Substituted 2-acylamino-cycloalkylthiophene-3-carboxylic acid arylamides as inhibitors of calcium-activated chloride channel TMEM16A

Provided herein are inhibitors of transmembrane protein 16A (TMEM 16A), a Ca2+-activated CI″ channel expressed widely in mammalian epithelia, as well as in vascular smooth muscle and some tumors and electrically excitable cells. TMEM16A inhibitors have potential utility for treatment or management of disorders of epithelial fluid and mucus secretion, hypertension, some cancers, pain, and other diseases.

BACKGROUND

Technical Field

This disclosure is related to inhibitors of chloride conductance via calcium-activated chloride channels and use thereof.

Description of the Related Art

BRIEF SUMMARY

Provided herein are 2-acylamino-cycloalkylthiophene-3-carboxylic acid arylamide (AACT) class of TMEM16A inhibitors with substantially improved inhibition potency and metabolic stability than currently known inhibitors.

One embodiment provides a compound having the structure represented by Formula I:

X is S, O, or NR;

R is hydrogen or C1-C6alkyl;

each R2is the same or different and independently hydrogen, halo, C1-C6alkyl, C1-C6alkoxy, C1-C6haloalkyl, or C1-C6; haloalkoxy; and

R3is C1-C6alkyl, or C1-C6haloalkyl, with the proviso that the compound of Formula (I) is not 2-(2,2,2-Trifluoro-acetylamino)-5,6,1,8-tetrahydro4H-cyclohepta[b]thiophene-3-carboxylic acid o-tolylamide.

Another related embodiment provides a pharmaceutical composition comprising a physiologically acceptable excipient and a compound of Formula (1).

Other embodiments provide a use of a composition or a compound of Formula (1) or any one of the substructures as represented by Formulae (I-1)-(I-14), for treating a condition, disease, or disorder associated with abnormally increased chloride ion secretion from a cell. Certain embodiments provide a use of a composition or a compound of Formula (I) or any one of the substructures as represented by Formulae (I-1)-(I-14), for reducing or managing pain, or for treating cancer, or for treating hypertension.

Another embodiment provides use of a composition or a compound of Formula (I) or any one of the substructures as represented by Formulae (I-1)-(I-14), for the manufacture of a medicament for treating a condition, disease, or disorder associated with abnormally increased chloride ion secretion from a cell.

These and other aspects of the invention, as well as advantages related to the same, will be apparent upon reference to the following detailed description.

DETAILED DESCRIPTION

A high-throughput screening assay was previously developed to identify small molecule inhibitors of TMEM16A. (See Namkung, W. et al.,Faseb J2011, 25, (11), 4048-4062.) The screen utilized FRT cells that were stably transfected with human TMEM16A and the iodide-sensitive fluorescent protein YFP-H148Q/1152L/F46L. The assay involved addition of test compounds to the cells for 10 min in a physiological chloride-containing solution, followed by addition of an iodide solution containing ATP. TMEM16A-facilitated iodide influx was determined from the initial time course of decreasing YFP fluorescence. TMEM16A inhibitors reduce iodide influx, resulting a reduced rate of decreasing fluorescence, Several classes of inhibitors with micromolar potency, including T16Ainh-A01, were discovered. (See Namkung, W. et al.,Faseb J2011, 25, (11), 4048-4062; Piechowicz, K. A. et al.,J Enzyme Inhib Med Chem2016, 1-7.) Screening of 50,000 drug-like synthetic small molecules identified 2-acylamino-cycloalkylthiophene-3-carboxylic acid arylamide (AACT) 6aa with IC50˜0.42 μM.

The only other reported biological activity AACTs is inhibition of the protozoan parasiteLeishmania donovani(EC50=6.4 μM), with no cytotoxicity seen against human macrophages (CC50>50 μM). (See Oh, S.,MedChemComm2014, 5, (2), 142-146.).

One embodiment provides a compound having the structure represented by Formula I:

X is S, O, or NR;

R is hydrogen or C1-C6alkyl;

each R2is the same or different and independently hydrogen, halo, C1-C6alkyl, C1-C6alkoxy, C1-C6haloalkyl, or C1-C6haloalkoxy; and

R3is C1-C6alkyl, or C1-C6haloalkyl, with the proviso that the compound of Formula (I) is not 2-(2,2,2-Trifluoro-acetylamino)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxylic acid o-tolylamide.

More specific embodiments provide a compound having one of the following structures Formula (I-1), Formula (I′-1), Formula (I-2), Formula (I-3), or Formula (I-4):

X is S, O, or NH;

In preferred embodiments, X is S in any one of Formulae (I-1), (I′-1), (I-2), (I-3) and (I-4); and the compounds are

Further embodiments provide a compound having one of the following structures Formula (I-5), Formula (I-6) or Formula (I′-6):

A further embodiment provides a compound of any one of Formulae (I), (I-5), (I-6) and (I′-6), wherein R1is C4alkylene, i.e., —(CH2)4—. More specifically, the compound has one of the following structures Formula (I-7), Formula (I-8) or Formula (I′-8).

Yet a further embodiment provides a compound of any one of Formulae (I), (I-5) and (I-6), wherein R1is C3alkylene, i.e., —(CH2)3—. More specifically, the compound has one of the following structures Formula (I-9) or Formula (I-10):

In more specific embodiments, the compound of any of one of Formulae (I), (I-2), (I-5), (I-6), (I-9) and (I-10) is:2-(2,2,2-trifluoro-acetylamino)-4,5,6,7-tetrahydro-benzo[b]thiophene-3-carboxylic acid phenylamide;2-(2,2,2-trifluoro-acetylamino)-4,5,6,7-tetrahydro-benzo[b]thiophene-3-carboxylic acid o-tolylamide;2-(2,2,2-trifluoro-acetylamino)-4,5,6,7-tetrahydro-benzo[b]thiophene-3-carboxylic acid p-tolylamide; or2-(2,2,2-trifluoro-acetylamino)-4,5,6,7-tetrahydro-benzo[b]thiophene-3-carboxylic acid (4-fluoro-phenyl)-amide.

A further embodiment provides a compound of any one of Formulae (I), (I-5) or (I-6), wherein R1is C2alkylene, i.e., —(CH2)2—. More specifically, the compound has one of the following structures Formula (I-11) or Formula (I-12):

In more specific embodiments, the compound of any of one of Formulae (I), (I-3), (I-5), (I-6), (I-11) and (I-12) is:2-(2,2,2-trifluoro-acetylamino)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylic acid o-tolylamide;2-(2,2,3,3,3-pentafluoro-propionylamino)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylic acid o-tolylamide;2-acetylamino-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylic acid o-tolylamide;2-propionylamino-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylic acid o-tolylamide;2-(2,2,2-trifluoro-acetylamino)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylic acid p-tolylamide;2-(2,2,2-trifluoro-acetylamino)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylic acid (2-chloro-phenyl)-amide;2-(2,2,2-trifluoro-acetylamino)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylic acid (2-trifluoromethoxy-phenyl)-amide;2-(2-chloro-2,2-difluoro-acetylamino)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylic acid (2-trifluoromethoxy-phenyl)-amide;2-(2-bromo-2,2-difluoro-acetylamino)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylic acid (4-methyl-2-trifluoromethoxy-phenyl)-amide; or2-(2,2-difluoro-2-iodo-acetylamino)-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylic acid (2-trifluoromethoxy-phenyl)-amide.

In still other embodiments, it is provided a compound of any one of Formulae (I), (I-5) and (I-6) wherein R1is C2heteroalkylene, including for example —(CH2—O—CH2)— and (CH2—CH2—O)—. In particular, one embodiment provides a compound having one of the following structures Formula (I-13) or Formula (I-14):

In more specific embodiments, the compound of any of one of Formulae (I), (I-4), (I-5), (I-6), (I-13) and (I-14) is:2-(2,2,3,3,3-pentafluoro-propionylamino)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxylic acid o-tolylamide;2-propionylamino-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxylic acid o-olylamide;2-(2,2,2-trifluoro-acetylamine)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxylic acid (2-chloro-phenyl)-amide;2-(2,2,2-trifluoro-acetylamino)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxylic acid p-tolylamide; or2-(2,2,2-trifluoro-acetylamino)-4,7-dihydro-5H-thieno[2,3-c]pyran-3-carboxylic acid (4-fluoro-phenyl)-amide.

Chemistry Definitions

“Alkyl” means a straight chain or branched, noncyclic, unsaturated or partially unsaturated aliphatic hydrocarbon containing from 1 to 12 carbon atoms. A lower alkyl refers to an alkyl that has any number of carbon atoms between 1 and 6 (i.e., C1-C6alkyl). Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like, while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, tert-pentyl, heptyl, n-octyl, isopentyl, 2-ethylhexyl and the like. Alkyl may be optionally substituted by one or more substituents as defined herein.

“Alkoxy” refers to the radical of —O-alkyl. Examples of alkoxy include methoxy, ethoxy, and the like. The alkyl moiety of alkoxy may be optionally substituted by one or more substituents as defined herein.

“Alkylene” refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule (e.g., forming a cycloalkyl ring), consisting solely of carbon and hydrogen, having from one to twelve carbon atoms, or more typically one to six carbons. Examples include methylene (C1alkylene), ethylene (C2alkylene), propylene (C3alkylene), or butylene (C4alkylene), and the like. The alkylene chain is attached to the rest of the molecule through respective single C—C bond. Alkylene may be optionally substituted by one or more substituents as defined herein.

“Arylalkyl” (e.g., phenylalkyl) means an alkyl having at least one alkyl hydrogen atom replaced with an aryl moiety, such as —CH2-phenyl, —CH═CH-phenyl, —C(CH3)═CH-phenyl, and the like.

“Heteroalkylene” refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule, wherein one or more of the carbon atoms in the divalent chain are replaced by one or more heteroatoms, including oxygen, nitrogen or sulfur. A heteroalkylene radical may comprise from one to eleven carbon atoms, or more typically one to five carbon atoms. Typically, heteroalkylene comprises one oxygen atom (—O—), one nitrogen (—NH—) or one sulfur (—O—) in the chain. The alkylene chain is attached to the rest of the molecule through respective single C—C bond or C—O bond, or C—NH bond, or C—S bond. Heteroalkylene may be optionally substituted by one or more substituents as defined herein.

“Haloalkyl” refers to a halo-substituted alkyl, i.e., alkyl in which at least one hydrogen atom is replaced with halogen. “Perhaloalkyl” refers to haloalkyl in which all of the hydrogens are replaced by halogens. Examples of haloalkyls include trifluoromethyl, chlorodifluoromethyl, bromodifluoromethyl, 1,1,2,2,3,3,3-heptafluoropropyl and the like. In certain embodiments, the halo substituents of a haloalkyl or perhaloalkyl may be the same (e.g., all of the halo substituents are fluoro) or different (e.g., the halo substituents may be a mixture of any two or more of fluoro, chloro, bromo or iodo). The alkyl moiety of a haloalkyl may be optionally substituted by one or more substituents as defined herein.

“Haloalkoxy” refers to a substituted alkoxy, means an alkoxy moiety having at least one hydrogen atom replaced with halogen, such as chloromethoxy and the like.

All the above groups may be “optionally substituted,” i.e., either substituted or unsubstituted. The term “substituted” as used herein means any of the above groups (i.e., alkyl, alkylene, alkoxy, alkoxyalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl and/or trifluoroalkyl), may be further functionalized wherein at least one hydrogen atom is replaced by a bond to a non-hydrogen atom substituent. Unless stated specifically in the specification, a substituted group may include one or more substituents selected from: oxo, nitrile, nitro, —CONH2, hydroxyl, thiooxy, alkyl, alkylene, alkoxy, alkoxyalkyl, alkylcarbonyl, alkyloxycarbonyl, aryl, aralkyl, arylcarbonyl, aryloxycarbonyl, aralkylcarbonyl, aralkylloxycarbonyl, aryloxy, cycloalkyl, cycloalkylalkyl, cycloalkyl carbonyl, cycloalkylalkylcarbonyl, cycloalkyloxycarbonyl, heterocyclyl, heteroaryl, dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides, imides, and enamines; a silicon atom in groups such as trialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups, thioalkyl triarylsilyl groups, perfluoroalkyl or perfluoroalkoxy, for example, trifluoromethyl or trifluoromethoxy. “Substituted” also means any of the above groups in which one or more hydrogen atoms are replaced by a higher-order bond (e.g., a double- or triple-bond) to a heteroatom such as oxygen in oxo, carbonyl, carboxyl, and ester groups; and nitrogen in groups such as imines, oximes, hydrazones, and nitriles. For example, “substituted” includes any of the above groups in which one or more hydrogen atoms are replaced with —NRgC(═O)NRgRh, —NRgC(═O)ORh, —NRgSO2Rh, —OC(═O)NRgRh, —ORg, —SRg, —SORg, —SO2Rg, —OSO2Rg, —SO2ORg, —NSO2Rg, and —SO2NRgRh. “Substituted” also means any of the above groups in which one or more hydrogen atoms are replaced with —C(═O)Rg, —C(═O)ORg, —CH2SO2Rg, —CH2SO2NRgRh, —SH, —SRgor —SSRg. In the foregoing, Rgand Rhare the same or different and independently hydrogen, alkyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and/or heteroarylalkyl. In addition, each of the foregoing substituents may also be optionally substituted with one or more of the above substituents.

Synthetic Schemes

The AACT derivatives of any one of Formulae (I) and (I-1)-(I-14) may be prepared using the modular synthetic strategy shown in Scheme 1.

The synthesis begins with the generation of substituted aryl cyanoacetamides, followed by a two-step Knoevenagel-Gewald sequence to generate 2-aminothiophenes, and coupling with simple electrophilic acylating agents. Substituted anilines (1a-1k) were coupled with cyanoacetic acid using EDCI-HCl to generate the library of cyanoacetamides (2a-2k). The substituent composition of this library, prepared typically in good yields, are also shown in Table 1, with some of the cyanoacetamides also being commercially available.

Next, the substituted aryl cyanoacetamides (2a-2k) were condensed with a small collection of cycloalkyl ketones (3a-3c) under buffered acid-catalyzed aldol conditions (AcOH:NH4OAc) to generate Knoevenagel adducts (4a-4v). While excess cyclic ketone was useful to obtain high conversion, we were pleased that this material could be removed by evaporation. The Knoevenagel adducts were subjected to the Gewald cyclization reaction in the presence of molecular octasulfur (Sg), to yield 2-amino-cycloalkylthiophene-3-carboxylic acid arylamides (5a-5v), which were typically crystalline and easily purified by trituration. The composition of the library and yields for the Knoevenagel and Gewald reactions are shown in Table 2, separated by the different cycloalkyl ketones.

Finally, coupling of the aminothiophenes (5a-5v) with alkyl and fluoroalkyl acyl chlorides, anhydrides, or EDCI-coupling was done to generate the final desired AACT compounds (6aa-6bw), also typically as crystalline solids, in fair to good yields (Table 3). After completion of a 1stgeneration of compounds (6aa-6bj) based on simple alkyl and fluoroalkyl groups at the R3position, we designed a 2ndgeneration library with halodifluoroalkyl (chloro, bromo, and iodo) and heptafluorobutyryl at R3, based on the most promising combinations of R1and R2(6bk-6bw). The synthesis of the difluoroiodoacetyl inhibitors (6bn, 6bq, 6bt, and 6bw) was accomplished by EDCI-mediated coupling of aminothiophenes with difluoroiodoacetic acid. In total, 49 inhibitor candidates were prepared by variations at the R1, R2, and R3positions. The structure and purity of the final products were confirmed by1H-NMR, ESI-LCMS (UV absorption detection at 254 nm), with purities estimated to be >95%.

The 1stgeneration library (6aa-6bj) showed several compounds with apparent IC50of 0.2-0.3 μM. 6baa (initial inhibitor), 6ae, 6ak, 6au, 6av, 6aw. These results showed that 5-, 6-, and 7-member rings were tolerated at the R1position, while compounds based on tetrahydro-4H-pyran-4-one (6ay-6bc) were inactive. The best inhibitors contained H, 2- or 4-(CH3), or 4-F on the aromatic ring (R2), and CF3as the acylamido substituent (R3). Inhibitors with differing groups at R2, such as 2-F, 2-Cl, 3-Cl, 4-Cl, 4-(CF3), 2-(OCF3), were less potent. Likewise, compounds with alternative substituents at R3, including CF2CF3, CH3, and CH2CH3, also had reduced potency.

Based on results that favored CF3at the R3position, we designed a 2nd-generation library (6bk-6bw) that incorporated novel groups such as chlorodifluoro, bromodifluoro, or difluoroiodo, probing steric and electronic effects at that position. Additionally, we prepared one compound that incorporated a heptafluorobutyryl substituent (6bl) to evaluate the effect of a multi-carbon fluoroalkyl group. Gratifyingly, we found three 2nd-generation compounds with lower apparent IC50of 0.08-0.18 μM: 6bk, 6bm, and 6bt.

The most potent TMEM16A inhibitors identified using the semi-quantative plate reader assay were then studied using a definitive short-circuit current assay in which measured current is a direct, quantitative measure of TMEM16A Cl−conductance. Compounds 6aa (original inhibitor from screen), 6ae, 6bk, 6bm, 6bn and 6bt were tested, and compared with previously reported inhibitors MONNA, Ani-9 and T16Ainh-A01. Concentration-dependence for selected compounds is shown inFIG. 2, with IC50values summarized in Table 4. By short-circuit current assay, 6aa showed an IC50of 0.26 μM, similar to the chlorodifluoroacetamide 6bk with IC50of 0.23 μM. Difluoroiodoacetamides 6bn and 6bt were less potent with IC50of 0.73 and 0.60 μM, respectively. Notably, bromodifluoroacetamide 6bm had IC50of 0.030 μM.

Table 4 shows certain characterizations of AACT derivatives disclosed herein. Concentration-dependent inhibition of TMEM16A measured by short-circuit current assay; TMEM16B and non-TMEM16 anion conductance measured using a fluorescence plate reader assay using FRT and HT-29 cells respectively; cell viability measured in FRT null cells.

Ion channel specificity and cytotoxicity were determined for the six most potent AACT compounds (Table 4). Selectivity was studied for TMEM16B, an isoform of TMEM16A that also functions as a Ca2+-activated Cl−channel. The AACT inhibitors were relatively non-selective against TMEM16B, with IC50from 0.4-1.4 μM. Two of the more potent TMEM16A inhibitors (6bk and 6bm) were also among the more potent against TMEM16B with IC50˜0.4 μM. We further assayed the compound potency on endogenous non-TMEM16A. Ca2+-activated Cl−channel in HT-29 cells. (See De La Fuente, R. et al.,Mol Pharmacol2008, 73, (3), 758-68.) In general, the AACT compounds were weak inhibitors of CaCCs in HT-29 cells (IC503.5-9.5 μM). None of the compounds examined showed significant toxicity using an Alamar blue assay at concentrations up to 5 μM. Additionally, none of the compounds inhibited the cAMP-activated Cl−channel cystic fibrosis transmembrane conductance regulator (CFTR) (data not shown).

In vitro metabolic stability was determined using a hepatic microsome assay for the most potent inhibitor 6bm (FIG. 3(A)) and previously reported Ani-9. These compounds were incubated with rat liver microsomes and NADPH, and non-metabolized compounds were quantified by ESI-LCMS.FIG. 3(B)shows near complete degradation of Ani-9 at 180 min, whereas for the same incubation time ˜30% of 6bm remained.FIG. 3Csummarizes the time course of compound degradation showing remarkably greater stability of 6bm compared to Ani-9. 6bm could be potentially metabolized by amide-bond hydrolysis or oxidation of the benzene or aryl methyl. It is speculated that Ani-9 could be oxidized at the aryl methyl or N—N bond; or hydrolyzed at the amide or hydrazone linkages.

To demonstrate one predicted biological action of TMEM16A inhibition, we measured intestinal smooth muscle contraction. The effect of 6bm was determined when added to the bath in an ex vivo preparation of mouse ileum. As is shown inFIG. 4, 6bm strongly inhibited spontaneous isometric contractions of ileum in a concentration-dependent manner.

Pharmaceutical Composition

Also provided herein is a pharmaceutical composition comprising a physiologically acceptable excipient and a compound of Formula (I):

X is S, O, or NR;

R is hydrogen or C1-C6alkyl;

each R2is the same or different and independently hydrogen, halo, C1-C6alkyl, C1-C6alkoxy, C1-C6haloalkyl, or C1-C6haloalkoxy; and

In various embodiments, the pharmaceutical composition comprising a compound of Formula (I) may have substructures as represented by any one of Formulae (I-1), (I′-1), (I-2), (I-3), (I-4), (I-5), (I-6), (I′-6), (I-7), (I-8), (I′-8), (I-9), (I-10), (I-11), (I-12), (I-13) or (I-14), or any one of Formulae (I-1a), (I′-1a), (I-2a), (I-3a), or (I-4a).

A pharmaceutical composition may be a sterile aqueous or non-aqueous solution, suspension or emulsion, which additionally comprises a physiologically acceptable excipient (also called a pharmaceutically acceptable or suitable excipient or carrier) (i.e., a non-toxic material that does not interfere with the activity of the active ingredient). Such compositions may be in the form of a solid, liquid, or gas (aerosol). Alternatively, compositions described herein may be formulated as a lyophilizate, or compounds may be encapsulated within liposomes using technology known in the art. Pharmaceutical compositions may also contain other components, which may be biologically active or inactive. Such components include, but are not limited to, buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, stabilizers, dyes, flavoring agents, and suspending agents and/or preservatives.

Use and Method of Treatment

Also provided herein is a method of inhibiting a calcium-activated chloride channel comprising: contacting (a) a cell that comprises the calcium-activated chloride channel and (b) a pharmaceutical composition comprising a compound of Formula (I) or any one of the substructures as represented by Formulae (I-1)-(I-14), in an amount effective and under conditions and for a time sufficient to inhibit activation of the channel. In a specific embodiment, the cell is an epithelial cell. In a particular embodiment, the epithelial cell is an intestinal epithelial cell or a lung epithelial cell. In a specific embodiment, the calcium-activated chloride channel is TMEM16A, and in other specific embodiments, the TMEM16A calcium-activated chloride channel is a human TMEM16A calcium-activated chloride channel. In a specific embodiment, the compound is 2-(2-bromo-2,2-difluoro-acetylamino)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxylic acid o-tolylamide.

In one embodiment, provided herein is a method of inhibiting fluid secretion from a cell comprising administering to a subject of a pharmaceutical composition comprising a physiologically acceptable excipient and a compound of Formula (I) or any one of the substructures as represented by Formulae (I-1)-(I-14), in an amount effective to inhibit conductance of chloride through a calcium-activated chloride channel, thereby inhibiting fluid secretion from the cell, wherein the subject has a condition, disease or disorder that is treatable by inhibiting conductance of chloride through a calcium-activated chloride channel. In certain embodiments, the disease or disorder is selected from abnormally increased intestinal fluid secretion, secretory diarrhea, asthma, chronic obstructive pulmonary disease, bronchiectasis, or cystic fibrosis. In other embodiments, a condition that is treatable by inhibiting conductance of chloride through a calcium-activated chloride channel includes abnormally increased mucus secretion, which in certain embodiments is a condition of a disease or disorder that is a pulmonary disorder (e.g., asthma, chronic obstructive pulmonary disease, bronchiectasis, or cystic fibrosis). In a specific embodiment, the compound is 2-(2-bromo-2,2-difluoro-acetylamino)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxylic acid o-tolylamide.

In another embodiment, a method of treating a condition, disease, or disorder associated with abnormally increased chloride ion secretion is provided, wherein the method comprises administering to a subject a pharmaceutical composition comprising a physiologically acceptable excipient and a compound of Formula (I) or any one of the substructures as represented by Formulae (I-1)-(I-14), in an amount effective to inhibit a calcium-activated chloride channel, thereby inhibiting chloride ion secretion. In one certain embodiment, the disease or disorder is abnormally (i.e., aberrantly) increased intestinal fluid secretion. In a particular embodiment, the disease or disorder is secretory diarrhea. In another particular embodiment, the condition, which may be a condition of the disease or disorder described herein is abnormally increased mucus secretion. In certain embodiments, a disease or disorder that comprises the condition of abnormally increased mucus secretion is asthma, chronic obstructive pulmonary disease, bronchiectasis, or cystic fibrosis. In certain embodiments, the method of treating a disease or disorder further comprising administering to the subject an agent that inhibits ion transport by a cystic fibrosis transmembrane conductance regulator (CFTR). In a specific embodiment, the compound is 2-(2-bromo-2,2-difluoro-acetylamino)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxylic acid o-tolylamide.

Another embodiment provides a method for reducing pain in a subject in need thereof comprising administering to the subject an effective amount of a pharmaceutical composition comprising a physiologically acceptable excipient and a compound of Formula (I) or any one of the substructures as represented by Formulae (I-1)-(I-14).

Another embodiment provides a method for treating cancer in a subject in need thereof comprising administering to the subject an effective amount of a pharmaceutical composition comprising a physiologically acceptable excipient and a compound of Formula (I) or any one of the substructures as represented by Formulae (I-1)-(I-14). In various embodiments, the cancer may be gastrointestinal stromal, esophageal cancer, PR-positive or HER2-negative breast cancer.

Also provided herein is a use of a composition or a compound of Formula (I) or any one of the substructures as represented by Formulae (I-1)-(I-14), for treating a condition, disease, or disorder associated with abnormally increased chloride ion secretion from a cell. In specific embodiments, the disease or disorder is secretory diarrhea, asthma, chronic obstructive pulmonary disease, bronchiectasis, or cystic fibrosis. In a specific embodiment, the compound is 2-(2-bromo-2,2-difluoro-acetylamine)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxylic acid o-tolylamide.

Also provided herein is a use of a composition or a compound of Formula (I) or any one of the substructures as represented by Formulae (I-1)-(I-14), for reducing or managing pain, or for treating cancer. In a specific embodiment, the compound is 2-(2-bromo-2,2-difluoro-acetylamino)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxylic acid o-tolylamide.

Another embodiment provides use of a composition or a compound of Formula (I) or any one of the substructures as represented by Formulae (I-1)-(I-14), for the manufacture of a medicament for treating a condition, disease, or disorder associated with abnormally increased chloride ion secretion from a cell. In a certain embodiment, the cell is an epithelial cell. In a particular embodiment, the epithelial cell is an intestinal or lung epithelial cell. In specific embodiments, the disease or disorder is secretory diarrhea, asthma, chronic obstructive pulmonary disease, bronchiectasis, or cystic fibrosis. In a specific embodiment, the compound is 2-(2-bromo-2,2-difluoro-acetylamino)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxylic acid o-tolylamide.

EXAMPLES

Abbreviations

Unless otherwise indicated, all reaction solvents were anhydrous and obtained as such from commercial sources. Difluoroiodoacetic acid was purchased from Synquest Laboratories (Alachua, Fla.). All other reagents were used as supplied. RP-HPLC analysis was performed using a Dionex Ultimate 3000 system, using a C18column [3×150 mm]. Low resolution ESI-LCMS was carried out with an Agilent 1100 HPLC coupled to an Agilent 1956B MSD. RP-HPLC runs typically employed gradients of two solvents: [A]=H2O (0.05% TFA) and [B] CH3CN (0.05% TFA); RP-LCMS used the same solvent system using the modifier formic acid (88% aq). The standard HPLC and LCMS gradients proceeded with [A:B]=95:5 to [A:B]=5:95 over 10 minutes. HRMS was performed using a hybrid quadrupole orbitrap mass analyzer, QExactive (Thermo, Bremen, Germany), with an electrospray ionization source. The mass resolution was set as 70,000 at m/z 200 and the mass accuracy was less than 3 ppm.1H and13C NMR spectra were recorded on a Bruker 500 MHz instrument.1H NMR chemical shifts are relative to TMS (δ=0.00 ppm), CDCl3(δ 7.26), CD3OD (δ=4.87 and 3.31), acetone-d6(δ 2.05), or DMSO-d6(δ 2.5).13C NMR chemical shifts are relative to CD3OD (δ 49.2) or CDCl3(δ 77.2).

General procedure 1: substituted cyanoacetamides (2a-2k) prepared from coupling of substituted anilines (1a-1k) with cyanoacetic acid. Substituted aniline (typical scale 6 mmol) (1a-1c) was dissolved in DCM (0.5M), followed by addition of cyanoacetic acid (1.0 eq) and EDCI-HCl (1.2 eq) and was stirred under argon at RT for 1h. LCMS indicated consumption of starting material and formation of product. The mixture was concentrated in vacuo. The crude product was treated with HCl (0.1M aq; 100 ml), transferred to a separatory funnel and extracted with ethyl acetate. The organic phase was then washed with additional HCl (0.1M aq), water, NaCl (satd aq), and was then dried over Na2SO4and concentrated in vacuo, to yield the title products (2a-2k) (shown below) typically as a colorless to pink solids:

Table 1 shows the synthesis yields for EDCI-mediated cyanoacetamide according to the General procedure 1; yields (%) are of the isolated or purified products. Purity of compounds was >95% based on HPLC-LCMS analysis at 254 nm, and absence of impurities was confirmed by1H NMR spectra.

General procedure 2: 2-aminocycloalkylthiophene-3-carboxylic acid arylamides (5a-5v) prepared in a two step process from cyclic ketones (3a-3d) and substituted cyanoacetamides (2a-2k). Substituted cyanoacetamides (typical scale 3 mmol) (2a-2k) was dissolved in toluene (0.1 M), followed by the addition of the appropriate cyclic ketone (3a-3d) (7.0 eq), ammonium acetate (5.0 eq), glacial acetic acid (7.0 eq), and Na2SO4(5.0 eq). This reaction mixture was refluxed at 100° C. for 1 h. LCMS indicated consumption of starting material and the formation of the desired Knoevenagel product (4a-4v), as well as excess ketone starting material. The mixture was then cooled to RT and treated with NaHCO3(5% aq), and transferred into a separatory funnel for extraction of product with ethyl acetate (3×10 ml). The organic mixture was then washed with additional NaHCO3(5% aq), water, and NaCl (5% aq), and then then dried over Na2SO4and concentrated in vacuo, to yield the title products (4a-4v) typically as a dark to red orange oils. The identity and purity of the Knoevenagel product was confirmed by LCMS, and the materials were typically used without additional purification or characterization.

Knoevenagel products (4a-4v) (2.674 mmol) were dissolved in ethanol (0.1 M) and treated with molecular octasulfur (“S8”) (2.0 eq) and morpholine (3.0 eq). The mixture was refluxed at 90° C. for 5 h. LCMS was used to follow the progress of the reaction. The mixture was then left to cool to RT. Upon reaching RT, the reaction mixture was filtered by using a Buchner funnel, to remove excess precipitated inorganic sulfur materials. The filtrate was then concentrated in vacuo, and the 2-aminocycloalkylthiophene intermediates (5a-5v) (shown below) were isolated as orange to brown solids.

Table 2 shows the synthesis yields for Knoevenagel reaction and subsequent aminothiophene formation reactions. Yields (%) are of the isolated or purified products. Purity of compounds was >95% based on HPLC-LCMS analysis at 254 nm, and absence of impurities was confirmed by1H NMR spectra.

General procedure 3: 2-acylamino-cycloalkylthiophene-3-carboxylic acid arylamides (6aa-6bv; with difluoroiodoacetyl compounds prepared by General Procedure 4) prepared from acylation of 2-aminocycloalkylthiophenes (5a-5v). 2-Aminocycloalkylthiophenes (typically 0.782 mmol) were dissolved in DCM (0.1 M), treated with triethylamine (1.3 eq) and an appropriate acylating agent (e.g. trifluoroacetic anhydride, pentafluoric propionic anhydride, acetic anhydride, propionic anhydride, chlorodifluoroacetic, bromodifluoroacetic, or heptafluorobutyric anhydride) (1.3 eq). The reactions were stirred at RT typically for 10 min, until LCMS confirmed consumption of starting material and formation of product. The mixture was then transferred into a separatory funnel with ethyl acetate. The organic layer was then washed with water, NaCl (satd aq), dried over Na2SO4, and then concentrated in vacuo to yield colorless to light yellow solids (shown below):

Table 3 shows the coupling yields and TMEM16A inhibition of AACT compounds (6aa-6bw). Yields (%) are of the isolated or purified products. IC50(μM) for inhibition of TMEM16A anion conductance using a fluorescence plate reader (FPR) assay. Purity of active compounds was >95% based on HPLC-LCMS analysis at 254 nm, and absence of impurities was confirmed by inspection of1H NMR spectra.

General procedure 4: 2-difluoroiodoacetylamino-cycloalkylthiophene-3-carboxylic acid arylamides (6bn, 6bq, 6bt, 6bw) prepared from EDCI-mediated coupling of 2-amino-cycloalkylthiophenes (5) with difluoroiodoacetic acid. 2-Amino-cycloalkylthiophenes (typically 0.164 mmol) were dissolved in 1.6 mL DCM (0.1 M), treated with 4-DMAP (0.1 eq) and iododifluoroacetic acid (1.2 eq), followed by EDCI-HCl (1.5 eq). The reactions were stirred at RT for 60 min, until LCMS confirmed consumption of starting material and formation of product. Reaction mixtures were diluted with ethyl acetate and was washed with HCl (0.1 M aq) (×3) followed by NaCl (satd aq). Organic layers were dried in vacuo and solids were triturated from diethyl ether. The products were isolated as off-white solids.

N-(5-chloro-2-methoxyphenyl)-2-(2,2,2-trifluoroacetamido)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxamide (am1_47) was Prepared According to the General Procedure 3 as Disclosed Herein

TMEM16A functional assay. TMEM16A functional plate-reader assay was done as previously described. (See Namkung, W. et al.,J Biol Chem2011, 286, (3), 2365-74.) Briefly, each well of 96-well plate containing the TMEM16A-expressing FRT cells was washed twice with phosphate buffer saline (PBS) leaving 50 μl. Test compounds (0.5 μl in DMSO) were added to each well at specified concentration. After 10 min each well was assayed individually for TMEM16A-mediated I−influx by recording fluorescence continuously (400 ms/point) for 2 s (baseline), then 50 μl of 140 mM I−solution containing 300 μM ATP was added at 2 s, and fluorescence was further read for 12 s. The initial rate of I− influx following each of the solution additions was computed from fluorescence data by nonlinear regression. TMEM16B activity was assayed similarly as described using FRT cells co-expressing YFP and TMEM16B. (See Namkung, W. et al.,J Biol. Chem2011, 286, (3), 2365-74.)

In Vitro Metabolic Stability

Compounds (each 10 uM) were incubated for specific time points (2, 5, 15, 30, 60, 180 min) with shaking at 37° C. with rat liver microsomes (1 mg protein/mL, Sigma-Aldrich, St. Louis, Mo.) in potassium phosphate butler (100 mM) containing 1 mM NADPH. The mixture was then chilled on ice, and 0.5 mL of ice-cold ethyl acetate was added. Samples were centrifuged for 15 min at 3000 RPM. The supernatant was evaporated to dryness, and the residue was dissolved in 80 μL of mobile phase (acetonitrile:/water, 3:1, containing 0.1% formic acid) for LC/MS. Reverse-phase HPLC separation was carried out using a Waters C18column (2.1 mm×100 mm, 3.5 mm particle size) equipped with a solvent delivery system (Waters model 2690, Milford, Mass.). The solvent system consisted of a linear gradient from 5% to 95% acetonitrile run over 16 min (0.2 mL/min flow rate).

Plate reader assays of chloride channel function, CFTR inhibition was assayed as described. (See Tradtrantip, L.,J Med Chem2009, 52, (20), 6447-55.) Briefly, FRT cells co-expressing YFP and wildtype CFTR were washed with phosphate-buffered saline (PBS) and then incubated for 15 min with test compounds in PBS containing 20 μM forskolin. I−influx was measured in a plate reader with initial baseline read for 2 s and then for 12 s after rapid addition of an I−containing solution. Activity of non-TMEM16A CaCC was assayed as described in HT-29 cells expressing YFP. (See De La Fuente, R. et al.,Mol Pharmacol2008, 73, (3), 758-68.) In each assay initial rates of I− influx were computed as a linear measure of channel function.

Cytotoxicity. FRT cells were cultured overnight in black 96-well Costar microplates and incubated with 5 μM test compounds for 8 h. Cytotoxicity was measured by Alamar Blue assay (Invitrogen, Carlsbad, Calif.) as per the manufacturer's instructions.

FIG. 3Csummarizes the time course of compound degradation showing remarkably greater stability of 6bm compared to Ani-9. 6bm could be potentially metabolized by amide-bond hydrolysis or oxidation of the benzene or aryl methyl. It is speculated that Ani-9 could be oxidized at the aryl methyl or N—N bond; or hydrolyzed at the amide or hydrazone linkages.

Ex Vivo Intestinal Contractility

The effect of a compound according to one embodiment on intestinal contractility was determined in an ex vivo preparation of mouse ileum. Adult mice (CD1 genetic background) were euthanized by avertin overdose (200 mg/kg, 2,2,2-tribromethanol, Sigma-Aldrich) and ileal segments of ˜2 cm length were isolated and washed with Krebs-Henseleit buffer (pH 7.4, in mM: 118 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, 2.5 CaCl2, 11 D-glucose). The ends of the ileal segments were tied, connected to a force transducer (Biopac Systems, Goleta, Calif.) and tissues were transferred to an organ chamber (Biopac Systems) containing Krebs-Henseleit buffer at 37° C. aerated with 95% O2, 5% CO2. Tissues were stabilized for 60 min with resting tension of 0.5 g and solutions were changed every 15 min. Effects of 6bm on baseline isometric intestinal contractions were recorded. As is shown inFIG. 4, 6bm strongly inhibited spontaneous isometric contractions of ileum in a concentration-dependent manner.

Inhibition Study of Tumor Cell Lines

Compounds according to the present disclosure (structures shown inFIG. 5B) were tested for their inhibitory effect on proliferation of SW-480 cell line.

SW-480 cells were plated into black 96-well microplates (Corning-Costar Corp., New York, N.Y.) at 10% seeding. The cells were treated with test compounds dissolved in DMSO in serial dilution while maintaining the final concentration of DMSO at 0.5%. Plated cells were then incubated for 72 h at 37° C. Cell proliferation was quantified using the AlamarBlue assay (Invitrogen, Carlsbad, Calif.) as per the manufacturer's instructions. Data was normalized against untreated control cells that were lysed for 10 minutes using Triton-X100 (0.1% in PBS).

Compound am1_47 was shown as considerably more potent than CaCCinh-A01 (6-t-butyl-2-(furan-2-carboxamido)-4,5,6,7-tetrahydrobenzo[b] thiophene-3-carboxylic acid) in inhibiting proliferation of SW480 cells. The compound CaCCinh-A01 has been reported to inhibit proliferation of several types of TMEM16A-expressing tumor cell lines, including SW480, albeit at relatively high concentrations (10-20 μM).

Pharmacokinetic Studies

The pharmacokinetics of compounds according to certain embodiments was studied in rats. Rats (Wistar males, 250-300 g) were purchased from Charles River Laboratories (Wilmington, Mass.) and were treated with 4 mg/kg 6bm (in 5% DMSO, 10% Kolliphor HS in saline) either intraperitoneally or by oral gavage. After treatments blood samples were collected by tail vein puncture at specified time points (15, 30, 60, 120 and 180 min). Serum was separated by centrifuging blood samples at 5000 rpm for 15 min. 6bm concentration were quantified as follows; A 190 μL aliquot plasma, containing different concentrations of bromodifluoroacetyl compound 6bm, was spiked with 10 μL of the chlorodifluoroacetyl-containing internal standard 6bk. The aliquot was then diluted to a total of 2 mL in PBS. A C18solid-phase extraction (SPE) column (Thermo Fisher, Waltham, Mass.) was conditioned with methanol (2 mL) and PBS (2 mL). The aliquot was loaded onto the conditioned SPE column, and washed with PBS (2 mL) followed by water (2 mL), and the column then dried under vacuum for 5 min. Analytes were eluted with ethyl acetate (3 mL), and concentrated by rotary evaporation. The residues were reconstituted into methanol (150 μL), and then analyzed using a hybrid quadrupole Q-Exactive Orbitrap mass analyzer (Thermo Fisher, Waltham, Mass.), with an electrospray ionization source, employing parallel-reaction monitoring (PRM) to provide high analytical sensitivity.

Intraperitoneal administration of 6bm at 4 mg/kg yielded serum concentrations greater than 3.5 μM for up to 2 h (peak 6.5 μM at 60 min); while oral administration at the same dose produced serum concentrations greater than 0.5 μM for 3 h (peak 1.9 μM at 2 h). Both administration methods produced serum concentrations well above the IC50of 6bm (30 nM) for inhibition of TMEM16A. No toxic effect was seen.

FIG. 6shows that compound 6bm is detectable at pharmacologically relevant concentrations after intraperitoneal (IP) or oral administration (PO).

Hypertension Indication

TMEM16A is expressed in vascular smooth muscle and its inhibition may reduce blood pressure. In a proof of concept study, AACT inhibitor 6bm was administered acutely to rats made hypertensive with phenylephrine. More specifically, a wild-type anesthetized rat was made hypertensive with intravenous (IV) phenylephrine (PE, 0.25 mg) and then administered IV vehicle (Veh, 5% DMSO-10% Kolliphor HS in saline) and then 0.3 mg 6bm (IV, in vehicle). Blood pressure was measured by femoral artery catheter.

As shown inFIG. 7, the compound 6bm demonstrated acute reduction in blood pressure, indicating that the compounds according to the present disclosure can be suitable for treating hypertension.