WO2014047212A1 - Compositions and methods comprising oxadiazole derivatives - Google Patents

Abstract

Provided herein are pharmaceutical compositions of compounds of Formula I, metabolites thereof, and pharmaceutically acceptable salts of each thereof containing an effective amount of the compound of Formula I, and methods of their use for treating diarrhea in humans.

Description

COMPOSITIONS AND METHODS COMPRISING OXADIAZOLE DERIVATIVES

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. provisional application number 61/704,380, filed September 21 , 2012, which is incorporated herein in its entirety by reference.

FIELD OF THE INVENTION

[0002] This disclosure relates to pharmaceutical compositions of oxadiazole-containing compounds and methods of their use in treating diarrhea.

BACKGROUND

[0003] Diarrhea is commonly caused by a variety of bacteria, parasites and viruses. It is a fundamental health-threat in regions lacking potable water. One way to avert diarrhea is to prevent exposure to the responsible pathogens. Unfortunately, this requires massive

improvement in both sanitation and nutritional status in developing countries, which is difficult to put in place in a short term. Furthermore, once diarrhea strikes, therapeutic interventions are required to treat the diarrhea. Thus, it is a continuing threat to the developing countries and especially to the health of children who may lack a robust immune response.

[0004] Second only to respiratory infection, diarrheal diseases are responsible for

approximately two million deaths in children under five years of age annually. Many who do survive have lasting health problems due to the effects of recurrent infections and malnutrition. Diarrheal diseases also are the major cause of childhood hospitalization, primarily for dehydration. Each year in developing countries, roughly four billion episodes of acute diarrhea, or approximately 3.2 episodes per child, occur among children under five years of age. See, in general, Diarrheal Diseases Fact Sheet, available at www.oneworldhealth.org.

[0005] Diarrheal episodes can be either acute or persistent (lasting two weeks or more). Of all childhood infectious diseases, diarrheal diseases are thought to have the greatest effect on growth, by reducing appetite, altering feeding patterns, and decreasing absorption of nutrients. The number of diarrheal episodes in the first two years of life has been shown not only to affect growth but also fitness, cognitive function, and school performance.

SUMMARY

[0006] In one aspect, provided herein is a pharmaceutical composition comprising at least a pharmaceutically acceptable excipient and about 10 mg to about 1500 mg of a compound of Formula I:

Formula I or a metabolite thereof, or a pharmaceutically acceptable salt of each thereof, wherein r is 1, 2, or 3;

each R¹ independently is hydrogen, halo, substituted amino, C₁-C₃ alkoxy, C₁-C₃ alkoxy substituted with 1-3 halo, Ci-C₆ alkyl, or Ci-C₆ alkyl substituted with 1-3 halo;

R is hydrogen, Ci-C₆ alkyl, or substituted Ci-C₆ alkyl substituted with an oxo or a nitrogen containing monocyclic 6 membered heteroaryl; and

R³ and R⁴ and independently halo.

[0007] Various embodiments of the compounds of Formula I utilized herein are illustrated as follows. In one embodiment, r is 1. In another embodiment, R² is hydrogen. In another embodiment, R 2 is or substi ·tuted 3 4

C₁-C₃ alkyl C₁-C₃ alkyl. In another embodiment, R and R are independently the same or different and are chloro or bromo. In another embodiment, R¹ is hydrogen. [0008] In one embodiment, the compound of Formula I is:

Compound 2

[0009] In another embodiment, the metabolite is a hyroxylated metabolite, i.e. , a metabolite where a hydrogen atom is substituted with a hydroxy group. In another embodiment, the metabolite is a glucuroni dated metabolite, i.e. , a metabolite which comprises a glucuronic acid moiety.

[0010] In another embodiment, the composition comprises about 10 mg, or about 30 mg, or about 100 mg, or about 300 mg, or about 500 mg, or about 1000, or about 1500 mg of the compound of Formula I, or a pharmaceutically acceptable salt thereof. In another embodiment, the pharmaceutical composition is provided in a unit dosage or a unit dose form. In one embodiment, the unit dosage forms comprise about 10 mg, or about 30 mg, or about 100 mg, about 250 mg, or about 300 mg, or about 500 mg, or about 1000 mg, or about 1500 of the compound of Formula I, or a pharmaceutically acceptable salt thereof.

[0011] In another embodiment, the composition is formulated for oral administration. In one embodiment, the composition comprises a solid form of the compound of Formula (I). In one embodiment, the solid form is in a micronized crystalline form. Methods of preparing micronized solid forms, such as milling, bashing, grinding, and the likes are well known in the art and can be adapted for the present technology by the skilled artisan. In another embodiment, the composition is formulated for parenteral administration. In a further aspect, the composition is formulated for pediatric administration. [0012] In another aspect, provided herein is a method of treating diarrhea in a human patient in need thereof, the method comprising administering a daily amount of about 10 mg to about 1500 mg of a compound of Formula I :

Formula I or a metabolite thereof, or a pharmaceutically acceptable salt of each thereof, wherein r is 1 , 2, or 3;

each R¹ independently is hydrogen, halo, amino, substituted amino, C₁-C₃ alkoxy, substituted d- C₃ alkoxy, C₁-C₃ alkyl, or substituted C₁-C₃ alkyl;

R is hydrogen, C₁-C₃ alkyl, or substituted C₁-C₃ alkyl; and

R³ and R⁴ and independently halo.

[0013] In another aspect, provided herein is a method of treating diarrhea in a human patient in need thereof, the method comprising administering a compound of Formula I or a metabolite thereof, or a pharmaceutically acceptable salt of each thereof, wherein Formula (I) is defined as above, so as to maintain in the human patient a C_max of about 1 ,000 nanogram (ng)/mL to 10,000 ng/mL, or about 2,000 ng/mL to 9,000 ng/mL, or about 3,000 ng/mL to 8,000 ng/mL, or about or about 4,000 ng/mL to 7,000 ng/mL, or about 5,000 ng/mL to 6,000 ng/mL or ranges

therebetween of the compound of Formula I, or a metabolite thereof, or a pharmaceutically acceptable salt of each thereof. In one embodiment, the compound of Formula I is administered at a daily amount of about 10 mg to about 1500 mg.

[0014] In another aspect, provided herein is a method of treating diarrhea in a human patient in need thereof, the method comprising administering a compound of Formula I or a metabolite thereof, or a pharmaceutically acceptable salt of each thereof, wherein Formula (I) is defined as above, so as to maintain in the human patient a plasma exposure as measured by an area under the curve for a dosing interval (AU au) of about 20,000 ng-h/mL to about 100,000 ng-h/mL, about 40,000 ng-h/mL to about 80,000 ng-h/mL, or about 50,000 ng-h/mL to about 70,000 ng'h/mL or ranges therebetween of the compound of Formula I, or a metabolite thereof, or a pharmaceutically acceptable salt of each thereof. In one embodiment, the compound of Formula I is administered at a daily amount of about 10 mg to about 1500 mg.

[0015] As used herein, diarrhea refers to diarrhea or another associated disease such as cholera. In one embodiment, the diarrhea is caused by enterotoxigenic Escherichia coli (ETEC). In one embodiment, the diarrhea is caused by Vibrio cholerae such as Vibrio cholerae 01. In another embodiment, the disease is severe cholera. In another embodiment, the disease is acute cholera.

[0016] In one embodiment, r is 1. In another embodiment, R is C1-C3 alkyl or substituted Ci- C3 alkyl. In another embodiment, R³ and R⁴ are independently the same or different and are chloro or bromo. In another embodiment, R¹ is hydrogen.

[0017] In another embodiment, the compound of Formula I is Compound 2:

Compound 2

[0018] In another embodiment, the method comprises administering a daily amount of about 10 mg, or about 30 mg, or about 100 mg, or about 300 mg, or about 500 mg, or about 1000 mg, or about 15000 mg of the compound of Formula I in the same or differing doses. In another embodiment, the compound of Formula I is administered for at least 3 days in the same or differing doses. In another embodiment, the compound of Formula I is administered for about 14 days to about 30 days in the same or differing doses. In another embodiment, the compound of Formula I is administered two or three times a day in the same or differing doses.

[0019] In another embodiment, the compound of Formula I is administered once a day. Within this embodiment, in other embodiments, a daily amount of 30 mg, or 100 mg, or 300 mg, or 500 mg, or 1000 mg of compound 2 is administered.

[0020] In another embodiment, the compound of Formula I is administered once every twelve hours, or twice a day, or BID. Within this embodiment, in other embodiments, 50 mg, or 100 mg, or 250 mg, or 300 mg, or 500 mg of Compound 2 is administered in each administration.

[0021] In another embodiment, the compound of Formula I is administered once every eight hours, or thrice a day, or TID. Within this embodiment, in other embodiments, 300 mg or 500 mg of Compound 2 is administered in each administration.

[0022] In some embodiments, the compound of Formula I is administered at a dose of 100 mg, 300 mg, or 500 mg every 12 hours (or twice daily, or BID) for three days, plus a single dose is administered on Day 4 (seven doses total). In some embodiments, the compound of Formula I is administered at a dose of 300 mg or 500 mg every 8 hours (or thrice daily, or TID) for three days, plus a single dose is administered on Day 4 (ten doses total).

[0023] In another embodiment, the method comprises administering about 1000 mg of the compound of Formula I. In another embodiment, the method comprises administering about 500 mg of the compound of Formula I three times a day. In another embodiment, the compound of Formula I is administered orally.

[0024] In certain other embodiments, the compound of Formula I is administered in a single ascending dose or in multiple ascending doses.

[0025] In some embodiments, the method of treatment further comprises administering a standard diarrhea management protocol that comprises intravenous and/or oral rehydration therapy (ORT). In some embodiments, the compounds of Formula I are administered with food. In some embodiments, the compounds of Formula I are administered without food.

[0026] The pharmaceutical compositions provided herein are useful for administration in accordance with the treatment and other related methods provided herein. According to this disclosure, it is also contemplated that the compounds administered in the method aspects and embodiments of this disclosure are formulated with at least one pharmaceutically acceptable excipient.

[0027] In some embodiments, a metabolite of Formula I or Compound 2, or a pharmaceutically acceptable salt of each thereof, is formulated for use in the dosage forms provided herein and administered in accordance with the methods provided herein in amounts the same as disclosed herein for compounds of Formula I.

DETAILED DESCRIPTION

[0028] This disclosure relates to pharmaceutical compositions of compounds of Formula I and methods of use thereof and is described in more detail below.

[0029] Throughout this disclosure, the text refers to various embodiments of the utilized compounds, and of the compositions and methods provided herein. The embodiments discussed herein are merely illustrative and are not meant to limit the scope of the present disclosure.

[0030] Also, throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure in their entirety to more fully describe the state of the art to which this disclosure pertains. A. Definitions

[0031] As used in the specification and claims, the singular form "a," "an" and "the" include plural references unless the context clearly dictates otherwise.

[0032] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but not excluding others. "Consisting essentially of when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude, e.g., trace pharmaceutically acceptable byproducts from the isolation and purification method and, e.g., preservatives, and the like. "Consisting of shall mean excluding more than trace elements of other ingredients. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0033] All numerical designations, e.g., amounts, pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied ( + ) or ( - ) by increments of 10%. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term "about." It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0034] "C_x-Cy" refers to a group containing x-y carbon atoms.

[0035] "Alkyl" refers to monovalent saturated aliphatic hydrocarbyl groups having from 1 to 10 carbon atoms and preferably 1 to 6 carbon atoms. This term includes, by way of example, linear and branched hydrocarbyl groups such as methyl (CH₃-), ethyl (CH₃CH₂-), n-propyl (CH₃CH₂CH₂-), isopropyl ((CH₃)₂CH-), n-butyl (CH₃CH₂CH₂CH₂-), isobutyl ((CH₃)₂CHCH₂-), sec-butyl ((CH₃)(CH₃CH₂)CH-), t-butyl ((CH₃)₃C-), n-pentyl (CH₃CH₂CH₂CH₂CH₂-), and neopentyl ((CH₃)₃CCH₂-). [0036] "Alkoxy" refers to the group -O-alkyl wherein alkyl is defined herein. Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, sec-butoxy, and n-pentoxy.

[0037] "Substituted amino" refers to the group -NR⁴⁸R⁴⁹ where R⁴⁸ and R⁴⁹ are independently selected from the group consisting of hydrogen and Ci-C₆ alkyl, such that they are both not hydrogen.

[0038] "Halo" or "halogen" refers to fluoro, chloro, bromo and iodo.

[0039] "Heteroaryl" refers to an aromatic ring of from 1 to 10 carbon atoms and 1 to 4 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur within the ring. Such heteroaryl groups can have a single ring (e.g. , pyridinyl or furyl) or multiple condensed rings (e.g. , indolizinyl or benzothienyl) wherein the condensed rings may or may not be aromatic and/or contain a heteroatom provided that the point of attachment is through an atom of the aromatic heteroaryl group. In one embodiment, the nitrogen and/or the sulfur ring atom(s) of the heteroaryl group are optionally oxidized to provide for the N-oxide (N→0), sulfinyl, or sulfonyl moieties. Preferred heteroaryls include pyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl.

[0040] "Oxo" refers to the atom (=0).

[0041] "Pharmaceutically acceptable" refers to nontoxic and safe for administration in vivo, particularly to humans.

[0042] "Pharmaceutically acceptable salt" refers to pharmaceutically acceptable salts of a compound, which salts are derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, and tetraalkylammonium; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, and oxalate (see Stahl and Wermuth, eds., "HANDBOOK OF

PHARMACEUTICALLY ACCEPTABLE SALTS," (2002), Verlag Helvetica Chimica Acta, Zurich, Switzerland), for a discussion of pharmaceutical salts, their selection, preparation, and use.

[0043] Generally, pharmaceutically acceptable salts are those salts that retain substantially one or more of the desired pharmacological activities of the parent compound and which are suitable for administration to humans. Pharmaceutically acceptable salts include acid addition salts formed with inorganic acids or organic acids. Inorganic acids suitable for forming

pharmaceutically acceptable acid addition salts include, by way of example and not limitation, hydrohalide acids (e.g., hydrochloric acid, hydrobromic acid, hydroiodic acid, etc.), sulfuric acid, nitric acid, phosphoric acid, and the like.

[0044] Organic acids suitable for forming pharmaceutically acceptable acid addition salts include, by way of example and not limitation, acetic acid, trifluoroacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, oxalic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, palmitic acid, benzoic acid, 3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, alkylsulfonic acids (e.g., methanesulfonic acid, ethanesulfonic acid, 1 ,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, etc.), arylsulfonic acids (e.g., benzenesulfonic acid,

4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid,

camphorsulfonic acid, etc.), 4-methylbicyclo[2.2.2]-oct-2-ene-l -carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like.

[0045] Pharmaceutically acceptable salts also include salts formed when an acidic proton present in the parent compound is either replaced by a metal ion (e.g. , an alkali metal ion, an alkaline earth metal ion, or an aluminum ion) or coordinates with an organic base (e.g., ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, morpholine, piperidine, dimethylamine, diethylamine, triethylamine, and ammonia). [0046] The pharmaceutically acceptable salts utilized in the present disclosure can be formed by conventional means, such as by reacting the free base form of the product with one or more equivalents of the appropriate acid in a solvent or medium in which the salt is insoluble or in a solvent such as water which is removed in vacuo, by freeze drying, or by exchanging the anions of an existing salt for another anion on a suitable ion exchange resin.

[0047] As used herein, "treating" or "treatment" of a disease in a patient refers to (1) preventing the symptoms or disease from occurring in the patient that is predisposed or does not yet display symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression of the disease or the symptoms of the disease. As understood in the art, "treatment" is an approach for obtaining beneficial or desired results, including clinical results. For the purposes of this disclosure, beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of a condition (including a disease), stabilized (i.e., not worsening) state of a condition (including disease), delay or slowing of condition (including disease), progression, amelioration or palliation of the condition (including disease), states and remission (whether partial or total), whether detectable or undetectable. Preferred are compounds that are potent and can be administered locally at very low doses, thus minimizing systemic adverse effects.

B. Compounds utilized

[0048] This disclosure utilizes compounds of Formula I. In some embodiments, a metabolite of Formula I or a pharmaceutically acceptable salt thereof is utilized. IN some embodiments, "compounds utilized herein" refer to compounds of Formula I, metabolites thereof, and pharmaceutically acceptable salts of each thereof. Certain non-limiting examples of compounds utilized in this disclosure are tabulated below.

Table 1

[0049] The compounds utilized in this disclosure can be synthesized via a variety of different synthetic routes using commercially available starting materials and/or starting materials prepared by conventional synthetic methods. It will also be appreciated by those skilled in the art that in the processes described below, the functional groups of intermediate compounds may need to be protected by suitable protecting groups.

[0050] The exact identity of any protecting group(s) used will depend upon the identity of the functional group being protected, and will be apparent to those of skill in the art.

Guidance for selecting appropriate protecting groups, as well as synthetic strategies for their attachment and removal, can be found, for example, in Greene & Wuts, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, 3d Edition, John Wiley & Sons, Inc., New York (1999) and the references cited therein. Examples of functional groups include hydroxy and amino.

[0051] As used herein, "protecting group" refers to a group of atoms that, when attached to a reactive functional group in a molecule, mask, reduce or prevent the reactivity of the functional group. Typically, a protecting group can be selectively removed as desired during the course of a synthesis. Examples of protecting groups can be found in Greene and Wuts, as mentioned above, and, additionally, in Harrison et al, COMPENDIUM OF SYNTHETIC ORGANIC METHODS, Vols. 1-8, 1971-1996, John Wiley & Sons, NY. Representative amino protecting groups include, but are not limited to, formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl ("CBZ"), tert-butoxycarbonyl ("Boc"), trimethylsilyl ("TMS"),

2-trimethylsilyl-ethanesulfonyl ("TES"), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl ("FMOC"), nitro-veratryloxycarbonyl ("NVOC"), and the like. Representative hydroxyl protecting groups include, but are not limited to, those where the hydroxyl group is either acylated to form acetate and benzoate esters or alkylated to form benzyl, substituted benzyl where the phenyl ring is substituted with 1-3, Ci-C₆ alkyl and Ci- C₆ alkoxy groups, and trityl ethers, as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers {e.g., TMS or TIPPS groups), aryl silyl ethers {e.g., triphenylsilyl ether), mixed alkyl and aryl substituted silyl ethers, and ally! ethers. [0052] It is understood that one of ordinary skill in the art would be able to make the compounds utilized herein by similar methods or by methods known to one skilled in the art. In general, starting components may be obtained from sources such as Aldrich, or synthesized according to sources known to those of ordinary skill in the art (see, e.g., Smith and March, MARCH'S ADVANCED ORGANIC CHEMISTRY: REACTIONS,

MECHANISMS, AND STRUCTURE, 5th edition (Wiley Interscience, New York)).

Moreover, the various substituted groups {e.g., R^R⁵, see below) of the compounds utilized herein may be attached to the starting components, intermediate components, according to methods known to those of ordinary skill in the art.

[0053] A variety of exemplary synthetic routes that can be used to synthesize the compounds utilized herein are described in Schemes I and II below. For example, and without limitation, various compounds of Formula I, can be synthesized from nitriles 1-1 as illustrated in Scheme I, below:

[0054] In Scheme I, the groups R -R⁴ and r are as defined herein and R⁵ is hydrogen or Ci- C₃ alkyl optionally substituted with 1-3 phenyl groups, one or more of which phenyl groups are optionally substituted with 1-3 C₁-C₃ alkyl and C₁-C₃ alkoxy groups. The starting benzonitriles I-l can be purchased from commercial sources or prepared using standard techniques of organic chemistry. For example, the starting benzonitriles I-l can be prepared from suitable unsubstituted amides via dehydration under standard dehydration conditions using a dehydrating reagent such as phosphorous pentoxide.

[0055] Compound 1-2 is prepared by conventional methods. Typically, such methods include reaction of compound I-l with at least an equimolar amount of hydroxylamine 1-2 and preferably a slight excess thereof in a suitable diluent such as methanol, ethanol and the like. The reaction is typically conducted at elevated temperatures and preferably at the reflux temperature of the selected solvent. The reaction is continued until substantially complete (as evidenced by, e.g., thin layer chromatography or high performance liquid chromatography) which typically occurs within 1 to 12 hours and preferably 2-4 hours. Compound 1-2 is recovered by conventional methods such as evaporation, chromatography, precipitation, crystallization, and the like or, alternatively, used in the next step without purification and/or isolation. In a preferred embodiment, compound 1-2 is recovered by cold filtration of the reaction mixture.

[0056] Compound 1-2 is converted to an oxadiazole, compound 1-3, by conventional condensation reaction conditions in the presence of ethyl 2-chloro-2-oxoacetate and pyridine. Specifically, approximately equimolar amounts of compound 1-3 and ethyl 2-chloro-2- oxoacetate are combined in pyridine and stirred at room temperature for about 1 hour and then at 60 °C for about 2 hours. The reaction is continued until substantially complete (as evidenced by, e.g., thin layer chromatography or high performance liquid chromatography) which typically occurs within 1 to 12 hours and preferably 2-5 hours. The resulting oxadiazole, compound 1-3, is recovered by conventional methods such as evaporation, chromatography, precipitation, crystallization, and the like. In one embodiment, compound 1-3 is recovered by chromatography followed by crystallization using toluene.

[0057] Compound 1-3 is converted to an oxadiazole of Formula 1-5 under standard substitution conditions using at least an equimolar amount of amine 1-4 and preferably a slight excess thereof in a suitable diluent such as methanol, ethanol and the like (Method 1). The reaction is typically conducted at elevated temperatures and preferably at the reflux temperature of the selected solvent. The reaction is continued until substantially complete (as evidenced by, e.g., thin layer chromatography or high performance liquid chromatography) which typically occurs within 1 to 12 hours and preferably 4-8 hours. Alternatively, the substitution reaction can be performed in the presence of a Lewis acid, such as aluminum(III) under prolonged reaction conditions (Method 2). The reaction typically occurs within 1 to 5 days and preferably 3 days. The compound of Formula 1-5 is deprotected using various art known methods to provide the compound of Formula B. Using either of these methods, compounds of Formula 1-5 and I can be recovered by conventional methods such as evaporation, chromatography, precipitation, crystallization, and the like.

[0058] The reactions depicted in Scheme I may proceed more quickly when the reaction solutions are rapidly heated by, e.g., microwave radiation. Compounds 1-4 can be purchased from commercial sources or prepared using standard techniques of organic chemistry. For example, amines 1-4 can be synthesized from suitable alkyl or aryl halide precursors under substitution or amination reaction conditions using standard synthetic organic chemistry. See also Vogel, 1989, PRACTICAL ORGANIC CHEMISTRY, Addison Wesley Longman, Ltd. and John Wiley & Sons, Inc.

[0059] Skilled artisans will recognize that in some instances, compounds 1-1 and 1-4 may include functional groups that require protection during synthesis. The exact identity of any protecting group(s) used will depend upon the identity of the functional group being protected, and will be apparent to those of skill in the art. Guidance for selecting appropriate protecting groups, as well as synthetic strategies for their attachment and removal, can be found, for example, in Greene & Wuts, PROTECTIVE GROUPS IN ORGANIC

SYNTHESIS, 3d Edition, John Wiley & Sons, Inc., New York (1999) and the references cited therein (hereinafter "Greene & Wuts").

[0060] Compounds utilized herein are also synthesized as shown in scheme II.

(Ri )r

Deprotection

Compound C is synthesized as compound 1-3 and reacted with 4-hydroxybenzyl amine to provide compound D. Compound D can be separated following various art known methods or used directly in the next step, where it is coupled with a substituted phenyl boronic acid in presence of Cu (II) or Pd (0) catalysts to provide a compound of Formula 1-6, which can be deprotected following art known methods to provide a compound of Formula I. The reactions are carried out in a solvent that is inert to the reaction conditions. Preferably equimolar amounts of the reactants are used, as will be apparent to the skilled artisan upon reading this disclosure.

[0061] In vitro and in vivo activity of the compounds utilized in this disclosure are determined by well known methods, certain of which are illustrated below. Diarrhea can involve CFTR protein and the effect of the compounds utilized herein on CFTR protein can be useful to determine the anti- diarrheal activity of these compounds. For the purpose of illustration only, Fisher Rat Thyroid (FRT) cells stably co-expressing wildtype human CFTR and a reporter protein such as green fluorescent protein (GFP) or a mutant such as the yellow 31

fluorescent protein-based CI IY halide sensor e.g. YFP-H148Q can be cultured on 96-well plates as described in Gruenert (2004), supra or Ma et al. (2002) J. Clin. Invest. 110: 1651- 1658. Following a 48 hour incubation confluent FRT-CFTR-YFP-H 148Q cells in 96-well plates are washed three times with phosphate buffered saline (PBS) and then CFTR halide conductance is activated by incubation for 5 minutes with a cocktail containing 5 μΜ, forskolin, 25 μΜ apigenin and 100 μΜ IB MX. Test compounds at a final concentration of 10 μΜ and 20 μΜ are added five minutes prior to assay of iodide influx in which cells are exposed to a 100 mM inwardly-directed iodide gradient. Baseline YFP fluorescence is recorded for two seconds followed by 12 seconds of continuous recording of fluorescence after rapid addition of the T containing solution, to create a T gradient. Initial rates of T influx can be computed from the time course of decreasing fiuorescence after the T gradient as known to those skilled in the art and described in Yang et al. (2002) J. Biol. Chem.:

35079-35085.

[0062] Activity of the CFTR channel can also be measured directly using

electrophysiological methods. An exemplary protocol for measuring CFTR current is described as whole cell patch clamp method. As an illustration, recordings are conducted at room temperature (~21°C) using a HEKA EPC-10 amplifier. Electrodes are fabricated from 1.7 mm capillary glass with resistances between 2 and 3 ΜΩ using a Sutter P-97 puller. For recording the CFTR channels, the extracellular solution can contain (in mM) 150 NaCl, 1 CaCl₂, 1 MgCl₂, 10 glucose, 10 mannitol, and 10 TES (pH 7.4), and the intracellular (pipette) solution can contain 120 CsCl, MgCl₂, 10 TEA-C1, 0.5 EGTA, 1 Mg-ATP and 10 HEPES (pH 7.3).

[0063] The CFTR channels are activated by forskoin (5μΜ) in the extracellular solution. The cells are held at a potential of 0 mV and currents are recorded by a voltage ramp protocol from -120 mV to +80 mV over 500 ms every 10 seconds. Compounds are superfused to individual cells using a Biologic MEV-9/EVH-9 rapid perfusion system.

[0064] Furthermore, human colonic T84 cells can be acquired from the European

Collection of Cell Cultures (ECACC) and grown in standard culture conditions as described by the supplier. On the day before assay 25,000 T84 cells per well are plated into standard black walled, clear bottom 384-well assay plates in standard growth medium consisting of DMEM:F12 with 10% FBS and incubated overnight. On the day of the assay the plates are washed using a standard assay buffer (HBSS with 10 mM Hepes) and incubated for 15 minutes in serum free cell culture medium before the addition of a commercially available membrane potential sensitive fluorescent dye (FLIPR Red membrane potential dye,

Molecular Devices Corporation). T84 cells are incubated with the FLIPR Red membrane potential dye for 45 minutes in the presence and absence of test compound before being transferred to a commercially available fluorescence imaging plate reader (FLIPR384, Molecular Devices Corporation). Fluorescence levels are monitored continuously every second for 150 seconds; after an initial 10 second baseline, CFTR channel activity is stimulated through the addition of 10 μΜ forskolin in the presence of 100 μΜ of the phosphodiesterase inhibitor iso-butyl-methylxanthine (IB MX). Addition of the forskolin leads to the activation of intracellular adenylyl cylase 1, elevating cAMP levels and results in the phosphorylation and opening of CFTR anion channels. CFTR channel opening causes chloride ion efflux and subsequent depolarization of the cells, which is measured by an increase in fluorescence. CFTR inhibitor compounds prevent cell depolarization and the associated increase in fluorescence.

[0065] For in vivo tests for treatment of diarrhea, mice (CD1 strain, 25-35 g) are deprived of food prior to surgery and can be anaesthetized with any suitable agent such as

intraperitoneal ketamine (40 mg/kg) and xylazine (8 mg/kg). Body temperature is maintained at 36-38° C using a heating pad. A small abdominal incision is made and 3 closed intestinal (ileal and/or duodenum/jejunum) loops (length 15-30 mm) proximal to the cecum are isolated by sutures. Loops are injected with 100 of PBS or PBS containing cholera toxin (^g) with or without test compound at appropriate doses. The abdominal incision is closed with suture and mice are allowed to recover from anesthesia. Approximately four to six hours later, the mice are anesthetized, intestinal loops are removed, and loop length and weight are measured to quantify net fluid secretion to be measured as g/cm of loop. C. Pharmaceutical compositions

[0066] In one aspect, this disclosure provides a pharmaceutical composition comprising an effective amount of a compound utilized herein. As used herein, an "effective amount" refers to an amount of the compound of Formula I, that is useful for (1) preventing diarrhea or one or more of its symptoms from occurring in the patient that is predisposed or does not yet display symptoms of the disease; (2) inhibiting diarrhea or arresting its development; or (3) ameliorating or causing regression of the diarrhea or one or more of its symptoms. In one embodiment, the diarrhea results from cholera, such that he effective amount refers to an amount that is useful for preventing, inhibiting, or ameliorating cholera or one or more of its symptoms. In one embodiment, an effective daily amount comprises about 10 mg to about 1500 mg of the compound of Formula I.

[0067] In certain embodiments the compounds utilized herein are formulated alone. In certain other embodiments the compounds utilized herein are formulated together with one or more pharmaceutically acceptable adjuvant, binding agent, carrier, coloring agent, diluent, excipient, fillers, flavoring agent, lubricants, preservative, pH sensitive polymers, stabilizer, sweetening agent, vehicle, wetting agents, and the likes. In some embodiments, the pharmaceutical compositions may include, for example, troches, lozenges, aqueous, or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. In one embodiment, the pharmaceutical composition is a microsuspension, such as an aqueous microsuspension.

[0068] In another embodiment, the composition comprises about 10 mg of the compound of Formula I. In another embodiment, the composition comprises about 30 mg of the compound of Formula I. In another embodiment, the composition comprises about 100 mg of the compound of Formula I. In another embodiment, the composition comprises about 300 mg, of the compound of Formula I. In another embodiment, the composition comprises about 500 mg of the compound of Formula I. In another embodiment, the composition comprises about 1000 mg of the compound of Formula I. In another embodiment, the composition comprises about 1500 mg of the compound of Formula I. [0069] In one embodiment, the compound of Formula I is formulated at least with a gum or a polysorbate. In another embodiment, the compound of Formula I thus formulated further comprises an aqueous carrier or excipient. Examples of gums and polysorbates useful in these formulations are well known to the skilled artisan. Non-limiting examples of the gums include xanthan gum, guar gum, acacia gum, and locust bean gum. Examples of polysorbates include without limitation Polysorbates 20, 40, 60, and 80.

[0070] In another embodiment, provided herein is a pharmaceutical composition comprising a compound of Formula I, a gum and/or a polysorbate as the pharmaceutically acceptable excipient(s), and optionally an aqueous carrier. In another embodiment, the pharmaceutical composition comprises an aqueous suspension. In another embodiment, the pharmaceutical composition comprises about 0.1% (w/v) to about 1% (w/v) of the gum. In another embodiment, the gum is present in about 0.2% (w/v). In another embodiment, the gum is xanthan gum. In another embodiment, the pharmaceutical composition comprises about 0.1%) (w/v) to about 1% (w/v) polysorbate. In another embodiment, the polysorbate is present in about 0.26% (w/v). In another embodiment, the polysorbate is polysorbate 80. In another embodiment, the compound of Formula I is present in an amount of 30 mg, or 100 mg, or 300, mg, or 500 mg, or 1000 mg. In another embodiment, the compound of Formula I is Compound 2.

[0071] In one embodiment, the pharmaceutical compositions provided herein can be for oral administration, including buccal and sublingual administration.

[0072] For oral administration, the pharmaceutical compositions may take the form of, for example, lozenges, tablets, or capsules prepared by conventional means with

pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, starch, gelatin, acacia, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents (e.g., corn starch or alginic acid); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potato starch or sodium starch glycolate); and/or wetting agents (e.g., sodium lauryl sulfate). [0073] The tablets can be left uncoated or they can be coated, for example, by sugars, films, or enteric coatings, and agents that delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.

[0074] Liquid compositions provided herein for oral administration may take the form of, for example, elixirs, solutions, syrups, or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or

hydrogenated edible fats); emulsifying agents (e.g., lecithin, or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, cremophore™, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl -p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, preservatives, flavoring, coloring, and sweetening agents as appropriate.

[0075] Compositions provided herein for oral administration can be suitably formulated to give controlled release or sustained release of the active compound, as is well known. For example, a time delay material such as glyceryl monostearate or glyceryl distearate can be employed. They may also be coated by the techniques described in the U.S. Pat. Nos.

4,256,108; 4,166,452; and 4,265,874 to form osmotic therapeutic tablets for control release. The sustained release compositions provided herein can be in the form of a compressed tablet comprising an intimate mixture of compounds utilized herein and a partially neutralized pH- dependent binder that controls the rate of compound dissolution in aqueous media across the range of pH in the stomach (typically approximately 2) and in the intestine (typically approximately about 5.5).

[0076] Many materials known in the pharmaceutical art as "enteric" binders and coating agents have a desired pH dissolution properties. The examples include phthalic acid derivatives such as the phthalic acid derivatives of vinyl polymers and copolymers, hydroxyalkylcelluloses, alkylcelluloses, cellulose acetates, hydroxyalkylcellulose acetates, cellulose ethers, alkylcellulose acetates, and the partial esters thereof, and polymers and copolymers of lower alkyl acrylic acids and lower alkyl acrylates, and the partial esters thereof. One or more pH-dependent binders present in the sustained release composition provided herein are in an amount ranging from about 1 to about 20 wt %, more preferably from about 5 to about 12 wt % and most preferably about 10 wt %.

[0077] One or more pH-independent binders may be in used in oral sustained release compositions provided herein. The pH-independent binders can be present in the

composition provided herein in an amount ranging from about 1 to about 10 wt %, and preferably in amount ranging from about 1 to about 3 wt % and most preferably about 2 wt %.

[0078] In one embodiment, the compound is admixed with about 0.2 % to about 5.0 % w/v solution of a pharmaceutically-acceptable polymer. In other embodiments, the amount of pharmaceutically-acceptable polymer is between about 0.25% and about 5.0 %; between about l%o and about 4.5%; between about 2.0% and about 4.0 %; between about 2.5% and about 3.5%; or alternatively about 0.2%; about 0.25%; about 0.3%; about 0.35%; about 0.4%; about 0.45%; about 0.5%, about 1.0%, about 2.0%, about 3.0%, or about 4.0%, of the polymer.

[0079] The sustained release composition provided herein may also contain pharmaceutical excipients intimately admixed with the compound and the pH-dependent binder.

Pharmaceutically acceptable excipients may include, for example, pH-independent binders or film-forming agents such as hydroxypropyl methylcellulose, hydroxypropyl cellulose, methylcellulose, polyvinylpyrrolidone, neutral poly(meth)acrylate esters, starch, gelatin, sugars, carboxymethylcellulose, and the like. Other useful pharmaceutical excipients include diluents such as lactose, mannitol, dry starch, microcrystalline cellulose and the like; surface active agents such as polyoxyethylene sorbitan esters, sorbitan esters and the like; and coloring agents and flavoring agents. Lubricants (such as talc and magnesium stearate) and other tableting aids can also be optionally present.

[0080] The sustained release compositions provided herein have a compound utilized herein in the range of about 50% by weight to about 95% or more by weight, and preferably between about 70% to about 90% by weight; a pH-dependent binder content of between 5% and 40%), preferably between 5% and 25%, and more preferably between 5% and 15%; with the remainder of the dosage form comprising pH-independent binders, fillers, and other optional excipients.

[0081] For buccal administration, the pharmaceutical compositions can take the form of tablets or lozenges formulated in the conventional manner.

[0082] In another embodiment, parenteral or systemic compositions, e.g., compositions for intramuscular, intraperitoneal, intravenous, or subcutaneous infusion or injection are also contemplated. Useful injectable preparations include sterile suspensions, solutions, or emulsions of the active compound(s) in aqueous or oily vehicles. The pharmaceutical compositions can also contain excipients, such as suspending, stabilizing, and/or dispersing agents. The compositions for injection can be presented in unit dosage form, e.g., in ampules or in multidose containers, and may contain added preservatives.

[0083] Alternatively, the injectable composition can be provided in powder form for reconstitution with a suitable vehicle, including but not limited to sterile pyrogen free water, buffer, and dextrose solution, before use. To this end, the active compound(s) can be dried by any art-known technique, such as lyophilization, and reconstituted prior to use.

[0084] The pharmaceutical compositions can be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, and isotonic sodium chloride solution. The compounds may also be administered in the form of suppositories for rectal or urethral administration of the drug.

[0085] For prolonged delivery, the compounds utilized herein can be formulated as a depot preparation for administration by implantation or intramuscular injection. The active ingredient can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt). [0086] Alternatively, other pharmaceutical delivery systems can be employed. Liposomes and emulsions are well-known examples of delivery vehicles that can be used to deliver active compound. Certain organic solvents such as dimethylsulfoxide (DMSO) may also be employed, although usually at the cost of greater toxicity.

[0087] The pharmaceutical compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active compound(s). The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device can be accompanied by instructions for administration.

[0088] The pharmaceutical compositions can be, for example, prepared by uniformly and intimately bringing the active ingredient into association with a liquid carrier, a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired composition. Accordingly, pharmaceutical compositions comprising the compounds described herein can be prepared by means of conventional mixing, dissolving, granulating, dragee-making levigating, emulsifying, encapsulating, entrapping, or lyophilization processes.

[0089] Also provided are kits for administration of the pharmaceutical compositions provided herein comprising an unit dosage amount of the compound. Kits may further comprise suitable packaging and/or instructions for use of the compound. Kits may also comprise a means for the delivery of the compound or the pharmaceutical composition, such as a syringe for injection, or pressure pack for capsules, tables, suppositories, or other device as described herein.

[0090] Other types of kits provide the compound and reagents to prepare a pharmaceutical compositions provided herein for administration. The pharmaceutical compositions can be in a dry or lyophilized form or in a solution, particularly a sterile solution. When the pharmaceutical compositions is in a dry form, the reagent may comprise a pharmaceutically acceptable diluent for preparing a liquid composition. The kit may contain a device for administration or for dispensing the pharmaceutical compositions, including, but not limited to a syringe or a pipette. [0091] The kits may include other therapeutic compounds for use in conjunction with the compounds described herein. These compounds can be provided in a separate form or mixed with the compounds of Formula I, metabolites thereof, and pharmaceutically acceptable salts of each thereof, as utilized herein. In one embodiment, the compound of Formula I is Compound 2. The kits will include appropriate instructions for preparation and administration of the pharmaceutical compositions, side effects of the pharmaceutical compositions, and any other relevant information. The instructions can be in any suitable format, including, but not limited to, printed matter, videotape, computer readable disk, or optical disc.

[0092] In one embodiment, this disclosure provides a kit comprising a compound selected from the compounds provided herein, packaging, and instructions for use.

[0093] In another embodiment, provided herein is a kit comprising the pharmaceutical composition provided herein and at least one pharmaceutically acceptable excipient, such as, e.g., a diluent, preservative, stabilizer, and the like, or a mixture thereof, packaging, and instructions for use. In another embodiment, kits for treating an individual who suffers from or is susceptible to the conditions described herein are provided, comprising a container comprising a dosage amount of a compound utilized herein or a pharmaceutical composition provided herein, and instructions for use. The container can be any of those known in the art and appropriate for storage and delivery of oral, intravenous, topical, rectal, urethral, or inhaled compositions.

[0094] Kits may also be provided that contain sufficient dosages of the pharmaceutical compositions to provide effective treatment for an individual for an extended period, such as a week, 2 weeks, 3, weeks, 4 weeks, 6 weeks, or 8 weeks or more.

D. Treatment methods

[0095] In one aspect, effective amounts of the compounds utilized herein and their pharmaceutical compositions are administered or delivered to treat diarrhea and associated symptoms in a human patient in need of such treatment. In one embodiment, the patient is an infant (i.e., less than 2 years old, or alternatively, less than one year old, or alternatively, less than 6 months old, or alternatively, less than 3 months old, or alternatively, less than 2 months old, or alternatively, less than 1 one month old, or alternatively, less than 2 weeks old), a newborn (e.g. , less than one week old, or alternatively, less than one day old), a pediatric patient (e.g., less than 18 years old or alternatively less than 16 years old) or yet further, a geriatric patient (e.g., greater than 65 years old). In another embodiment, the diarrhea is, infectious diarrhea, inflammatory diarrhea or diarrhea associated with chemotherapy. In one embodiment, the treatment of secretory diarrhea involves use of a compounds utilized herein to inhibit the CFTR chloride channel.

[0096] In one embodiment, the compounds utilized herein and their compositions are used in the treatment of the conditions associated with aberrantly increased intestinal secretion, particularly acute aberrantly increased intestinal secretion. Such intestinal secretion can result in intestinal inflammatory disorders and diarrhea, particularly secretory diarrhea.

[0097] As used herein, "diarrhea" refers to a disorder characterized by the primary symptom of diarrhea and secondary clinical symptoms that may result from a secretory imbalance and without regard to the underlying cause and therefore includes exudative (inflammatory), decreased absorption (osmotic, anatomic derangement, and motility disorders) and secretory. Diarrheas generally have a secretory component. Diarrheal symptoms include, but are not limited to impaired colonic absorption, ulcerative colitis, shigellosis, and amebiasis. Osmotic diarrhea can occur as a result of digestive abnormalities such as lactose intolerance. Anatomic derangement results in a decreased absorption surface caused by such procedures as subtotal colectomy and gastrocolic fistula. Motility disorders result from decreased contact time resulting from such diseases as hyperthyroidism and irritable bowel syndrome. Secretory diarrhea is characterized by the hypersecretion of fluid and electrolytes from the cells of the intestinal wall. In classical form, the hypersecretion is due to changes which are independent of the permeability, absorptive capacity and exogenously generated osmotic gradients within the intestine.

[0098] Diarrhea amenable to treatment following the methods provided herein can result from exposure to a variety of pathogens or agents including, without limitation, cholera toxin (Vibrio cholera), E. coli (particularly enterotoxigenic (ETEC)), Salmonella, e.g.Cryptosporidiosis, diarrheal viruses (e.g., rotavirus)), food poisoning, or toxin exposure that results in increased intestinal secretion mediated by CFTR.

[0099] Other diarrheas that can be treated by the compounds utilized herein include diarrhea associated with AIDS {e.g., AIDS-related diarrhea), diarrheas caused by anti-AIDS medications such as protease inhibitors and inflammatory gastrointestinal disorders, such as ulcerative colitis, inflammatory bowel disease (IBD), Crohn's disease, chemotherapy, and the like. It has been reported that intestinal inflammation modulates the expression of three major mediators of intestinal salt transport and may contribute to diarrhea in ulcerative colitis both by increasing transepithelial CI^" secretion and by inhibiting the epithelial NaCl absorption. See, e.g., Lohi et al. (2002) Am. J. Physiol. Gastrointest. Liver Physiol

283(3):G567-75).

[0100] In another embodiment, provided herein are methods for treating cholera or one more symptoms, such as secretory symptoms thereof.

[0101] Effective amounts of the compounds utilized herein and pharmaceutical

compositions provided herein can be administered alone or combined with other suitable therapy such as Oral Rehydration Therapy (ORT), supportive renal therapy, administration of an antiviral, vaccine, or other compound to treat the underlying infection or by administering an effective amount of an oral glucose-electrolyte solution to the patient. In another embodiment, compounds utilized herein and pharmaceutical compositions provided herein are co-administered with micronutrients, e.g., zinc, iron, and vitamin A. The therapies may be administered simultaneously or concurrently. Administration is by any appropriate route and varies with the disease or disorder to be treated and the age and general health of the human patient.

[0102] Effective amounts of the compounds utilized herein and pharmaceutical

compositions provided herein can be administered on a mucosal surface of the

gastrointestinal tract {e.g., by an enteral route, such as oral, intraintestinal, intraluminally, rectal as a suppository, and the like) or to a mucosal surface of the oral cavity {e.g. , buccal, sublingual, and the like). In one embodiment, effective amounts of the compounds utilized herein and pharmaceutical compositions provided herein are administered orally. In another embodiment, compounds utilized herein and pharmaceutical compositions provided herein are administered in a pharmaceutical composition suitable for sustained release.

[0103] In one embodiment, the compound is administered in a sustained release composition (see, e.g., supra) which comprises the compound and an effective amount of a pharmaceutically-acceptable polymer. Such sustained release compositions have a modified pharmacokinetic profile that is suitable for treatment as described herein. In one

embodiment, the sustained release composition provides decreased C_ma_x and increased T_max without altering bioavailability of the drug.

[0104] When used to treat diarrhea as provided herein, the compounds utilized herein can be administered singly, as mixtures of one or more compounds provided herein, or in mixture or combination with other agents useful for treating such diseases and/or the symptoms associated with such diseases. The compounds utilized herein may also be administered in mixture or in combination with agents useful to treat other disorders or maladies, such as steroids, membrane stabilizers, 5 -lipoxygenase (5LO) inhibitors, leukotriene synthesis and receptor inhibitors, inhibitors of IgE isotype switching or IgE synthesis, IgG isotype switching or IgG synthesis, β-agonists, tryptase inhibitors, aspirin, cyclooxygenase (COX) inhibitors, methotrexate, anti-TNF drugs, retuxin, PD4 inhibitors, p38 inhibitors, PDE4 inhibitors, and antihistamines, to name a few. As used herein, administration in

"combination" or "co-administration" refers to administering a compound utilized herein and another agent useful in treating diarrhea in such a manner that the pharmacological effects of both are manifest in the patient at the same time. Accordingly, such a combination does not necessarily require administering in a single dosage form or at the same instance.

[0105] The following examples are intended to illustrate, and not limit, the various embodiments of this disclosure.

EXAMPLES

[0106] In the examples below as well as throughout the disclosure, the following abbreviations have the following meanings. If not defined, the terms have their generally accepted meanings. AcOH = acetic acid

APCI = atmospheric pressure chemical ionization

ATP = adenosine tri-phospate

DMEM = Dulbecco's modified eagle's medium

DMSO = dimethylsulfoxide

EGTA = ethylene glycol tetraacetic acid

Et = ethyl

EtOAc = ethyl acetate

EtOH = ethanol

FBS = fetal bovine serum

g = gram

LC = liquid chromatography

LCMS = liquid chromatography mass spectrometry m = multiplet

m/z = mass/Charge

Me = methyl

Me₃Al = trimethylaluminium

mg = milligram

MHz = megahertz

min = minute

mL = milliliter

mm = millimeter

mM = milimolar

mmol = millimole

ms = millisecond

MS = mass spectrum

mV = millivolt

N = normal

NH₂OH = hydroxylamine

nM = nanomolar

nm = nanometer NMR = nuclear magnetic resonance

ppm = parts per million

Rt = retention time

rt = room temperature

SSC = standard saline citrate

TEA = triethylamine

UV = ultraviolet

v/v = volume/volume

μ = microgram

= microliter

μηι = micrometer

μΜ = micromolar

General Synthetic Methods

[0107] Unless otherwise stated, all chemicals are purchased from commercial suppliers and used without further purification.

Standard acidic LC-MS conditions: (10cm esci formic or 10cm apci formic):

[0108] A Phenomenex Luna 5μιη CI 8 (2), 100 x 4.6 mm (plus guard cartridge) column using an acetonitrile (Far UV grade) with 0.1% (v/v) formic acid: Water (High purity via Elga UHQ unit) with 0.1% formic acid gradient is used. The flow rate is 2 mL/min. UV detection is done using a Waters diode array detector (start range 210 nm, end range 400 nm, range interval 4.0 nm). Mass detection is via a single quadrapole LCMS instrument.

Ionization is either ESCi™ or APCI dependent on compound types. The gradient runs from 95% of aqueous solvent at time 0.00 min to 5% of aqueous solvent at 3.50 min. This percentage is then held for a further 2 min. Standard basic LC-MS conditions: (lOcm esci bicarb or lOcm apci bicarb):

[0109] A Waters Xterra MS 5μηι CI 8 , 100 x 4.6 mm (plus guard cartridge) column using an acetonitrile (far UV grade): water (high purity via Elga UHQ unit) with lOmM ammonium bicarbonate (ammonium hydrogen carbonate) gradient is used. The flow rate is 2 mL/min. UV detection is done using a Waters diode array detector (start range 210 nm, end range 400 nm, range interval 4.0 nm). Mass detection is via a single quadrapole LCMS instrument. Ionization is either ESCi™ or APCI dependent on compound types. The gradient runs from 95% of aqueous solvent at time 0.00 min to 5% of aqueous solvent at 3.50 min. This percentage is then held for a further 2 min.

Example 1

Preparation of intermediates A and B

ine

ine, EtOH

od 1)

Step 1: 3,5-Dibromo- V',4-dihydroxybenzimidamide (Compound A)

[0110] Hydroxylamine (10 mL of a 50% solution in water) is added in one portion to a stirred suspension of 3,5-dibromo-4-hydroxybenzonitrile (30 g, 110 mmol) in ethanol (100 mL) at room temperature and the mixture is heated to reflux for 3 hours before cooling back down to room temperature. The solid is filtered, washed with cold ethanol and dried to yield the title compound as a colourless powder. Step 2: Ethyl 3-(3,5-dibromo-4-hydroxyphenyl)-l,2,4-oxadiazole-5-carboxylate

(Compound B)

[0111] Ethyl 2-chloro-2-oxoacetate (12.3 g, 82 mmol) is added dropwise to a stirred solution of 3,5-dibromo-N',4-dihydroxybenzimidamide (25.5 g, 82 mmol) in pyridine (120 mL) and the mixture is stirred at room temperature for 1 hour and then at 60 °C for 2 hours. The resulting suspension is poured onto water (1.5 L) and extracted with ethyl acetate (2 x 400 mL). The combined extracts are washed with saturated sodium chloride, dried (MgS0₄) and evaporated in vacuo to give an oily solid that is purified by flash chromatography to give a colourless powder. Crystallisation from toluene (400 mL) gives the title compound as colourless crystals.

Example 2

Preparation of 3-(3,5-Dichloro-4-hydroxyphenyl)- V-(4-(4-fluoro-3- (trifluoromethyl)phenoxy)benzyl)-l,2,4-oxadiazole-5-carboxamide (Compound 23)

compound 23 Ethyl 3-(3,5-dichloro-4-(4-methoxybenzyloxy)phenyl)-l,2,4-oxadiazole-5-carboxylate (Compound C)

[0112] Ethyl 3-(3,5-dichloro-4-(4-methoxybenzyloxy)phenyl)-l,2,4-oxadiazole-5- carboxylate (compound C) is prepared starting from commercially available 3,5-dichloro-4- hydroxybenzonitrile.

3-(3,5-Dichloro-4-(4-methoxybenzyloxy)phenyl)-N-(4-hydroxybenzyl)-l,2,4-oxadiazole- 5-carboxamide (Compound D)

[0113] Ethyl 3-(3,5-dichloro-4-(4-methoxybenzyloxy)phenyl)-l,2,4-oxadiazole-5- carboxylate (4 g, 9.46 mmol) and 4-hydroxybenzylamine (2.33 g, 18.91 mmol) are heated in EtOH (150 mL) at 80 °C forming a yellow solution. After 1 h a colourless precipitate forms and the mixture is cooled to room temperature. The solid is filtered off, washed with EtOH and dried in a vacuum oven giving the title compound as a colourless solid.

3-(3,5-Dichloro-4-hydroxyphenyl)- V-(4-(4-fluoro-3-(trifluoromethyl)phenoxy)benzyl)- l,2,4-oxadiazole-5-carboxamide (Compound 25)

[0114] 3-(3,5-Dichloro-4-(4-methoxybenzyloxy)phenyl)-N-(4-hydroxybenzyl)-l,2,4- oxadiazole-5-carboxamide (compound D, 60 mg, 0.12 mmol), 4-fluoro-3- (trifluoromethyl)phenylboronic acid (50 mg, 0.24 mmol), Cu(OAc)₂ (44 mg, 0.24 mmol) and 3 A powdered molecular sieves (70 mg) are stirred in an open tube in dichloromethane (3 mL). Pyridine (57 mg, 0.72 mmol) is added and the dark green suspension is stirred vigorously at room temperature in an open tube for 1 d. The molecular sieves are filtered, washed with dichloromethane and the filtrate concentrated in vacuo. The residue is dissolved in dichloromethane (3 mL) and trifluoroacetic acid (0.3 mL) is added. The dark solution is stirred at room temperature for 5 h, then methanol (1 mL) is added and the solution concentrated in vacuo. The residue is purified by preparative HPLC providing the title compound.

[0115] Other compounds of the invention were prepared following these methods and appropariately modifying reactants and reaction conditions as will be apparrent to the skilled artisan. Example 3

Preparation of an aqueous micro-suspension of Compound 2

[0116] The formulation used for human administration was as follows. A suspension of compound 2 in a vehicle of 0.2% (w/v) Xantural® xanthan gum, and 0.26% (w/v) polysorbate 80 was prepared. Suspensions of 30, 100, 300, 500, or 1000 mg of Compound 2 in a total volume 50 mL were used for administration.

Example 4

Human administration of compound 2 4a

[0117] Compound 2, in amounts of 10, 30, 100, 300, and 1000 mg, was orally administered as an aqueous microsuspension to healthy human subjects. The following amounts were administered as a single ascending daily dose: 30 mg, 100 mg, 300 mg, 500 mg, or 1000 mg. The following amounts were administered as multiple ascending doses: 7 doses each of 100 mg, 300 mg, and 500 mg BID (every 12 h), and 7 doses each of 300 mg, 500 mg TID (every 8 h). Pharmacokinetic parameters were determined. Based on the results, it is contemplated that Compound 2 can be administered, e.g., and without limitation, 1000 mg once daily and 500 mg thrice daily for treating diarrhea in humans.

4b

[0118] Healthy humans were orally administered Compound 2 at a dose of 100 mg, 300 mg, and 500 mg every 12 hours for three days, plus a single morning dose on Study Day 4 (seven doses total), and at a dose of 300 mg and 500 mg every 8 hours for three days, plus a single morning dose on Study Day 4 (ten doses total).

[0119] Concentrations of Compound 2 were quantifiable (greater than 1 ng/mL) in plasma in all subjects out to the last collection time point (96 hours after the last dose on Day 4). [0120] The median T_max ranged from 4.00 to 6.00 hours following single-dose administration on Day 1 and from 3.00 to 4.00 hours following multiple-dose administration on Day 4. Following T_max on Day 4, Compound 2 plasma concentrations declined in a mostly monophasic manner. The elimination half-life of Compound 2 (10.3 to 11.7 hours) was independent of the administered dose. After single doses, geometric mean values ranged from 2,000 ng/niL (100 mg every 12 hours) to 5,210 ng/mL (500 mg every 12 hours) and geometric mean AUC_tau values ranged from 14,900 ng-h/mL (100 mg every 12 hours) to 43,200 ng-h/mL (500 mg every 12 hours). After multiple dosing, geometric mean C_max values ranged from 2,520 ng/mL (100 mg every 12 hours) to 8,730 ng/mL (500 mg every 8 hours) and geometric mean AUC_taU values ranged from 23,000 ng-h/mL (100 mg every 12 hours) to 72,000 ng-h/mL (500 mg every 12 hours).

[0121] The dose-exposure regression line for AUC_tau is represented by the following linear equation: Log(AUC_taU) = 0.6497 Log (dose) + 7.026; R (coefficient of determination) 0.686. The point estimate for slope (0.6497) indicates a less than proportional increase over the tested dose range.

[0122] It is to be understood that the foregoing description and examples are intended to illustrate and not limit the scope of the disclosure. Other aspects, advantages and

modifications within the scope of the disclosure will be apparent to those skilled in the art to which the disclosure pertains.

[0123] It should be understood that although the present invention has been specifically disclosed by certain aspects, embodiments, and optional features, modification, improvement and variation of such aspects, embodiments, and optional features can be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this disclosure.

[0124] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims

WHAT IS CLAIMED IS:

1. A pharmaceutical composition comprising at least a pharmaceutically acceptable excipient and about 10 mg to about 1500 mg of a compound of Formula I:

Formula I or a metabolite thereof, or a pharmaceutically acceptable salt of each thereof, wherein r is 1, 2, or 3;

R¹ is hydrogen, halo, amino, substituted amino, C₁-C₃ alkoxy, substituted C₁-C₃ alkoxy, Ci- C₃ alkyl, or substituted C₁-C₃ alkyl;

R is H, C₁-C₃ alkyl, or substituted C₁-C₃ alkyl; and

R³ and R⁴ and independently halo.

2. The composition of claim 1 , wherein r is 1.

3. The composition of claim 1 or 2, wherein R is hydrogen.

4. The composition of any one of claims 1-3, wherein R is C₁-C₃ alkyl or substituted C₁-C₃ alkyl.

5. The composition of any one of claims 1-4, wherein R³ and R⁴ are independently the same or different and are chloro or bromo.

6. The composition of any one of claims 1-5, wherein R¹ is hydrogen.

7. The composition of claim 1, wherein the compound of Formula I is of formula:

or a pharmaceutically acceptable salt thereof.

8. The composition of any one of claims 1-7, which comprises about 10 mg, or about 30 mg, or about 100 mg, or about 300 mg, or about 500 mg, or about 1000, or about 1500 mg of the compound of Formula I.

9. The composition of any one of claims 1-8, which is formulated for oral or parenteral administration.

10. A method of treating diarrhea or an associated disorder in a human patient in need thereof, the method comprising administering a daily amount of about 10 mg to about 1500 mg of a compound of Formula I:

Formula I or a metabolite thereof, or a pharmaceutically acceptable salt of each thereof, wherein r is 1, 2, or 3;

R¹ is hydrogen, halo, amino, substituted amino, C₁-C₃ alkoxy, substituted C₁-C₃ alkoxy, Ci- C₃ alkyl, or substituted C₁-C₃ alkyl;

R is H, C₁-C₃ alkyl, or substituted C₁-C₃ alkyl; and

R³ and R⁴ and independently halo.

11. A method of treating diarrhea in a human patient in need thereof, the method comprising administering a compound of Formula I :

Formula I or a metabolite thereof, or a pharmaceutically acceptable salt of each thereof, wherein r is 1, 2, or 3;

R¹ is hydrogen, halo, amino, substituted amino, C₁-C₃ alkoxy, substituted C₁-C₃ alkoxy, Ci- C₃ alkyl, or substituted C₁-C₃ alkyl;

R is H, C₁-C₃ alkyl, or substituted C₁-C₃ alkyl; and

R³ and R⁴ and independently halo, to maintain in the human patient a C_max of about 1 ,000 nanogram (ng)/mL to 10,000 ng/niL, or about 2,000 ng/mL to 9,000 ng/niL, or about 3,000 ng/mL to 8,000 ng/mL, or about or about 4,000 ng/mL to 7,000 ng/mL, or about 5,000 ng/mL to 6,000 ng/mL or to maintain in the human patient a plasma exposure as measured by an area under the curve for a dosing interval (AUC_tau) of about 20,000 ng'h/mL to about 100,000 ng'h/mL, about 40,000 ng'h/mL to about 80,000 ng'h/mL, or about 50,000 ng'h/mL to about 70,000 ng'h/mL, of the compound of Formula I, or a metabolite thereof, or a pharmaceutically acceptable salt of each thereof.

12. The method of claim 11, wherein the compound of Formula I is administered at a daily amount of about 10 mg to about 1500 mg.

13. The method of claim 11 or 12, wherein r is 1.

14. The method of any one of claims 10-13, wherein R is hydrogen.

15. The method of any one of claims 10-14, wherein R is C1-C3 alkyl or substituted Ci- C₃ alkyl.

16. The method of any one of claims 10-15, wherein R³ and R⁴ are independently the same or different and are chloro or bromo.

17. The method of any one of claims 10-16, wherein R¹ is hydrogen.

18. The method of any one of claims 10-17, wherein the compound of Formula I is of formula:

or a pharmaceutically acceptable salt thereof.

19. The method of any one of claims 10-18, which comprises administering a daily amount of about 10 mg, or about 30 mg, or about 100 mg, or about 300 mg, or about 500 mg, or about 1000 mg, or about 1500 mg of the compound of Formula I.

20. The method of any one of claims 10-19, wherein the compound of Formula I is administered for at least 3 days.

21. The method of any one of claims 10-19, wherein the compound of Formula I is administered for about 14 days to about 30 days.

22. The method of any one of claims 10-19, wherein the compound of Formula I is administered once a day.

23. The method of any one of claims 10-19, which comprises administering a daily amount of about 1000 mg of the compound of Formula I.

24. The method of any one of claims 10-19, wherein the compound of Formula I is administered three times a day.

25. The method of claim 24, which comprises administering a daily amount of about 300 mg or about 500 mg of the compound of Formula I.

26. The compound of any one of claims 10-23, wherein the compound of Formula I is administered orally or parenterally.

27. A method of treating diarrhea in a human patient in need thereof, comprising administering a daily amount of the pharmaceutical composition of any one of claims 1-9 to the patient.

28. The pharmaceutical composition of claim 1, comprising a gum and/or a polysorbate as the pharmaceutically acceptable excipient(s).

29. The pharmaceutical composition of claim 26 which is an aqueous suspension.

30. The pharmaceutical composition of claim 28 or 29, which comprises about 0.1% (w/v) to about 1% (w/v) of the gum.

31. The pharmaceutical composition of any one of claims 28-30, which comprises about 0.1% (w/v) to about 1%> (w/v) polysorbate.

32. The pharmaceutical composition of any one of claims 28-31 , wherein the gum is xanthan gum.

33. The pharmaceutical composition of any one of claims 28-32, wherein the polysorbate is polysorbate 80.

34. The pharmaceutical composition of claim 32, wherein the gum is present in about 0.2% (w/v).

35. The pharmaceutical composition of claim 32, wherein the polysorbate is present in about 0.26% (w/v).

36. The pharmaceutical composition of any one of claims 28-35, wherein the compound of Formula I is Compound 2.

37. The pharmaceutical composition of any one of claims 28-36, wherein the compound of Formula I is present in an amount of 30 mg, or 100 mg, or 300, mg, or 500 mg, or 1000 mg.

38. Use of a compound of Formula I:

Formula I or a metabolite thereof, or a pharmaceutically acceptable salt of each thereof, wherein r is 1, 2, or 3;

R¹ is hydrogen, halo, amino, substituted amino, C₁-C₃ alkoxy, substituted C₁-C₃ alkoxy, Ci- C₃ alkyl, or substituted C₁-C₃ alkyl;

R is H, C₁-C₃ alkyl, or substituted C₁-C₃ alkyl; and

R³ and R⁴ and independently halo, in a daily amount of about 10 mg to about 1500 mg for the treatment of diarrhea or an associated disorder in a human patient.

39. Use of a compound of Formula I:

Formula I or a metabolite thereof, or a pharmaceutically acceptable salt of each thereof, wherein r is 1, 2, or 3;

R¹ is hydrogen, halo, amino, substituted amino, C₁-C₃ alkoxy, substituted C₁-C₃ alkoxy, Ci- C₃ alkyl, or substituted C₁-C₃ alkyl;

R is H, C₁-C₃ alkyl, or substituted C₁-C₃ alkyl; and

R³ and R⁴ and independently halo, for the treatment of diarrhea or an associated disorder in a human patient, wherein the compound of Formula I, or a metabolite thereof, or a pharmaceutically acceptable salt of each thereof is maintained in the human patient: at a C_max of about 1,000 nanogram (ng)/mL to 10,000 ng/mL, or about 2,000 ng/mL to 9,000 ng/mL, or about 3,000 ng/mL to 8,000 ng/mL, or about or about 4,000 ng/mL to 7,000 ng/mL, or about 5,000 ng/mL to 6,000 ng/mL, or at a plasma exposure as measured by an area under the curve for a dosing interval (AUCau) of about 20,000 ng-h/mL to about 100,000 ng-h/mL, about 40,000 ng-h/mL to about 80,000 ng-h/mL, or about 50,000 ng-h/mL to about 70,000 ng-h/mL.