WO2010148177A2 - Compounds, compositions, methods of synthesis, and methods of treatment - Google Patents

Abstract

Embodiments of the present disclosure provide for chloride channel or transporter compounds or compositions (inhibitors or agents), methods of synthesizing these compounds or compositions, methods of treatment using these compounds or compositions, and the like.

Description

COMPOUNDS, COMPOSITIONS, METHODS OF SYNTHESIS, AND METHODS OF TREATMENT

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application Ser. No. 61/187,765, entitled "COMPOUNDS, COMPOSITIONS, METHODS OF SYNTHESIS, AND METHODS OF TREATMENT" filed on June 17^th, 2009, which is hereby incorporated by reference.

STATEMENT OF FEDERALLY SPONSORED RESEARCH This invention was made with government support under grant number GM070773, awarded by the U.S. National Institutes of Health of the United States government. The government has certain rights in the invention.

BACKGROUND

Members of the CLC "chloride channel" family are expressed in almost every organism. The nine mammalian homologs contribute to a remarkable array of physiological processes including epithelial ion transport, neuronal and skeletal muscle excitability, hippocampal development, cardiac pacemaker activity, endocytosis, and lysosomal acidification. The widespread physiological importance of the CLCs is highlighted by the observation that every CLC knockout mouse displays a significant phenotype. In humans, defects in CLC proteins are directly responsible for human diseases of kidney, muscle, and bone, for disorders of blood- pressure regulation, and may act as susceptibility factors for epilepsy.

SUMMARY

Embodiments of the present disclosure provide for chloride channel or transporter compounds or compositions (inhibitors or agents), methods of synthesizing these compounds or compositions, methods of treatment using these compounds or compositions, and the like. One exemplary compound, among others, includes: a compound of formula (I),

_? wherein: R¹ is chosen from:

and O; R² is independently chosen from: H and CH₃;

R³ is independently chosen from: H,

and

R⁴ is chosen from: alkyl, halogen, -SSO₂CH₃, -SeCH₃, -NH₃, -NH₂CH₃, -SH, -S-alkyl, -OH, and -O-alkyl;

R⁵ and R⁶ are chosen from: H and CH₃; and n is 1 to 10.

One exemplary compound, among others, includes: a compound of formula (I),

R R N

, wherein:

R¹ is chosen from:

and O; R² is independently chosen from: H and CH₃;

R³ is independently chosen from: H,

R⁴ is chosen from: alkyl, halogen, -SSO₂CH₃, -SeCH₃, -NH₃, -NH₂CH₃, -SH, -S-alkyl, -OH, and -O-alkyl;

R⁵ and R⁶ are chosen from: H and CH₃; and n is 1 to 10.

One exemplary compound, among others, includes: a compound of formula (1),

, wherein R⁴ is selected from: methyl, halogen, -SSO₂CH₃, -SeCH₃, -NH₃, -SH, and -OH, wherein n is 1 to 10.

One exemplary compound, among others, includes: a compound of formula (2),

, wherein R⁴ is selected from: methyl, halogen, -SSO₂CH₃, -SeCH₃, -NH₃, -SH, and -OH, wherein n is 1 to 10. One exemplary compound, among others, includes: a compound of formula (3),

, wherein R⁴ is selected from: methyl, halogen, -SSO₂CH₃, -SeCH₃, -NH₃, -SH, and -OH, wherein n is 1 to 10.

One exemplary compound, among others, includes: a compound of formula (4),

wherein R⁴ is selected from: methyl, halogen, -SSO₂CH₃, -SeCH₃, -NH₃, -SH, and -OH, wherein n is 1 to 10. One exemplary compound, among others, includes: a compound of formula (5),

, wherien R⁴ is selected from: methyl, halogen, -SSO₂CH₃, -SeCH₃, -NH₃, -SH, and -OH, wherein n is 1 to 10. One exemplary compound, among others, includes: a compound of formula (6),

, wherein R is selected from: methyl, halogen, -SSO₂CH₃, -SeCH₃, -NH₃, -SH, and -OH, wherein n is 1 to 10.

One exemplary pharmaceutical composition, among others, includes: a compound, radiolabeled isotopes, racemic mixtures, and stereoisomers, and pharmaceutically acceptable salts thereof, and combinations thereof of any one of a compound noted above or mixtures thereof, and a pharmaceutically acceptable carrier.

One exemplary method of inhibiting a chloride channel or transporter in a cell, among others, includes: contacting (e.g., administering) the chloride channel or transporter with a compound noted above.

One exemplary method of disrupting a chloride channel or transporter in a cell, among others, includes: contacting (e.g., administering) the chloride channel or transporter with a compound noted above.

One exemplary method of treating symptoms associated with diseases or disorders associated with a chloride channel or transporter, among others, includes: contacting (e.g., administering) the chloride channel or transporter with a therapeutically effective amount of a compound noted above.

All of the compounds described above can also include radiolabeled isotopes, racemic mixtures, and stereoisomers, and pharmaceutically acceptable salts thereof, and combinations thereof.

Other compounds, compositions, methods of synthesis, methods of treatment, features, and advantages of this disclosure will be or become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional compounds, compositions, methods of synthesis, methods of treatment, features, and advantages be included within this description, be within the scope of this disclosure, and be protected by the accompanying claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Further aspects of the present disclosure will be more readily appreciated upon review of the detailed description of its various embodiments, described below, when taken in conjunction with the accompanying drawings.

FIG. 1 includes graphs that illustrate inhibition of CIC-ec1 activity using chloride flux assays by purified DIDS hydrolysis products. Vesicles were pre-equilibrated in various concentrations of the polythioureas. Error bars represent the SEM from 3-6 separate assays. Fits are to the Hill equation: (F/F_o) = (F/F₀)min + [(F/F₀)max-(F/F₀)min)]/[1 + (K1/2/[lnhibitor])n], where F is the chloride flux, F₀ is the chloride flux in the absence of inhibitor, K1/2 is the apparent affinity, and n is the Hill coefficient. For the dimer, trimer, tetramer, and pentamer, K1/2 = 100 μM, 10 μM, 5 μM, and 1 μM respectively.

FIG. 2 is a graph that illustrates inhibition of CIC-Ka by purified DIDS hydrolysis products. 1/I₀ is the fraction of current remaining at +40 mV. Fits were to the Hill equation, (1/I₀)= (l/l_o)min + [1-(l/lo)min]/[1+(K1/2/[lnhibitor])]. For the tetramer and pentamer, K1/2 = 1 μ and 0.5 μM respectively. The K1/2 of CIC-Ka for DIDS is -100 mM (Picollo et al, 2004).

FIG. 3A illustrates OADS (4,4'-octanamidostilbene-2,2'-disulfonate) that incorporates elements from the fatty acid inhibitors and the stilbene disulfonates.

FIG 3B illustrates the inhibition of CLC-ec1 by OADS. CLC-ec1 was reconstituted into phospholipid vesicles, and activity was assayed using a Cl^"-flux assay. Inhibition was fit to a Hill equation with n=3 and half maximal inhibition (K₁) of 60 μM. Experiments were performed in triplicate, and error bars are s.e.m.

FIG. 4A illustrates current tracings of CLC-Ka before, during, and after application of 15 μM OADS.

FIG. 4B illustrates a summary of OADS inhibition of CLC channels. Currents were recorded using two-electrode voltage of channels expressed in Xenopus oocytes. The graph shows inhibition of the current at +60 mV by the indicated concentration of inhibitor OADS. Error bars indicate the standard deviation of 2-4 data points. CIC-Ka is inhibited with an apparent affinity of 2 μM. The N68D mutation reduces the affinity to 8 μM. OADS does not detectably inhibit the skeletal muscle homolog CIC-O at concentrations up to 30 μM. FIG. 5 illustrates CIC-ec1 and CIC-Ka apparent affinity for various derivatives. FIG. 5A: The parent compound OADS exhibits an apparent affinity of 2 μM for CLC-Ka and 60 μM for CLC-ec1. FIG. 5B: lsopropyl DADS was hydrogenated before coupling with octanoyl chloride. The increased flexibility afforded by hydrogenated OADS has a negligible affect on the affinity of the inhibitor. FIG. 5C: Adding a hydroxyl at the center of the molecule greatly increases selectivity: there is no effect on potency towards CLC-Ka and a five-fold decrease in potency towards CLC-ec1. FIG. 5D: Adding one equivalent of octanoyl chloride to isopropyl DADS ultimately affords this "mono OADS" product, which is 40-fold reduced in potency towards CLC- Ka and only 3-fold reduced towards CLC-ec1. FIG. 5E: Decreasing the alkyl chain-length greatly decreases affinity of the inhibitor towards both CLC-Ka and CLC-ec1.

FIG. 6A illustrates various functional groups on OADS can be targeted for modification.

FIG. 6B illustrates various functional groups on OADS can be targeted for modification.

FIG. 7 illustrates the efficacy of OADS in vivo. OADS dissolved in water to 1.6 mM was injected into rats intraperitoneal^ at 5 mg/kg. FIG 7A shows the increase in urine output in OADS-injected rats versus control rats. FIG 7B shows the decrease in urine osmolality in OADS rats versus controls.

DETAILED DESCRIPTION

This disclosure is not limited to particular embodiments described, and as such may, of course, vary. The terminology used herein serves the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

Where a range of values is provided, each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the compositions and compounds disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ⁰C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 ⁰C and 1 atmosphere.

Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, dimensions, frequency ranges, applications, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence, where this is logically possible. It is also possible that the embodiments of the present disclosure can be applied to additional embodiments involving measurements beyond the examples described herein, which are not intended to be limiting. It is furthermore possible that the embodiments of the present disclosure can be combined or integrated with other measurement techniques beyond the examples described herein, which are not intended to be limiting.

It should be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a support" includes a plurality of supports. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

Each of the applications and patents cited in this text, as well as each document or reference cited in each of the applications and patents (including during the prosecution of each issued patent; "application cited documents"), and each of the PCT and foreign applications or patents corresponding to and/or claiming priority from any of these applications and patents, and each of the documents cited or referenced in each of the application cited documents, are hereby expressly incorporated herein by reference. Further, documents or references cited in this text, in a Reference List before the claims, or in the text itself; and each of these documents or references ("herein cited references"), as well as each document or reference cited in each of the herein-cited references (including any manufacturer's specifications, instructions, etc.) are hereby expressly incorporated herein by reference. Prior to describing the various embodiments, the following definitions are provided and should be used unless otherwise indicated.

Definitions

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of molecular biology. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described herein.

As used in the specification and the appended claims, the singular forms "a," "an," and "the" may include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a support" includes a plurality of supports. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

As used herein, "alkyl" or "alkyl group" refers to a saturated aliphatic hydrocarbon radical which can be straight or branched, having 1 to 20 carbon atoms, wherein the stated range of carbon atoms includes each intervening integer individually, as well as sub-ranges. Examples of alkyl groups include, but are not limited to, methyl, ethyl, /-propyl, /7-propyl, n-butyl, f-butyl, pentyl, hexyl, septyl, octyl, nonyl, decyl, and the like.

As used herein, "alkenyl" or "alkenyl group" refers to an aliphatic hydrocarbon radical which can be straight or branched, containing at least one carbon-carbon double bond, having 2 to 20 carbon atoms, wherein the stated range of carbon atoms includes each intervening integer individually, as well as sub-ranges. Examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, n-butenyl, i-butenyl, 3-methylbut-2-enyl, n-pentenyl, heptenyl, octenyl, decenyl, and the like.

As used herein, "halo", "halogen", or "halogen radical" refers to a fluorine, chlorine, bromine, and iodine, radicals. Further, when used in compound words, such as "haloalkyl" or "haloalkenyl", "halo" refers to an alkyl or alkenyl radical in which one or more hydrogens are substituted by halogen radicals.

As used herein, "agent", "active agent", "inhibitor or the like, can include a compound of the present disclosure. The agent or inhibitor can be disposed in a composition or a pharmaceutical composition.

As used herein, "pharmaceutical composition" refers to the combination of an active agent with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable salt" refers to a salt of a compound, which possesses the desired pharmacological activity of the parent compound, for example (1) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, and the like; or (2) salts formed when an acidic proton present in the parent compound is replaced by a metal ion such as an alkali metal ion, an alkaline earth ion an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, and triethanolamine.

As used herein, "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipients, or carrier with which a compound or pharmaceutically acceptable salt is administered.

As used herein, "treat", "treatment", "treating", and the like refer to acting upon a disease or disorder with an agent to effect the disease or disorder by improving or altering it. The improvement or alteration may include an improvement in symptoms or an alteration in the physiologic pathways associated with the disease or disorder. "Treatment," as used herein, covers any treatment of a disease in a host (e.g., a mammal, typically a human or non-human animal of veterinary interest), and includes: (a) reducing the risk of occurrence of the disease in a subject determined to be predisposed to the disease but not yet diagnosed as infected with the disease (b) impeding the development of the disease, and (c) relieving the disease, i.e., causing regression of the disease and/or relieving one or more disease symptoms. "Treatment" is also meant to encompass delivery of an agent to provide a pharmacologic effect, even in the absence of a disease or condition. For example, "treatment" encompasses delivery of an agent that provides for enhanced or desirable effects in the subject {e.g., reduction of disease symptoms, etc.).

As used herein, the terms "prophylactically treat" or "prophylactically treating" refers completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.

As used herein, "inhibit", "inhibits", "inhibiting", and the like refer to acting upon a biological target with an agent to moderate or limit the effect of the target. In certain embodiments, the effect of the biological target can be inhibited up to 100%. Examples of inhibiting a biological target, e.g., a chloride channel or transporter, include, but are not limited to, acting upon a chloride channel or transporter with a compound of formula (I), as described herein, thereby moderating or limiting the effect of the chloride channel or transporter. As used herein, "disrupt", "disrupts", "disrupting", and the like refer to acting upon a biological target with an agent to interrupt the ordinary course of the biological target. Examples of inhibiting a biological target, e.g. a chloride channel or transporter, include, but are not limited to, acting upon a chloride channel or transporter with a compound of formula (I), as described herein, thereby interrupting the ordinary course of the chloride channel or transporter.

The term "unit dosage form," as used herein, refers to physically discrete units suitable as unitary dosages for human and/or animal subjects, each unit containing a predetermined quantity of a compound calculated in an amount sufficient (e.g., weight of host, disease, severity of the disease, etc) to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for unit dosage forms depend on the particular compound employed, the route and frequency of administration, and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

The term "therapeutically effective amount" as used herein refers to that amount of an embodiment of the agent (which may be referred to as a compound) being administered that will relieve to some extent one or more of the symptoms of the disease being treated, and/or that amount that will prevent, to some extent, one or more of the symptoms of the disease that the host being treated has or is at risk of developing.

The term "prodrug" refers to an inactive precursor of an agent that is converted into a biologically active form in vivo. Prodrugs are often useful because, in some situations, they may be easier to administer than the parent compound. They may, for instance, be bioavailable by oral administration whereas the parent compound is not. The prodrug may also have improved solubility in pharmaceutical compositions over the parent drug. A prodrug may be converted into the parent drug by various mechanisms, including enzymatic processes and metabolic hydrolysis. Harper, N.J. (1962). Drug Latentiation in Jucker, ed. Progress in Drug Research, 4:221-294; Morozowich et al. (1977). Application of Physical Organic Principles to Prodrug Design in E. B. Roche ed. Design of Biopharmaceutical Properties through Prodrugs and Analogs, APhA; Acad. Pharm. ScL; E. B. Roche, ed. (1977). Bioreversible Carriers in Drug in Drug Design, Theory and Application, APhA; H. Bundgaard, ed. (1985) Design of Prodrugs, Elsevier; Wang et al. (1999) Prodrug approaches to the improved delivery of peptide drug, Curr. Pharm. Design. 5(4):265-287; Pauletti et al. (1997). Improvement in peptide bioavailability: Peptidomimetics and Prodrug Strategies, Adv. Drug. Delivery Rev. 27:235-256; Mizen et al. (1998). The Use of Esters as Prodrugs for Oral Delivery of β-Lactam antibiotics, Pharm. Biotech. 11 ,:345-365; Gaignault et al. (1996). Designing Prodrugs and Bioprecursors I. Carrier Prodrugs, Pract. Med. Chem. 671-696; M. Asghamejad (2000). Improving Oral Drug Transport Via Prodrugs, in G. L. Amidon, P. I. Lee and E. M. Topp, Eds., Transport Processes in Pharmaceutical Systems, Marcell Dekker, p. 185-218; Balant et al. (1990) Prodrugs for the improvement of drug absorption via different routes of administration, Eur. J. Drug Metab. Pharmacokinet., 15(2): 143-53; Balimane and Sinko (1999). Involvement of multiple transporters in the oral absorption of nucleoside analogues, Adv. Drug Delivery Rev., 39(1 -3): 183-209; Browne (1997). Fosphenytoin (Cerebyx), Clin. Neuropharmacol. 20(1): 1-12; Bundgaard (1979). Bioreversible derivatization of drugs-principle and applicability to improve the therapeutic effects of drugs, Arch. Pharm. Chemi. 86(1): 1-39; H. Bundgaard, ed. (1985) Design of Prodrugs, New York: Elsevier; Fleisher et al. (1996). Improved oral drug delivery: solubility limitations overcome by the use of prodrugs, Adv. Drug Delivery Rev. 19(2): 115-130; Fleisher et al. (1985). Design of prodrugs for improved gastrointestinal absorption by intestinal enzyme targeting, Methods Enzymol. 112: 360-81 ; Farquhar D, et al. (1983). Biologically Reversible Phosphate-Protective Groups, J. Pharm. ScL, 72(3): 324-325; Han, H. K. et al. (2000). Targeted prodrug design to optimize drug delivery, AAPS PharmSci., 2(1): E6; Sadzuka Y. (2000). Effective prodrug liposome and conversion to active metabolite, Curr. Drug Metab., 1 (1):31-48; D. M. Lambert (2000) Rationale and applications of lipids as prodrug carriers, Eur. J. Pharm. Sci., 11 Suppl 2:S15-27; Wang, W. et al. (1999) Prodrug approaches to the improved delivery of peptide drugs. Curr. Pharm. Des., 5(4):265-87.

As used herein, the term "host," "subject," "patient," or "organism" includes humans and mammals (e.g., mice, rats, pigs, cats, dogs, and horses). Typical hosts to which compounds of the present disclosure can be administered will be mammals, particularly primates, especially humans. For veterinary applications, a wide variety of subjects will be suitable, e.g., livestock such as cattle, sheep, goats, cows, swine, and the like; poultry such as chickens, ducks, geese, turkeys, and the like; and domesticated animals particularly pets such as dogs and cats. For diagnostic or research applications, a wide variety of mammals will be suitable subjects, including rodents (e.g., mice, rats, hamsters), rabbits, primates, and swine such as inbred pigs and the like. The term "living host" refers to a host noted above or another organism that is alive. The term "living host" refers to the entire host or organism and not just a part excised (e.g., a liver or other organ) from the living host.

As used herein the phrase, "compound . . . as well as radiolabeled isotopes, racemic mixtures, and stereoisomers, and pharmaceutically acceptable salts thereof, and combinations thereof, refers to compounds, radiolabeled isotopes, racemic mixtures, and stereoisomers, according to a given formula or genus, as well as their pharmaceutically acceptable salts. Use of the phrase "radiolabeled isotopes" can mean that the compound or the R group (depending on the use of radiolabeled isotopes) can include one or more radiolabeled isotopes. In addition, this phrase encompasses a compound that includes one or more radiolabeled isotopes in one of the compounds in a racemic mixture, a stereoisomer, or a pharmaceutically acceptable salt. Furthermore, this phase encompasses various combinations of compounds, radiolabeled isotopes, racemic mixtures, stereoisomers, and pharmaceutically acceptable salts according to a given formula or genus of formulae. For example, claim 1 encompasses compounds, radiolabeled isotopes, racemic mixtures, and stereoisomers, according to formula (I), as well as their pharmaceutically acceptable salts, and various combinations of compounds, radiolabeled isotopes, racemic mixtures, stereoisomers, and pharmaceutically acceptable salts according to formula (I). In addition, this phrase includes pharmaceutically acceptable salts of one or more of the compound, a racemic mixture, and a stereoisomer.

Discussion

Embodiments of the present disclosure provide compounds which can be used to treat the symptoms associated with diseases or disorders associated with a chloride channel or transporter. Diseases and disorders associated with chloride channel or transporter are associated with defects in CLC proteins are directly responsible for human diseases of kidney, muscle, and bone, for disorders of blood-pressure regulation, and may act as susceptibility factors for epilepsy. In particular, the diseases and disorders associated with chloride channel or transporter can include hypertension, heart failure, osteoporosis, inappropriate water retention, and food poisoning.

Embodiments of the present disclosure provide for chloride channel or transporter compounds or compositions (inhibitors or agents), methods of synthesizing these compounds or compositions, methods of treatment using these compounds or compositions, and the like. In particular, embodiments of the present disclosure provide stilbene compound derivatives, methods of synthesizing these compounds, intermediate compounds, pharmaceutical compositions including one or more of these compounds, methods of treatment using these compounds, and the like.

An embodiment of the present disclosure includes compounds according to formula (I):

R as well as their radiolabeled isotopes, racemic mixtures, and stereoisomers, and pharmaceutically acceptable salts thereof, and combinations

thereof. R¹ can be selected from D2

R* can be independently selected from: H and CH₃. R³ can be independently selected from: H,

and

. R⁴ can be selected from: alkyl, halogen, -

SSO₂CH₃, -SeCH₃, -NH₃, -NH₂CH₃, -SH, -S-alkyl, -OH, and -O-alkyl. n can be 1 to 10. R⁵ and R⁶ can be selected from H and CH₃. In certain embodiments, -NR²R³ may be different or the same. For example, R² can be H at both positions, and one R³ can be H, while the other R³

can be

. Accordingly, certain embodiments of the present disclosure include asymmetrical stilbene derivatives according to formula (I).

An embodiment of the present disclosure includes compounds of formula (I), as well as their radiolabeled isotopes, racemic mixtures, and stereoisomers, and pharmaceutically

in certain

embodiments, R² can be H, and R³ can be

. Accordingly, an embodiment of the present disclosure includes a class of compounds according to formula (I), such as formula

(1), , radiolabeled isotopes, racemic mixtures, and stereoisomers, and pharmaceutically acceptable salts thereof, and combinations thereof. These embodiments include examples in which R⁴ can be selected from: methyl, halogen, -SSO₂CH₃, -SeCH₃, -NH₃, -SH, and -OH. An embodiment of the present disclosure includes compounds of formula (I), as well as their radiolabeled isotopes, racemic mixtures, and stereoisomers, and pharmaceutically

acceptable salts thereof, and combinations thereof, in which R¹ can be S ^^5 . in certain

embodiments, R² and R⁶ can be H, and R³ can be

^R . Accordingly, an embodiment of the present disclosure includes a class of compounds according to formula (I),

such as formula (5),

radiolabeled isotopes, racemic mixtures, and stereoisomers, and pharmaceutically acceptable salts thereof, and combinations thereof. These embodiments include examples in which R⁴ can be selected from: methyl, halogen, -SSO₂CH₃, -SeCH₃, -NH₃, -SH, and -OH.

An embodiment of the present disclosure includes compounds of formula (I), as well their radiolabeled isotopes, racemic mixtures, and stereoisomers, and pharmaceutically

acceptable salts thereof, and combinations thereof, in which R¹ can be ζ ^^O . In certain

embodiments, R² can be H, and R³ can be H at one position and

at the other position. Accordingly, an embodiment of the present disclosure includes a class of compounds according to formula (I), such as formula (6),

, radiolabeled isotopes, racemic mixtures, and stereoisomers, and pharmaceutically acceptable salts thereof, and combinations thereof. These embodiments include examples in which R⁴ can be selected from: methyl, halogen, - SSO₂CH₃, -SeCH₃, -NH₃, -SH, and -OH. An embodiment of the present disclosure includes compounds of formula (I), as well as their radiolabeled isotopes, racemic mixtures, and stereoisomers, and pharmaceutically

acceptable salts thereof, and combinations thereof, in which R¹ can be

. In certain

embodiments, R² can be H, and R³ can be

. Accordingly, an embodiment of the present disclosure includes a class of compounds according to formula (I), such as formula

(2), , radiolabeled isotopes, racemic mixtures, and stereoisomers, and pharmaceutically acceptable salts thereof, and combinations thereof. These embodiments include examples in which R⁴ can be selected from: methyl, halogen, -SSO₂CH₃, -SeCH₃, -NH₃, -SH, and -OH.

An embodiment of the present disclosure includes compounds of formula (I), as well as their radiolabeled isotopes, racemic mixtures, and stereoisomers, and pharmaceutically

acceptable salts thereof, and combinations thereof, in which R¹ can be

In certain

embodiments, R² and R⁵ can be H, and R³ can be

^{L J}n . Accordingly, an embodiment of the present disclosure includes a class of compounds according to formula (I),

such as formula (3),

radiolabeled isotopes, racemic mixtures, and stereoisomers, and pharmaceutically acceptable salts thereof, and combinations thereof. These embodiments include examples in which R⁴ can be: methyl, halogen, -SSO₂CH₃, -SeCH₃, -NH₃, -SH, and -OH. An embodiment of the present disclosure includes compounds of formula (I), as well as their radiolabeled isotopes, racemic mixtures, and stereoisomers, and pharmaceutically acceptable salts thereof, and combinations thereof, in which R¹ can be O. In certain

embodiments, R² can be H, and R³ can be

. Accordingly, an embodiment of the present disclosure includes a class of compounds according to formula (I), such as formula

(4),

radiolabeled isotopes, racemic mixtures, and stereoisomers, and pharmaceutically acceptable salts thereof, and combinations thereof. These embodiments include examples in which R⁴ can be selected from: methyl, halogen, -SSO₂CH₃, -SeCH₃, -NH₃, -SH, and -OH.

Exemplar embodiments of preparing compounds of the present disclosure are described at least in Examples 5-9.

An embodiment of the present disclosure includes compounds according to any of the formulae described herein in which the compounds are isotopically labeled including, but not limited to, ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁸F, ⁷⁵Br, ⁷⁶Br, ¹²³I, ¹²⁴1, ¹²⁵I, ¹³¹I.

An embodiment of the present disclosure includes compounds according to any of the formulae described herein, which are enantiomers, stereoisomers, diastereomers, racemic mixtures, and cis/trans isomers of these compounds.

An embodiment of the present disclosure includes prodrugs of the compounds according to any of the formulae described herein.

An embodiment of the present disclosure includes compositions comprising a compound of formula (I) or formula (1-6) or a pharmaceutically acceptable salt thereof. An embodiment of the present disclosure includes compositions such as those described in formula (I) or formula (1-6) in a therapeutically effective amount (e.g., a unit dosage form). An embodiment further comprises a pharmaceutically acceptable carrier. Embodiments including compounds of formula (I) or formula (1-6), their radiolabeled isotopes, racemic mixtures, stereoisomers, pharmaceutically acceptable salts, and mixtures thereof, together with a pharmaceutically acceptable carrier can be referred to as pharmaceutical compositions of formula (I) or formula (1-6). An embodiment of the present disclosure includes a method of inhibiting, disrupting, and/or effecting chloride channels or transporters comprising contacting (e.g., administration) chloride channels or transporters with a compound of formula (I), its radiolabeled isotopes, racemic mixtures, stereoisomers, pharmaceutically acceptable salts, and/or mixtures thereof. The step of contacting a chloride channel or transporter can be confined to a cell or can be used as a method of treating hosts or subjects (e.g., humans) with diseases or disorders that are associated with the chloride channel or transporter, such that inhibition or disruption of the chloride channel or transporter alleviates or ameliorates the disease or disorder associated with the chloride channel or transporter or its symptoms. Diseases or disorders that benefit from chloride channel or transporter disruption or inhibition include, but are not limited to, hypertension, heart failure, osteoporosis, inappropriate water retention, and food poisoning.

Embodiments of the compounds of the present disclosure are typically administered to a patient in the form of a pharmaceutical composition or formulation. Such pharmaceutical compositions can be administered to the patient by any acceptable route of administration including, but not limited to, oral, rectal, vaginal, nasal, inhaled, topical (including transdermal) and parenteral modes of administration.

Accordingly, an embodiment of the present disclosure is directed to a pharmaceutical composition comprising a pharmaceutically-acceptable carrier or excipient and a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof. Optionally, such pharmaceutical compositions may contain other therapeutic and/or formulating agents if desired. When discussing compositions, the "compound of the present disclosure" may also be referred to herein as the "active agent" or "agent". As used herein, the term "compound of the present disclosure" is intended to include a compound of formula (I) or a class of compounds embodied in formulae (1)-(6).

The pharmaceutical compositions of the present disclosure typically contain a therapeutically effective amount of a compound of the present disclosure or a pharmaceutically- acceptable salt thereof. Typically, such pharmaceutical compositions can contain about 0.1 to about 95% by weight of the active agent; preferably, about 5 to about 70% by weight; and more preferably about 10 to about 60% by weight of the active agent.

A conventional carrier or excipient can be used in the pharmaceutical compositions of the present disclosure. The choice of a particular carrier or excipient, or combinations of carriers or excipients, will depend on the mode of administration being used to treat a particular patient or type of medical condition or disease state. In this regard, the preparation of a suitable pharmaceutical composition for a particular mode of administration is well within the scope of those skilled in the pharmaceutical arts. Additionally, the carriers or excipients used in the pharmaceutical compositions of this present disclosure are commercially-available. By way of further illustration, conventional formulation techniques are described in Remington: The Science and Practice of Pharmacy, 20^th Edition, Lippincott Williams & White, Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7^th Edition, Lippincott Williams & White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, the following: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, such as microcrystalline cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical compositions.

Pharmaceutical compositions are typically prepared by thoroughly and intimately mixing or blending the active agent with a pharmaceutically-acceptable carrier and one or more optional ingredients. The resulting uniformly blended mixture can then be shaped or loaded into tablets, capsules, pills and the like using conventional procedures and equipment.

The pharmaceutical compositions of the present disclosure are preferably packaged in a unit dosage form. The term "unit dosage form" refers to a physically discrete unit suitable for dosing a patient, e.g., each unit containing a predetermined quantity of active agent calculated to produce the desired therapeutic effect either alone or in combination with one or more additional units. For example, such unit dosage forms can be capsules, tablets, pills, and the like, or unit packages suitable for parenteral administration.

In an embodiment, the pharmaceutical compositions of the present disclosure are suitable for oral administration. Suitable pharmaceutical compositions for oral administration can be in the form of capsules, tablets, pills, lozenges, cachets, dragees, powders, granules; or as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil liquid emulsion; or as an elixir or syrup; and the like; each containing a predetermined amount of a compound of the present disclosure as an active ingredient. When intended for oral administration in a solid dosage form (i.e., as capsules, tablets, pills and the like), the pharmaceutical compositions of the present disclosure will typically include the active agent and one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate. Optionally or alternatively, such solid dosage forms may also comprise: fillers or extenders, such as starches, microcrystalline cellulose, lactose, sucrose, glucose, mannitol, and/or silicic acid; binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, such as glycerol; disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and/or sodium carbonate; solution retarding agents, such as paraffin; absorption accelerators, such as quaternary ammonium compounds; wetting agents, such as cetyl alcohol and/or glycerol monostearate; absorbents, such as kaolin and/or bentonite clay; lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and/or mixtures thereof; coloring agents; and buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the pharmaceutical compositions of the present disclosure. Examples of pharmaceutically-acceptable antioxidants include: water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfate, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin, propyl gallate, alpha- tocopherol, and the like; and metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid, sorbitol, tartaric acid, phosphoric acid, and the like. Coating agents for tablets, capsules, pills and like, include those used for enteric coatings, such as cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate, methacrylic acid-methacrylic acid ester copolymers, cellulose acetate trimellitate, carboxymethyl ethyl cellulose, hydroxypropyl methyl cellulose acetate succinate, and the like.

Pharmaceutical compositions of the present disclosure may also be formulated to provide slow or controlled release of the active agent using, by way of example, hydroxypropyl methyl cellulose in varying proportions; or other polymer matrices, liposomes and/or microspheres. In addition, the pharmaceutical compositions of the present disclosure may optionally contain opacifying agents and can be formulated so that they release the active ingredient only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active agent can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients. Suitable liquid dosage forms for oral administration include, by way of illustration, pharmaceutically-acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. Liquid dosage forms typically comprise the active agent and an inert diluent, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, oils (e.g., cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Suspensions, in addition to the active ingredient, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

The compounds of the present disclosure can also be administered parenterally (e.g., by intravenous, subcutaneous, intramuscular or intraperitoneal injection). For parenteral administration, the active agent is typically admixed with a suitable vehicle for parenteral administration including, by way of example, sterile aqueous solutions, saline, low molecular weight alcohols such as propylene glycol, polyethylene glycol, vegetable oils, gelatin, fatty acid esters such as ethyl oleate, and the like. Parenteral formulations may also contain one or more anti-oxidants, solubilizers, stabilizers, preservatives, wetting agents, emulsifiers, buffering agents, or dispersing agents. These formulations can be rendered sterile by use of a sterile injectable medium, a sterilizing agent, filtration, irradiation, or heat.

Alternatively, the pharmaceutical compositions of the present disclosure are formulated for administration by inhalation. Suitable pharmaceutical compositions for administration by inhalation will typically be in the form of an aerosol or a powder. Such compositions are generally administered using well-known delivery devices, such as a metered-dose inhaler, a dry powder inhaler, a nebulizer or a similar delivery device.

When administered by inhalation using a pressurized container, the pharmaceutical compositions of the present disclosure will typically comprise the active ingredient and a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. Additionally, the pharmaceutical composition can be in the form of a capsule or cartridge (made, for example, from gelatin) comprising a compound of the present disclosure and a powder suitable for use in a powder inhaler. Suitable powder bases include, by way of example, lactose or starch.

The compounds of the present disclosure can also be administered transdermal^ using known transdermal delivery systems and excipients. For example, the active agent can be admixed with permeation enhancers, such as propylene glycol, polyethylene glycol monolaurate, azacycloalkan-2-ones and the like, and incorporated into a patch or similar delivery system. Additional excipients including gelling agents, emulsifiers and buffers, can be used in such transdermal compositions if desired.

If desired, the compounds of this present disclosure can be administered in combination with one or more other therapeutic agents. In this embodiment, a compound of this present disclosure is either physically mixed with the other therapeutic agent to form a composition containing both agents; or each agent is present in separate and distinct compositions which are administered to the patient simultaneously or sequentially.

For example, a compound of formula (I) can be combined with a second therapeutic agent using conventional procedures and equipment to form a composition comprising a compound of formula (I) and a second therapeutic agent. Additionally, the therapeutic agents can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical composition comprising a compound of formula (I), a second therapeutic agent and a pharmaceutically acceptable carrier. In this embodiment, the components of the composition are typically mixed or blended to create a physical mixture. The physical mixture is then administered in a therapeutically effective amount using any of the routes described herein. Alternatively, the therapeutic agents may remain separate and distinct before administration to the patient. In this embodiment, the agents are not physically mixed together before administration but are administered simultaneously or at separate times as separate compositions. Such compositions can be packaged separately or can be packaged together as a kit. The two therapeutic agents in the kit can be administered by the same route of administration or by different routes of administration. Any therapeutic agent compatible with the compounds of the present disclosure can be used as the second therapeutic agent.

In an embodiment, multiple doses of the agent are contacted (e.g., administered). The frequency of administration of the agent can vary depending on any of a variety of factors, e.g., severity of the symptoms, and the like. For example, in an embodiment, the agent is administered once per month, twice per month, three times per month, every other week (qow), once per week (qw), twice per week (biw), three times per week (tiw), four times per week, five times per week, six times per week, every other day (qod), daily (qd), twice a day (qid), or three times a day (tid). As discussed above, in an embodiment, the agent is administered continuously.

The duration of contacted (e.g., administered) of the agent, e.g., the period of time over which the agent is administered, can vary, depending on any of a variety of factors, e.g., patient response, etc. For example, the agent can be administered over a period of time of about one day to one week, about two weeks to four weeks, about one month to two months, about two months to four months, about four months to six months, about six months to eight months, about eight months to 1 year, about 1 year to 2 years, or about 2 years to 4 years, or more.

The amount of the agent contacted (e.g., administered) can vary according to factors such as the degree of susceptibility of the individual, the age, sex, and weight of the individual, idiosyncratic responses of the individual, the dosimetry, and the like. Detectably effective amounts of the agent of the present disclosure can also vary according to instrument and film- related factors. Optimization of such factors is well within the level of skill in the art.

While embodiments of the present disclosure are described in connection with the Examples and the corresponding text and figures, there is no intent to limit the disclosure to the embodiments in these descriptions. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of embodiments of the present disclosure.

EXAMPLES

The following synthetic and biological examples are offered to illustrate the present disclosure, and are not to be construed in any way as limiting the scope of the present disclosure. In the examples below, abbreviations have their generally accepted meanings.

Example 1

Three homologs: (1) CIC-ec1 a prokaryotic chloride/proton antiporter; (2) CIC-Ka, a human chloride channel found in the thin ascending limb of the kidney, and (3) CIC-Kb, a human chloride channel found in the thick ascending limb of the kidney are therapeutic targets for food poisoning, hyponatremia (which occurs with heart failure, liver disease, and syndrome of inappropriate anti-diuretic hormone secretion (SIADH), and hypertension, respectively. Because CIC-ec1 may act as a model for CLC homologs, protein/inhibitor interactions gleaned from the structure allow design of more potent inhibitors of mammalian transporters and channels. For example, a class of inhibitors can be created utilizing a 4,4'-

diisothiocyanatostilbene-2,2'-disulfonate (DIDS),

, scaffold. DIDS is an identified inhibitor of CIC-ec1 , CIC-Ka, and CIC-kb. Example 2

In addition, hydrolysis and oligomerization of DIDS also produces chloride channel or transporter inhibitors. Water hydrolyzes the isothiocyanate, releasing carbonyl sulfide gas. The resulting aniline then nucleophilically attacks a subsequent isothiocyanate to form a dimeric structure.

The remaining isothiocyanates can react further to form higher order oligomers, as demonstrated below.

DIPS Hydrolysis Products

Pentamer:

Data for inhibition of CIC-ec1 and CIC-Ka by DIDS hydrolysis products are shown in Figs. 1 and 2.

Example 3

The core structure of the oligomers is DADS, which has three sites for derivatization:

Namely the amine, sulfonate, and olefin sites can be derivatized. Sulfonates can be deleted or exchanged for other anionic moieties. The olefin can be hydrogenated to increase flexibility. Alkyl chains can be incorporated into the DADS core structure by coupling acid chlorides with the terminpal amines of isopropyl protected DADS:

C₇H₁₅. The alkyl chain length, R, can be varied, and the terminal groups, as well as carbons in the alkyl chain, can be changed to introduce additional charge or steric interactions with the target protein.

The following synthesis applies to Example 3 as well as to Example 8. Synthesis Scheme of 4,4'-octanamidostilbene-2,2'-disulfonate:

R - C₇H₁₅

Experimental:

2,20-[(E)-1 ,2-Ethenediyl]bis[5-aminobenzenesulfonic acid], bis(1-methylethyl)ester (A) was prepared as described in Wrobel, J. et al. Bioorg. Med. Chem 2002, 10, 639, which is incorporated herein by reference.

R - C₇H₁₅ R = CsH^Br

General Method for (bis/mono)acetylation:

To a solution of A (1 mmol) and potassium carbonate (3 mmol) in THF was added the corresponding acid chloride (2.2 mmol for bis, 0.9 mmol for mono) dropwise. The reaction was stirred in the absence of light until complete as determined by TLC analysis. The reaction was then poured into a separatory funnel containing 15 ml. water and 15 ml. EtOAc. The aqueous fraction was extracted with 3 x 20 ml_ EtOAc. The combined organic extracts were washed with 2 x 10 mL water, 1 x 20 ml. brine and dried over MgSO₄. Purification of the white solid via column chromatography (hexanes/DCM/EtOAc) afforded the desired product as a white solid.

General procedure for deprotection to sulfonates:

The corresponding isopropyl stilbene disulfonate (1 mmol) and sodium bromide (5 mmol) were dissolved in an acetone/water (5 ml_/1 mL) solution. The reaction was stirred at 55 ⁰C for 12 h and then cooled to room temperature. A rotoary evaporator was used to evaporate the acetone from solution resulting in precipitation of the product out of the aqueous layer. MeOH was then added until the compound dissolved back into solution. The reaction mixture was then filtered and purified using reversed-phase HPLC. HPLC purification and analysis of the compound was carried out at 23⁰C using a C18 semipreparative column and UV detection at 254 nm. A multi-step linear MeCN gradient in 50 mM ammonium acetate pH 7.0 was used. All samples were filtered through 0.2 μM PTFE syringe filters before injection.

Other subclasses of formula (I) may be prepared incorporating the following reaction mechanisms in Examples 4-9 below.

Example 4

The following discusses the synthesis in a step by step manner.

lsopropyl 4_>4'-diiaminodihydrostilbene-2,2¹-disulfonate:

To a solution of A (1 mmol) in THF (5 mL) was added 10% palladium on carbon (0.1 mmol). The suspension was first flushed with N₂ and the suspenstion was then exposed to a hydrogen-filled balloon until TLC analysis indicated the reaction was complete. The suspension was then diluted with 20 mL EtOAc and filtered over celite. The organic solution was then concentrated and purified (hexanes, DCM, EtOAc) via column chromatography to afford a white solid.

(see the discussion in Example 3 above for deprotection of sulfonates)

Example 5

octanoyl chloride

NaHCO₃ H₂O

The following discusses the synthesis in a step by step manner.

2-Fluoro-5-nitrobenzenesulfonyl chloride

1-Fluoro-4-nitrobenzene (848 ml, 8.00 mmol) was suspended in chlorosulfonic acid (3.19 ml, 48.0 mmol). The mixture was heated to 100 ⁰C in a sealed tube, stirred for 3 days, poured into crashed ice, extracted with AcOEt. The organic layer was washed with sat. NaCI, dried over anhydrous Na₂SO₄ and concentrated in vacuo. Purification of the residue with silica gel column chromatography (hexanes: ethyl acetate = 12: 1) gave the title compound (717 mg, 37%) as a brown oil. ¹H-NMR (CDCI₃, 400 MHz) d 8.89 (dd, 1 H, J = 6.0, 2.8 Hz), 8.65 (ddd, 1 H₁ J = 9.0, 4.0, 2.8 Hz), 7.57 (t, 1 H, J = 9.0 Hz) ppm.

2,2-Dimethylpropyl 2-fluoro-5-nitrobenzenesulfonate

To a solution of 2-fluoro-5-nitrobenzenesulfonyl chloride (475 mg, 1.98 mmol) in dichloroethane (20 ml) were added neopentylalcohol (1.74 g, 19.8 mmol) and pyridine (323 ml, 3.96 mmol) in order. The mixture was heated to 70 °C, stirred for 6 h and then, cooled to room temperature (rt). To the mixture were added sat. NaCI and dichloromethane. The organic layer was dried over anhydrous Na₂SO₄ and concentrated in vacuo. Purification of the residue with silica gel column chromatography (hexanes: ethyl acetate = 20: 1) gave the title compound (451 mg, 78%) as a pale yellow solid. ¹H-NMR (CDCI₃, 400 MHz) d 8.83 (dd, 1 H, J = 5.6, 2.8 Hz), 8.54 (ddd, 1 H₁ J = 9.0, 4.0, 2.8 Hz), 7.46 (t, 1 H₁ J = 9.0 Hz), 3.91 (s, 2H), 0.97 (s, 9H) ppm.

2,2-Dimethylpropyl 2-amino-5-nitrobenzenesulfonate

2,2-Dimethylpropyl 2-fluoro-5-nitrobenzenesulfonate (400 mg, 1.38 mmol) was solved in NH3 in MeOH solution (7N, 4.0 ml). The mixture was stirred at rt for overnight and concentrated. To the residue were added AcOEt (10 ml) and CHCI3 (10 ml) and sat. NaCI (10 ml). The organic layer was dried over anhydrous Na2SO4. Evaporation of organic solvents gave the title compound (393 mg, 99%) as a pale yellow solid.

¹H-NMR (CDCI₃, 400 MHz) d 8.66 (d, 1 H, J = 2.4 Hz), 8.23 (dd, 1 H, J = 9.0, 2.4 Hz),

6.79 (d, 1 H, J = 9.0 Hz), 5.69 (br s, 2H), 3.70 (s, 2H), 0.92 (s, 9H) ppm.

Sodium 2-({2-[(2,2-dimethylpropoxy)sulfonyl]-4-nitrophenyl}carbamoyl) -5- Nitrobenzenesulfonate

To the suspension of dried NaH (33 mg, 1.35 mmol) in THF (15 ml) was added 2,2- dimethylpropyl 2-amino-5-nitrobenzenesulfonate (390 mg, 1.35 mmol). The mixture was stirred at ice-cooling temperature for 5 min. To the mixture was added 6-nitro-3/-/-2,1- benzoxathiol-3-one 1 ,1-dioxide (300 mg, 1.31 mmol). The mixture was stirred at ice cooling temperature for 10 min and concentrated in vacuo. Purification of the residue with silica gel column chromatography (dichloromethane: methanol = 20: 1 - 10: 1) gave the title compound (228 mg, 51 %) as a colorless amorphous solid.

¹H-NMR (DMSO-c/6, 400 MHz) d 10.37 (br s, 1 H), 8.69 (dd, 1 H, J = 9.2, 2.8 Hz), 8.58 (d, 1 H, J = 2.8 Hz), 8.53 (d, 1 H, J = 2.4 Hz), 8.38 (d, 1 H₁ J = 9.2 Hz), 8.33 (dd, 1 H, J = 8.4, 2.4 Hz)₁ 7.73 (d, 1 H₁ J = 8.4 Hz), 3.76 (s, 2H)₁ 0.79 (S₁ 9H) ppm.

Sodium 5-amino-2-({4-amino-2-[(2,2-dimethylpropoxy)sulfonyl]phenyl}carbamoyl)benzenesulfonate

Sodium 2-({2-[(2,2-dimethylpropoxy)sulfonyl]-4-nitrophenyl}carbamoyl) -5- nitrobenzenesulfonate (40 mg, 0.074 mmol) in MeOH (1 ml) was added dried 10 wt% Pd-C (15 mg). The mixture was vigorously stirred at ambient temperature and pressure under H₂ atmosphere for overnight, and then filtered. Concentration of the filtrate gave the title compound (31 mg, 87%) as a colorless amorphous solid.

¹H-NMR (D2O, 400 MHz) d 7.57 (d, 1 H, J = 8.4 Hz), 7.45 (d, 1 H, J = 8.4 Hz), 7.38 (d,

1 H, J = 2.8 Hz), 7.30 (d, 1 H, J = 2.4 Hz), 7.18 (dd, 1 H₁ J = 8.4, 2.8 Hz), 6.95 (dd, 1 H₁ J = 8.4,

2.4 Hz), 3.76 (s, 2H), 0.84 (s, 9H) ppm.

Sodium 2-({2-[(2,2-dimethylpropoxy)sulfonyl]-4- (octanoylaminoJphenylJcarbamoyO-δ^octanoylaminoJbenzenesulfonate

To a solution of sodium 5-amino-2-({4-amino-2-[(2,2-dimethylpropoxy) sulfonyl]phenyl}carbamoyl)benzenesulfonate (29 mg, 0.060 mmol) in H₂O (1.2 ml) were added NaHCO₃ (51 mg, 0.60 mmol) and octanoyl chloride (51 ml, 0.30 mmol) in order at rt. The mixture was stirred for 1 h, and then extracted with dichloromethane. The organic layer was washed with sat. NaCI and dried over anhydrous Na₂SO₄. Purification of the residue with silica gel column chromatography (dichloromethane: methanol = 20: 1 - 10: 1) gave the title compound (23 mg, 52%) as a colorless amorphous solid. 1H-NMR (DMSO-d6, 400 MHz) d 10.26 (br s, 2H), 10.18 (s, 1 H), 8.32 (d, 1 H₁ J = 2.4 Hz), 7.94 (d, 1 H, J = 2.0 Hz), 7.88-7.83 (m, 2H), 7.77 (d, 1 H, J = 8.8 Hz), 7.43 (d, 1 H, J = 8.4 Hz), 3.61 (S₁ 2H), 2.35-2.28 (m, 4H), 1.64-1.54 (m, 4H), 1.33-1.22 (m, 16H), 0.88-0.83 (m, 6H), 0.80 (s, 9H) ppm.

Disodium 5-(octanoylamino)-2-{[4-(octanoylamino)-2- sulfonatophenyl]carbamoyl}benzenesulfonate

To a solution of sodium 2-({2-[(2,2-dimethylpropoxy)sulfonyl]-4-(octanoylamino) phenylJcarbamoyO-S-CoctanoylaminoJbenzenesulfonate (17 mg, 0.023 mmol) in DMF (250 ml) was added NaI (7.0 mg, 0.046 mmol). The mixture was heated to 100 ⁰C, stirred for 12 h then cooled to rt and concentrated in vacuo. To the residue was added H₂O (5 ml). The suspension was sonicated for 3 min and filterated. The filtrate was concentrated in vacuo. To the residue was added H₂O (300 ml). Collecting of precipitate gave tittle compound (6.0 mg, 38%) as a brown amorphous solid.

¹H-NMR (DMSO-d6, 400 MHz) d 10.08 (s, 2H), 9.85 (s, 1 H), 8.16 (d, 1 H, J = 8.8 Hz), 7.96 (d, 1 H₁ J = 2.4 Hz), 7.85 (d, 1 H₁ J = 2.4 Hz), 7.73 (dd, 1 H₁ J = 8.2, 2.4 Hz), 7.64 (dd, 1 H, J = 8.8, 2.4 Hz), 7.29 (d, 1 H₁ J = 8.2 Hz)₁ 2.33-2.24 (m, 4H), 1.62-1.54 (m, 4H), 1.32-1.22 (m, 16H), 0.88-0.83 (m, 6H) ppm.

O₉N

2 steps 27%

6-Nitro-3H-2, 1 -benzoxathiol-3-one 1 , 1 -dioxide

To a mixture of 5-nitro-o-toluenesulfonic acid monohydrate (5.00 g, 19.8 mmol) and 5N NaOH (4.16 ml, 20.8 mmol) in H₂O (70 ml) was added KMnO4 (20.3 g, 128 mmol) in portions. The mixture was heated to 90 ⁰C and stirred for 3 h. After cooling to rt, the mixture was filtered. The filtrate was acidified to pH 1-2 with c. HCI (4.25 ml) and concentrated. To the residue was added thionylchloride (15.2 ml, 128 mmol). The mixture was refluxed for overnight, cooled to rt, and then concentrated. To the residue was added toluene. Collection of the solid gave the title compound (1.24 g, 27%) as a colorless solid. 1H-NMR (CDCI3, 400 MHz) d 8.84 (dd, 1 H, J = 2.0, 0.4 Hz), 8.78 (dd, 1 H₁ J = 8.4, 2.0 Hz)₁ 8.39 (dd, 1 H, J = 8.4, 0.4 Hz) ppm. Example 6

The following discusses the synthesis in a step by step manner.

(See Macromolecule 2002, 35, 9022, which is incorporated herein by reference)

4,4'Diaminodiphenyl ether (0.2Og, 1.0 mmol) was dissolved in cone. H2SO4 (0.4 ml) and stirred at room temperature to dissolve. The solution was cooled to O⁰C and added 20% SO₃ in H₂SO₄ (1.5 ml) and stirred at 50⁰C for 2 hours. The mixture was poured into crashed ice and white precipitates were formed. The precipitates was collected by filtration and rinsed with water and methanol to give disulfonic acid (0.31 g, 0.86 mmol, 86%).

Disulfonic acid (0.14g, 0.39 mmol) and NaOH (0.2g, 5 mmol) was dissolved in water (10 ml). The solution was stirred at O⁰C and octanoyl chloride (0.4 ml) was added over 5 minutes. After 1 hour, formed precipitates were collected by filtration and rinsed with water and hexanes to give a sodium salt of diamide (77 mg, 0.12 mmol, 30%). Example 7

The following discusses the synthesis in a step by step manner.

4,4'-(3-hexylthioureidostilbene-2,2'-disulfonate:

To a solution of DIDS (0.012 mmol) in THF (6 mL) was added hexylamine (0.048 mmol). The mixture was heated to 60 ⁰C at which point DIDS dissolved in solution. The reaction was stirred in the absence of light for 18 h, at which point it was cooled to room temperature and concentrated. The crude product was dissolved in water, filtered and purified using reversed- phase HPLC. HPLC purification and analysis of the compound was carried out at 23 ⁰C using a C18 semipreparative column and UV detection at 254 nm. A multi-step linear MeCN gradient in 10 mM ammonium acetate pH 7.0 was used. All samples were filtered through 0.2 μM PTFE syringe filters before injection.

Example 8

Example 9

One possible end product of the aforementioned syntheses is OADS,

Data for inhibition of CIC-ec1 by OADS are shown in Fig.

3.

Example 10

Therapeutic potential of CLC inhibitors: CLC-7, CLC-ec1 , CLC-Ka, and CLC-Kb:

At least four CLC homologs are potential therapeutic targets for treating human disease. The role of CLC-7 in osteoclast function suggests this homolog as a potential new drug target for treatment of osteoporosis, a crippling disorder in which osteoclasts resorb too much bone.

CLC-Ka and Kb are expressed predominantly in two regions of the body: in the inner ear and in the kidney. In the inner ear, they are expressed in the basolateral membrane of the stria vascularis, where they are required for maintaining the K⁺ secretion performed by these cells, and their functions are redundant. (Disruption of both homologs results in deafness, while disruption of either individually has no effect.) In the kidney, CLC-Kb is expressed in the thick ascending limb of the nephron, where it is responsible for Cl^" re-absorption. Defective CLC-Kb causes the salt-wasting nephropathy called Bartter's syndrome; the low blood pressure observed in patients with this disease suggests CLC-Kb is a potential target for drugs to combat hypertension. The potential of CLC-Kb inhibitors to act as antihypertensives is confirmed by a CLC-Kb polymorphism found in 20% of the population that increases CLC-Kb activity and causes a predisposition to hypertension.

CLC-Ka is expressed in the thin ascending limb of the nephron, where it catalyzes the Cl^" flux necessary for maintaining the steep solute gradient in the kidney medulla and provides the driving force for water absorption from the urine to the blood. Targeted disruption of CLC- Ka dissipates this medullary concentration gradient, leading to an increased water excretion. This suggests CLC-Ka inhibitors as therapeutics to treat hyponatremia, which develops in heart failure patients when the kidneys fail to excrete free water. Diuretics are the classical therapy but can also cause worsening hyponatremia, because sodium is excreted in excess of free water. Thus, a new class of pharmacologic agents that can selectively enhance free water excretion by the kidney (termed "aquaretics") is being developed. To date, antagonists that inhibition vasopressin-2 receptors are the only class of aquaretics that have been approved by the FDA for the treatment of heart failure-associated hyponatremia. The response failure observed in 10-20% of patients with hyponatremia has been attributed to increased thirst, increased responsiveness of the receptor to its ligand, and the presence of activating mutations in the receptor gene in the general population. A CLC-Ka inhibitor would work by dissipating the medullary concentration gradient (the driving force for water movement) and would thus circumvent vasopressin-2 receptor-dependent treatment failures. Such an aquaretic could also have even wider clinical indications for the treatment of hyponatremia associated with liver disease, syndrome of inappropriate anti-diuretic hormone secretion (SIADH), and diuretic use.

CLC-ec1 is a prokaryotic homolog from E. coli. It is necessary for the organism's tolerance of extreme acid conditions, such as those found in the human stomach. Inhibition of prokaryotic CLCs from E. coli and other pathogenic bacteria could be used to prevent food poisoning.

CLC inhibitors

We recently reported our discovery of the highest-affinity CLC-K inhibitors yet known {ACS Chem Biol 3, 419-428 (2008), which is incorporated herein by reference). These inhibitors, which are also the first known inhibitors of CLC-ec1 , are polythiourea oligomers based on a stilbene dilsulfonate building block. While the discovery of these new -Ka inhibitors is exciting, the DIDS oligomers are relatively large molecules (1600 - 2000 g/mol) and thus not ideal for structure-function studies or medicinal chemistry. As a next step, we reasoned that by combining the structures of weak chloride-channel inhibitors, it might be possible to synergistically create an improved inhibitor. We achieved this goal by combining the stilbene- disulfonate building block of the oligomers with a fatty acid amphophilic blocker (J Gen Physiol 133, 43-58 (2009)) to generate the novel compound 4,4'-octanamidostilbene-2,2'-disulfonate (OADS) (FIG.3A). While neither element alone (the stilbene disulfonate or the octanoic acid) inhibits CLC-ec1 at mM concentrations, the combined structure OADS inhibits CLC-ec1 with an apparent affinity of 60 μM (FIG. 3B).

This incredible synergy motivated us to test OADS on two of the eukaryotic CLC homologs which were known to be inhibited at high-micromolar concentrations by stilbene disulfonates (CLC-Ka) or by both stilbene disulfonates and octanoic acid (CLC-O). We found that OADS inhibits CLC-Ka with an apparent affinity of 2 μM (FIGS. 4A and B) - making it the most potent small-molecule CLC inhibitor in efficacy to the polythiourea inhibitors. Moreover, while the polythiourea inhibitors reversed only very slowly, inhibition by OADS is rapidly and completely reversible.

While 30 μM OADS maximally inhibits CLC-Ka, this concentration has absolutely no effect on CLC-O. This selectivity is remarkable and suggests the possibility of developing single- isoform specific inhibitors. We additionally tested OADS on the N68D CLC-Ka mutant, because this mutation greatly reduces inhibition by the larger first-generation inhibitors (Nat Genet 21 , 95-98 (1999)). With OADS, there is also a reduction in affinity, by approximately four-fold (FIGS. 4A and B). Since the CLC-Kb channel, which differs from CLC-Ka at only 60 amino acids, has an aspartate at the 68 position, this result suggests that OADS will likely inhibit CLC- Kb with less efficacy than CLC-Ka. Such selectivity is exactly what is needed for drugs to treat hyponatremia without causing deafness.

The OADS molecule can be derivatized as illustrated in FIG. 6A and 6B. Such derivitizations will likely lead to altered selectivity between the various CLCs, which could be useful for different therapies as described above.

The results of experiments from derivatives that have been synthesized thus far are summarized in FIG. 5.

Proof-of-concept that OADS has efficacy in animals is shown in FIGS. 7A and B. The observed four-fold increase in urine dilution and output is consistent with the role and function of CLC-Ka in urine concentration and supports the hypothesis that OADS can acutely inhibit CLC- Ka function in vivo. FIG. 7A illustrates that OADS (5 mg/kg) was administered to five rats. Rats were maintained in metabolic cages and urine output (FIG. 15A, OADS administered at t=0) or urine osmolality (FIG. 7B, OADS administered at t=24 hrs) was determined. The increase in urine output is nearly identical to that observed in rats injected with Lasix, a commonly prescribed diuretic (data not shown). It should be noted that ratios, concentrations, amounts, and other numerical data may be expressed herein in a range format. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a concentration range of "about 0.1% to about 5%" should be interpreted to include not only the explicitly recited concentration of about 0.1 wt% to about 5 wt%, but also include individual concentrations (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5%, 1.1 %, 2.2%, 3.3%, and 4.4%) within the indicated range. In an embodiment, the term "about" can include traditional rounding according to significant figures of the numerical value. In addition, the phrase "about 'x' to 'y'" includes "about 'x' to about 'y"\

Many variations and modifications may be made to the above-described embodiments. All such modifications and variations are intended to be included herein within the scope of this disclosure and protected by the following claims.

Claims

CLAIMSWe claim:

1. A compound of formula (I), R

, wherein: R¹ is

chosen from _:

and O; each R² is independently chosen from: H and CH₃;

each R³ is independently chosen from: H,

and

each R⁴ is independently chosen from: alkyl, halogen, -SSO₂CH₃, -SeCH₃, -NH₃, - NH₂CH₃, -SH, -S-alkyl, -OH, and -O-alkyl;

R⁵ and R⁶ are independently chosen from: H and CH₃; and n is 1 to 10; as well as radiolabeled isotopes, racemic mixtures, and stereoisomers, and pharmaceutically acceptable salts thereof, and combinations thereof.

2. The compound according to claim 1 , wherein R¹ is S ^^^o , as well as radiolabeled isotopes, racemic mixtures, and stereoisomers, and pharmaceutically acceptable salts thereof, and combinations thereof.

3. The compound according to claim 2, wherein one or both R²S is H and one of both R³S is

, as well as radiolabeled isotopes, racemic mixtures, and stereoisomers, and pharmaceutically acceptable salts thereof, and combinations thereof.

4. The compound according to claim 2, wherein one or both R²S is H and one or both R³S is

, and R⁶ is H, as well as radiolabeled isotopes, racemic mixtures, and stereoisomers, and pharmaceutically acceptable salts thereof, and combinations thereof.

5. The compound according to claim 2, wherein one or both R²S is H, an R³ is H, and the

other R³ is

, as well as radiolabeled isotopes, racemic mixtures, and stereoisomers, and pharmaceutically acceptable salts thereof, and combinations thereof.

6. The compound according to any one of claims 3-5, wherein each R⁴ is independently chosen from: methyl, halogen, -SSO₂CH₃, -SeCH₃, -NH₃, -SH, and -OH, as well as radiolabeled isotopes, racemic mixtures, and stereoisomers, and pharmaceutically acceptable salts thereof, and combinations thereof.

7. The compound according to claim 1 , wherein R¹ is ^ ^ , as well as radiolabeled isotopes, racemic mixtures, and stereoisomers, and pharmaceutically acceptable salts thereof, and combinations thereof.

8. The compound according to claim 7, wherein R² is H and one or both R³S is

, as well as radiolabeled isotopes, racemic mixtures, and stereoisomers, and pharmaceutically acceptable salts thereof, and combinations thereof.

9. The compound according to claim 8, wherein each R⁴ is independently chosen from: methyl, halogen, -SSO₂CH₃, -SeCH₃, -NH₃, -SH, and -OH, as well as radiolabeled isotopes, racemic mixtures, and stereoisomers, and pharmaceutically acceptable salts thereof, and combinations thereof.

10. The compound according to claim 1 , wherein R¹ is

R , as well as radiolabeled isotopes, racemic mixtures, and stereoisomers, and pharmaceutically acceptable salts thereof, and combinations thereof.

11. The compound according to claim 10, wherein one or both R²S is H and one or both R³S

is

, and R⁵ is H, as well as radiolabeled isotopes, racemic mixtures, and stereoisomers, and pharmaceutically acceptable salts thereof, and combinations thereof.

12. The compound according to claim 11 , wherein each R⁴ is independently chosen from: methyl, halogen, -SSO₂CH₃, -SeCH₃, -NH₃, -SH, and -OH, as well as radiolabeled isotopes, racemic mixtures, and stereoisomers, and pharmaceutically acceptable salts thereof, and combinations thereof.

13. The compound according to claim 1 , wherein R¹ is O, as well as radiolabeled isotopes, racemic mixtures, and stereoisomers, and pharmaceutically acceptable salts thereof, and combinations thereof.

14. The compound according to claim 13, wherein one or both R²S is H and one or both R³S

is

, as well as radiolabeled isotopes, racemic mixtures, and stereoisomers, and pharmaceutically acceptable salts thereof, and combinations thereof.

15. The compound according to claim 14, wherein each R⁴ is independently chosen from: methyl, halogen, -SSO₂CH₃, -SeCH₃, -NH₃, -SH, and -OH, as well as radiolabeled isotopes, racemic mixtures, stereoisomers, and pharmaceutically acceptable salts thereof, and combinations thereof.

16. A compound of formula (I), R

R N NR R , wherein: R¹ is

chosen from ,:

, and O; each R² is independently chosen from: H and CH₃;

each R³ is independently chosen from: H,

and

each R⁴ is independently chosen from: alkyl, halogen, -SSO₂CH₃, -SeCH₃, -NH₃, - NH₂CH₃, -SH, -S-alkyl, -OH, and -O-alkyl;

R⁵ and R⁶ are independently chosen from: H and CH₃; and n is 1 to 10.

17. A compound of formula (1 ),

, wherein each R⁴ is independently chosen from: methyl, halogen, -SSO₂CH₃, -SeCH₃, -NH₃, -SH, and -OH, wherein n is 1 to 10.

18. A compound of formula (2),

, wherein each R⁴ is independently chosen from: methyl, halogen, -SSO₂CH₃, -SeCH₃, -NH₃, -SH, and -OH, wherein n is 1 to 10.

19. A compound of formula (3),

, wherein each R⁴ is independently chosen from: methyl, halogen, -SSO₂CH₃, -SeCH₃, -NH₃, -SH, and -OH, wherein n is 1 to 10.

20. A compound of formula (4),

each R⁴ is independently chosen from: methyl, halogen, -SSO₂CH₃, -SeCH₃, -NH₃, -SH, and - OH, wherein n is 1 to 10.

21. A compound of formula (5),

_{t wh}erein each R⁴ is independently chosen from: methyl, halogen, -SSO₂CH₃, -SeCH₃, -NH₃, -SH, and -OH, wherein n is 1 to 10.

22. A compound of formula (6),

, wherein each R⁴ is independently chosen from: methyl, halogen, -SSO₂CH₃, -SeCH₃, -NH₃, -SH, and -OH, wherein n is 1 to 10.

23. A pharmaceutical composition comprising a compound, radiolabeled isotopes, racemic mixtures, and stereoisomers, and pharmaceutically acceptable salts thereof, and combinations thereof of any one of claims 1-22, or mixtures thereof, and a pharmaceutically acceptable carrier.

24. A method of inhibiting a chloride channel or transporter in a cell, comprising contacting the chloride channel or transporter with a compound according to any one of claims 1-22.

25. A method of disrupting a chloride channel or transporter in a cell, comprising contacting the chloride channel or transporter with a compound according to any one of claims 1-22.

26. A method of treating symptoms associated with diseases or disorders associated with a chloride channel or transporter, comprising contacting the chloride channel or transporter with a therapeutically effective amount of a compound according to any one of claims 1-22.