin was injected into the intestinal loop. The basic intestinal fluid volume (dotted line) was almost zero when measured in a loop not injected with cholera toxin. Liquid accumulating in intestinal loops injected with cholera toxin inhibited 90% by CFTR _inh -172, was CFTR _inh -172 (150μg / kg) at 50% inhibition of ～5Myug. The duration of inhibition was measured as in the dose response study except that 20 μg of a single dose of CFTR _inh -172 was administered at different time points before and after cholera toxin. FIG. 11, panel B shows significant inhibition of luminal fluid retention when CFTR _inh -172 is administered 3 hours before and after cholera toxin. However, the inhibition observed at 6 hours prior to cholera toxin was much lower. Considering the 6 hr duration of the cholera toxin induction test, the t _1/2 for the persistence of CFTR _inh -172 inhibition was ˜9-10 hr.

Biological Example 6
To test the antidiarrheal efficacy of CFTR _inh -172 oral bioavailability orally administered CFTR _inh -172, to determine the CFTR _inh -172 pharmacokinetics in mice, CFTR _inh -172 transport across Caco-2 monolayers Was measured. CFTR _inh -172 is a fairly non-polar weak acid (pKa = 5.5) that is thought to precipitate in the stomach, so two drugs commonly used to solubilize drugs for oral administration, vitamin E TPGS and Oral administration was performed using cyclodextrin. Measured using ¹⁴ C-labeled CFTR _inh -172.

FIG. 11, panel C shows the pharmacokinetics of ¹⁴ C-labeled CFTR _inh -172 after oral versus intravenous administration in mice. Intravenous administration increased the initial serum concentration and decreased over ˜30 minutes (tissue redistribution), but serum radioactivity was low immediately after oral administration, peaking at ˜60-90 minutes, and then decreased. FIG. 11, panel D summarizes the organ distribution of ¹⁴ C-labeled CFTR _inh -172 6 hr after oral and intravenous administration, showing accumulation in the gastrointestinal tract and liver and kidney. ¹⁴ C radioactivity is concentrated ˜10-fold in bile vs. serum, with little radioactivity excreted in the stool (<10% of total radioactivity excreted over 24 hours), which results in CFTR _inh − It is suggested that the accumulation of 172 is promoted by enterohepatic circulation. Comparison of oral versus intravenous CFTR _inh -172 administration (tissue / serum volume at 4-6 hr) showed 15-20% CFTR _inh -172 oral bioavailability of the TPGS preparation.

FIG. 11, panel F shows a linear increase in the appearance of CFTR _inh -172 on the transformer side of the Caco-2 monolayer, resulting in an estimated CFTR _inh -172 transmission coefficient of 16 × 10 ⁻⁶ cm / s. This value is within the range found for various orally administered drugs (e.g., pindolol, 36 × 10 ⁻⁶ cm / s, sildenafil, 48 × 10 ⁻⁶ cm / s) (Stenberg et al. J. Med. Chem. 2001 44: 1927-1937).

Biological Example 7
cGMP- and cAMP-mediated fluid secretion An in vivo rat intestinal loop model was used to determine the efficacy of CFTR _inh -172 in inhibiting cGMP- and cAMP-mediated fluid secretion, and CFTR _inh -172 in another animal model Was tested for effectiveness. Guanilyl cyclase C receptor is expressed in rat intestinal cells, allowing STa toxin binding and cytoplasmic cGMP elevation (Mann et al. Biochem Biophs Res commun 1997 239: 463-466). STa toxin was found to cause fluid secretion in the rat ileum after 3 hr (Cohen et al. Am J Physiol 1989 257: G118-123). CFTR _inh -172 inhibited cholera toxin-induced fluid secretion in the rat intestinal loop at an effective dose (600 μg / kg) in mice (FIG. 12, panel A). For fluid secretion induced by STa toxin, STa toxin (300 μl of PBS containing 0.1 μg) was injected into the intestinal loop, and the loop weight was measured after 3 hours. FIG. 12, panel B shows ˜75% inhibition of intestinal fluid secretion by CFTR _inh -172.

Short circuit current measurements were performed on mouse and human intestinal epithelial sheets to evaluate CFTR _inh -172 inhibition of transepithelial ion secretion. FIG. 13, Panel A shows CFTR _inh -172 dose-dependent inhibition of short-circuit current in mouse ileum after stimulation with forskolin or STa toxin (inset). 50% inhibition was found with ˜5 μM CFTR _inh -172 for both cAMP and cGMP-dependent chloride secretion. FIG. 12, panel B shows similar CFTR _inh -172 potency for inhibition of short circuit currents in the human colon.

Unexpected observations show that the apparent efficacy of intestinal short-circuit current (2-5 μM) on CFTR _inh -172 inhibition includes CFTR-expressing FRT cells (0.2-0.5 μM) and Calu-3 cells (0.5 μM) It was substantially lower than that found in electrophysiological studies performed on some cell lines. There are several possible explanations for this difference, including differences in cell types, limited contact of CFTR _inh -172 to intact intestinal enterocytes, membrane potential effects (cells with negative cell potential on the inside) Of [CFTR _inh -172]), and ATP competition with CFTR _inh -172.

Short circuit current measurements were performed on T84 colonic epithelial cells to investigate this phenomenon. FIG. 14, in a representative experiment in panel A, cAMP agonist forskolin (left), cell-permeable cGMP analog 8-Br-cGMP (middle) in unpermeabilized T84 cell monolayers After stimulation with a direct activator of CFTR chloride conductance CFTR _act -16, identified by high-throughput screening, or ˜3 μM CFTR _inh -172 inhibited 50% of the short circuit current. To CFTR _inh -172 relative reduction in potency in T84 cells to determine whether require intact cells, after the basolateral membrane was permeabilized with amphotericin B, Cl ^- gradient (measurable current Short-circuit current measurement was performed in the presence of FIG. 14, panel B (left) shows that the CFTR _inh -172 potency for inhibition of short circuit current after permeabilization is substantially increased. The dose response data summarized in FIG. 14, panel B (middle) shows that the apparent KI for CFTR _inh -172 inhibition decreases from ˜3 to 0.3 μM after cell permeabilization. To test whether the decreased CFTR _inh -172 potency in intact cells is due to a negative membrane potential on the inside (decrease in cytoplasm vs external [CFTR _inh -172]), short-circuit current measurement, high K ⁺ Performed on T84 cells after depolarization with basolateral soaking solution. FIG. 14, Panel C shows that increased CFTR _inh -172 potency (KI-0.3 μM) was preserved in depolarized cells, indicating that cell membrane potential plays an important role in CFTR _inh -172 potency .

Based on the above data, as exemplified by CFTR _inh -172, the thiazolidine compounds of the present invention are enterotoxins caused by enterotoxigenic organisms such as V. cholerae, travelers and AIDS-complex associated diarrhea in E. coli and cholera. It can be predicted to have an antidiarrheal effect in induced secretory diarrhea. Although CFTR inhibition may be beneficial in adjunct therapy of diarrhea caused by enteroinvasive bacteria such as Clostridium difficile and Salmonella species, mucosal damage caused by these organisms is not alleviated by CFTR inhibition. Similarly, CFTR inhibition is not expected to correct the underlying pathology in inflammatory bowel disease, but it reduces the volume of intestinal fluid secretion. Recent evidence suggests that fluid secretion caused by viral diarrhea such as rotavirus may involve other mechanisms such as Ca ²⁺ -mediated Cl ^- channels, but the role of CFTR in fluid secretion is unknown And can be tested by using the compounds of the invention in a suitable animal model.

In summary, the thiazolidinone CFTR blocker CFTR _inh -172 inhibited ion / fluid secretion induced by cAMP and cGMAP in the rodent and human intestine without affecting intestinal fluid absorption. Its advantageous pharmacological and activity profile supports further development for antidiarrheal applications.

  Although the invention has been described with reference to specific embodiments thereof, various modifications can be made by those skilled in the art without departing from the true spirit and scope of the invention, and equivalents may be substituted. It should be understood that it is good. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, method, method step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the appended claims.

(A) Schematic of screening technique used to detect CTFR inhibitors. CFTR was maximally stimulated by multiple agonists in stably transfected epithelial cells co-expressing human CFTR and yellow fluorescent protein (YFP) with Cl ⁻ / I ⁻ sensitive fluorescence. After a test compound was added, I ^- induced influx ^- I by adding a solution containing. (B) A graph of representative fluorescence data from individual wells using the screening technique of FIG. 1A. Control (no activator, no test compound), inactive compound and active CFTR inhibitor compound are shown. (C) A diagram showing a compound structure of a CFTR inhibitor identified by the screening technique of FIG. 1A. (D) shows the chemical structure of ring 2 of a thiazolidinone derivative having the largest CFTR inhibitory activity. The complete thiazolidine derivative structure is shown in FIG. 1C. Relative potency is 0.2 (CFTR _inh -020), 0.3 (CFTR _inh -029), 1.0 (CFTR _inh -172), 0.2 (CFTR _inh -185), 0.1 (CFTR _inh -214) and 0.1 (CFTR _inh -236) Met. (A) CFTR inhibitor 3-[(3-trifluoromethyl) phenyl] -5-[(4-carboxyphenyl) methylene] -2-thioxo-4-thiazolidinone (referred to herein as CFTR _inh -172) 1B is a graph of relative fluorescence versus time using the screening technique of FIG. (B) 2 [mu] M of CFTR _inh -172 different time after the addition CFTR- mediated I ^- is a graph of the time course of inhibition indicating a transport rate. Inset is a graph showing the time course of inhibition reversal showing the I ^- transport rate at different times after flushing with 1 μM CFTR _inh -172. Mean ± SE was obtained from 3 sets of experiments. (C) Different agonists such as benzoflavone and benzimidazolone UCCF compounds (UCCF-029 (2- (4-pyridinium) benzo [h] -4H-chromen-4-one bisulphate) and UCCF-853 (Galietta et al. al., 2001, J. Biol. Chem. 276: 19723-19728)), genistein, CPT-cAMP, 8-methoxypsoralen (8-MPO), 8-cyclopentyl-1,3-dipropylxanthine (CPX) FIG. 6 is a graph showing inhibition of CFTR after stimulation by (all 50 μM) (± SE, obtained from 3 sets of experiments). Black bars indicate agonist and open bars indicate agonist with 5 μM CFTR _inh -172 added. (A) CFTR _inh -172 inhibition of short circuit current in permeabilized FRT cells expressing human CFTR. CFTR was stimulated with 100 μM CPT-cAMP. (B) Summary of dose-inhibition data for CFTR _inh -172 (circles) and glibenclamide (squares) (SE, 3 sets of experiments). (C) is a graph showing CFTR _inh -172 inhibition of short-circuit current in primary culture of human bronchial epithelial cells (not permeabilized). Inhibitors were added to the apical bath solution (left panel) or basolateral apical solution (right panel). (D) is a graph showing whole cell patch clam of CFTR-expressing FRT cells showing membrane currents induced at +80 mV (open circles) and -100 mV (filled circles). CFTR was stimulated by adding 5 μM forskolin followed by 2 μM CFTR _inh -172. (E) A graph showing that alternating stimulation is interrupted (ac) and a stepwise membrane potential is applied. (F) Shows the current-voltage relationship under basic conditions (control, open circles), after forskolin stimulation (filled circles), and after addition of 0.2 μM CFTR _inh -172 (open triangles) providing ~ 50% inhibition. It is a graph. (A) in the absence and presence of 5μM of CFTR _inh -172, Ca ²⁺ dependency Cl stimulated UTP- was measured in short-circuit current measurement on airway epithelial cells (100 [mu] M) ^- is a graph of secretion. (B) FRT cell volume activation was determined in whole cell patch clamp experiments to Cl ^- is a graph showing current (hypotonic 250mosM / kg H ₂ O). The absence and presence of 5 μM CFTR _inh -172 was recorded. (C) Graph showing ³ H-vincristine accumulation in 9HTEo-Dx cells with upregulated MDR-1 expression. Intracellular vincristine was measured with and without verapamil (100 μM) or CFTR _inh -172 (5 μM) (SE, n = 3). (D) Representative membrane potential recordings derived from pancreatic β cells (INS-1) perfused extracellularly with CFTR _inh -172, diazoxide (100 μM) and glibenclamide (10 μM). FIG. 4E is a graph showing an average change (ΔmV) in membrane potential caused by the procedure shown in FIG. 4D. (A) A photograph of the isolated ileal loop of an isolated mouse 6 hours after luminal injection of 1 μg of cholera toxin without (top) and with (center) CFTR _inh -172 (150 μg / kg). is there. Saline controls (no cholera toxin, bottom) are shown for comparison. (B) Graph illustrating ileal loop weight at 6 hours, with 14-16 loops studied, with mean ± SE (n = 6-8 mice). In the inert analogue, the 4-carboxyphenyl group in CFTR _inh -172 was replaced with 3-methoxy-4-methoxyvinylphenyl (SE, 6-8 mice / group, p <0.001, ANOVA). (C) A graph showing the weight ratio of the entire small intestine 6 hours after oral gavage before and after removal of luminal fluid (SE, 4 mice per group, p <0.001). (D) Representative CFTR _inh -172 inhibitory short circuit currents after amiloride addition and forskolin (20 μM) stimulation in isolated rat colon mucus. As indicated, CFTR _inh -172 was added to the serosa and then to the mucosal surface (n = 4). FIG. 2 is a schematic diagram showing the synthesis of ¹⁴ C-labeled CFTR _inh -172. ¹⁴ C was incorporated into the thiazolidinone core using ¹⁴ C-labeled Br acetic acid as the starting material. FIG. 6 is a set of graphs showing the pharmacokinetic analysis results of CFTR _inh -172 in rats after a single intravenous bolus injection of 50 μCi ¹⁴ C-labeled CFTR _inh -172. Data shown as mean ± SE (n = 3-6 rats) for serum radioactivity. The fitted curve corresponds to the 2-compartment model, with a redistribution half-life of 0.14 hr, removal half-life of 10.3 hr, maximum serum concentration of 3.2 μg / mL, and area under the curve of 3.8 μghr / mL, distribution The volume was 1.2 L and the clearance was 99 mL / hr. FIG. 6 is a set of graphs showing organ distribution of ¹⁴ C-labeled CFTR _inh -172 after bolus injection. Panel A results are from a mouse that received a single intravenous bolus injection of 2 μCi ¹⁴ C-labeled CFTR _inh -172, was sacrificed at the indicated time, and had an organ taken for measurement of ¹⁴ C-radioactivity. Yes, data presented as total organ ¹⁴ C-radioactivity at the indicated times after injection (excluding skeletal muscle reported as per gram of tissue) (mean ± SE, 4 mice per time point). Panel B results are from rats that received a bolus injection of 50 μCi ¹⁴ C-labeled CFTR _inh -172 and total organ CFTR _inh -172 measured 60 minutes after injection (3 rats). A set of photographs showing the results of CFTR _inh -172 metabolism analysis by thin layer chromatography of body fluid and liver homogenate from mice injected with ¹⁴ C-labeled CFTR _inh -172, as shown in panel A of FIG. . ^{The 14} C-labeled CFTR _inh -172 standards were 1, 3 and 6 nCi (left panel), and 10, 30 and 60 nCi (right panel). The film was irradiated for 48 hr (left panel) and 12 hr (right panel) for autoradiography. It is a set of graphs showing the results of characterization of a mouse intestinal loop model. Panel A: 200 μL of buffer was injected into the intestinal loop and loop weight was measured at the indicated times (mean ± SEM, 4 mice per hour point). Inset (bottom)% absorption with or without CFTR _inh -172 (20 μg, IP, n = 4). Inset (top) Chemical structure of CFTR _inh -172. Panel B: Time course of fluid secretion induced by cholera toxin in a mouse closed loop model. The dotted line shows the control (saline infusion) loop. Data as mean ± SEM (4-6 mice) for injection loop (1 μg cholera toxin / loop). 1 is a set of graphs showing CFTR _inh -172 inhibition of intestinal secretion after injecting cholera toxin into mice. Panel A: Dose response to inhibition of fluid retention in a mouse loop model. CFTR _inh -172 was administered once by intraperitoneal administration to mice, and the loop weight (mean ± SEM, 4-6 mice per dose) was measured at 6 hr. The dotted line shows the average weight in the saline infusion control loop of the same mouse. Panel B: Persistence of CFTR _inh -172 inhibition. Mice were given 20 μg CFTR _inh -172 (IP) before and after cholera toxin administration (4-6 mice per hour) at designated times. Panel C: Time course of plasma ¹⁴ C-labeled CFTR _inh -172 radioactivity after iv infusion (tail vein, left coordinate) and oral administration (CFTR _inh -172 in TPGS, right coordinate). Data are presented as counts / min / injection μCi (4 mice). Panel D: ¹⁴ C-labeled CFTR _inh -172 accumulation in gastrointestinal organs 6 hours after iv and oral ¹⁴ C-labeled CFTR _inh -172 administration (4 mice). Panel E: Inhibition of cholera toxin-induced fluid secretion by orally administered CFTR _inh -172 (200 μg in TPGS) in a mouse open loop model. Data are presented as the weight ratio of total small intestine 6 hours after oral gavage before and after luminal fluid removal (mean ± SEM, 4 mice / group, ^* p <0.01). Panel F: CFTR _inh -172 permeability across the Caco-2 monolayer (mean ± SEM, 18 inserts), Papp = 16 × 10 ⁻⁶ cm / s. 1 is a set of graphs showing CFTR _inh -172 inhibition of cholera toxin (panel A) and STa toxin (panel B) induced fluid secretion in a rat closed loop model. Data are shown as mean ± SEM (4 rats per group). ^* p <0.01. 1 is a set of graphs showing CFTR _inh -172 inhibition of forskolin- and STa toxin-stimulated short circuit currents in mouse ileum (panel A) and human colon (panel B). STa toxin is shown as an inset. Data are representative of a study of 5 mice and 2 sets of human tissues. Add CFTR _inh -172 to both sides of the tissue. Amiloride (10 μM) was present in the apical solution. FIG. 5 is a set of graphs showing a short-circuit analysis of CFTR _inh −172 inhibition of Cl ⁻ secretion in T84 colonic epithelial cells. Panel A: Data are shown as a representative follow-up from experiments on 5-12 inserts per condition. CFTR _inh -172 was added to both sides of the total cell. CFTR agonists include forskolin (left), 8-Br-cGMP (middle), and CFTR _act- 16 (right). Panel B: (Left) CFTR _inh -172 inhibition of forskolin-stimulated short circuit current after basolateral permeabilization with amphotericin B (250 μg / mL). 6 insert experiments are shown. (Middle) Mean dose response (mean ± SEM) to CFTR _inh -172 inhibition of forskolin-stimulated (mal) and 8-Br-cGMP-stimulated (triangle) short-circuit currents in permeabilized versus non-permeabilized T84 cells 6-12 inserts). (Right) CFTR _inh -172 inhibition of forskolin-stimulated short-circuit current in the presence of low Cl ^{− in} apical solution and high K ⁺ (68 mM) in basolateral solution. Representative of 4 experiments.

Claims (64)

A method of treating a patient having a condition or sign mediated by a cystic fibrosis membrane conductance regulator (CFTR) protein comprising:
Administering to a subject a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable derivative thereof as individual stereoisomers or mixtures thereof:

(Where
X ₁ , X ₂ and X ₃ are each selected from hydrogen, organic group, halo group, nitro group, azo group, hydroxyl group and mercapto group; Y ₁ , Y ₂ and Y ₃ are each hydrogen, organic group, Selected from halo, nitro, azo, hydroxyl and mercapto groups; A ₁ and A ₂ are each selected from oxygen and sulfur, A ₃ is selected from sulfur and selenium; A ₄ is one or more Having carbon atoms or heteroatoms, which may or may not be present).   2. The method of claim 1, wherein the condition and signs are associated with abnormally increased intestinal secretion.   3. The method of claim 2, wherein the condition and sign is secretory diarrhea. The compound of formula (I) is a compound in which A ₄ is absent, A ₁ and A ₃ are each sulfur and A ₂ is oxygen, ie 3-aryl-5-arylmethylene-2-thioxo-4 2. The method of claim 1 which is thiazolidinone. 2. The method of claim 1, wherein the compound of formula (I) is selected from:
3-[(3-trifluoromethyl) phenyl] -5-[(4-nitrophenyl) methylene] -2-thioxo-4-thiazolidinone; 3-[(3-trifluoromethyl) phenyl] -5-[( 4-oxycarboxyphenyl) methylene] -2-thioxo-4-thiazolidinone; 3-[(3-trifluoromethyl) phenyl] -5-[(4-carboxyphenyl) methylene] -2-thioxo-4-thiazolidinone; 3-[(3-trifluoromethyl) phenyl] -5-[(3,4-dihydroxyphenyl) methylene] -2-thioxo-4-thiazolidinone; 3-[(3-trifluoromethyl) phenyl] -5- [(3,5-dibromo-4-hydroxyphenyl) methylene] -2-thioxo-4-thiazolidinone; and 3-[(3-trifluoromethyl) phenyl] -5-[(3-bromo-4-hydroxy- 5-Nitrophenyl) methylene] -2-thioxo-4-thiazolidinone. 2. The method of claim 1, wherein the compound of formula (I) is a compound of formula (Ia):

(Where
X ₁ , X ₂ and X ₃ are each selected from hydrogen, organic group, halo group, nitro group, azo group, hydroxyl group and mercapto group; Y ₁ , Y ₂ and Y ₃ are each hydrogen, organic group, Selected from halo, nitro, azo, hydroxyl and mercapto groups; A ₁ and A ₂ are each selected from oxygen and sulfur). The method according to claim 6, wherein X ₁ is an electron-withdrawing group. X ₁ is selected from the group consisting of perfluoroalkyl groups and fluoro groups, The process of claim 7 wherein. Y ₂ is alkyl, hydroxyl, carboxyl, nitro, carbonate, carbamate, alkoxy, alkylcarbonyl, and halo groups, The method of claim 8. The method according to claim 7, wherein X ₁ is a 3-trifluoromethyl group. The method according to claim 6, wherein Y ₂ is a hydroxyl group. 12. The method according to claim 11, wherein Y ₁ is a hydroxyl group. 12. The method according to claim 11, wherein Y ₁ is a bromo group. Y ₃ is a nitro group, The method of claim 11. 2. The method of claim 1, wherein the compound of formula (I) is a compound of formula (Ib):

(Where
X ₁ , X ₂ and X ₃ are each selected from hydrogen and an organic group; Y ₁ , Y ₂ and Y ₃ are each selected from hydrogen and an organic group). 2. The method of claim 1, wherein the compound of formula (I) is a compound of formula (Ib):

(Where
At least one of X ₁ , X ₂ and X ₃ is an electron withdrawing group; Y ₁ , Y ₂ and Y ₃ are each hydrogen, alkyl, hydroxyl, carboxyl, nitro, carbonate, carbamate, alkoxy, alkylcarbonyl, And a halo group). The method according to claim 16, wherein X ₁ is a trifluoromethyl group. 18. The method according to claim 17, wherein X ₁ is a 3-trifluoromethyl group. 2. The method of claim 1, wherein the compound of formula (I) is selected from:

A method for inhibiting the activity of a cystic fibrosis membrane conductance regulator protein in a subject cell comprising the step of treating the cell with formula (I) or a pharmaceutically acceptable derivative thereof and the individual stereoisomer or The method comprising the step of contacting as a mixture of a sufficient amount to inhibit intracellular transport of CFTR ions:

(Where
X ₁ , X ₂ and X ₃ are each selected from hydrogen, organic group, halo group, nitro group, azo group, hydroxyl group and mercapto group; Y ₁ , Y ₂ and Y ₃ are each hydrogen, organic group, Selected from halo, nitro, azo, hydroxyl and mercapto groups; A ₁ and A ₂ are each selected from oxygen and sulfur, A ₃ is selected from sulfur and selenium; A ₄ is one or more Having carbon atoms or heteroatoms, which may or may not be present). The compound of formula (I) is a compound in which A ₄ is absent, A ₁ and A ₃ are each sulfur and A ₂ is oxygen, ie 3-aryl-5-arylmethylene-2-thioxo-4 21. The method of claim 20, which is a thiazolidinone. The method of claim 21, wherein the compound of formula (I) is selected from:
3-[(3-trifluoromethyl) phenyl] -5-[(4-nitrophenyl) methylene] -2-thioxo-4-thiazolidinone; 3-[(3-trifluoromethyl) phenyl] -5-[( 4-oxycarboxyphenyl) methylene] -2-thioxo-4-thiazolidinone; 3-[(3-trifluoromethyl) phenyl] -5-[(4-carboxyphenyl) methylene] -2-thioxo-4-thiazolidinone; 3-[(3-trifluoromethyl) phenyl] -5-[(3,4-dihydroxyphenyl) methylene] -2-thioxo-4-thiazolidinone; 3-[(3-trifluoromethyl) phenyl] -5- [(3,5-dibromo-4-hydroxyphenyl) methylene] -2-thioxo-4-thiazolidinone; and 3-[(3-trifluoromethyl) phenyl] -5-[(3-bromo-4-hydroxy- 5-Nitrophenyl) methylene] -2-thioxo-4-thiazolidinone. 21. The method of claim 20, wherein the compound of formula (I) is a compound of formula (Ia):

(Where
X ₁ , X ₂ and X ₃ are each selected from hydrogen, organic group, halo group, nitro group, azo group, hydroxyl group and mercapto group; Y ₁ , Y ₂ and Y ₃ are each hydrogen, organic group, Selected from halo, nitro, azo, hydroxyl and mercapto groups; A ₁ and A ₂ are each selected from oxygen and sulfur). X ₁ is an electron withdrawing group, The method of claim 23. X ₁ is selected from perfluoroalkyl groups and fluoro groups, 25. The method of claim 24. The method according to claim 24, wherein X ₁ is a 3-trifluoromethyl group. Y ₂ is alkyl, hydroxyl, carboxyl, nitro, carbonate, carbamate, alkoxy, alkylcarbonyl, and halo groups, The method of claim 23. Y ₂ is a hydroxyl group, The method of claim 23. Y ₁ is a hydroxyl group, the process according to claim 28. Y ₁ is a bromo group, The method of claim 28. Y ₃ is a nitro group, The method of claim 28. 21. The method of claim 20, wherein the compound of formula (I) is a compound of formula (Ib):

(Where
X ₁ , X ₂ and X ₃ are each selected from hydrogen and an organic group; Y ₁ , Y ₂ and Y ₃ are each selected from hydrogen and an organic group). 21. The method of claim 20, wherein the compound of formula (I) is a compound of formula (Ib):

(Where
At least one of X ₁ , X ₂ and X ₃ is an electron withdrawing group; Y ₁ , Y ₂ and Y ₃ are each hydrogen, alkyl, hydroxyl, carboxyl, nitro, carbonate, carbamate, alkoxy, alkylcarbonyl, And a halo group). X ₁ is a trifluoromethyl group, The method of claim 33. X ₁ is 3-trifluoromethyl group, The method of claim 34, wherein. 21. The method of claim 20, wherein the compound of formula (I) is selected from:

  21. The method of claim 20, wherein the step of contacting the cell comprises the step of the subject taking the compound of formula (I).   38. The method of claim 37, wherein the ingesting step comprises ingesting a pharmaceutically acceptable carrier with the compound of formula (I). A method for inhibiting the activity of a cystic fibrosis membrane conductance regulator protein in a cell in an in vivo assay, wherein the cell is treated with a compound of formula (I) or a pharmaceutically acceptable derivative thereof and the individual steric Contacting the isomer or a mixture thereof in an amount sufficient to inhibit intracellular CFTR ion transport:

(Where
X ₁ , X ₂ and X ₃ are each selected from hydrogen, organic group, halo group, nitro group, azo group, hydroxyl group and mercapto group; Y ₁ , Y ₂ and Y ₃ are each hydrogen, organic group, Selected from halo, nitro, azo, hydroxyl and mercapto groups; A ₁ and A ₂ are each selected from oxygen and sulfur, A ₃ is selected from sulfur and selenium; A ₄ is one or more Having carbon atoms or heteroatoms, which may or may not be present). A method for creating a cystic fibrosis (CF) phenotype in a non-human animal, wherein the non-human animal is administered a compound of formula (I) or a pharmaceutically acceptable derivative thereof in an individual stereotype. Administering as an isomer or a mixture thereof in an amount sufficient to induce a cystic fibrosis (CF) phenotype in a non-human animal:

(Where
X ₁ , X ₂ and X ₃ are each selected from hydrogen, organic group, halo group, nitro group, azo group, hydroxyl group and mercapto group; Y ₁ , Y ₂ and Y ₃ are each hydrogen, organic group, Selected from halo, nitro, azo, hydroxyl and mercapto groups; A ₁ and A ₂ are each selected from oxygen and sulfur, A ₃ is selected from sulfur and selenium; A ₄ is one or more Having carbon atoms or heteroatoms, which may or may not be present). A method of treating a subject having a condition associated with abnormal ion transport by cystic fibrosis transmembrane conductance regulator (CFTR) in the subject, comprising:
Administering to the subject an effective amount of a thiazolidinone compound,
A method wherein CFTR ion transport is inhibited and the condition is treated.   42. The method of claim 41, wherein abnormally increased CFTR ion transport is associated with diarrhea.   43. The method of claim 42, wherein the diarrhea is secretory diarrhea.   Each of the thiazoline compounds selected from: 3-[(3-trifluoromethyl) phenyl] -5-[(4-nitrophenyl) methylene] -2-thioxo-4-thiazolidinone; 3-[(3-tri Fluoromethyl) phenyl] -5-[(4-oxycarboxyphenyl) methylene] -2-thioxo-4-thiazolidinone; 3-[(3-trifluoromethyl) phenyl] -5-[(4-carboxyphenyl) methylene ] -2-thioxo-4-thiazolidinone; 3-[(3-trifluoromethyl) phenyl] -5-[(3,4-dihydroxyphenyl) methylene] -2-thioxo-4-thiazolidinone; 3-[(3 -Trifluoromethyl) phenyl] -5-[(3,5-dibromo-4-hydroxyphenyl) methylene] -2-thioxo-4-thiazolidinone; and 3-[(3-trifluoromethyl) phenyl] -5- [(3-bromo-4-hydroxy-5-nitrophenyl) methylene] -2-thioxo-4-thiazolidinone; and at least one pharmaceutically acceptable A pharmaceutical composition comprising an acceptable carrier, a pharmaceutically acceptable diluent, a pharmaceutically acceptable excipient and a pharmaceutically acceptable adjuvant.   45. The composition of claim 44, wherein the composition is free of detectable dimethyl sulfoxide. Compounds of formula (I), or pharmaceutically acceptable derivatives thereof, as individual stereoisomers or mixtures thereof:

(Where
X ₁ , X ₂ and X ₃ are each selected from hydrogen, organic group, halo group, nitro group, azo group, hydroxyl group and mercapto group; Y ₁ , Y ₂ and Y ₃ are each hydrogen, organic group, halo group, a nitro group, an azo group, selected from hydroxyl and mercapto groups; each a ₁ and a ₂ are selected from oxygen and sulfur, a ₃ is selected from oxygen, sulfur and selenium; a ₄ is 1 or more Has carbon atoms or heteroatoms and may or may not be present provided that:
1) A ₄ is absent, A ₁ and A ₂ are each oxygen, A ₃ is sulfur, and one of X ₁ , X ₂ and X ₃ is trifluoromethyl or chloro in the 4-position And the remainder of X ₁ , X ₂ and X ₃ are each hydrogen, and one of Y ₁ , Y ₂ and Y ₃ cannot be 4-methylpiperazin-1-yl at the 2-position, Y ₁ , the remainder of Y ₂ and Y ₃ are each hydrogen;
2) A ₄ is not present, A ₁ and A ₃ are each sulfur, A ₂ is oxygen, one of X ₁ , X ₂ and X ₃ is carboxyl at the 4-position, X ₁ , X ₂ and X ₃ are each hydrogen, and each of Y ₁ , Y ₂ and Y ₃ cannot be hydrogen;
3) A ₄ is not present, A ₁ and A ₃ are each sulfur, A ₂ is oxygen, and one of X ₁ , X ₂ and X ₃ is in the 2-, 3- or 4-position Hydroxy, ethoxy in the 4-position, the rest of X ₁ , X ₂ and X ₃ are each hydrogen, one of Y ₁ , Y ₂ and Y ₃ is hydrogen, Y ₁ , Y ₂ And another one of Y ₃ is hydroxy or methoxy at the 4-position and the remaining one of Y ₁ , Y ₂ and Y ₃ cannot be methoxy at the 3-position; and
4) A ₄ is not present, A ₁ and A ₃ are each sulfur, A ₂ is oxygen, one of X ₁ , X ₂ and X ₃ is methyl at the 4-position, X ₁ , X ₂ and X ₃ are chloro in the 3-position, one of Y ₁ , Y ₂ and Y ₃ is methoxy in the 4-position, and the rest of Y ₁ , Y ₂ and Y ₃ are Each cannot be hydrogen)
And at least one pharmaceutically acceptable carrier, a pharmaceutically acceptable diluent, a pharmaceutically acceptable excipient and a pharmaceutically acceptable adjuvant. 47. The composition of claim 46, wherein the compound of formula (I) is a compound of formula (Ia):

(Where
X ₁ , X ₂ and X ₃ are each selected from hydrogen, organic group, halo group, nitro group, azo group, hydroxyl group and mercapto group; Y ₁ , Y ₂ and Y ₃ are each hydrogen, organic group, Selected from halo, nitro, azo, hydroxyl and mercapto groups; A ₁ and A ₂ are each selected from oxygen and sulfur). X ₁ is an electron withdrawing group, The composition of claim 47. X ₁ is selected from perfluoroalkyl groups and fluoro groups The method of claim 48, wherein. Y ₂ is alkyl, hydroxyl, carboxyl, nitro, carbonate, carbamate, alkoxy, alkylcarbonyl, and halo groups, The composition of claim 47,. X ₁ is 3-trifluoromethyl group, The composition of claim 47. Y ₂ is a hydroxyl group The composition of claim 47,. Y ₁ is a hydroxyl group, according to claim 52 placing the composition. Y ₁ is a bromo group, claim 52 composition. Y ₃ is a nitro group, The composition of claim 54. 47. The composition of claim 46, wherein the compound of formula (I) is a compound of formula (Ib):

(Where
X ₁ , X ₂ and X ₃ are each selected from hydrogen and an organic group; Y ₁ , Y ₂ and Y ₃ are each selected from hydrogen and an organic group). 47. The composition of claim 46, wherein the compound of formula (I) is a compound of formula (Ib):

(Where
At least one of X ₁ , X ₂ and X ₃ is an electron withdrawing group; Y ₁ , Y ₂ and Y ₃ are each hydrogen, alkyl, hydroxyl, carboxyl, nitro, carbonate, carbamate, alkoxy, alkylcarbonyl, And a halo group). X ₁ is a trifluoromethyl group, claim 57 composition. X ₁ is 3-trifluoromethyl group, claim 57 composition.   48. The composition of claim 46, wherein the composition is free of detectable dimethyl sulfoxide.   A non-human animal having a cystic fibrosis membrane conductance regulator (CFTR) deficiency, wherein the deficiency is caused by administration of a thiazoline compound to the animal in an amount effective to inhibit CFTR ion transport; Non-human animals.   62. The non-human animal according to claim 61, wherein the animal is a mammal.   63. The non-human animal of claim 62, wherein the mammal is a non-human primate, rodent, ungulate, or bird.   62. The non-human animal of claim 61, wherein the animal has a phenotype similar to cystic fibrosis.

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